Publikationen des SFB 765

377 Publikation(en)

Density-based cluster algorithms for the identification of core sets

Oliver Lemke and Bettina G. Keller

J. Chem. Phys. 2016, 145, 164104 ...

The core-set approach is a discretization method for Markov state models of complex molecular dynamics. Core sets are disjoint metastable regions in the conformational space, which need to be known prior to the construction of the core-set model. We propose to use density-based cluster algorithms to identify the cores. We compare three different density-based cluster algorithms: the CNN, the DBSCAN, and the Jarvis-Patrick algorithm. While the core-set models based on the CNN and DBSCAN clustering are well-converged, constructing core-set models based on the Jarvis-Patrick clustering cannot be recommended. In a well-converged core-set model, the number of core sets is up to an order of magnitude smaller than the number of states in a conventional Markov state model with comparable approximation error. Moreover, using the density-based clustering one can extend the core-set method to systems which are not strongly metastable. This is important for the practical application of the core-set method because most biologically interesting systems are only marginally metastable. The key point is to perform a hierarchical density-based clustering while monitoring the structure of the metric matrix which appears in the core-set method. We test this approach on a molecular-dynamics simulation of a highly flexible 14-residue peptide. The resulting core-set models have a high spatial resolution and can distinguish between conformationally similar yet chemically different structures, such as register-shifted hairpin structures.

Intradomain Allosteric Network Modulates Calcium Affinity of the C‑Type Lectin Receptor Langerin

Jonas Hanske, Stevan Aleksić, Martin Ballaschk, Marcel Jurk, Elena Shanina, Monika Beerbaum, Peter Schmieder, Bettina G. Keller, and Christoph Rademacher

J. Am. Chem. Soc., 2016, 138 (37), 12176–12186 ...

Antigen uptake and processing by innate immune cells is crucial to initiate the immune response. Therein, the endocytic C-type lectin receptors serve as pattern recognition receptors, detecting pathogens by their glycan structures. Herein, we studied the carbohydrate recognition domain of Langerin, a C-type lectin receptor involved in the host defense against viruses such as HIV and influenza as well as bacteria and fungi. Using a combination of nuclear magnetic resonance and molecular dynamics simulations, we unraveled the molecular determinants underlying cargo capture and release encoded in the receptor architecture. Our findings revealed receptor dynamics over several time scales associated with binding and release of the essential cofactor Ca2+ controlled by the coupled motions of two loops. Applying mutual information theory and site-directed mutagenesis, we identified an allosteric intradomain network that modulates the Ca2+ affinity depending on the pH, thereby promoting fast ligand release.

19F NMR-Guided Design of Glycomimetic Langerin Ligands

E.-C. Wamhoff, J. Hanske, L. Schnirch, J. Aretz, M. Grube, D. Varón Silva, and C. Rademacher

ACS Chem. Biol., 2016, 11 (9), 2407–2413 ...

C-type lectin receptors (CLRs) play a pivotal role in pathogen defense and immune homeostasis. Langerin, a CLR predominantly expressed on Langerhans cells, represents a potential target receptor for the development of anti-infectives or immunomodulatory therapies. As mammalian carbohydrate binding sites typically display high solvent exposure and hydrophilicity, the recognition of natural monosaccharide ligands is characterized by low affinities. Consequently, glycomimetic ligand design poses challenges that extend to the development of suitable assays. Here, we report the first application of 19F R2-filtered NMR to address these challenges for a CLR, i.e., Langerin. The homogeneous, monovalent assay was essential to evaluating the in silico design of 2-deoxy-2-carboxamido-α-mannoside analogs and enabled the implementation of a fragment screening against the carbohydrate binding site. With the identification of both potent monosaccharide analogs and fragment hits, this study represents an important advancement toward the design of glycomimetic Langerin ligands and highlights the importance of assay development for other CLRs.

Inhibition of key enzyme of sialic acid biosynthesis by C6-Se modified N-acetylmannosamine analogs

Olaia Nieto-Garcia, Paul R. Wratil, Long D. Nguyen, Verena Böhrsch, Stephan Hinderlich, Werner Reutter and Christian P. R. Hackenberger

Chem. Sci., 2016, 7, 3928-3933 ...

Synthetically accessible C6-analogs of N-acetylmannosamine (ManNAc) were tested as potential inhibitors of the bifunctional UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase (GNE/MNK), the key enzyme of sialic acid biosynthesis. Enzymatic experiments revealed that the modification introduced at the C6 saccharide position strongly influences the inhibitory potency. A C6-ManNAc diselenide dimer showed the strongest kinase inhibition in the low μM range among all the substrates tested and successfully reduced cell surface sialylation in Jurkat cells.

From hydration repulsion to dry adhesion between asymmetric hydrophilic and hydrophobic surfaces

Matej Kanduč and Roland R. Netz

Proc. Natl. Acad. Sci. USA, 2016, 112(40), 12338–12343 ...

Using all-atom molecular dynamics (MD) simulations at constant water chemical potential in combination with basic theoretical arguments, we study hydration-induced interactions between two overall charge-neutral yet polar planar surfaces with different wetting properties. Whether the water film between the two surfaces becomes unstable below a threshold separation and cavitation gives rise to long-range attraction, depends on the sum of the two individual surface contact angles. Consequently, cavitation-induced attraction also occurs for a mildly hydrophilic surface interacting with a very hydrophobic surface. If both surfaces are very hydrophilic, hydration repulsion dominates at small separations and direct attractive force contribution can-if strong enough-give rise to wet adhesion in this case. In between the regimes of cavitation-induced attraction and hydration repulsion we find a narrow range of contact angle combinations where the surfaces adhere at contact in the absence of cavitation. This dry adhesion regime is driven by direct surface-surface interactions. We derive simple laws for the cavitation transition as well as for the transition between hydration repulsion and dry adhesion, which favorably compare with simulation results in a generic adhesion state diagram as a function of the two surface contact angles.

Hydration force fluctuations in hydrophilic planar systems

Matej Kanduč and Roland R. Netz

Biointerphases, 2016, 11, 019004 ...

Utilizing all-atom simulations with explicit solvent, the authors model hydrophilicsurfacesinteracting across water at a fixed chemical potential. They extract the hydration forces acting between the surfaces and assess force fluctuations as well as interlamellar water number fluctuations. The trends obtained from the simulations are captured by a continuum-based description with effective model parameters. The significance of fluctuations depends on surfacehydrophilicity and rigidity. The authors show that the force fluctuations play an important role in kinetic processes in systems with lateral sizes smaller than several tens of nanometers.

Bis(arylmethyl)-substituted unsymmetrical phosphites for the synthesis of lipidated peptides via Staudinger-phosphite reactions

Nicole Nischan, Marc-André Kasper, Thresen Mathew, and Christian P. R. Hackenberger

Org. Biomol. Chem., 2016,14, 7500-7508 ...

With this study we introduce new unsymmetrical phosphites to obtain lipidated peptide-conjugates starting from easily accessible azide-modified amino acid or peptide precursors. For this purpose, we investigated which substituents at alkyl phosphites lead to the highest formation of mono-alkylated phosphoramidate peptides. We found that phosphites containing one alkyl-chain and two picolyl or benzyl-substituents delivered alkyl phosphoramidate-conjugates in high yields, which also allowed a chemoselective lipidation of an unprotected azido polypeptide. Finally, monolipidated phosphoramidate peptides obtained by the unsymmetrical Staudinger phosphite reaction led to the formation of micelle-like structures and cellular uptake.

Congeneric lipases with improved catalytic activity and substrate accessibility / Engineering lipases with an expanded genetic code.

Alessandro De Simone,Michael Georg Hoesl, and Nediljko Budisa

Applied Biocatalysis - From Fundamental Science to Industrial Applications. Wiley-VCH. 2016, ISBN: 978-3-527-33669-2 ...

This reference book originates from the interdisciplinary research cooperation between academia and industry. In three distinct parts, latest results from basic research on stable enzymes are explained and brought into context with possible industrial applications. Downstream processing technology as well as biocatalytic and biotechnological production processes from global players display the enormous potential of biocatalysts. Application of "extreme" reaction conditions (i.e. unconventional, such as high temperature, pressure, and pH value) - biocatalysts are normally used within a well defined process window - leads to novel synthetic effects. Both novel enzyme systems and the synthetic routes in which they can be applied are made accessible to the reader. In addition, the complementary innovative process technology under unconventional conditions is highlighted by latest examples from biotech industry.

Incorporation of Amino Acids with Long-Chain Terminal Olefins into Proteins

Matthias P. Exner, Sebastian Köhling, Julie Rivollier, Sandrine Gosling, Puneet Srivastava, Zheni I. Palyancheva, Piet Herdewijn, Marie-Pierre Heck, Jörg Rademann, and Nediljko Budisa

Molecules 2016, 21(3), 287

The increasing need for site-specific protein decorations that mimic natural posttranslational modifications requires access to a variety of noncanonical amino acids with moieties enabling bioorthogonal conjugation chemistry. Here we present the incorporation of long-chain olefinic amino acids into model proteins with rational variants of pyrrolysyl-tRNA synthetase (PylRS). Nε-heptenoyl lysine was incorporated for the first time using the known promiscuous variant PylRS(Y306A/Y384F), and Nε-pentenoyl lysine was incorporated in significant yields with the novel variant PylRS(C348A/Y384F). This is the only example of rational modification at position C348 to enlarge the enzyme’s binding pocket. Furthermore, we demonstrate the feasibility of our chosen amino acids in the thiol-ene conjugation reaction with a thiolated polysaccharide.

Towards Reassignment of the Methionine Codon AUG to Two Different Noncanonical Amino Acids in Bacterial Translation

Alessandro De Simone, Carlos G. Acevedo-Rocha, Michael Georg Hoesl, Nediljko Budisa

Croat. Chem. Acta 2016, 89(2)

Genetic encoding of noncanonical amino acids (ncAAs) through sense codon reassignment is an efficient tool for expanding the chemical functionality of proteins. Incorporation of multiple ncAAs, however, is particularly challenging. This work describes the first attempts to reassign the sense methionine (Met) codon AUG to two different ncAAs in bacterial protein translation. Escherichia coli methionyl-tRNA synthetase (MetRS) charges two tRNAs with Met: tRNAfMet initiates protein synthesis (starting AUG codon), whereas elongator tRNAMet participates in protein elongation (internal AUG codon(s)). Preliminary in vitro experiments show that these tRNAs can be charged with the Met analogues azidohomoalanine (Aha) and ethionine (Eth) by exploiting the different substrate specificities of EcMetRS and the heterologous MetRS / tRNAMet pair from the archaeon Sulfolobus acidocaldarius, respectively. Here, we explored whether this configuration would allow a differential decoding during in vivo protein initiation and elongation. First, we eliminated the elongator tRNAMet from a methionine auxotrophic E. coli strain, which was then equipped with a rescue plasmid harboring the heterologous pair. Although the imported pair was not fully orthogonal, it was possible to incorporate preferentially Eth at internal AUG codons in a model protein, suggesting that in vivo AUG codon reassignment is possible. To achieve full orthogonality during elongation, we imported the known orthogonal pair of Methanosarcina mazei pyrrolysyl-tRNA synthetase (PylRS) / tRNAPyl and devised a genetic selection system based on the suppression of an amber stop codon in an important glycolytic gene, pfkA, which restores enzyme functionality and normal cellular growth. Using an evolved PylRS able to accept Met analogues, it should be possible to reassign the AUG codon to two different ncAAs by using directed evolution.

Towards Biocontained Cell Factories: An Evolutionarily Adapted Escherichia coli Strain Produces a New-to-nature Bioactive Lantibiotic Containing Thienopyrrole-Alanine

Anja Kuthning, Patrick Durkin, Stefan Oehm, Michael G. Hoesl, Nediljko Budisa and Roderich D. Süssmuth

Scientific Reports 2016, 6, 33447

Genetic code engineering that enables reassignment of genetic codons to non-canonical amino acids (ncAAs) is a powerful strategy for enhancing ribosomally synthesized peptides and proteins with functions not commonly found in Nature. Here we report the expression of a ribosomally synthesized and post-translationally modified peptide (RiPP), the 32-mer lantibiotic lichenicidin with a canonical tryptophan (Trp) residue replaced by the ncAA L-β-(thieno[3,2-b]pyrrolyl)alanine ([3,2]Tpa) which does not sustain cell growth in the culture. We have demonstrated that cellular toxicity of [3,2]Tpa for the production of the new-to-nature bioactive congener of lichenicidin in the host Escherichia coli can be alleviated by using an evolutionarily adapted host strain MT21 which not only tolerates [3,2]Tpa but also uses it as a proteome-wide synthetic building block. This work underscores the feasibility of the biocontainment concept and establishes a general framework for design and large scale production of RiPPs with evolutionarily adapted host strains.

Anomeric thiols from unprotected sugars in water for anomeric ligations

S. Köhling, M. P. Exner, S. Nojoumi, J. Schiller, N. Budisa, J. Rademann

Angew. Chem. Int. Ed. 2016, 55, in press

Design of an S-Allylcysteine in situ production and incorporation system based on a novel pyrrolysyl-tRNA synthetase variant

M. P. Exner, T. Kuenzl, S. Schwagerus, T. To, Z. Ouyang, M. G. Hoesl, M. C. Lensen, C. P. R. Hackenberger, S. Panke, and N. Budisa

ChemBioChem 2016, in press

Residue-specific Incorporation of Noncanonical Amino Acids into Model Proteins Using an Escherichia coli Cell-free Transcription-translation System

Emanuel G. Worst, Matthias P. Exner, Alessandro De Simone, Marc Schenkelberger, Vincent Noireaux, Nediljko Budisa, Albrecht Ott

J. Vis. Exp., 2016, 114, e54273 ...

The canonical set of amino acids leads to an exceptionally wide range of protein functionality. Nevertheless, the set of residues still imposes limitations on potential protein applications. The incorporation of noncanonical amino acids can enlarge this scope. There are two complementary approaches for the incorporation of noncanonical amino acids. For site-specific incorporation, in addition to the endogenous canonical translational machineries, an orthogonal aminoacyl-tRNA-synthetase-tRNA pair must be provided that does not interact with the canonical ones. Consequently, a codon that is not assigned to a canonical amino acid, usually a stop codon, is also required. This genetic code expansion enables the incorporation of a noncanonical amino acid at a single, given site within the protein. The here presented work describes residue-specific incorporation where the genetic code is reassigned within the endogenous translational system. The translation machinery accepts the noncanonical amino acid as a surrogate to incorporate it at canonically prescribed locations, i.e., all occurrences of a canonical amino acid in the protein are replaced by the noncanonical one. The incorporation of noncanonical amino acids can change the protein structure, causing considerably modified physical and chemical properties. Noncanonical amino acid analogs often act as cell growth inhibitors for expression hosts since they modify endogenous proteins, limiting in vivo protein production. In vivo incorporation of toxic noncanonical amino acids into proteins remains particularly challenging. Here, a cell-free approach for a complete replacement of L-arginine by the noncanonical amino acid L-canavanine is presented. It circumvents the inherent difficulties of in vivo expression. Additionally, a protocol to prepare target proteins for mass spectral analysis is included. It is shown that L-lysine can be replaced by L-hydroxy-lysine, albeit with lower efficiency. In principle, any noncanonical amino acid analog can be incorporated using the presented method as long as the endogenous in vitro translation system recognizes it.

Synthesis, photophysical and biological evaluation of sulfated polyglycerol dendronized perylenebisimides (PBIs) – a promising platform for anti-inflammatory theranostic agents?

T Heek, C. Kuehne, H. Depner, K. Achazi, J. Dernedde, and R. Haag

Bioconjugate Chem.,2016, 27(3), 727–736 ...

A set of four water-soluble perylene bisimides (PBI) based on sulfated polyglycerol (PGS) dendrons were developed, their photophysical properties determined via UV/vis and fluorescence spectroscopy, and their performance as possible anti-inflammatory agents evaluated via biological in vitro studies. It could be shown that in contrast to charge neutral PG–PBIs the introduction of the additional electrostatic repulsion forces leads to a decrease in the dendron generation necessary for aggregation suppression, allowing the preparation of PBIs with fluorescence quantum yields of >95% with a considerable decreased synthetic effort. Furthermore, the values determined for L-selectin binding down to the nanomolar range, their limited impact on blood coagulation, and their minor activation of the complement system renders these systems ideal for anti-inflammatory purposes.

Restoring the Oncosuppressor Activity of MicroRNA-34a in Glioblastoma Using a Polyglycerol-based Polyplex.

Paula Ofek, Marcelo Calderón, Fatemeh Sheikhi Mehrabadi, Adva Krivitsky, Shiran Ferber, Galia Tiram, Noga Yerushalmi, Sharon Kredo-Russo, Rachel Grossm, Zvi Ram, Rainer Haag, Ronit Satchi-Fainaro

Nanomedicine: NBM, 2016, 12 (7), 2201–2214 ...

Glioblastoma multiforme (GBM) is the most common and aggressive primary neoplasm of the brain. Poor prognosis is mainly attributed to tumor heterogeneity, invasiveness, and drug resistance. microRNA-based therapeutics represent a promising approach due to their ability to inhibit multiple targets. In this work, we aim to restore the oncosuppressor activity of microRNA-34a (miR-34a) in GBM. We developed a cationic carrier system, dendritic polyglycerolamine (dPG-NH2), which remarkably improves miRNA stability, intracellular trafficking, and activity. dPG-NH2 carrying mature miR-34a targets C-MET, CDK6, Notch1 and BCL-2, consequently inhibiting cell cycle progression, proliferation and migration of GBM cells. Following complexation with dPG-NH2, miRNA is stable in plasma and able to cross the blood–brain barrier. We further show inhibition of tumor growth following treatment with dPG-NH2–miR-34a in a human glioblastoma mouse model. We hereby present a promising technology using dPG-NH2–miR-34a polyplex for brain-tumor treatment, with enhanced efficacy and no apparent signs of toxicity.

Synthesis of pH-Cleavable dPG-Amines for Gene Delivery Application

Mathias Dimde, Dirk Steinhilber, Falko Neumann, Yan Li, Florian Paulus, Nan Ma, and Rainer Haag

Macromol. Biosci.,2016, accepted ...

The development of effective nonviral vectors for gene therapy is still a challenge in research, due to the high toxicity of many existing polycationic nanocarriers. In this paper, the development of two pH-cleavable polyglycerol-amine-based nanocarriers is described. The benz­acetal bond represents the pH-sensitive cleavage site between dendritic polyglycerol (dPG) and glycerol-based 1,2-diamines that can complex genetic material. Due to the acid lability of the acetal moiety, the cleavable dPG-amines are less toxic in vitro. Cell-mediated degradation results in non-toxic dPG with low amine functionalization and low molecular weight cleavage products (cp). The genetic material is released because of the loss of multivalent amine groups. Interestingly, the release kinetics at the endosomal pH could be controlled by simple chemical modification of the acetals. In vitro experiments demonstrate the ability of the cleavable dPG-amine to transfect HeLa cells with GFP-DNA, which resulted in cell-compatible cleavage products.

Pathogen Inhibition by Multivalent Ligand Architectures.

S. Bhatia, J. L. Cuellar Camacho and R. Haag

J. Am. Chem. Soc.,2016, 138 (28), 8654–8666 ...

Interfacial multivalent interactions at pathogen–cell interfaces can be competitively inhibited by multivalent scaffolds that prevent pathogen adhesion to the cells during the initial stages of infection. The lack of understanding of complex biological systems makes the design of an efficient multivalent inhibitor a toilsome task. Therefore, we have highlighted the main issues and concerns associated with blocking pathogen at interfaces, which are dependent on the nature and properties of both multivalent inhibitors and pathogens, such as viruses and bacteria. The challenges associated with different cores or carrier scaffolds of multivalent inhibitors are concisely discussed with selected examples.

Highly Efficient Multivalent 2D Nanosystems for Inhibition of Orthopoxvirus Particles

B. Ziem, H. Thien, K. Achazi, C. Yue, D. Stern, K. Silberreis, M. Fardin Gholami, F. Beckert, D. Gröger, R. Mülhaupt, J. P. Rabe, A. Nitsche, and R. Haag

Adv. Healthcare Mater.,2016, accepted ...

Efficient inhibition of cell–pathogen interaction to prevent subsequent infection is an urgent but yet unsolved problem. In this study, the synthesis and functionalization of novel multivalent 2D carbon nanosystems as well as their antiviral efficacy in vitro are shown. For this reason, a new multivalent 2D flexible carbon architecture is developed in this study, functionalized with sulfated dendritic polyglycerol, to enable virus interaction. A simple “graft from” approach enhances the solubility of thermally reduced graphene oxide and provides a suitable 2D surface for multivalent ligand presentation. Polysulfation is used to mimic the heparan sulfate-containing surface of cells and to compete with this natural binding site of viruses. In correlation with the degree of sulfation and the grafted polymer density, the interaction efficiency of these systems can be varied. In here, orthopoxvirus strains are used as model viruses as they use heparan sulfate for cell entry as other viruses, e.g., herpes simplex virus, dengue virus, or cytomegalovirus. The characterization results of the newly designed graphene derivatives demonstrate excellent binding as well as efficient inhibition of orthopoxvirus infection. Overall, these new multivalent 2D polymer nanosystems are promising candidates to develop potent inhibitors for viruses, which possess a heparan sulfate-dependent cell entry mechanism.

Carbohydrates in Supramolecular Chemistry

Martina Delbianco, Priya Bharate, Silvia Varela-Aramburu, and Peter H. Seeberger

Chem. Rev.,2016, 116 (4), 1693–1752 ...

Carbohydrates are involved in a variety of biological processes. The ability of sugars to form a large number of hydrogen bonds has made them important components for supramolecular chemistry. We discuss recent advances in the use of carbohydrates in supramolecular chemistry and reveal that carbohydrates are useful building blocks for the stabilization of complex architectures. Systems are presented according to the scaffold that supports the glyco-conjugate: organic macrocycles, dendrimers, nanomaterials, and polymers are considered. Glyco-conjugates can form host–guest complexes, and can self-assemble by using carbohydrate–carbohydrate interactions and other weak interactions such as π–π interactions. Finally, complex supramolecular architectures based on carbohydrate–protein interactions are discussed.

Analysis of carbohydrate-carbohydrate interactions using sugar-functionalized silicon nanoparticles for cell imaging

Chian-Hui Lai, Julia Hütter, Chien-Wei Hsu, Hidenori Tanaka, Silvia Varela-Aramburu, Luisa De Cola, Bernd Lepenies, and Peter H. Seeberger

Nano Lett., 2016, 16 (1), pp 807–811 ...

Chemo-enzymatic Synthesis of Perfluoroalkyl-functionalized Dendronized Polymers as Cyto-compatible Nanocarriers for Drug Delivery Applications

Badri Parshad, Meena Kumari, Katharina Achazi, Christoph Bӧttcher, Rainer Haag, and Sunil K. Sharma

Polymers 2016, 8(8), 311

Among amphiphilic polymers with diverse skeletons, fluorinated architectures have attracted significant attention due to their unique property of segregation and self-assembly into discrete supramolecular entities. Herein, we have synthesized amphiphilic copolymers by grafting hydrophobic alkyl/perfluoroalkyl chains and hydrophilic polyglycerol [G2.0] dendrons onto a co-polymer scaffold, which itself was prepared by enzymatic polymerization of poly[ethylene glycol bis(carboxymethyl) ether]diethylester and 2-azidopropan-1,3-diol. The resulting fluorinated polymers and their alkyl chain analogs were then compared in terms of their supramolecular aggregation behavior, solubilization capacity, transport potential, and release profile using curcumin and dexamethasone drugs. The study of the release profile of encapsulated curcumin incubated with/without a hydrolase enzyme Candida antarctica lipase (CAL-B) suggested that the drug is better stabilized in perfluoroalkyl chain grafted polymeric nanostructures in the absence of enzyme for up to 12 days as compared to its alkyl chain analogs. Although both the fluorinated as well as non-fluorinated systems showed up to 90% release of curcumin in 12 days when incubated with lipase, a comparatively faster release was observed in the fluorinated polymers. Cell viability of HeLa cells up to 95% in aqueous solution of fluorinated polymers (100 μg/mL) demonstrated their excellent cyto-compatibility.

A Toolset with Different Linker Strategies for Application in Bioconjugation of Functionalized Porphyrins

M. H. Staegemann, S. Gräfe, R. Haag and A. Wiehe

Org. Biomol. Chem., 2016, 14, 9114-9132 ...

The reaction of amines with pentafluorophenyl-substituted A3B-porphyrins has been used to obtain different useful reactive groups for further functionalization and/or conjugation of these porphyrins to other substrates or materials. Porphyrins with alkenyl, alkynyl, amino, azido, epoxide, hydroxyl, and maleimido groups have thus been synthesized. For the first time such functionalized porphyrins have been conjugated to hyperbranched polyglycerol (hPG) as a biocompatible carrier system for photodynamic therapy (PDT) using the copper(I)-catalyzed 1,3-dipolar cycloaddition (CuAAC). The photocytotoxicity of selected porphyrins as well as of the porphyrin-hPG-conjugates has been assessed in cellular assays with human epidermoid carcinoma A-253 and squamous carcinoma CAL-27 cells. For several biomedical applications a release of the active drug and/or fluorescent dye is desired. Therefore, additionally, the synthesis of A3B-porphyrins with cleavable linker moieties is presented, namely disulfide, cleavable in a reductive environment, and acetal linkers whose cleavage is pH triggered.

Controlling the Interaction and Non-Close-Packed Arrangement of Nanoparticles on Large Areas

Madlen Schmudde, Christian Grunewald, Christian Goroncy, Christelle N. Noufele, Benjamin Stein, Thomas Risse, and Christina Graf

ACS Nano, 2016, 10 (3), 3525–3535 ...

In light of the importance of nanostructured surfaces for a variety of technological applications, the quest for simple and reliable preparation methods of ordered, nanometer ranged structures is ongoing. Herein, a versatile method to prepare ordered, non-close-packed arrangements of nanoparticles on centimeter sized surfaces by self-assembly is described using monodisperse (118–162 nm Ø), amino-functionalized silica nanoparticles as an exploratory example. It is shown that the arrangement of the particles is governed by the interplay between the electrostatic repulsion between the particles and the interaction between particles and surfaces. The latter is tuned by the properties of the particles such as their surface roughness as well as the chemistry of the linkage. Weak dispersive interactions between amino groups and gold surfaces are compared to a covalent amide linkage of the amino groups with carboxylic acid functionalized self-assembled monolayers. It was shown that the order of the former systems may suffer from capillary forces between particles during the drying process, while the covalently bonded systems do not. In turn, covalently bonded systems can be dried quickly, while the van der Waals bonded systems require a slow drying process to minimize aggregation. These highly ordered structures can be used as templates for the formation of a second, ordered, non-close-packed layer of nanoparticles exemplified for larger polystyrene particles (Ø 368 ± 14 nm), which highlights the prospect of this approach as a simple preparation method for ordered arrays of nanoparticles with tunable properties.

Ordered Structures of Functionalized Silica Nanoparticles on Gold Surfaces: Correlation of Quartz Crystal Microbalance with Structural Characterization

Christian Grunewald, Madlen Schmudde, Christelle N. Noufele, Christina Graf, and Thomas Risse

Anal. Chem., 2015, 87 (20), 10642–10649 ...

Quartz crystal microbalance (QCM) is frequently used to investigate adsorption of nanometer-sized objects such as proteins, viruses, or organic as well as inorganic nanoparticles from solution. The interpretation of the data obtained for heterogeneous adsorbate layers is not straightforward in particular if the systems exhibit sizable amounts of dissipation. In this study we investigate the deposition of monodisperse, amine functionalized silica nanoparticles on gold surfaces using QCM with dissipation (QCM-D) to obtain frequency and dissipation changes during adsorption from the liquid phase. These investigations are combined with ex situ scanning electron microscopy (SEM) measurements to study both coverage as well as lateral arrangement of the particles. An ordered layer of particles is found at saturation coverage due to the charged particle surface resulting in a repulsive interaction between the particles. The repulsion ensures a minimal distance between the particles, which leads to a saturation coverage of 15% for particles of 137 nm diameter. The frequency shift is shown to be a linear function of coverage which is a behavior expected for an elastic medium according to the Sauerbrey equation. However, the system shows a strong dependence of the normalized frequency shift on the overtones as well as a large dissipation, which is a clear indication for a system with viscoelastic properties. The analysis of the data show that a reliable determination of the adsorbed mass solely on the basis of QCM-D results is not possible, but additional information as determined by SEM in the present case is required to determine the coverage. From a correlation of the QCM-D results with the structural characterization it is possible to infer that the dissipation is a long ranged phenomenon. A lower boundary of the interaction length could be derived being twice the particle diameter for the particles studied here. In contrast to that the frequency response behaves like local phenomenon.

Ultrafast Dynamics of Photoisomerization and Subsequent Unfolding of an Oligo-Azobenzene Foldamer

Sabrina Steinwand, Zhilin Yu, Stefan Hecht, Josef Wachtveitl

J. Am. Chem. Soc., 2016, 138 (39), 12997–13005 ...

Investigating and deciphering the dynamics of photoswitchable foldamers provides a detailed understanding of their photoinduced conformational transitions, resembling similar processes in photoresponsive biomacromolecules. We studied the ultrafast dynamics of the photoisomerization of azobenzene moieties embedded in a foldamer backbone and the resulting conformational helix–coil transition by time-resolved femtosecond/picosecond pump–probe spectroscopy in the visible and infrared region. During E → Z photoisomerization of the azobenzenes, the complexity of the photoinduced conformational transition of the pentameric foldamer 105 is reflected in distinct spectral characteristics and a 2-fold slower decay of the excited-state absorption bands compared to the monomer M (τ4,foldamer = 20 ps, τ4,monomer = 9 ps). Time-resolved IR experiments reveal the vibrational features of the monomer and the foldamer after photoexcitation, with an additional time constant for the foldamer (τ = 150 ps), indicating the initial steps of unfolding of the helical conformation, which are supported by density functional theory calculations. Our results record the overall sequence of photoinduced structural changes in the foldamer, starting from the initial ultrafast isomerization of the azobenzene unit(s) and ending with the complete unfolding on a later time scale. From our experiments, we could gain insight into the coupling of primary photoisomerization events (“cause”) and secondary unfolding processes (“effect”) in these oligoazobenzene foldamers.

Remote-controlling Imine Exchange Kinetics with Photoswitches to Modulate Self-healing in Polysiloxane Networks by Light

Michael Kathan, Petr Kovaříček, Christoph Jurissek, Antti Senf, Andre Dallmann, Andreas F. Thünemann, Stefan Hecht

Angew. Chem. Int. Ed. 2016, 55, 13882-13886 ...

Various aldehyde-containing photoswitches have been developed whose reactivity toward amines can be controlled externally. A thermally stable bifunctional diarylethene, which in its ring-closed form exhibits imine formation accelerated by one order of magnitude, was used as a photoswitchable crosslinker and mixed with a commercially available amino-functionalized polysiloxane to yield a rubbery material with viscoelastic and self-healing properties that can be reversibly tuned by irradiation.

Anti-Hemagglutinin Antibody Derived Lead Peptides for Inhibitors of Influenza Virus Binding

Henry Memczak, Daniel Lauster, Parimal Kar, Santiago Di Lella, Rudolf Volkmer, Volker Knecht, Andreas Herrmann, Eva Ehrentreich-Förster, Frank F. Bier, Walter F. M. Stöcklein

PLoS ONE, 2016, 11(7), e0159074 ...

Allosteric and Chelate Cooperativity in Divalent Crown Ether/Ammonium Complexes with Strong Binding Enhancement

Larissa K. S. von Krbek, Andreas J. Achazi, Marthe Solleder, Marcus Weber, Beate Paulus, and Christoph A. Schalley

Chem. Eur. J., 2016, 22, 15475 – 15484 ...

A thorough thermodynamic analysis by isothermal titration calorimetry of allosteric and chelate cooperativity effects in divalent crown ether/ammonium complexes is combined with DFT calculations including implicit solvent on the one hand and large-scale molecular dynamics simulations with explicit solvent molecules on the other. The complexes studied exhibit binding constants up to 2×106 m−1 with large multivalent binding enhancements and thus strong chelate cooperativity effects. Slight structural changes in the spacers, that is, the exchange of two ether oxygen atoms by two isoelectronic methylene groups, cause significantly stronger binding and substantially increased chelate cooperativity. The analysis is complemented by the examination of solvent effects and allosteric cooperativity. Such a detailed understanding of the binding processes will help to efficiently design and construct larger supramolecular architectures with multiple multivalent building blocks.

Enzyme-responsive polymer-substituted pillar[5]arene amphiphiles: synthesis, self-assembly in water, and application in controlled drug release

Lingyan Gao, Bo Zheng, Wei Chen and Christoph A. Schalley

Chem. Commun., 2015, 51, 14901-14904 ...

An enzyme-responsive drug delivery system was constructed from a pillar[5]arene-based polyethyleneglycol-substituted amphiphile which self-assembles into micelles in water. These micelles exhibit superior drug encapsulation capability, and display drug release behaviour in response to enzyme catalysis, in particular to L-asparaginase. Doxorubicin-loaded micelles show significant cytotoxicity against MCF-7 cancer cells.

Potential of proapoptotic peptides to induce formation of giant plasma membrane vesicles with lipid domains

D. Lauster, O. Vazquez, R. Schwarzer, O. Seitz, A. Herrmann

ChemBioChem, 2015, 11, 589–595 ...

We have established a method of preparing giant plasma membrane vesicles (GPMVs) by using cysteine mutants of the proapoptotic peptide (PAP) Ac-R7-GG-KLAKLAKKLAKLAK. A cysteine scan revealed that cytotoxicity and GPMV formation were dependent on the cysteine position within the PAP sequence. In comparison to GPMVs prepared by extensive treatment with paraformaldehyde (PFA) and dithiothreitol (DTT), our GPMVs were produced from HeLa cells at much lower concentrations of the blebbing agent. We found that only GPMVs derived from cysteine-containing PAP showed lipid phase separation. This membrane model was applied to investigate the phase partitioning of two relevant membrane proteins: influenza virus hemagglutinin (HA) and tetherin, which clamps budding HIV to infected cells. For tetherin, we show for the first time exclusion from cholesterol-rich domains in a GPMV model, thus documenting the potential of our approach for membrane-partitioning studies.

Probing heterobivalent binding to the endocytic AP-2 adaptor complex by DNA-based spatial screening

F. Diezmann, L. Kleist, V. Haucke, O. Seitz

Org. Biomol. Chem., 2015, 13, 8008-8015 ...

The double helical DNA scaffold offers a unique set of properties, which are particularly useful for studies of multivalency in biomolecular interactions: (i) multivalent ligand displays can be formed upon nucleic acid hybridization in a self-assembly process, which facilitates spatial screening (ii) valency and spatial arrangement of the ligand display can be precisely controlled and (iii) the flexibility of the ligand display can be adjusted by integrating nick sites and unpaired template regions. Herein we describe the use of DNA-based spatial screening for the characterization of the adaptor complex 2 (AP-2), a central interaction hub within the endocytic protein network in clathrin-mediated endocytosis. AP-2 is comprised of a core domain and two, so-called appendage domains, the α- and the β2-ear, which associate with cytoplasmatic proteins required for the formation or maturation of clathrin/AP-2 coated pits. Each appendage domain has two binding grooves which recognize distinct peptide motives with micromolar affinity. This provides opportunities for enhanced interactions with protein molecules that contain two (or more) different peptide motives. To determine whether a particular, spatial arrangement of binding motifs is required for high affinity binding we probed the distance-affinity relationships by means of DNA-programmed spatial screening with self-assembled peptide-DNA complexes. By using trimolecular and tetramolecular assemblies two different peptides were positioned in 2–22 nucleotide distance. The binding data obtained with both recombinant protein in well-defined buffer systems and native AP-2 in brain extract suggests that the two binding sites of the AP-2 α-appendage can cooperate to provide up to 40-fold enhancement of affinity compared to the monovalent interaction. The distance between the two recognized peptide motives was less important provided that the DNA duplex segments were connected by flexible, single strand segments. By contrast, the experiments with a more rigid, duplex-spaced assembly revealed marked distance dependencies. Consequences for the function of adaptor proteins are discussed.

Efficient Self-Assembly of Di-, Tri-, Tetra- and Hexavalent Hosts with Predefined Geometries for the Investigation of Multivalency

Igor Linder, Stefan Leisering, Rakesh Puttreddy, Nadine Rades, Kari Rissanen, and Christoph A.Schalley

Chem. Eur. J., 2015, 21,13035 –13044 ...

Coordination-driven self-assembly of differently shaped di- to hexavalent crown-ether host molecules is described. A series of [21]crown-7- and [24]crown-8-substituted bipyridine and terpyridine ligands was synthetized in a “toolbox” approach. Subsequent coordination to 3d transition metal and ruthenium(II) ions provides an easy and fast access to host assemblies with variable valency and pre-defined orientations of the crown-ether moieties. Preliminary isothermal calorimetry (ITC) titrations provided promising results, which indicated the host complexes under study to be suitable for the future investigation of multivalent and cooperative binding. The hosts described herein will also be suitable for the construction of various multiply threaded mechanically interlocked molecules.

High Affinity Fluorescent Ligands for the Estrogen Receptor

F. Abendroth, M Solleder, D. Mangoldt, P. Welker, K. Licha, M. Weber, O.Seitz

Eur. J. Org. Chem., 2015, 2157–2166 ...

Fluorescent binders of the estrogen receptor (ER) are used in binding assays and in detection or imaging studies. However, fluorescence labelling of ER ligands usually leads to substantial decreases in binding affinity. In this study, we describe the development of high affinity fluorescent ER ligands. Cyanine dyes of the MiDye series were directly attached to the SERMs 4-hydroxytamoxifen (OHT) and raloxifene (Ral); linkers were deliberately omitted. This approach yielded conjugates with ERα binding affinities superior to the natural ligand estradiol. The OHT- and Ral-MiDye conjugates emitted in the 600–800 nm range. First round staining experiments showed that the conjugates, but not the dyes alone, accumulate in cells expressing estrogen-binding receptors.

A basis set for peptides for the variational approach to conformational kinetics

Francesca Vitalini, Frank Noé, and Bettina G. Keller

J. Chem. Theory Comput., 2015, 11 (9), 3992–4004 ...

Although Markov state models have proven to be powerful tools in resolving the complex features of biomolecular kinetics, the discretization of the conformational space has been a bottleneck since the advent of the method. A recently introduced variational approach, which uses basis functions instead of crisp conformational states, opened up a route to construct kinetic models in which the discretization error can be controlled systematically. Here, we develop and test a basis set for peptides to be used in the variational approach. The basis set is constructed by combining local residue-centered kinetic modes that are obtained from kinetic models of terminally blocked amino acids. Using this basis set, we model the conformational kinetics of two hexapeptides with sequences VGLAPG and VGVAPG. Six basis functions are sufficient to represent the slow kinetic modes of these peptides. The basis set also allows for a direct interpretation of the slow kinetic modes without an additional clustering in the space of the dominant eigenvectors. Moreover, changes in the conformational kinetics due to the exchange of leucine in VGLAPG to valine in VGVAPG can be directly quantified by comparing histograms of the basis set expansion coefficients.

Multivalent polyglycerol supported imidazolidin-4-one organocatalysts for enantioselective Friedel–Crafts alkylations

T. Pecchioli, M. K. Muthyala, R. Haag, M. Christmann

Beilstein J. Org. Chem. 2015, 11, 730–738 ...

The first immobilization of a MacMillan’s first generation organocatalyst onto dendritic support is described. A modified tyrosine-based imidazolidin-4-one was grafted to a soluble high-loading hyperbranched polyglycerol via a copper-catalyzed alkyne–azide cycloaddition (CuAAC) reaction and readily purified by dialysis. The efficiency of differently functionalized multivalent organocatalysts 4a–c was tested in the asymmetric Friedel–Crafts alkylation of N-methylpyrrole with α,β-unsaturated aldehydes. A variety of substituted enals was investigated to explore the activity of the catalytic system which was also compared with monovalent analogues. The catalyst 4b showed excellent turnover rates and no loss of activity due to immobilization, albeit moderate enantioselectivities were observed. Moreover, easy recovery by selective precipitation allowed the reuse of the catalyst for three cycles.

Shell Cleavable Dendritic Polyglycerol Sulfates Show High Anti-Inflammatory Properties by Inhibiting L-Selectin Binding and Complement Activation

Sabine Reimann, Dominic Gröger, Christian Kühne, Sebastian B. Riese, Jens Dernedde, and Rainer Haag

Adv. Healthc. Mater., 2015, 4, 2154–2162 ...

A new class of fully synthetic shell cleavable multivalent polysulfates is prepared by introducing degradable linkers into a stable biocompatible dendritic polyglycerol scaffold and subsequent sulfation. The sulfated polymers show different degradation profiles, low anticoagulant and high anti-inflammatory properties, are able to efficiently bind to L-selectin and inhibit the complement activation at very low concentrations in vitro.

Systematic adjustment of charge densities and size of polyglycerol amines reduces cytotoxic effects and enhances cellular uptake.

Markus Hellmund, Katharina Achazi, Falko Neumann, Bala N. S. Thota, Nan Ma and Rainer Haag

Biomater. Sci., 2015, 3, 1459-1465 ...

Excessive cationic charge density of polyplexes during cellular uptake is still a major hurdle in the field of non-viral gene delivery. The most efficient cationic vectors such as polyethylene imine (PEI) or polyamidoamine (PAMAM) can be highly toxic and may induce strong side effects due to their high cationic charge densities. Alternatives like polyethylene glycol (PEG) are used to ‘shield’ these charges and thus to reduce the cytotoxic effects known for PEI/PEG-core–shell architectures. In this study, we compared the ability of hyperbranched polyglycerol amines (hPG amines) with different amine densities and molecular weights as non-viral cationic vectors for DNA delivery. By adjusting the hydroxyl to amine group ratio on varying molecular weights, we were able to perform a systematic study on the cytotoxic effects caused by the effective charge density in correlation to size. We could demonstrate that carriers with moderate charge density have a higher potential for effective DNA delivery as compared to high/low charged ones independent of their size, but the final efficiency can be optimized by the molecular weight. We analyzed the physicochemical properties and cellular uptake capacity as well as the cytotoxicity and transfection efficiency of these new vector systems.

Potential of acylated peptides to target the influenza A virus

D. Lauster, D. Pawolski, J. Storm, K. Ludwig, R. Volkmer, H. Memczak, A. Herrmann, S. Bhatia

Beilstein J. Org. Chem. 2015, 11, 589–595 ...

For antiviral drug design, especially in the field of influenza virus research, potent multivalent inhibitors raise high expectations for combating epidemics and pandemics. Among a large variety of covalent and non-covalent scaffold systems for a multivalent display of inhibitors, we created a simple supramolecular platform to enhance the antiviral effect of our recently developed antiviral Peptide B (PeBGF), preventing binding of influenza virus to the host cell. By conjugating the peptide with stearic acid to create a higher-order structure with a multivalent display, we could significantly enhance the inhibitory effect against the serotypes of both human pathogenic influenza virus A/Aichi/2/1968 H3N2, and avian pathogenic A/FPV/Rostock/34 H7N1 in the hemagglutination inhibition assay. Further, the inhibitory potential of stearylated PeBGF (C18-PeBGF) was investigated by infection inhibition assays, in which we achieved low micromolar inhibition constants against both viral strains. In addition, we compared C18-PeBGF to other published amphiphilic peptide inhibitors, such as the stearylated sugar receptor mimicking peptide (Matsubara et al. 2010), and the “Entry Blocker” (EB) (Jones et al. 2006), with respect to their antiviral activity against infection by Influenza A Virus (IAV) H3N2. However, while this strategy seems at a first glance promising, the native situation is quite different from our experimental model settings. First, we found a strong potential of those peptides to form large amyloid-like supramolecular assemblies. Second, in vivo, the large excess of cell surface membranes provides an unspecific target for the stearylated peptides. We show that acylated peptides insert into the lipid phase of such membranes. Eventually, our study reveals serious limitations of this type of self-assembling IAV inhibitors.

Chelate cooperativity effects on the formation of di- and trivalent pseudo[2]rotaxanes with diketopiperazine threads and tetralactam wheels

Nora L. Traulsen, Christoph H.-H. Traulsen, Peter M. Deutinger, Sebastian Müller, Deborah Schmidt, Igor Linder and Christoph A. Schalley

Org. Biomol. Chem., 2015, 13, 10881-10887 ...

The formation of singly, doubly and triply threaded pseudo[2]rotaxanes with diketopiperazine threads and tetralactam wheels is investigated with respect to chelate cooperativity effects on multivalent binding. Two series of guest molecules are prepared which differ with respect to their spacers, one with preorganised centrepieces with di- or tripodal roof-like structures, one with more flexible spacers. The thermodynamics of pseudorotaxane formation is examined using isothermal titration calorimetry and 1H NMR spectroscopy. Force-field calculations provide more detailed structural insight and help rationalizing the thermodynamic data. All di- and trivalent pseudorotaxanes exhibit positive chelate cooperativity presumably arising from spacer–spacer interactions. Higher cooperativity factors are observed for the more preorganised threads.

Thermodynamic Analysis of Allosteric and Chelate Cooperativity in Di- and Trivalent Ammonium/Crown-Ether Pseudorotaxanes

Karol Nowosinski, Larissa K. S. von Krbek, Nora L. Traulsen, and Christoph A. Schalley

Org. Lett., 2015, 17 (20), 5076–5079 ...

A detailed thermodynamic analysis of the axle-wheel binding in di- and trivalent secondary ammonium/[24]crown-8 pseudorotaxanes is presented. Isothermal titration calorimetry (ITC) data and double mutant cycle analyses reveal an interesting interplay of positive as well as negative allosteric and positive chelate cooperativity thus providing profound insight into the effects governing multivalent binding in these pseudorotaxanes.

Single-virus force spectroscopy unravels molecular details of virus infection

A. Herrmann, C. Sieben

Integr. Biol., 2015, 7, 620-632 ...

Virus infection is a multistep process that has significant effects on the structure and function of both the virus and the host cell. The first steps of virus replication include cell binding, entry and release of the viral genome. Single-virus force spectroscopy (SVFS) has become a promising tool to understand the molecular details of those steps. SVFS data complemented by biochemical and biophysical, including theoretical modeling approaches provide valuable insights into molecular events that accompany virus infection. Properties of virus–cell interaction as well as structural alterations of the virus essential for infection can be investigated on a quantitative level. Here we review applications of SVFS to virus binding, structure and mechanics. We demonstrate that SVFS offers unexpected new insights not accessible by other methods.

Kovalente Verknüpfung cyclischer TAT-Peptide mit GFP resultiert in der direkten Aufnahme in lebende Zellen mit sofortiger biologischer Verfügbarkeit

N. Nischan, H. D. Herce, F. Natale, N. Bohlke, N. Budisa, M. C. Cardoso, C. P. R. Hackenberger

Angew. Chem., 2015, 127, 1972–1976 ...

Der Transport von freien Molekülen in das Zytoplasma und den Zellkern mittels Arginin-reicher zellpenetrierender Peptide (CPPs) ist auf kleine Substanzen beschränkt, während große Frachten wie Proteine mittels Endozytose aufgenommen werden und nicht aus den Endosomen entkommen können. Vor kurzem entdeckten wir, dass sich die Transduktionseffizienz Arginin-reicher CPPs durch Cyclisierung maßgeblich erhöhen lässt, und prüfen hier ob cyclische CPPs auch den Transport ganzer Proteine, hier GFP, in das Zytosol von lebenden Zellen ermöglichen. GFP-Konjugate cyclischer und linearer CPPs wurden ausgehend von orthogonalen Azid-funktionalisierten CPPs und Alkin-GFP erhalten. Unsere Ergebnisse zeigen, dass nur die cyclischen CPP-GFP-Konjugate von lebenden Zellen in Zytosol und Nukleoli mit sofortiger biologischer Verfügbarkeit aufgenommen werden. Die vorgestellte Methode erweitert die Anwendung von cyclischen CPPs mit dem effizienten Transport von ganzen, funktionalen Proteinen in lebenden Zellen.

Covalent Attachment of Cyclic TAT Peptides to GFP Results in Protein Delivery into Live Cells with Immediate Bioavailability

N. Nischan, H. D. Herce, F. Natale, N. Bohlke, N. Budisa, M. C. Cardoso, C. P. R. Hackenberger

Angew. Chem. Int. Ed., 2015, 54, 1950–1953 ...

The delivery of free molecules into the cytoplasm and nucleus by using arginine-rich cell-penetrating peptides (CPPs) has been limited to small cargoes, while large cargoes such as proteins are taken up and trapped in endocytic vesicles. Based on recent work, in which we showed that the transduction efficiency of arginine-rich CPPs can be greatly enhanced by cyclization, the aim was to use cyclic CPPs to transport full-length proteins, in this study green fluorescent protein (GFP), into the cytosol of living cells. Cyclic and linear CPP-GFP conjugates were obtained by using azido-functionalized CPPs and an alkyne-functionalized GFP. Our findings reveal that the cyclic-CPP-GFP conjugates are internalized into live cells with immediate bioavailability in the cytosol and the nucleus, whereas linear CPP analogues do not confer GFP transduction. This technology expands the application of cyclic CPPs to the efficient transport of functional full-length proteins into live cells.

Traceless Purification and Desulfurization of Tau Protein Ligation Products

O. Reimann, C. Smet-Nocca, Christian P. R. Hackenberger

Angew. Chem. Int. Ed., 2015, 54, 306–310 ...

We present a novel strategy for the traceless purification and synthetic modification of peptides and proteins obtained by native chemical ligation. The strategy involves immobilization of a photocleavable semisynthetic biotin–protein conjugate on streptavidin-coated agarose beads, which eliminates the need for tedious rebuffering steps and allows the rapid removal of excess peptides and additives. On-bead desulfurization is followed by delivery of the final tag-free protein product. The strategy is demonstrated in the isolation of a tag-free Alzheimer's disease related human tau protein from a complex EPL mixture as well as a triphosphorylated peptide derived from the C-terminus of tau.

Dense or Porous Packing? Two-Dimensional Self-Assembly of Star-Shaped Mono-, Bi-, and Terpyridine Derivatives

D.Trawny, P. Schlexer, K. Steenbergen, J. P. Rabe, B. Paulus, H.-U. Reissig

ChemPhysChem 2015, 16, 949 – 953 ...

The self-assembly behavior of five star-shaped pyridyl-functionalized 1,3,5-triethynylbenzenes was studied at the interface between an organic solvent and the basal plane of graphite by scanning tunneling microscopy. The mono- and bipyridine derivatives self-assemble in closely packed 2D crystals, whereas the derivative with the more bulky terpyridines crystallizes with porous packing. DFT calculations of a monopyridine derivative on graphene, support the proposed molecular model. The calculations also reveal the formation of hydrogen bonds between the nitrogen atoms and a hydrogen atom of the neighboring central unit, as a small nonzero tunneling current was calculated within this region. The title compounds provide a versatile model system to investigate the role of multivalent steric interactions and hydrogen bonding in molecular monolayers.

Spurlose Aufreinigung und Desulfurierung von Ligationsprodukten des Tau-Protein

O. Reimann, C. Smet-Nocca, Christian P. R. Hackenberger

Angew. Chem., 2015, 127, 311–315 ...

Wir präsentieren eine neue Strategie zur spurlosen Aufreinigung und synthetischen Modifikation von durch native chemische Ligation erzeugten Peptiden und Proteinen. Die Strategie umfasst die Immobilisierung eines photospaltbaren semisynthetischen Biotin-Protein-Konjugats an Streptavidin-Agarosepartikel, wodurch aufwendiges Umpuffern vermieden und überschüssiges Peptid und Additive entfernt werden können. Eine Desulfurierung der immobilisierten Peptide und Proteine liefert nach Abspaltung die gewünschten Tag-freien Produkte. Mit dieser Methode konnte Alzheimer-relevantes Tau-Protein aus einer komplexen EPL-Mischung und dreifach phosphoryliertes C-terminales Tau-Peptid isoliert werden.

C3-Symmetric Pyridine and Bipyridine Derivatives: Simple Preparation by Cyclocondensation and 2D Self-Assembly at a Solution-Graphite Interface

J. Dash, D. Trawny, J. P. Rabe, H.-U. Reissig

Synlett 2015, 26(11), 1486-1489 ...

The efficient preparation of four C3-symmetric (star-shaped) pyridine and bipyridine derivatives is reported. The key steps are Suzuki couplings of 4-pyridyl nonaflates with 4-acetyl-phenylboronic acid followed by an acid-promoted cyclocondensation reaction converting the methyl ketone moiety into the central benzene ring of the target compounds. Based on STM studies at a graphite–solution interface the two-dimensional arrangements of the compounds are discussed, showing the influence of the pyridine substitution pattern.

Live-cell MRI with Xenon Hyper-CEST Biosensors Targeted to Metabolically Labeled Cell-Surface Glycans

C.Witte, V. Martos, H. M. Rose, S. Reinke, S. Klippel, L. Schröder, C. P. R. Hackenberger

Angew. Chem. Int. Ed., 2015, 54, 2806–2810 ...

The targeting of metabolically labeled glycans with conventional MRI contrast agents has proved elusive. In this work, which further expands the utility of xenon Hyper-CEST biosensors in cell experiments, we present the first successful molecular imaging of such glycans using MRI. Xenon Hyper-CEST biosensors are a novel class of MRI contrast agents with very high sensitivity. We designed a multimodal biosensor for both fluorescent and xenon MRI detection that is targeted to metabolically labeled sialic acid through bioorthogonal chemistry. Through the use of a state of the art live-cell bioreactor, it was demonstrated that xenon MRI biosensors can be used to image cell-surface glycans at nanomolar concentrations.

Xenon-MRT an lebenden Zellen mit Hyper-CEST-Biosensoren für metabolisch markierte Glykane an der Zelloberfläche

C.Witte, V. Martos, H. M. Rose, S. Reinke, S. Klippel, L. Schröder, C. P. R. Hackenberger

Angew. Chem., 2015, 127, 2848–2852 ...

Das Targeting von metabolisch markierten Glykanen mit herkömmlichen MRT-Kontrastmitteln erschien bislang als nicht realisierbar. Hier erweitern wir die Anwendungen von Xenon-Hyper-CEST-Biosensoren in Zellen und präsentieren damit die erste erfolgreiche molekulare Bildgebung dieser Glykane mittels MRT. Hyper-CEST-Biosensoren sind eine neue Klasse von MRT-Kontrastmitteln mit sehr hoher Empfindlichkeit. Darauf basierend wurde ein multimodaler Biosensor für sowohl Fluoreszenz- als auch Xenon-MRT-Detektion entwickelt, der selektiv mit metabolisch markierten Sialinsäuren durch bioorthogonale Chemie verknüpft wurde. Die Verwendung eines kürzlich entwickelten Bioreaktors zur Untersuchung lebender Zellen ermöglicht somit den Nachweis von Zelloberflächenglykanen mittels Xenon-MRT bei nanomolaren Konzentrationen.

The GYF domain protein CD2BP2 is critical for embryogenesis and podocyte function

G. I. Albert, C. Schell, K. M. Kirschner, S. Schäfer, R. Naumann, A. Müller, O. Kretz, B. Kuropka, N. Hübner, M. Girbig, E. Krause, H. Scholz, T. Huber, K.-P. Knobeloch, C. Freund

J. Mol. Cell. Biol., 2015, 7(5), 402-14 ...

Scaffolding proteins play pivotal roles in the assembly of macromolecular machines such as the spliceosome. The adaptor protein CD2BP2, originally identified as a binding partner of the adhesion molecule CD2, is a pre-spliceosomal assembly factor that utilizes its glycine-tyrosine-phenylalanine (GYF) domain to co-localize with spliceosomal proteins. So far, its function in vertebrates is unknown. Using conditional gene targeting in mice, we show that CD2BP2 is crucial for embryogenesis, leading to growth retardation, defects in vascularization, and premature death at embryonic day 10.5 when absent. Ablation of the protein in bone marrow-derived macrophages indicates that CD2BP2 is involved in the alternative splicing of mRNA transcripts from diverse origins. At the molecular level, we identified the phosphatase PP1 to be recruited to the spliceosome via the N-terminus of CD2BP2. Given the strong expression of CD2BP2 in podocytes of the kidney, we use selective depletion of CD2BP2, in combination with next-generation sequencing, to monitor changes in exon usage of genes critical for podocyte functions, including VEGF and actin regulators. CD2BP2-depleted podocytes display foot process effacement, and cause proteinuria and ultimately lethal kidney failure in mice. Collectively, our study defines CD2BP2 as a non-redundant splicing factor essential for embryonic development and podocyte integrity.

A modular toolkit to inhibit proline-rich motif–mediated protein–protein interactions

R. Opitz, M. Müller, C. Reuter, M. Barone, A. Soicke, Y. Roskec, K. Piotukha, P. Huy, M. Beerbaum, B. Wiesner, M. Beyermann, P.Schmieder, C. Freund, R.Volkmer, H. Oschkinat, H.-G. Schmalz, R. Kühne

Proc. Natl. Acad. Sci. USA, 2015, 112, 5011-5016

Protein–protein interactions mediated by proline-rich motifs are involved in regulation of many important signaling cascades. Protein domains specialized in recognition of these motifs expose a flat and relatively rigid binding site that preferentially interacts with sequences adopting a left-handed polyproline helix II. Here, we present a toolkit of new chemical entities that enables rational construction of selective small-molecule inhibitors for these protein domains. As proof of principle, we developed a selective, cell-permeable inhibitor of Drosophila enabled (Ena)/vasodilator-stimulated phosphoprotein (VASP) homology 1 (EVH1) domains of the Ena/VASP protein family. Invasive breast-cancer cells treated with our EVH1 inhibitor showed strongly reduced cell invasion.

Exploring monovalent and multivalent peptides for the inhibition of FBP21-tWW

L. M. Henning, S. Bhatia, M. Bertazzon, M. Marczynke, O. Seitz, R. Volkmer, R. Haag, C.Freund

Beilstein J. Org. Chem. 2015, 11, 701–706 ...

The coupling of peptides to polyglycerol carriers represents an important route towards the multivalent display of protein ligands. In particular, the inhibition of low affinity intracellular protein–protein interactions can be addressed by this design. We have applied this strategy to develop binding partners for FBP21, a protein which is important for the splicing of pre-mRNA in the nucleus of eukaryotic cells. Firstly, by using phage display the optimized sequence WPPPPRVPR was derived which binds with KDs of 80 μM and 150 µM to the individual WW domains and with a KD of 150 μM to the tandem-WW1–WW2 construct. Secondly, this sequence was coupled to a hyperbranched polyglycerol (hPG) that allowed for the multivalent display on the surface of the dendritic polymer. This novel multifunctional hPG-peptide conjugate displayed a KD of 17.6 µM which demonstrates that the new carrier provides a venue for the future inhibition of proline-rich sequence recognition by FBP21 during assembly of the spliceosome.

Chemoselective Bioconjugation of Triazole Phosphonites in Aqueous Media

M.R.J. Vallée, P. Majkut, D. Krause, M. Gerrits, C.P.R. Hackenberger

Chem. Eur. J. 2015, 21(3), 970-974 ...

Readily accessible and versatile phosphonite building blocks with improved stability against hydrolysis were used for the efficient metal-free functionalization of peptides and proteins in aqueous buffers at low micromolar concentrations. The application of this protocol to the immobilization of a Rasa1-SH2 domain revealed high binding affinity to the human T-cell protein ADAP and supports the applicability of triazole phosphonites for protein modifications without harming their function.

Side-Chain Cysteine-Functionalized Poly(2-oxazoline)s for Multiple Peptide Conjugation by Native Chemical Ligation

M. Schmitz, M. Kuhlmann, O. Reimann, C. P. R. Hackenberger, J. Groll

Biomacromolecules, 2015, 16 (4), 1088–1094 ...

We prepared statistical copolymers composed of 2-methyl-2-oxazoline (MeOx) in combination with 2-butenyl-2-oxazoline (BuOx) or 2-decenyl-2-oxazoline (DecOx) as a basis for polymer analogous introduction of 1,2-aminothiol moieties at the side chain. MeOx provides hydrophilicity as well as cyto- and hemocompatibility, whereas the alkene groups of BuOx and DecOx serve for functionalization with a thiofunctional thiazolidine by UV-mediated thiol–ene reaction. After deprotection the cysteine content in functionalized poly(2-oxazoline) (POx) is quantified by NMR and a modified trinitrobenzenesulfonic acid assay. The luminescent cell viability assay shows no negative influence of cysteine-functionalized POx (cys-POx) concerning cell viability and cell number. cys-POx was used for multiple chemically orthogonal couplings with thioester-terminated peptides through native chemical ligation (NCL), which was performed and confirmed by NMR and MALDI-ToF measurements.

Native chemical ligation between asparagine and valine: Application and limitations for the synthesis of tri-phosphorylated C-terminal tau

O. Reimann, M. Glanz, C.P.R. Hackenberger

Bioorg. Med. Chem., 2015, 23, 2890–2894 ...

We present the successful native chemical ligation (NCL) at an Asn-Val site employing β-mercaptovaline and subsequent desulfurization in the synthesis of native phosphorylated C-terminal tau, relevant for Alzheimer’s disease related research. Despite recent progress in the field of NCL we illustrate limitations of this ligation site that stem from thioester hydrolysis and predominantly aspartimide formation. We systematically investigated the influence of pH, temperature, peptide concentration and thiol additives on the outcome of this ligation and identified conditions under which the ligation can be driven toward complete conversion, which required the deployment of a high surplus of thioester. Application of the optimized conditions allowed us to gain access to challenging tri-phosphorylated C-terminal tau peptide in practical yields.

Cysteine-Functional Polymers via Thiol-ene Conjugation

M. Kuhlmann, O. Reimann, C. P. R. Hackenberger, J. Grol

Macromol. Rapid Commun., 2015, 36, 472–476 ...

A thiofunctional thiazolidine is introduced as a new low-molar-mass building block for the introduction of cysteine residues via a thiol-ene reaction. Allyl-functional polyglycidol (PG) is used as a model polymer to demonstrate polymer-analogue functionalization through reaction with the unsaturated side-chains. A modified trinitrobenzenesulfonic acid (TNBSA) assay is used for the redox-insensitive quantification and a precise final cysteine content can be predetermined at the polymerization stage. Native chemical ligation at cysteine-functional PG is performed as a model reaction for a chemoselective peptide modification of this polymer. The three-step synthesis of the thiofunctional thiazolidine reactant, together with the standard thiol-ene coupling and the robust quantification assay, broadens the toolbox for thiol-ene chemistry and offers a generic and straightforward approach to cysteine-functional materials.

Kinetics of Aggregation and Growth Processes of PEGStabilised Mono- and Multivalent Gold Nanoparticles in Highly Concentrated Halide Solutions

B. Stein, D. Zopes, M. Schmudde, R. Schneider, A. Mohsen, S. Mathur, C. Graf

Faraday Discussions, 2015, 181, 85-102 ...

5–6 nm gold nanoparticles were prepared by hydrolytic decomposition of [NMe4][Au(CF3)2] and functionalized in situ with mono- and multivalent thiolated PEG ligands. Time-dependent changes of the nanoparticles were monitored in aqueous NaCl, NaBr, and NaI solutions by UV-Vis spectroscopy, TEM, and HRTEM. The purely sterically protected particles are stable in ≤1 M NaCl and NaBr solutions, regardless of the valence of the ligands. At higher concentrations (≥2 M), the monovalent stabilized particles show minor reaction limited colloidal aggregation. In NaBr but not in NaCl solutions a minor Ostwald ripening also occurs. The divalent stabilized particles remain colloidally stable in both halide solutions, even if the temperature is raised or the concentration is increased above 2 M. In ≤1 M aqueous NaI solutions the particles remain stable. Above, the monovalent stabilized particles undergo an oxidative reaction, resulting in a time-dependent shift and broadening of the absorbance spectrum. Finally, this process slows down while the width of the spectra slightly narrows. The kinetics of this process can be described by a two-step sigmoidal process, comprising a slow induction period where active species are formed, followed by a fast growth and aggregation process. The increasing concentration of fused structures from the aggregates during this process results in a narrowing of the size distributions. The divalent stabilized particles show only some minor broadening and a slight shift of the absorbance spectra in ≤3 M NaI solutions. These observations confirm the excellent stability of the multivalent stabilized particles from this chloride-free particle synthesis.

Supramolecular hydrophobic guest transport system based on pillar[5]arene

Z. Qi, K. Achazi, R. Haag, S. Dong, C.A. Schalley

Chem. Commun., 2015, 51, 10326-10329 ...

A pillar[5]arene-based bioactive guest loading system has been developed, which can increase the solubility of the drug norharmane in aqueous medium, and also enable its pH-stimulated release. Furthermore, this supramolecular transport system reduces the toxicity of loaded guest.

Orthogonal Translation Meets Electron Transfer: In Vivo Labeling of Cytochrome c for Probing Local Electric Fields

J. Völler, H. Biava, B. Koksch, P. Hildebrandt, N. Budisa

ChemBioChem, 2015, 16, 742–745 ...

Cytochrome c (cyt c), a redox protein involved in diverse fundamental biological processes, is among the most traditional model proteins for analyzing biological electron transfer and protein dynamics both in solution and at membranes. Studying the role of electric fields in energy transduction mediated by cyt c relies upon appropriate reporter groups. Up to now these had to be introduced into cyt c by in vitro chemical modification. Here, we have overcome this restriction by incorporating the noncanonical amino acid p-cyanophenylalanine (pCNF) into cyt c in vivo. UV and CD spectroscopy indicate preservation of the overall protein fold, stability, and heme coordination, whereas a small shift of the redox potential was observed by cyclic voltammetry. The C≡N stretching mode of the incorporated pCNF detected in the IR spectra reveals a surprising difference, which is related to the oxidation state of the heme iron, thus indicating high sensitivity to changes in the electrostatics of cyt c.

Mechanical Rupture of Mono- and Bivalent Transition Metal Complexes in Experiment and Theory

M. Gensler, C. Eidamshaus, A. Galstyan, E.-W. Knapp, H.-U. Reissig, J. P. Rabe

J. Phys. Chem. C, 2015, 119(8), 4333–4343 ...

Biomolecular systems are commonly exposed to a manifold of forces, often acting between multivalent ligands. To understand these forces, we studied mono- and bivalent model systems of pyridine coordination complexes with Cu2+ and Zn2+ in aqueous environment by means of scanning force microscopy based single-molecule force spectroscopy in combination with ab initio DFT calculations. The monovalent interactions show remarkably long rupture lengths of approximately 3 Å that we attribute to a dissociation mechanism involving a hydrogen-bound intermediate state. The bivalent interaction with copper dissociates also via hydrogen-bound intermediates, leading to an even longer rupture length between 5 and 6 Å. Although the bivalent system is thermally more stable, the most probable rupture forces of both systems are similar over the range of measured loading rates. Our results prove that already in small model systems the dissociation mechanism strongly affects the mechanical stability. The presented approach offers the opportunity to study the force-reducing effects also as a function of different backbone properties.

First principle investigation of the linker length effects on the thermodynamics of divalent pseudorotaxanes

A. J. Achazi, D. Mollenhauer, B. Paulus

Beilstein J. Org. Chem. 2015, 11, 687–692 ...

The Gibbs energies of association (Gibbs free (binding) energies) for divalent crown-8/ammonium pseudorotaxanes are determined by investigating the influence of different linkers onto the binding. Calculations are performed with density functional theory including dispersion corrections. The translational, rotational and vibrational contributions are taken into account and solvation effects including counter ions are investigated by applying the COSMO-RS method, which is based on a continuum solvation model. The calculated energies agree well with the experimentally determined ones. The shortest investigated linker shows an enhanced binding strength due to electronic effects, namely the dispersion interaction between the linkers from the guest and the host. For the longer linkers this ideal packing is not possible due to steric hindrance.

Identifying Multivalent Binding Kinetics of Precision Glycomacromolecules: A Kinetic Study Using kinITC

Sinaida Igde, Hendrik Wölk, Susanna Röblitz, Marco Reidelbach, Marcus Weber, Laura Hartmann ...

Multivalent sugar/protein interactions are well-known to proceed through different binding modes 1-5 which in turn can be described by their binding kinetics 3-5. This study provides additional insight into the association and dissociation reaction rates of complex multivalent sugar/protein interactions. Binding kinetics of recently introduced multivalent precision glycomacromolecules 6-8 to Concanavalin A (Con A) were studied by " kinetic Isothermal Titration Calorimetry " (kinITC) 9-11. The effect of multivalency is evaluated by comparing rate constants of glycomacromolecules obtaining the same and different valency of mannose ligands and by variation of the overall backbone properties, such as hydrophilic/ hydrophoboc. In addition, binding kinetics were studied using different conformations of Con A (homodimer vs.-tetramer) and thus a different protein valency. Our results show that precision glycomacromolecule/Con A binding proceeds non-cooperatively. Further, association and dissociation rates are mainly described by intermolecular complex formation. Together with the so-called functional valency, we can discriminate between " bound " and " unbound " states for macroscopic on-and off-rates, even for such complex glycooligomer/protein systems. By comparing e.g. a mono-to a divalent glycomacromolecule for their binding to dimeric Con A, we see a lower dissociation rate for the latter. As both bind monovalently to Con A, this is a strong indication for a statistical rebinding event. Further, there is a strong dependence of multivalent binding kinetics on the ligand density of glycomacromolecules as well as the Con A conformation and thus the overall on-and off-rates.

Applying γ-Substituted Prolines in the Foldon Peptide: Polarity Contradicts Preorganization

D. Dietz, V. Kubyshkin, N. Budisa.

ChemBioChem, 2015, 16, 403–406 ...

Rational choice of chemical modifications to proline residues allows the preorganization principle to be exploited for more stable assembly of the foldon domain as a tag for trimerization. With systematic knowledge of how chemical and steric variations of the ring substituents affect the relative stabilities of exo and endo puckers, the preorganization principle should then be usable in biotechnologically synthesized foldon mutants and applicable for protein tagging elsewhere.

Site-Directed and Global Incorporation of Orthogonal and Isostructural Noncanonical Amino Acids into the Ribosomal Lasso Peptide Capistruin

R. S. Al Toma, A. Kuthning, M. P. Exner, A. Denisiuk, J. Ziegler, N. Budisa, R. D. Süssmuth,

ChemBioChem, 2015, 16, 503–509 ...

Expansion of the structural diversity of peptide antibiotics was performed through two different methods. Supplementation-based incorporation (SPI) and stop-codon suppression (SCS) approaches were used for co-translational incorporation of isostructural and orthogonal noncanonical amino acids (ncAAs) into the lasso peptide capistruin. Two ncAAs were employed for the SPI method and five for the SCS method; each of them probing the incorporation of ncAAs in strategic positions of the molecule. Evaluation of the assembly by HR-ESI-MS proved more successful for the SCS method. Bio-orthogonal chemistry was used for post-biosynthetic modification of capistruin congener Cap_Alk10 containing the ncAA Alk (Nε-Alloc-L-lysine) instead of Ala. A second-generation Hoveyda–Grubbs catalyst was used for an in vitro metathesis reaction with Cap_Alk10 and an allyl alcohol, which offers options for post-biosynthetic modifications. The use of synthetic biology allows for the in vivo production of new peptide-based antibiotics from an expanded amino acid repertoire.

A Self-Assembling Peptide Scaffold for the Multivalent Presentation of Antigens

E. Zacco, C. Anish, C. E. Martin, H. v. Berlepsch, E. Brandenburg, P. H. Seeberger, B. Koksch

Biomacromolecules, 2015, 16 (7), 2188–2197 ...

Self-assembling peptides can be used to create tunable higher-order structures for the multivalent presentation of a variety of ligands. We describe a novel, fiber-forming coiled-coil-based peptide that assembles to display, simultaneously, carbohydrate and peptide ligands recognized by biomacromolecules. Preassembly decoration of the scaffold with a diphtheria toxin peptide epitope or a mannose motif did not interfere with self-assembly of the nanostructure. The resulting multivalent display led to tighter binding by antidiphtheria toxin antibodies and mannose-specific carbohydrate binding proteins, respectively. The potential of this self-assembling peptide to display ligands in bioanalytical assays is illustrated by its decoration with a disaccharide glycotope from the Leishmania parasite. Carbohydrate-specific antibodies produced in response to a Leishmania infection are detected more sensitively in human and canine sera due to the multivalent presentation on the self-assembled scaffold. Thus, nanofibers based on coiled-coil peptides are a powerful tool for the development of bioassays and diagnostics.

Orthogonal dual-modification of proteins for the engineering of multivalent protein scaffolds

M. Mühlberg, M. G. Hoesl, C. Kuehne, J. Dernedde, N. Budisa, C. P. R. Hackenberger

Beilstein J. Org. Chem. 2015, 11, 784–791 ...

To add new tools to the repertoire of protein-based multivalent scaffold design, we have developed a novel dual-labeling strategy for proteins that combines residue-specific incorporation of unnatural amino acids with chemical oxidative aldehyde formation at the N-terminus of a protein. Our approach relies on the selective introduction of two different functional moieties in a protein by mutually orthogonal copper-catalyzed azide–alkyne cycloaddition (CuAAC) and oxime ligation. This method was applied to the conjugation of biotin and β-linked galactose residues to yield an enzymatically active thermophilic lipase, which revealed specific binding to Erythrina cristagalli lectin by SPR binding studies.

Orthogonal Translation Meets Electron Transfer: In Vivo Labeling of Cytochrome c for Probing Local Electric Fields

J. Völler, H. Biava, B. Koksch, P. Hildebrandt, N. Budisa

ChemBioChem, 2015, 16, 742–745 ...

Cytochrome c (cyt c), a redox protein involved in diverse fundamental biological processes, is among the most traditional model proteins for analyzing biological electron transfer and protein dynamics both in solution and at membranes. Studying the role of electric fields in energy transduction mediated by cyt c relies upon appropriate reporter groups. Up to now these had to be introduced into cyt c by in vitro chemical modification. Here, we have overcome this restriction by incorporating the noncanonical amino acid p-cyanophenylalanine (pCNF) into cyt c in vivo. UV and CD spectroscopy indicate preservation of the overall protein fold, stability, and heme coordination, whereas a small shift of the redox potential was observed by cyclic voltammetry. The C≡N stretching mode of the incorporated pCNF detected in the IR spectra reveals a surprising difference, which is related to the oxidation state of the heme iron, thus indicating high sensitivity to changes in the electrostatics of cyt c.

Dendritic polyglycerol sulfate as a novel platform for paclitaxel delivery: pitfalls of ester linkage

A. Sousa-Herves, P. Würfel, P. Welker, K. Licha, J. Khandare, R. Haag, M. Calderón

Nanoscale, 2015, 7, 3923-3932 ...

In this study, dendritic polyglycerol sulfate (dPGS) is evaluated as a delivery platform for the anticancer, tubulin-binding drug paclitaxel (PTX). The conjugation of PTX to dPGS is conducted via a labile ester linkage. A non-sulfated dendritic polyglycerol (dPG) is used as a control, and the labeling with an indocarbocyanine dye (ICC) renders multifunctional conjugates that can be monitored by fluorescence microscopy. The conjugates are characterized by 1H NMR, UV-vis measurements, and RP-HPLC. In vitro cytotoxicity of PTX and dendritic conjugates is evaluated using A549 and A431 cell lines, showing a reduced cytotoxic efficacy of the conjugates compared to PTX. The study of uptake kinetics reveals a linear, non saturable uptake in tumor cells for dPGS-PTX-ICC, while dPG-PTX-ICC is hardly taken up. Despite the marginal uptake of dPG-PTX-ICC, it prompts tubulin polymerization to a comparable extent as PTX. These observations suggest a fast ester hydrolysis and premature drug release, as confirmed by HPLC measurements in the presence of plasma enzymes.

Polyglycerol based coatings to reduce non-specific protein adsorption in sample vials and on SPR sensors

T. Becherer, C. Grunewald, V. Engelschalt, G. Multhaup, T. Risse, R. Haag

Anal. Chim. Acta, 2015, 867, 47-55 ...

Coatings based on dendritic polyglycerol (dPG) were investigated for their use to control nonspecific protein adsorption in an assay targeted to analyze concentrations of a specific protein. We demonstrate that coating of the sample vial with dPG can significantly increase the recovery of an antibody after incubation. First, we determine the concentration dependent loss of an antibody due to nonspecific adsorption to glass via quartz crystal microbalance (QCM). Complementary to the QCM measurements, we applied the same antibody as analyte in an surface plasmon resonance (SPR) assay to determine the loss of analyte due to nonspecific adsorption to the sample vial. For this purpose, we used two different coatings based on dPG. For the first coating, which served as a matrix for the SPR sensor, carboxyl groups were incorporated into dPG as well as a dithiolane moiety enabling covalent immobilization to the gold sensor surface. This SPR-matrix exhibited excellent protein resistant properties and allowed the immobilization of amyloid peptides via amide bond formation. The second coating which was intended to prevent nonspecific adsorption to glass vials comprised a silyl moiety that allowed covalent grafting to glass. For demonstrating the impact of the vial coating on the accuracy of an SPR assay, we immobilized amyloid beta (Aβ) 1-40 and used an anti-Aβ 1-40 antibody as analyte. Alternate injection of analyte into the flow cell of the SPR device from uncoated and coated vials, respectively gave us the relative signal loss (1 − RUuncoated/RUcoated) caused by the nonspecific adsorption. We found that the relative signal loss increases with decreasing analyte concentration. The SPR data correlate well with concentration dependent non-specific adsorption experiments of the analyte to glass surfaces performed with QCM. Our measurements show that rendering both the sample vial and the sensor surface is crucial for accurate results in protein assays.

Tolerogenic Modulation of the Immune Response by Oligoglycerol– and Polyglycerol–Peptide Conjugates

S. Gupta, J. Pfeil, S. Kumar, C. Poulsen, U. Lauer, A. Hamann, U. Hoffmann, R. Haag

Bioconjug Chem. 2015, 26(4), 669-79 ...

Peptide-based therapy is a promising strategy for antigen-specific immunosuppression to treat or even heal autoimmune diseases with significantly reduced adverse effects compared to conventional therapies. However, there has been no major success due to the drawbacks of native peptides, i.e., limited bioavailability. Considering the importance and limitations of peptide-based therapies for treatment of autoimmune diseases, we designed and constructed oligoglycerol (OG)- and polyglycerol (PG)-based peptide conjugates. They were evaluated for their biological activity (in vitro and in vivo), bioavailability, and tolerogenic potential. Among the OG- and PG-peptide constructs, PG-peptide constructs exhibited an extended bioavailability compared to OG-peptide constructs and unconjugated peptide. Interestingly, size, structure, and linker chemistry played a critical role for the tolerogenic capacity of the constructs. The PG-peptide construct bound via an ester linkage was the most tolerogenic conjugate, while the PG-peptide construct bound via an amide induced stronger proliferation, but also higher TNF production and lower frequencies of Foxp3+ regulatory T-cells. Therefore, we conclude that PG–peptide conjugates bound via an ester linkage are not only promising candidates for tolerogenic vaccination, but also open a new avenue toward the application of peptides for the treatment of autoimmune diseases.

Biodistribution, cellular localization, and in vivo tolerability of 35S-labeled antiinflammatory dendritic polyglycerol sulfate amine

C. Holzhausen, D. Gröger, L. Mundhenk, C. K. Donat, J. Schnorr, R. Haag, and A. D. Gruber

J. Nanopart. Res., 2015, 17, 116 ...

Antiinflammatory dendritic polyglycerol sulfate (dPGS) holds great potential in the treatment and imaging of inflammatory processes. Here, we studied its biokinetic behavior, biodistribution, target cells, and in vivo toxicology. Following intravenous or subcutaneous application of 35sulfur-labeled dPGS amine with a molecular weight of 10.05 kDa and a hydrodynamic diameter of 5.7 ± 1.5 nm to mice, tissues were collected at specific time points (2, 15 min; 1, 24 h; 5, 21 days) and analyzed by liquid scintillation counting, autoradiography, radioluminography, and light microscopic autoradiography. The blood half-life of dPGS amine was 12 days. The major route of elimination was via the bile and feces. Elimination via the kidney and urine was only initially observed after i.v., but not after s.c. injection. Regardless of the administration mode, liver and spleen were late target organs where dPGS amine accumulated in phagocytic cells. Despite bioaccumulation, toxicological histopathology failed to identify any adverse effects at any time and in any tissues examined suggesting a high in vivo biocompatibility and encouraging future investigation for biomedical applications.

Size-dependence of steric shielding and multivalency effects for globular binding inhibitors

J. Vonnemann, S. Liese, C. Kuehne, K. Ludwig, J. Dernedde, C. Böttcher, R. R. Netz, R. Haag

J. Am. Chem. Soc., 2015, 137(7), 2572–2579 ...

Competitive binding inhibitors based on multivalent nanoparticles have shown great potential for preventing virus infections. However, general design principles of highly efficient inhibitors are lacking as the quantitative impact of factors such as virus concentration, inhibitor size, steric shielding, or multivalency effects in the inhibition process is not known. Based on two complementary experimental inhibition assays we determined size-dependent steric shielding and multivalency effects. This allowed us to adapt the Cheng–Prusoff equation for its application to multivalent systems. Our results show that the particle and volume normalized IC50 value of an inhibitor at very low virus concentration predominantly depends on its multivalent association constant, which itself exponentially increases with the inhibitor/virus contact area and ligand density. Compared to multivalency effects, the contribution of steric shielding to the IC50 values is only minor, and its impact is only noticeable if the multivalent dissociation constant is far below the virus concentration, which means if all inhibitors are bound to the virus. The dependence of the predominant effect, either steric shielding or multivalency, on the virus concentration has significant implications on the in vitro testing of competitive binding inhibitors and determines optimal inhibitor diameters for the efficient inhibition of viruses.

Dendronylation: Residue-specific chemoselective attachment of oligoglycerol dendrimers on proteins with noncanonical amino acids.

Ying Ma, Bala N.S. Thota, Rainer Haag, and Nediljko Budisa

Bioorg. Med. Chem. Lett.,2015, 25(22), 5247-5249 ...

Polyglycerol dendrimers as an important class of polymeric materials especially attractive for covalent attachment to therapeutic proteins as a useful alternative to traditional PEGylation procedures. Herein, we combine in vivo noncanonical amino acid (ncAA) incorporation and chemoselective conjugation in vitro to produce novel hybrid protein–dendrimer conjugates with the defined architectures. We incorporated Azidohomoalanine (Aha) as methionine substitute in vivo into various protein scaffolds to allow non-invasive dendrimer conjugations (dendronylation). Our approach makes recombinant proteins accessible for the design of multivalent dendrimer conjugates since it enables the preparation of many sequences with various positions for regioselective dendronylation.

Multivalent dendritic polyglycerolamine with arginine and histidine end groups for efficient siRNA transfection

F. Sheikhi Mehrabadi, H. Zeng, M. Johnson, C. Schlesener, Z. Guan, R. Haag

Beilstein J. Org. Chem. 2015, 11, 763–772 ...

The success of siRNA-based therapeutics highly depends on a safe and efficient delivery of siRNA into the cytosol. In this study, we post-modified the primary amines on dendritic polyglycerolamine (dPG-NH2) with different ratios of two relevant amino acids, namely, arginine (Arg) and histidine (His). To investigate the effects from introducing Arg and His to dPG, the resulting polyplexes of amino acid functionalized dPG-NH2s (AAdPGs)/siRNA were evaluated regarding cytotoxicity, transfection efficiency, and cellular uptake. Among AAdPGs, an optimal vector with (1:3) Arg to His ratio, showed efficient siRNA transfection with minimal cytotoxicity (cell viability ≥ 90%) in NIH 3T3 cells line. We also demonstrated that the cytotoxicity of dPG-NH2 decreased as a result of amino acid functionalization. While the incorporation of both cationic (Arg) and pH-responsive residues (His) are important for safe and efficient siRNA transfection, this study indicates that AAdPGs containing higher degrees of His display lower cytotoxicity and more efficient endosomal escape.

Integrative self-sorting: a versatile strategy for the construction of complex supramolecular architecture

Z. He, W. Jiang, C. A. Schalley

Chem. Soc. Rev., 2015, 44, 779-789 ...

Large protein-sized synthetic supramolecular architecture is rare and certainly has not yet achieved the structural and functional complexity of biomolecules. As multiple, identical copies of a few building blocks are repetitively used, a highly symmetrical architecture results with limitations in function. In marked contrast, functional structures in nature are often assembled with high geometric precision from many different building blocks. They cooperate in a complex way realizing energy conversion, mechanical motion or transport phenomena. Beyond self-assembly, the structurally and functionally complex biomolecular machines rely on self-sorting to correctly position all subunits through orthogonal recognition sites. Mimicking such self-sorting processes is a promising strategy for supramolecular synthesis – resulting in higher structural complexity and promising access to a more sophisticated function. The term “integrative self-sorting” was coined to describe the strategy to form well-defined assemblies with well-controlled subunit positions. The key process is the incorporation of two or more orthogonal binding motifs into at least some of the subunits. Modularity and programmability based on orthogonal yet similar binding motifs generate diversity and complexity. Integrative self-sorting is thus inherently related to systems chemistry. Depending on the individual binding motifs, (multi-)stimuli responsiveness can be achieved. When different recognition events en route to the final assembly occur on significantly different time scales, kinetic pathway selection is observed. In this account, we review the modularity, programmability, and emergent properties of integrative self-sorting, emphasizing its utility and perspective for complex supramolecular

Dynamic properties of force fields

F. Vitalini, A. S. J. S. Mey, F. Noé, B. G. Keller

J. Chem. Phys.2015, 142, 084101 ...

Molecular-dynamics simulations are increasingly used to study dynamic properties of biological systems. With this development, the ability of force fields to successfully predict relaxation timescales and the associated conformational exchange processes moves into focus. We assess to what extent the dynamic properties of model peptides (Ac-A-NHMe, Ac-V-NHMe, AVAVA, A10) differ when simulated with different force fields (AMBER ff99SB-ILDN, AMBER ff03, OPLS-AA/L, CHARMM27, and GROMOS43a1). The dynamic properties are extracted using Markov state models. For single-residue models (Ac-A-NHMe, Ac-V-NHMe), the slow conformational exchange processes are similar in all force fields, but the associated relaxation timescales differ by up to an order of magnitude. For the peptide systems, not only the relaxation timescales, but also the conformational exchange processes differ considerably across force fields. This finding calls the significance of dynamic interpretations of molecular-dynamics simulations into question.

Peptide-polymer ligands for a tandem WW-domain, an adaptive multivalent protein-protein interaction: lessons on the thermodynamic fitness of flexible ligands.

K. Koschek, V. Durmaz, O. Krylova, M. Wieczorek, S. Gupta, M. Richter, A. Bujotzek, C. Fischer, R. Haag, C. Freund, M. Weber, J. Rademann

Beilstein J. Org. Chem.,2015, 11, 837–847 ...

Influence of length and flexibility of spacers on the binding affinity of divalent ligands

S. Liese, R. R. Netz

Beilstein J. Org. Chem. 2015, 11, 804–816 ...

We present a quantitative model for the binding of divalent ligand–receptor systems. We study the influence of length and flexibility of the spacers on the overall binding affinity and derive general rules for the optimal ligand design. To this end, we first compare different polymeric models and determine the probability to simultaneously bind to two neighboring receptor binding pockets. In a second step the binding affinity of divalent ligands in terms of the IC50 value is derived. We find that a divalent ligand has the potential to bind more efficiently than its monovalent counterpart only, if the monovalent dissociation constant is lower than a critical value. This critical monovalent dissociation constant depends on the ligand-spacer length and flexibility as well as on the size of the receptor. Regarding the optimal ligand-spacer length and flexibility, we find that the average spacer length should be equal or slightly smaller than the distance between the receptor binding pockets and that the end-to-end spacer length fluctuations should be in the same range as the size of a receptor binding pocket.

Theoretical and Experimental Investigation of Crown/Ammonium Complexes in Solution

A. Achazi, L. K.S. von Krbek, C. A. Schalley, B. Paulus

J. Comput. Chem. 2015, 37(1), 18–24 ...

The Gibbs energies of association inline image between primary alkyl ammonium ions and crown ethers in solution are measured and calculated. Measurements are performed by isothermal titration calorimetry and revealed a strong solvent-dependent ion pair effect. Calculations are performed with density functional theory including Grimme's dispersion correction D3(BJ). The translational, rotational, and vibrational contributions to the Gibbs energy of association inline image are taken into account by a rigid-rotor-harmonic-oscillator approximation with a free-rotor approximation for low lying vibrational modes. Solvation effects inline image are taken into account by applying the continuum solvation model COSMO-RS. Our study aims at finding a suitable theoretical method for the evaluation of the host guest interaction in crown/ammonium complexes as well as the observed ion pair effects. A good agreement of theory and experiment is only achieved, when solvation and the effects of the counterions are explicitly taken into account.

Gating the photochromism of an azobenzene by strong host-guest interactions in a divalent pseudo[2]rotaxane

M. Lohse, K. Nowosinski, N.L. Traulsen, A.J. Achazi, L.K.S. von Krbek, B. Paulus, C.A. Schalley, S. Hecht

Chem.Commun. 2015, 51, 9777-9780 ...

The ability of an E-configured azobenzene guest to undergo photoisomerisation is controlled by the presence of a complementary host. Addition of base/acid allowed for a weakening/strengthening of the interactions in the divalent pseudo[2]rotaxane complex and hence could switch on/off photochromic activity.

SIGNR3-dependent immune regulation by Lactobacillus acidophilus surface layer protein A in colitis

Y. L. Lightfoot, K. Selle, T. Yang, Y. Jun Goh, B. Sahay, M. Zadeh, J. L. Owen, N. Colliou, E. Li, T. Johannssen B. Lepenies, T. R. Klaenhammer, M. Mohamadzadeh

The EMBO Journal, 2015, 34, 881–895 ...

Intestinal immune regulatory signals govern gut homeostasis. Breakdown of such regulatory mechanisms may result in inflammatory bowel disease (IBD). Lactobacillus acidophilus contains unique surface layer proteins (Slps), including SlpA, SlpB, SlpX, and lipoteichoic acid (LTA), which interact with pattern recognition receptors to mobilize immune responses. Here, to elucidate the role of SlpA in protective immune regulation, the NCK2187 strain, which solely expresses SlpA, was generated. NCK2187 and its purified SlpA bind to the C-type lectin SIGNR3 to exert regulatory signals that result in mitigation of colitis, maintenance of healthy gastrointestinal microbiota, and protected gut mucosal barrier function. However, such protection was not observed in Signr3−/− mice, suggesting that the SlpA/SIGNR3 interaction plays a key regulatory role in colitis. Our work presents critical insights into SlpA/SIGNR3-induced responses that are integral to the potential development of novel biological therapies for autoinflammatory diseases, including IBD.

Carbohydrate-Based Vaccines - Methods and Protocols

B. Lepenies

Methods in Molecular Biology, 2015, Vol. 1331 ...

This volume summarizes current cutting-edge methods related to carbohydrate-based vaccines, from the identification of a suitable carbohydrate antigen via the preparation of glycoconjugate vaccines to the characterization of vaccine candidates for their use in pre-clinical and clinical studies. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and key tips on troubleshooting and avoiding known pitfalls.

The C-Type Lectin Receptor Mincle Binds to Streptococcus pneumoniae but Plays a Limited Role in the Anti-Pneumococcal Innate Immune Response

A. Rabes, S. Zimmermann, K. Reppe, R. Lang, P. H. Seeberger, N. Suttorp, M. Witzenrath, B. Lepenies, B. Opitz

PLoS ONE 2015, 10(2), e0117022 ...

The innate immune system employs C-type lectin receptors (CLRs) to recognize carbohydrate structures on pathogens and self-antigens. The Macrophage-inducible C-type lectin (Mincle) is a FcRγ-coupled CLR that was shown to bind to mycobacterial cord factor as well as certain fungal species. However, since CLR functions during bacterial infections have not yet been investigated thoroughly, we aimed to examine their function in Streptococcus pneumonia infection. Binding studies using a library of recombinantly expressed CLR-Fc fusion proteins indicated a specific, Ca2+-dependent, and serotype-specific binding of Mincle to S. pneumonia. Subsequent experiments with different Mincle-expressing cells as well as Mincle-deficient mice, however, revealed a limited role of this receptor in bacterial phagocytosis, neutrophil-mediated killing, cytokine production, and antibacterial immune response during pneumonia. Collectively, our results indicate that Mincle is able to recognize S. pneumonia but is not required for the anti-pneumococcal innate immune response.

Dendritic polyglycerol sulfate inhibits microglial activation and reduces hippocampal CA1 dendritic spine morphology deficits.

Dusica Maysinger, Dominic Gröger, Andrew Lake, Kai Licha, Marie Weinhart, Philip K.-Y. Chang, Rose Mulvey, Rainer Haag, and R. Anne McKinney

Biomacromolecules, 2015, 16(9), 3073–3082 ...

Hyperactivity of microglia and loss of functional circuitry is a common feature of many neurological disorders including those induced or exacerbated by inflammation. Herein, we investigate the response of microglia and changes in hippocampal dendritic postsynaptic spines by dendritic polyglycerol sulfate (dPGS) treatment. Mouse microglia and organotypic hippocampal slices were exposed to dPGS and an inflammogen (lipopolysaccharides). Measurements of intracellular fluorescence and confocal microscopic analyses revealed that dPGS is avidly internalized by microglia but not CA1 pyramidal neurons. Concentration and time-dependent response studies consistently showed no obvious toxicity of dPGS. The adverse effects induced by proinflammogen LPS exposure were reduced and dendritic spine morphology was normalized with the addition of dPGS. This was accompanied by a significant reduction in nitrite and proinflammatory cytokines (TNF-α and IL-6) from hyperactive microglia suggesting normalized circuitry function with dPGS treatment. Collectively, these results suggest that dPGS acts anti-inflammatory, inhibits inflammation-induced degenerative changes in microglia phenotype and rescues dendritic spine morphology.

Multivalency at Interfaces: Supramolecular Carbohydrate-Functionalized Graphene Derivatives for Bacterial Capture, Release, and Disinfection.

Zhenhui Qi, Priya Bharate, Chian-Hui Lai, Benjamin Ziem, Christoph Böttcher, Andrea Schulz, Fabian Beckert, Benjamin Hatting, Rolf Mülhaupt, Peter H. Seeberger, and Rainer Haag

Nano Lett., 2015, 15(9), 6051–6057 ...

A supramolecular carbohydrate-functionalized two-dimensional (2D) surface was designed and synthesized by decorating thermally reduced graphene sheets with multivalent sugar ligands. The formation of host–guest inclusions on the carbon surface provides a versatile strategy, not only to increase the intrinsic water solubility of graphene-based materials, but more importantly to let the desired biofunctional binding groups bind to the surface. Combining the vital recognition role of carbohydrates and the unique 2D large flexible surface area of the graphene sheets, the addition of multivalent sugar ligands makes the resulting carbon material an excellent platform for selectively wrapping and agglutinating Escherichia coli (E. coli). By taking advantage of the responsive property of supramolecular interactions, the captured bacteria can then be partially released by adding a competitive guest. Compared to previously reported scaffolds, the unique thermal IR-absorption properties of graphene derivatives provide a facile method to kill the captured bacteria by IR-laser irradiation of the captured graphene–sugar–E. coli complex.

Discrete Multiporphyrin Pseudorotaxane Assemblies from Di- and Tetravalent Porphyrin Building Blocks

M. Lohse, L.K.S. von Krbek, S. Radunz, S. Moorthy, C.A. Schalley, S. Hecht

Beilstein J. Org. Chem. 2015, 11, 748–762. ...

Two pairs of divalent and tetravalent porphyrin building blocks carrying the complementary supramolecular crown ether/secondary ammonium ion binding motif have been synthesized and their derived pseudorotaxanes have been studied by a combination of NMR spectroscopy in solution and ESI mass spectrometry in the gas phase. By simple mixing of the components the formation of discrete dimeric and trimeric (metallo)porphyrin complexes predominates, in accordance to binding stoichiometry, while the amount of alternative structures can be neglected. Our results illustrate the power of multivalency to program the multicomponent self-assembly of specific entities into discrete functional nanostructures.

Performance of dispersion corrected DFT for the weak interaction between aromatic molecules and extended carbon-based systems

D. Mollenhauer, C. Brieger, E. Voloshina, B. Paulus

J. Chem. Phys. C 2015, 119, 1898-1904 ...

The performance of different DFT approaches in combination with dispersion correction is studied for the interaction between aromatic molecules and extended carbon-based materials on the example of the pyridine–graphene system. The basic interaction is modeled using graphene fragments of increasing size as well as periodic boundary conditions. Different DFT-D2/D3 methods are tested for small and medium fragment systems in comparison to wave-function-based CCSD(T) and SCS-MP2 approaches. Furthermore, the adsorption energy between pyridine and extended graphene sheets or periodic modeled graphene calculated by DFT-D2/D3 or nonlocal correlation functionals (vdW-DF) is compared to experimental values. The study of DFT-D performance along different scales reveals the dispersion correction as too strong along increasing graphene fragment sizes. Finally, this leads to different methodology advice for small and extended pyridine–graphene systems.

Optimized effective charge density using polyglycerol amines leads to strong and target specific knockdown efficacy.

Anna Maria Staedtler, Markus Hellmund, Fatemeh Sheikhi Mehrabadi, Bala N. S. Thota, Thomas M. Zollner, Markus Koch, Rainer Haag and Nicole Schmidt

J. Mater. Chem. B, 2015, 3, 8993-9000 ...

RNA interference (RNAi)-based therapy extends the range of “druggable” targets beyond existing pharmacological drugs and enables the development of new treatment strategies for various diseases. A prerequisite are non-viral polyvalent gene delivery vectors capable for safe and effective siRNA delivery to cells in vivo allowing a broad clinical application. We synthesized hyperbranched polyglycerol amines (hPG amines) which varied in their charge density, multiplicity (absolute frequency of amine groups) and core size to successfully develop potent and safe siRNA transfer vectors. The characterization of hyperbranched polyglycerol amines with an invariable core size (8 kDa) but different amine loading revealed a correlation between the effective charge density and the transfection efficacy without impacting the cell viability in vitro. However, this correlation was not seen in tumor bearing mice in vivo treated with 8 kDa hPG amine–siRNA complexes. Improving the effective charge density and the multiplicity of amine functionalities by increasing the molecular weight (43 kDa) revealed comparable transfection efficacy in vitro but less toxic side effects after systemic administration in vivo compared to the respective hPG amine (8 kDa). In addition, in vivo delivery of 43 kDa hPG amine–siRNA–polyplexes in tumors resulted in a highly specific and significant knockdown effect. These findings demonstrate that hyperbranched polyglycerol amines with a balanced effective charge density, multiplicity and core size are promising gene delivery vectors for siRNA therapy which enable to address so far “undruggable” targets due to high tolerability and effective siRNA delivery.

Structure–activity relationship study of dendritic polyglycerolamines for efficient siRNA transfection.

Fatemeh Sheikhi Mehrabadi, Ole Hirsch, Reiner Zeisig, Paola Posocco, Erik Laurini, Sabrina Pricl, Rainer Haag, Wolfgang Kemmner and Marcelo Calderón

RSC Adv.,2015, 5, 78760-78770 ...

Structure–activity relationship studies are pivotal in the development of existing small interfering RNA (siRNA) nanocarriers and in designing new delivery systems. In this paper, we investigated the ability of four dendritic polyglycerolamines (dPG-NH2) with increasing amine degree of functionalization (DF) on dendritic polyglycerol (dPG) to complex DNA by a coupled in silico/in vitro approach. In parallel, we examined our dPG-NH2 analogues for siRNA complexation, cytotoxicity, and transfection efficiency in vitro and in vivo. Our simulation data indicate the most effective nucleic acid affinities for dPG-NH2 analogues with DF ≥ 50%. Concomitantly, the results of in vitro and in vivo transfection studies also demonstrate efficient siRNA transfection only for those dPG-NH2 analogues with DF ≥ 50%. Thus, both MD simulation and siRNA knockdown studies show that a minimum DF per dPG unit (namely 50% on a 10 kDa dPG) is needed to achieve efficient siRNA interaction and successful transfection.

In-depth analysis of switchable glycerol based polymeric coatings for cell sheet engineering

T. Becherer, S. Heinen, Q. Wei, R. Haag, and M. Weinhart

Acta Biomater.,2015, 25, 43–55 ...

Scaffold-free cell sheet engineering using thermoresponsive substrates provides a promising alternative to conventional tissue engineering which in general employs biodegradable scaffold materials. We have previously developed a thermoresponsive coating with glycerol based linear copolymers that enables gentle harvesting of entire cell sheets. In this article we present an in-depth analysis of these thermoresponsive linear polyglycidyl ethers and their performance as coating for substrates in cell culture in comparison with commercially available poly(N-isopropylacrylamide) (PNIPAM) coated culture dishes. A series of copolymers of glycidyl methyl ether (GME) and glycidyl ethyl ether (EGE) was prepared in order to study their thermoresponsive properties in solution and on the surface with respect to the comonomer ratio. In both cases, when grafted to planar surfaces or spherical nanoparticles, the applied thermoresponsive polyglycerol coatings render the respective surfaces switchable. Protein adsorption experiments on copolymer coated planar surfaces with surface plasmon resonance (SPR) spectroscopy reveal the ability of the tested thermoresponsive coatings to be switched between highly protein resistant and adsorptive states. Cell culture experiments demonstrate that these thermoresponsive coatings allow for adhesion and proliferation of NIH 3T3 fibroblasts comparable to TCPS and faster than on PNIPAM substrates. Temperature triggered detachment of complete cell sheets from copolymer coated substrates was accomplished within minutes while maintaining high viability of the harvested cells. Thus such glycerol based copolymers present a promising alternative to PNIPAM as a thermoresponsive coating of cell culture substrates.

Structural requirements of mono- and multivalent L-selectin blocking aptamers for enhanced receptor inhibition in vitro and in vivo

Sebastian B. Riese, Konrad Buscher, Sven Enders, Christian Kuehne, Rudolf Tauber, and Jens Dernedde

Nanomedicine 2015, 12 (4), 901–908 ...

L-selectin mediates extravasation of leukocytes from blood into the surrounding tissue during inflammation and is therefore a therapeutical target in certain overwhelming immune reactions. In this study, we characterized an L-selectin specific blocking DNA aptamer with respect to nucleotide composition and target binding. Introduction of deletions and nucleotide exchanges resulted in an optimized DNA sequence but preservation of the IC50 in the low nanomolar range. The inhibitory potential was significantly increased when the aptamer was displayed as a di- and trimer connected via appropriate linker length. Similar to monoclonal antibodies, trimer yielded picomolar IC50 values in a competitive binding assay. In comparison to the monovalent aptamer, the trivalent assembly reduced PBMC interactions to L-selectin ligands 90-fold under shear and exerted superior inhibition of PBMC rolling in vivo. In conclusion, our work demonstrates the feasibility of optimizing aptamer sequences and shows that multivalent ligand presentation enables superior adhesion receptor targeting.

Synthesis of multivalent carbohydrate mimetics with aminopolyol end groups and their evaluation as L-selectin inhibitors

J. Salta, J. Dernedde, H.-U. Reissig

Beilstein J. Org. Chem. 2015, 11, 638–646 ...

In this article a series of divalent and trivalent carbohydrate mimetics on the basis of an enantiopure aminopyran and of serinol is described. These aminopolyols are connected by amide bonds to carboxylic acid derived spacer units either by Schotten–Baumann acylation or by coupling employing HATU as reagent. The O-sulfation employing the SO3·DMF complex was optimized. It was crucial to follow this process by 700 MHz 1H NMR spectroscopy to ensure full conversion and to use a refined neutralization and purification protocol. Many of the compounds could not be tested as L-selectin inhibitor by SPR due to their insolubility in water, nevertheless, a divalent and a trivalent amide showed surprisingly good activities with IC50 values in the low micromolar range.

Understanding multivalent interactions

C.A. Schalley, B. Koksch, R. Haag

Int. Innov., 2015, 169, 99-101 ...

Impact of multivalent charge presentation on peptide–nanoparticle aggregation

D. Schöne, B. Schade, C. Böttcher, B. Koksch

Beilstein J. Org. Chem. 2015, 11, 792–803 ...

Strategies to achieve controlled nanoparticle aggregation have gained much interest, due to the versatility of such systems and their applications in materials science and medicine. In this article we demonstrate that coiled-coil peptide-induced aggregation based on electrostatic interactions is highly sensitive to the length of the peptide as well as the number of presented charges. The quaternary structure of the peptide was found to play an important role in aggregation kinetics. Furthermore, we show that the presence of peptide fibers leads to well-defined nanoparticle assembly on the surface of these macrostructures.

Supramolecular Polymers as Surface Coatings: Rapid Fabrication of Healable Superhydrophobic and Slippery Surfaces

Z. Qi, Q. Wei, C. Schlaich, S. Prévost, M. Gradzielski, R. Haag, C. A. Schalley

Adv. Mater., 2014, 26, 7358 - 7364

Full TextSupporting Information

Supramolecular polymerization for non-wetting surface coatings. Self-assembly of low-molecular-weight gelators (LMWGs) with perfluorinated side chains can be utilized to rapidly construct superhydrophobic as well as liquid-infused slippery surfaces within minutes. The lubricated slippery surface exhibits impressive repellency for biological liquids such as human serum and blood and very fast self-healing.

Self-sorting of crown ether/secondary ammonium ion hetero-[c2]daisy chain pseudorotaxanes

B. Zheng, F. Klautzsch, M. Xue, F. Huang, C. A. Schalley

Org. Chem. Frontiers 2014, 1, 532-540

Full TextSupporting Information

Four monomeric building blocks equipped with one crown ether and one secondary ammonium ion are synthesized and studied with respect to their ability to form daisy chain dimers. Two crown ethers with different cavity sizes – i.e. [21]crown-7 and [24]crown-8 – and two ammonium ions substituted with either a thin alkyl group or a more bulky benzyl group are used as the binding motifs. Self-sorting behaviour can be expected as the [21]crown-7/alkyl ammonium and [24]crown-8/benzyl ammonium binding motifs are orthogonal. Three homodimers are characterized by NMR, X-ray crystallo-graphy and ESI mass spectrometry. They are recognizable by the presence of signals for diastereotopic protons in the 1H NMR spectra. The formation of hetero-[c2]daisy chain dimers can be monitored by NMR spectroscopy and ESI mass spectrometry and show the expected self-sorting behaviour.

Evaluation of Multivalency as an Organization Principle for the Efficient Synthesis of Doubly and Triply Threaded Amide Rotaxanes

L. Kaufmann, N. L. Traulsen, A. Springer, H. V. Schröder, T. Mäkelä, K. Rissanen, C. A. Schalley

Org. Chem. Frontiers 2014, 1, 521-531

Full TextSupporting Information

Mono-, di- and trivalent pseudorotaxanes with tetralactam macrocycle hosts and axles containing diamide binding stations as the guests have been synthesised. Their threading behaviour was analyzed in detail by NMR experiments and isothermal titration calorimetry. An X-ray crystal structure of the monovalent pseudorotaxane confirms the binding motif. Double mutant cycle analysis provides the effective molarities and insight into the chelate cooperativity of multivalent binding. While the second binding event in a trivalent pseudorotaxane exhibits a slightly positive cooperativity, the third binding is nearly non-cooperative. Nevertheless, the enhanced binding affinities resulting from the multivalent interaction are the basis for a highly efficient synthesis of di- and trivalent rotaxanes through stoppering the axle termini by “click” chemistry. Evidence for the multiply threaded geometry comes from NMR spectroscopy as well as tandem mass-spectrometric fragmentation experiments of mass-selected rotaxane ions in the gas phase. Furthermore, the trivalent rotaxane can be controlled by external stimuli (chloride addition and removal) which lead to an elevator-type movement of the wheel along the axle.

Stimuli-Induced Folding Cascade of a Linear Oligomeric Guest Chain Programmed through Cucurbit[n]uril Self-Sorting (n = 6, 7, 8)

L. Cera, C. A. Schalley

Chem. Sci. 2014, 5, 2560-2567

Full TextSupporting Information

A six-station linear guest for cucurbit[7]uril and cucurbit[8]uril has been synthesized in order to implement a cascade of transformations driven by external stimuli. The guest chain is sequence-programmed with electron-deficient viologen and electron-rich naphthalene stations linked by either flexible or rigid spacers that affect the chain’s folding properties. Together with the orthogonal guest selectivity of the two cucurbiturils, these properties result in self-sorted cucurbituril pseudorotaxane foldamers. Each transformation is controlled by suitable chemical and redox inputs and leads not only to refolding of the guest chain, but also to the liberation of secondary messenger molecules which render the system presented here reminiscent of natural signaling cascades. The steps of the cascade are analyzed by UV/Vis, 1H NMR and electrospray (tandem) mass spectrometry to investigate the different pseudorotaxane structures in detail. With one guest oligomer, three different cucurbiturils, and several different chemical and redox inputs, a chemical system is created which exhibits complex behavior beyond the chemist’s paradigm of the pure chemical compound.

Peptide Desorption Kinetics from Single Molecule Force Spectroscopy Studies

Stefanie Krysiak, Susanne Liese, Roland R. Netz and Thorsten Hugel

J. Am. Chem. Soc., 2014, 136, 688?697

We use a combined experimental/theoretical approach to determine the intrinsic monomeric desorption rate k0 of polytyrosine and polylysine homopeptides from flat surfaces. To this end, single polypeptide molecules are covalently attached to an AFM cantilever tip and desorbed from hydrophobic self-assembled monolayers in two complementary experimental protocols. In the constant-pulling-velocity protocol, the cantilever is moved at finite velocity away from the surface and the distance at which the constant plateau force regime ends and the polymer detaches is recorded. In the waiting-time protocol, the cantilever is held at a fixed distance above the surface and the time until the polymer detaches is recorded. The desorption plateau force is varied between 10 and 90 pN, by systematically changing the aqueous solvent quality via the addition of ethanol or salt. A simultaneous fit of the experimental data from both protocols with simple two-state kinetic polymer theory allows to unambiguously disentangle and determine the model parameters corresponding to polymer contour length L, Kuhn length a, adsorption free energy λ, and intrinsic monomeric desorption rate k0. Crucial to our analysis is that a statistically significant number of single-polymer desorption experiments are done with one and the same single polymer molecule for different solvent qualities. The surprisingly low value of about k0 ≈ 105 Hz points to significant cooperativity in the desorption process of single polymers.

Multivalent anchored and crosslinked hyperbranched polyglycerol monolayers as antifouling coating for titanium oxide surfaces.

Q. Wei, S. Krysiak, K. Achazi, T. Becherer, P.L. Noeske, F. Paulus, H. Liebe, I. Grunwald, J. Dernedde, A. Hartwig, T. Hugel, R. Haag

‎Colloids Surf., B, 2014 122, 684–692 ...

A set of new catecholic monolayer coatings was developed to improve the antifouling performance of TiO2 surfaces. To solve the problem of the weak charge-transfer interaction between a single catechol anchor and TiO2, multiple catechol groups were combined with hyperbranched polyglycerol (hPG) which is a distinct dendritic scaffold that exposes its multivalent anchor groups on the surface. Thus, multivalent catecholic hPGs can be easily prepared for surface modification. The immobilization of the compounds was monitored by quartz crystal microbalance with dissipation monitoring. Surface properties of the coatings were analyzed by water contact angle, X-ray photoelectron spectroscopy, and atomic force microscopy. The antifouling ability and stability were investigated by protein adsorption and cell adhesion. By increasing the number of catechol groups on the hPG scaffold, the stability and surface coverage could be significantly enhanced. Moreover, the inner-layer crosslinking of the coatings by grafting and initiating vinyl groups clearly improved their long-term stability. As a result, hPG with a catecholic functional degree of 10% (hPG-Cat10) and hPG with both catecholic and vinylic functional degree of 5% (hPG-Cat5-V5) were identified as the best catecholic hPGs to prepare bioinert and stable monolayer coatings on TiO2.

Biological evaluation of multivalent Lewis X-MGL-1 interactions

M. Eriksson, S. Serna, M. Maglinao, M.K. Schlegel, P.H. Seeberger, N.C. Reichardt, B. Lepenies

ChemBioChem 2014, 15(6): 844-51

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Myeloid C-type lectin receptors (CLRs) expressed by antigen-presenting cells are pattern-recognition receptors involved in the recognition of pathogens as well as of self-antigens. The interaction of carbohydrate ligands with a CLR can trigger immune responses. Although several CLR ligands are known, there is limited insight into CLR targeting by carbohydrate ligands. The weak affinity of lectin–carbohydrate interactions often renders multivalent carbohydrate presentation necessary. Here, we have analyzed the impact of multivalent presentation of the trisaccharide Lewis X (LeX) epitope on its interaction with the CLR macrophage galactose-type lectin-1 (MGL-1). Glycan arrays, including N-glycan structures with terminal LeX, were prepared by enzymatic extension of immobilized synthetic core structures with two recombinant glycosyltransferases. Incubation of arrays with an MGL-1-hFc fusion protein showed up to tenfold increased binding to multiantennary N-glycans displaying LeX structures, compared to monovalent LeX trisaccharide. Multivalent presentation of LeX on the model antigen ovalbumin (OVA) led to increased cytokine production in a dendritic cell /T cell coculture system. Furthermore, immunization of mice with LeX-OVA conjugates modulated cytokine production and the humoral response, compared to OVA alone. This study provides insights into how multivalent carbohydrate–lectin interactions can be exploited to modulate immune responses.

A platform to screen for C-type lectin receptor-binding carbohydrates and their potential for cell-specific targeting and immune modulation

M. Maglinao, M. Eriksson, M.K. Schlegel, S. Zimmermann, T. Johannssen, S. Götze, P.H. Seeberger, B. Lepenies

J. Control. Release. 2014, 175: 36-42 ...

Myeloid C-type lectin receptors (CLRs) in innate immunity represent a superfamily of pattern recognition receptors that recognize carbohydrate structures on pathogens and self-antigens. The primary interaction of an antigen-presenting cell and a pathogen shapes the following immune response. Therefore, the identification of CLR ligands that can either enhance or modulate the immune response is of interest. We have developed a screening platform based on glycan arrays to identify immune modulatory carbohydrate ligands of CLRs. A comprehensive library of CLRs was expressed by fusing the extracellular part of each respective CLR, the part containing the carbohydrate-recognition domain (CRD), to the Fc fragment of human IgG1 molecules. CLR-Fc fusion proteins display the CRD in a dimeric form, are properly glycosylated, and can be detected by a secondary antibody with a conjugated fluorophore. Thus, they are valuable tools for high-throughput screening. We were able to identify novel carbohydrate binders of CLRs using the glycan array technology. These CLR-binding carbohydrates were then covalently attached to the model antigen ovalbumin. The ovalbumin neoglycoconjugates were used in a dendritic cell/T cell co-culture assay to stimulate transgenic T cells in vitro. In addition, mice were immunized with these conjugates to analyze the immune modulatory properties of the CLR ligands in vivo. The CLR ligands induced an increased Th1 cytokine production in vitro and modulated the humoral response in vivo. The platform described here allows for the identification of CLR ligands, as well as the evaluation of each ligand's cell-specific targeting and immune modulatory properties.

Development of 1,8-Naphthalimides as clathrin inhibitors

Macgregor, K.A., Robertson, M.J., Young, K.A., von Kleist, L., Stahlschmidt, W., Whiting, A., Chau, N., Robinson, P.J., Haucke V., McCluskey, A.

J. Med. Chem., 2014, 57 (1), pp 131–143 ...

We reported the first small molecule inhibitors of the interaction between the clathrin N-terminal domain (TD) and endocyctic accessory proteins (i.e., clathrin inhibition1). Initial screening of a ∼17 000 small molecule ChemBioNet library identified 1. Screening of an existing in-house propriety library identified four substituted 1,8-napthalimides as ∼80–120 μM clathrin inhibitors. Focused library development gave 3-sulfo-N-(4-aminobenzyl)-1,8-naphthalimide, potassium salt (18, IC50 ≈ 18 μM). A second library targeting the 4-aminobenzyl moiety was developed, and four analogues displayed comparable activity (26, 27, 28, 34 with IC50 values of 22, 16, 15, and 15 μM respectively) with a further four (24, 25, 32, 33) more active than 18 with IC50 values of 10, 6.9, 12, and 10 μM, respectively. Docking studies rationalized the structure–activity relationship (SAR) with the biological data. 3-Sulfo-N-benzyl-1,8-naphthalimide, potassium salt (25) with an IC50 ≈ 6.9 μM, is the most potent clathrin terminal domain–amphiphysin inhibitor reported to date.

Role of the C-type lectin receptors MCL and DCIR in experimental colitis

J. Hütter, M. Eriksson, T. Johannssen, R. Klopfleisch, D. von Smolinski, A.D. Gruber, P.H. Seeberger, B. Lepenies

PLOS One 2014, 9(7), e103281 ...

Inflammatory bowel disease (IBD) is a chronic inflammatory disorder of the gastrointestinal tract. Though its exact etiology is still unclear, it is proposed that an imbalance in the intestinal homeostasis leads to a disturbed interaction between commensal microbiota and the mucosal immune system. Previous studies have shown that both innate and adaptive immunity are involved in an overwhelming colon inflammation, and thus contribute to the pathogenesis of IBD. In innate immunity, several pattern recognition receptors such as Toll-like receptors, NOD-like receptors or C-type lectin receptors (CLRs) are involved in IBD pathogenesis. Myeloid CLRs are mainly expressed by antigen-presenting cells and bind to glycan structures present on self or foreign antigens. The Macrophage-restricted C-type lectin (MCL) and the Dendritic cell immunoreceptor (DCIR) are two poorly characterized members of the CLR family. In this study, we investigated the role of MCL and DCIR in the pathogenesis of murine colitis. Both CLRs bound to intestinal microbiota to a different extent. They modulated the production of pro-inflammatory cytokines by antigen-presenting cells upon stimulation with heat-killed microbiota and impacted subsequent T cell responses. To analyze whether MCL and DCIR contribute to the pathogenesis of IBD, the dextran sulfate sodium (DSS) murine colitis model was employed. MCL−/− as well as DCIR−/− mice exhibited only a slightly increased severity of disease compared to wild-type mice indicating a limited role for MCL and DCIR in the regulation of intestinal immunity.

Genetics and virulence role of the classical Group A Streptococcus Lancefield antigen

N.M. van Sorge, J.N. Cole, K. Kuipers, A. Henningham, R.K. Aziz, A. Kasirer-Friede, L. Lin, E.T. Berends, M.R. Davies, G. Dougan, F. Zhang, S. Dahesh, L. Shaw, J. Gin, M. Cunningham, J.A. Merriman, J. Hütter, B. Lepenies, S.H.M. Rooijakkers, R. Malley, M.J. Walker, S.J. Shattil, P.M. Schlievert, B. Choudhury, V. Nizet

Cell Host Microbe 2014, 15(6), 729-40 ...

Group A Streptococcus (GAS) is a leading cause of infection-related mortality in humans. All GAS serotypes express the Lancefield group A carbohydrate (GAC), comprising a polyrhamnose backbone with an immunodominant N-acetylglucosamine (GlcNAc) side chain, which is the basis of rapid diagnostic tests. No biological function has been attributed to this conserved antigen. Here we identify and characterize the GAC biosynthesis genes, gacA through gacL. An isogenic mutant of the glycosyltransferase gacI, which is defective for GlcNAc side-chain addition, is attenuated for virulence in two infection models, in association with increased sensitivity to neutrophil killing, platelet-derived antimicrobials in serum, and the cathelicidin antimicrobial peptide LL-37. Antibodies to GAC lacking the GlcNAc side chain and containing only polyrhamnose promoted opsonophagocytic killing of multiple GAS serotypes and protected against systemic GAS challenge after passive immunization. Thus, the Lancefield antigen plays a functional role in GAS pathogenesis, and a deeper understanding of this unique polysaccharide has implications for vaccine development.

Synthesis of Rigid p-Terphenyl-Linked Carbohydrate Mimetics

M. Kandziora, H.-U. Reissig

Beilstein J. Org. Chem. 2014, 10, 1749-1758 ...

An approach to β-D-2-aminotalose- and β-D-2-aminoidose-configured carbohydrate mimetics bearing a phenyl substituent is described. Unnatural divalent rigid p-terphenyl-linked C-aryl glycosides with 2.0 nm dimension are available using Suzuki cross-couplings. The key compound, a p-bromophenyl-substituted 1,2-oxazine, was prepared by a stereoselective [3 + 3]-cyclization of a D-isoascorbic acid-derived (Z)-nitrone and lithiated TMSE-allene. The Lewis acid-induced rearrangement of this heterocycle provided the corresponding bicyclic 1,2-oxazine derivative that may be regarded as internally protected amino sugar analogue. After subsequent reduction of the carbonyl group, the resulting bicyclic compound was used for Suzuki cross-couplings to form biphenyl aminopyran or p-terphenyl-linked dimers. Hydrogenolysis afforded new unnatural aminosugar mimetics. Zinc in the presence of acid or samarium diiodide were examined for the N–O bond cleavage in order to obtain the rigid p-terphenyl-linked C-glycosyl dimers.

Synthesis of Di- and Trivalent Carbohydrate Mimetics with Oxepane Substructure by Employing Copper-Catalyzed [3+2] Cycloadditions of Alkynes with Azidooxepanes

L. Bouché, H.-U. Reissig

Eur. J. Org. Chem. 2014, 3697-3703 ...

A series of enantiopure poly(hydroxy)aminooxepanes was converted into the corresponding azidooxepanes by a safe and efficient copper(II)-catalyzed diazo transfer reaction employing nonafluorobutanesulfonyl azide as nitrogen donor. These azidooxepanes underwent smooth copper(I)-catalyzed [3+2] cycloadditions with alkynes (click reaction) to provide a series of simple triazoles. With dialkynes and a trialkyne, bis- and tristriazoles containing oxepane substructures were prepared. Due to the polyhydroxylated end groups, these compounds are regarded as carbohydrate mimetics with potential biological activities, for example, as selectin inhibitors. In addition, unsymmetrical systems and macrocyclic compounds were prepared, again by employing [3+2] cycloadditions as key steps.

Attraction between hydrated hydrophilic surfaces

Matej Kanduc, Emanuel Schneck, Roland R. Netz

Chem. Phys. Lett.,2014, 610-611, 375-380 ...

According to common knowledge, hydrophilic surfaces repel via hydration forces while hydrophobic surfaces attract, but mounting experimental evidence suggests that also hydrophilic surfaces can attract. Using all-atom molecular dynamics simulations at prescribed water chemical potential we study the crossover from hydration repulsion to hydrophobic attraction for planar polar surfaces of varying stiffness and hydrogen-bonding capability. Rescaling the partial charges of the polar surface groups, we cover the complete spectrum from very hydrophobic surfaces (characterized by contact angles θ ≃ 135°) to hydrophilic surfaces exhibiting complete wetting (θ = 0°). Indeed, for a finite range θadh < θ < 90°, we find a regime where hydrophilic surfaces attract at sub-nanometer separation and stably adhere without intervening water. The adhesive contact angle θadh depends on surface type and lies in the range 65° < θadh < 80°, in good agreement with experiments. Analysis of the total number of hydrogen bonds (HBs) formed by water and surface groups rationalizes this crossover between hydration repulsion and hydrophilic attraction in terms of a subtle balance: Highly polar surfaces repel because of strongly bound hydration water, less polar hydrophilic surfaces attract because water–water HBs are preferred over surface–water HBs. Such solvent reorganization forces presumably underlie also other important phenomena, such as selective ion adsorption to interfaces as well as ion pair formation.

Iron oxide nanoparticles stabilized with dendritic polyglycerols as selective MRI contrast agents

D. Nordmeyer, P. Stumpf, D. Gröger, A. Hofmann, S. Enders, S.B. Riese, J. Dernedde, M. Taupitz, U. Rauch, R. Haag, E. Rühl, C. Graf

Nanoscale 2014, 6, 9646-9654


Monodisperse small iron oxide nanoparticles functionalized with dendritic polyglycerol (dPG) or dendritic polyglycerol sulfate (dPGS) are prepared. They are highly stable in aqueous solutions as well as physiological media. In particular, oleic acid capped iron oxide particles (core diameter = 11 ± 1 nm) were modified by a ligand exchange process in a one pot synthesis with dPG and dPGS bearing phosphonate as anchor groups. Dynamic light scattering measurements performed in water and different biological media demonstrate that the hydrodynamic diameter of the particles is only slightly increased by the ligand exchange process resulting in a final diameter of less than 30 nm and that the particles are stable in these media. It is also revealed by magnetic resonance studies that their magnetic relaxivity is reduced by the surface modification but it is still sufficient for high contrast magnetic resonance imaging (MRI). Additionally, incubation of dPGS functionalized iron oxide nanoparticles with human umbilical vein endothelial cells showed a 50% survival at 85 nM (concentration of nanoparticles). Surface plasmon resonance (SPR) studies demonstrate that the dPGS functionalized iron oxide nanoparticles inhibit L-selectin ligand binding whereas the particles containing only dPG do not show this effect. Experiments in a flow chamber with human myelogenous leukemia cells confirmed L-selectin inhibition of the dPGS functionalized iron oxide nanoparticles and with that the L-selectin mediated leukocyte adhesion. These results indicate that dPGS functionalized iron oxide nanoparticles are a promising contrast agent for inflamed tissue probed by MRI.

Synthesis of New Enantiopure Poly(hydroxy)aminooxepanes as Building Blocks for Multivalent Carbohydrate Mimetics

L. Bouché, M. Kandziora, H.-U. Reissig

Beilstein J. Org. Chem. 2014, 10, 213-223 ...

New compounds with carbohydrate-similar structure (carbohydrate mimetics) are presented in this article. Starting from enantiopure nitrones and lithiated TMSE-allene we prepared three 1,2-oxazine derivatives which underwent a highly stereoselective Lewis acid-induced rearrangement to give bicyclic products in good yield. Subsequent reductive transformations delivered a library of new poly(hydroxy)aminooxepane derivatives. The crucial final palladium-catalyzed hydrogenolysis of the 1,2-oxazine moiety was optimized resulting in a reasonably efficient approach to a series of new seven-membered carbohydrate mimetics.

Hydration repulsion betweenmembranes and polar surfaces: Simulation approaches versus continuum theories

Matej Kanduc, Alexander Schlaich, Emanuel Schneck, Roland R. Netz

Adv. Colloid Interface Sci., 2014, 208, 142-152 ...

A review of various computer simulation approaches for the study of the hydration repulsion between lipid membranes and polar surfaces is presented. We discuss different methods and compare their advantages and limitations. We consider interaction pressures, interaction thermodynamics, and interaction mechanisms. We take a close look at the influence of the experimental boundary conditions and at repulsion mechanisms due to the unfavorable overlap of interfacial water layers. To this end, we analyze several distinct water order parameters in simulations of interacting polar surfaces and compare the results to the predictions of simple continuum theories.

Exploring Macrocycles in Functional Supramolecular Gels: From Stimuli-Responsiveness to Systems Chemistry

Z. Qi, C. A. Schalley

Acc. Chem. Res., 2014, 47 (7), 2222–2233 ...

Supramolecular gels are ideal candidates for soft, stimuli-responsive materials, because they combine the elastic behavior of solids with the microviscous properties of fluids. The dynamic networks of fibers in supramolecular gels are reminiscent of the cytoskeleton of a cell and provide scaffolds to implement function. When gels are made responsive to stimuli, these mechanical properties can be controlled. Gel−sol transitions also open opportunities to immobilize molecules inside the gel’s cavities and to release them on demand. To establish selective responsiveness, suitable recognition sites are required influencing the properties of the fiber network depending on the presence of the stimulus. Supramolecular gels are expected to be stimuli-responsive per se, for example, to temperature, mechanical stress, or an environment that is competitive with the noncovalent interactions connecting the low-molecular weight gelators. Nevertheless, the opportunities for controlling the mechanical properties are rather limited, if one merely relies on interfering with these interactions. It would be much more promising to equip the gel with additional receptor sites that offer selectivity for a broader variety of chemical stimuli. Macrocycles often exhibit a distinct host−guest chemistry and thus are excellent candidates for this purpose. A broad variety of macrocycles differing with respect to structure, topology, solubility, or biocompatibility have been incorporated in gels and endow gels with responsiveness and function. Macrocycles can have different roles: They offer rather rigid scaffolds for the construction of structurally well-defined gelator molecules. Furthermore, their host−guest interactions can be integral to gel formation, if these interactions are required to build the gel fibers. Finally, macrocycles can also be functional groups with which gelators are equipped that would also form gels in the absence of the macrocycle. Here, the macrocycle can be used as a binding site to allow additional stimuli control. To combine different stimuli for triggering gel−sol transitions certainly expands the options for establishing stimuli responsiveness. If, for example, an agent trapped inside the gel is only liberated when two different stimuli are present simultaneously, its release can be controlled with much higher precision and selectivity compared with a gel that responds to one stimulus only. In this Account, the recent progress in the construction of functional macrocycle-containing supramolecular gels is summarized. First, recent strategies to engineer responsiveness into macrocycle-containing gels are discussed. Next, different functions are presented including applications as responsive reaction media, for controlled drug-delivery or tissue engineering, and as selfhealing materials. Finally, we highlight the recent progress in designing macrocycle-containing supramolecular gel materials exhibiting complex behavior. This field is part of systems chemistry and still in its infancy but appears to be one of the most promising routes to smart responsive materials.

Imaging of doxorubicin release from theranostic macromolecular prodrugs via fluorescence resonance energy transfer

Krüger HR, Schütz I, Justies A, Licha K, Welker P, Haucke V, Calderón M.

Journal of Controlled Release 194 (2014) 189–196 ...

Herein we present a FRET-based theranostic macromolecular prodrug (TMP) composed of (a) dendritic polyglycerol (PG) as polymeric nanocarrier, (b) doxorubicin (Dox) linked via a pH-sensitive hydrazone to (c) a tri-functional linker, and (d) an indodicarbocyanine dye (IDCC) attached in close proximity to Dox. The drug fluorescence is quenched via intramolecular FRET until the pH-sensitive hydrazone bond between the TMP and Dox is cleaved at acidic pH. By measuring its fluorescence, we characterized the TMP cleavage kinetics at different pH values in vitro. The intracellular release of Dox from the carrier was monitored in real time in intact cancer cells, giving more insight into the mode of action of a polymer drug conjugate.

Virus inhibition induced by polyvalent nanoparticles of different sizes

J. Vonnemann, C. Sieben, C. Wolff, K. Ludwig, C. Böttcher, A. Herrmann, R. Haag.

Nanoscale, 2014, 6, 2353-2360 ...

The development of antiviral agents is one of the major challenges in medical science. So far, small monovalent molecular drugs that inhibit the late steps in the viral replication cycle, i.e., virus budding, have not worked well which emphasizes the need for alternative approaches. Polyvalently presented viral receptors, however, show potential as good inhibitors of virus–cell binding, which is the first step in the viral infection cycle. By gradually increasing the size of ligand functionalized gold nanoparticles up to virus-like dimensions, we are now able to quantify the polyvalent enhancement of virus–cell binding inhibition and to identify varying mechanisms of virus inhibition with different efficacies: by employing a new binding assay we found that surface area-normalized polysulfated gold nanoparticles of diameters equal to and larger than the virus diameter (>50 nm) more efficiently inhibit the binding of vesicular stomatitis virus (VSV) to cells than smaller particles. On a per particle basis, larger sized gold nanoparticles were surprisingly shown to inhibit the viral infection up to two orders of magnitude more efficiently than smaller particles, which suggests different mechanisms of virus inhibition. Based on complementary electron microscopic data, we noticed that larger gold nanoparticles act as efficient cross-linkers between virions, whereas smaller gold nanoparticles decorate the surface of individual virus particles. Our systematic study accentuates the need for the design of biodegradable, virus-sized inhibitors capitalizing on polyvalent binding.

Dendritic polyglycerol cyclodextrin amphiphiles and their self-assembled architectures to transport hydrophobic guest molecules

A. Campo Rodrigo, S. Malhotra, C. Böttcher, M. Adeli, R. Haag

RSC Adv., 2014, 4, 61656-61659 ...

Here we report for the first time a microwave assisted synthesis of polyglycerol dendron functionalized cyclodextrins (CD) with hydrophobic tails. These amphiphilic CDs consist of seven polyglycerol dendrons and fourteen alkyl chains on the primary and secondary rims of the cyclodextrin core, respectively. They self-assemble to form nanostructures in aqueous solutions and efficiently encapsulate hydrophobic aromatic guests. The size and shape of the self-assemblies and also their ability to encapsulate guest molecules depend on the generation of conjugated polyglycerol dendrons.

Visualization of Real-Time Degradation of pH-Responsive Polyglycerol Nanogels via Atomic Force Microscopy

M. Richter, D. Steinhilber, R. Haag, R. von Klitzing

Macromol. Rapid Commun., 2014, 35, 2018–2022 ...

Polyglycerol nanogels (nPG) have a huge impact in biomedical applications as drug deliverer due to their high biocompability. For such nPG nanogels, particle degradation is widely used as drug delivery method. The knowledge of this degradation process is limited up to date. In this communication, a real time visualization of such a degradation process is presented for pH-responsive nPG nanogels via atomic force microscopy (AFM) under ambient and in liquid conditions. The particle height plays a major role in the degradation process and decays exponentially in the beginning of this process. The particle width increases during the process indicating a “decross-linking” step of the particles into their starting monomers. Measurements under ambient conditions confirm this assumption and provide further insight in the “decross-linking” step of the nanogels into individual dendritic particles. The present work gives a detailed insight in the particle degradation process, which is essential for further progress for the development of new drug delivery systems.

ZIBgridfree: Efficient Conformational Analysis by Partition-of-Unity Coupling

A. Bujotzek, O. Schütt, A. Nielsen, K. Fackeldey, M. Weber

A. Bujotzek, O. Schütt, A. Nielsen, K. Fackeldey, M. Weber | 2014

J. Math. Chem., 2014, 52, 781-804


Obtaining a sufficient sampling of conformational space is a common problem in molecular simulation. We present the implementation of an umbrella-like adaptive sampling approach based on function-based meshless discretization of conformational space that is compatible with state of the art molecular dynamics code and that integrates an eigenvector-based clustering approach for conformational analysis and the computation of inter-conformational transition rates. The approach is applied to three example systems, namely n-pentane, alanine dipeptide, and a small synthetic host-guest system, the latter two including explicitly modeled solvent.

Computing the Minimal Rebinding Effect Included in a Given Kinetics

M. Weber, K. Fackeldey

M. Weber, K. Fackeldey | 2014

Multiscale Model. Simul., 2014 12, 318–334


In this paper we show that the binding kinetics of a molecular system can be identified by a projection of a continuous process onto a finite number of macro states. We thus interpret binding kinetics as a projection. When projecting onto nonoverlapping macro states the Markovianity is spoiled. As a consequence, the description of, e.g., a receptor-ligand system by a two state kinetics, is not accurate. By assigning a degree of membership to each state, we abandon the nonoverlapping approach. This overlap is crucial for a correct mapping of binding effects by Markov state models with regard to their long time behavior. It enables us to describe the highly discussed rebinding effect, where the spatial arrangement of the system has the be included. By introducing a “degree of fuzziness,” we have an indicator for the strength of the rebinding effect such that the minimal rebinding effect can be derived from an optimization problem. The fuzziness also includes some new paradigms for molecular kinetics. These new model paradigms show good agreement with experimental data.

The effect of polyglycerol sulfate branching on inflammatory processes.

F. Paulus, R. Schulze, D. Steinhilber, M. Zieringer, I. Steinke, P. Welker, K. Licha, S. Wedepohl, J. Dernedde, R. Haag

Macromol. Biosci. 2014, 14, 643-654


In this study, the extent to which the scaffold architecture of polyglycerol sulfates affects inflammatory processes and hemocompatibility is investigated. Competitive L-selectin binding assays, cellular uptake studies, and blood compatibility readouts are done to evaluate distinct biological properties. Fully glycerol based hyperbranched polyglycerol architectures are obtained by either homopolymerization of glycidol (60% branching) or a new copolymerization strategy of glycidol with ethoxyethyl glycidyl ether. Two polyglycerols with 24 and 42% degree of branching (DB) are synthesized by using different monomer feed ratios. A perfectly branched polyglycerol dendrimer is synthesized according to an iterative two-step protocol based on allylation of the alcohol and subsequent catalytic dihydroxylation. All the polyglycerol sulfates are synthesized with a comparable molecular weight and degree of sulfation. The DB make the different polymer conjugates perform different ways. The optimal DB is 60% in all biological assays.

Controlled thioamide vs. amide formation in the thioacid–azide reaction under acidic aqueous conditions

M. Mühlberg, K.D. Siebertz, B. Schlegel, P. Schmieder, C. P. R. Hackenberger

Chem. Commun.,2014, 50, 4603--4606 ...

The thioacid–azide reaction and its chemoselectivity were probed with alkyl azides for a potential application to form amide bonds in aqueous solvents. Our results reveal that under acidic conditions thioamides were formed as major reaction products suggesting a competing mechanism, whereas reactions forming amides predominated at slightly higher pH values.

Site-Specifically Phosphorylated Lysine Peptides

J. Bertran-Vicente, R.A. Serwa, M. Schümann, P. Schmieder, E. Krause, C. P. R. Hackenberger

J. Am Chem. Soc. 2014, 136(39), 13622-13628 ...

Protein phosphorylation controls major processes in cells. Although phosphorylation of serine, threonine, and tyrosine and also recently histidine and arginine are well-established, the extent and biological significance of lysine phosphorylation has remained elusive. Research in this area has been particularly limited by the inaccessibility of peptides and proteins that are phosphorylated at specific lysine residues, which are incompatible with solid-phase peptide synthesis (SPPS) due to the intrinsic acid lability of the P(═O)–N phosphoramidate bond. To address this issue, we have developed a new synthetic route for the synthesis of site-specifically phospholysine (pLys)-containing peptides by employing the chemoselectivity of the Staudinger-phosphite reaction. Our synthetic approach relies on the SPPS of unprotected ε-azido lysine-containing peptides and their subsequent reaction to phosphoramidates with phosphite esters before they are converted into the natural modification via UV irradiation or basic deprotection. With these peptides in hand, we demonstrate that electron-transfer dissociation tandem mass spectrometry can be used for unambiguous assignment of phosphorylated-lysine residues within histone peptides and that these peptides can be detected in cell lysates using a bottom-up proteomic approach. This new tagging method is expected to be an essential tool for evaluating the biological relevance of lysine phosphorylation.

More than add-on: chemoselective reactions for the synthesis of functional peptides and proteins

D. Schumacher, C. P. R. Hackenberger

Curr. Opin. Chem. Biol. 2014, 22, 62-69 ...

The quest to enlarge the molecular space of functional biomolecules has led to the discovery of selective, mild and high-yielding chemical reactions for the modification of peptides and proteins. These conjugation methods have recently become even more advanced with the advent of modern biochemical techniques such as unnatural protein expression or enzymatic reactions that allow the site-specific modification of proteins. Within this overview, we will highlight recent examples that describe the site-specific functionalization of proteins. These examples go beyond the straightforward attachment of a given functional moiety to the protein backbone by employing either an innovative linker-design or by novel conjugation chemistry, where the modification reaction itself is responsible for the (altered) functional behaviour of the biomolecule. The examples covered herein include ‘turn-on’ probes for cellular imaging with low levels of background fluorescence, branched or cleavable polymer–protein conjugates of high stability within a cellular environment, the installation of natural occurring posttranslational modifications to help understand their role in complex cellular environments and finally the engineering of novel antibody drug conjugates to facilitate target specific drug release.

Site-specific PEGylation of Proteins: Recent Developments

N. Nischan, C.P.R. Hackenberger

J. Org. Chem., 2014, 79(22), 10727–10733 ...

The attachment of linear polyethylene glycol (PEG) to peptides and proteins for their stabilization for in vivo applications is a milestone in pharmaceutical research and protein–drug development. However, conventional methods often lead to heterogeneous PEGylation mixtures with reduced protein activity. Current synthetic efforts aim to provide site-specific approaches by chemoselective targeting of canonical and noncanonical amino acids and to improve the PEG architecture. This synopsis highlights recent work in this area, which also resulted in improved pharmacokinetics of peptide and protein therapeutics.

Synthesis of the Pitstop family of clathrin inhibitors

Robertson MJ, Deane FM, Stahlschmidt W, von Kleist L, Haucke V, Robinson PJ, McCluskey A.

Nat Protoc., 2014, 9(7), 1592-1606


This protocol describes the synthesis of two classes of clathrin inhibitors, Pitstop 1 and Pitstop 2, along with two inactive analogs that can be used as negative controls (Pitstop inactive controls, Pitnot-2 and Pitnot-2-100). Pitstop-induced inhibition of clathrin TD function acutely interferes with clathrin-mediated endocytosis (CME), synaptic vesicle recycling and cellular entry of HIV, whereas clathrin-independent internalization pathways and secretory traffic proceed unperturbed; these reagents can, therefore, be used to investigate clathrin function, and they have potential pharmacological applications. Pitstop 1 is synthesized in two steps: sulfonation of 1,8-naphthalic anhydride and subsequent reaction with 4-amino(methyl)aniline. Pitnot-1 results from the reaction of 4-amino(methyl)aniline with commercially available 4-sulfo-1,8-naphthalic anhydride potassium salt. Reaction of 1-naphthalene sulfonyl chloride with pseudothiohydantoin followed by condensation with 4-bromobenzaldehyde yields Pitstop 2. The synthesis of the inactive control commences with the condensation of 4-bromobenzaldehyde with the rhodanine core. Thioketone methylation and displacement with 1-napthylamine affords the target compound. Although Pitstop 1–series compounds are not cell permeable, they can be used in biochemical assays or be introduced into cells via microinjection. The Pitstop 2–series compounds are cell permeable. The synthesis of these compounds does not require specialist equipment and can be completed in 3–4 d. Microwave irradiation can be used to reduce the synthesis time. The synthesis of the Pitstop 2 family is easily adaptable to enable the synthesis of related compounds such as Pitstop 2-100 and Pitnot-2-100. The procedures are also simple, efficient and amenable to scale-up, enabling cost-effective in-house synthesis for users of these inhibitor classes.

Clathrin terminal domain-ligand interactions regulate sorting of mannose 6-phosphate receptors mediated by AP-1 and GGA adaptors

Stahlschmidt W, Robertson MJ, Robinson PJ, McCluskey A, Haucke V.

J Biol Chem., 2014, 289(8), 4906-4918


Clathrin plays important roles in intracellular membrane traffic including endocytosis of plasma membrane proteins and receptors and protein sorting between the trans-Golgi network (TGN) and endosomes. Whether clathrin serves additional roles in receptor recycling, degradative sorting, or constitutive secretion has remained somewhat controversial. Here we have used acute pharmacological perturbation of clathrin terminal domain (TD) function to dissect the role of clathrin in intracellular membrane traffic. We report that internalization of major histocompatibility complex I (MHCI) is inhibited in cells depleted of clathrin or its major clathrin adaptor complex 2 (AP-2), a phenotype mimicked by application of Pitstop® inhibitors of clathrin TD function. Hence, MHCI endocytosis occurs via a clathrin/AP-2-dependent pathway. Acute perturbation of clathrin also impairs the dynamics of intracellular clathrin/adaptor complex 1 (AP-1)- or GGA (Golgi-localized, γ-ear-containing, Arf-binding protein)-coated structures at the TGN/endosomal interface, resulting in the peripheral dispersion of mannose 6-phosphate receptors. By contrast, secretory traffic of vesicular stomatitis virus G protein, recycling of internalized transferrin from endosomes, or degradation of EGF receptor proceeds unperturbed in cells with impaired clathrin TD function. These data indicate that clathrin is required for the function of AP-1- and GGA-coated carriers at the TGN but may be dispensable for outward traffic en route to the plasma membrane.

Chemoenzymatic synthesis of functional sialyl lewis(x) mimetics with a heteroaromatic core

C. Schlemmer, C. Wiebe, D. Ferenc, D. Kowalczyk, S. Wedepohl, P. Ziegelmüller, J. Dernedde, T. Opatz

Chem. Asian J. 2014, 9, 2119- 2125


Functional mimetics of the sialyl LewisX tetrasaccharide were prepared by the enzymatic sialylation of a 1,3-diglycosylated indole and a glycosyl azide, which was subsequently transformed into a 1,4-diglycosylated 1,2,3-triazole, by using the trans-sialidase of Trypanosoma cruzi. These compounds inhibited the binding of E-, L-, and P-selectin-coated nanoparticles to polyacrylamide-bound sialyl-LewisX-containing neighboring sulfated tyrosine residues (sTyr/sLeX-PAA) at low or sub-millimolar concentrations. Except for E-selectin, the mimetics showed higher activities than the natural tetrasaccharide.

Sortase A mediated site-specific immobilization for identification of protein interactions in affinity purification–mass spectrometry experiments

B. Kuropka, N. Royla, C.Freund, E. Krause

Proteomics, 2014, 15, 1230–1234 ...

Proteomics approaches using MS in combination with affinity purification have emerged as powerful tools to study protein-protein interactions. Here we make use of the specificity of sortase A transpeptidation reaction to prepare affinity matrices in which a protein bait is covalently linked to the matrix via a short C-terminal linker region. As a result of this site-directed immobilization, the bait remains functionally accessible to protein interactions. To apply this approach, we performed SILAC-based pull-down experiments and demonstrate the suitability of the approach.

Polyglycerolsulfate functionalized gold nanorods as optoacoustic signal nanoamplifiers for in vivo bioimaging of rheumatoid arthritis.

J. Vonnemann, N. Beziere, C. Böttcher, S.B. Riese, C. Kuehne, J. Dernedde, K. Licha, C. von Schacky, Y. Kosanke, M. Kimm, R. Meier, V. Ntziachristos, R. Haag

Theranostics, 2014, 4, 629-641


We have synthesized a targeted imaging agent for rheumatoid arthritis based on polysulfated gold nanorods. The CTAB layer on gold nanorods was first replaced with PEG-thiol and then with dendritic polyglycerolsulfate at elevated temperature, which resulted in significantly reduced cytotoxicity compared to polyanionic gold nanorods functionalized by non-covalent approaches. In addition to classical characterization methods, we have established a facile UV-VIS based BaCl2 agglomeration assay to confirm a quantitative removal of unbound ligand. With the help of a competitive surface plasmon resonance-based L-selectin binding assay and a leukocyte adhesion-based flow cell assay, we have demonstrated the high inflammation targeting potential of the synthesized gold nanorods in vitro. In combination with the surface plasmon resonance band of AuNRs at 780 nm, these findings permitted the imaging of inflammation in an in vivo mouse model for rheumatoid arthritis with high contrast using multispectral optoacoustic tomography. The study offers a robust method for otherwise difficult to obtain covalently functionalized polyanionic gold nanorods, which are suitable for biological applications as well as a low-cost, actively targeted, and high contrast imaging agent for the diagnosis of rheumatoid arthritis. This paves the way for further research in other inflammation associated pathologies, in particular, when photothermal therapy can be applied.

Influence of Linkage Chemistry on Folding, Self-assembly, and Photoresponse of Amphiphilic Azobenzene Main Chain Polymers

Z. Yu, S. Hecht

J. Polym. Sci., Part A: Polym. Chem., 2015, 53, 313-318


Two types of amphiphilic polymers composed of azobenzene repeat units in the main chain connected either via ethynylene (acetylene) or butadiynylene (diacetylene) linkages and carrying oligo(ethylene glycol) side chains were reported. Synthesis was accomplished by polycondensation involving Sonogashira–Hagihara cross coupling and Glaser coupling, respectively. Solvent titration experiments revealed that both polymers fold into stable helices in a polar environment. While the ethynylene-bridged polymer resembled the behavior of its oligomeric counterparts, introduction of the extended diacetylene unit strengthened π,π-stacking interactions in case of the butadiynylene-bridged polymer leading to a pronounced aggregation tendency and suppressing photoisomerization in the folded state. Our study demonstrates the importance of backbone connectivity to balance intra- and intermolecular forces for the successful design of photoresponsive polymers. © 2014 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2015, 53, 313–318

Modular Syntheses of Star-Shaped Pyridine, Bipyridine, and Terpyridine Derivatives by Employing Sonogashira Reactions

D. Trawny, V. Kunz, H.-U. Reissig

Eur. J. Org. Chem., 2014, 28, 6295–630 ...

A simple and flexible synthesis for a series of star-shaped pyridine, bipyridine, and terpyridine derivatives is reported by using a modular approach that combines the use of a ligand, spacer, and core unit. A fairly efficient method to prepare 4′-nonafloxy-functionalized terpyridine derivatives is described. The building blocks that contain the functionalized pyridine, bipyridine, or terpyridine derivatives were linked to different C3-symmetrical core units. In most cases, Sonogashira reactions were employed in the crucial final steps of the synthesis. A star-shaped dodecafluorinated compound was also prepared in a straightforward fashion. A simple procedure for the preparation of partially silylated 1,3,5-triethynylbenzene derivatives is presented, which provides an approach to C2-symmetrical star-shaped compounds that have only one terpyridine and two terphenyl units as “dummy” ligands. The absorption and emission spectra of the fully conjugated C3-symmetrical pyridine derivatives were systematically investigated, and fairly large Stokes shifts were observed.

The Side Chain Makes the Difference: Investigation of the 2D Self-Assembly of 1,3,5-Tris[4-(4-pyridinyl)phenyl]benzene Derivatives by Scanning Tunneling Microscopy

D. Trawny, L. Vandromme, J. P. Rabe, H.-U. Reissig

Eur. J. Org. Chem. 2014, 4985-4992


Flexible and straightforward syntheses of a series of D3h- or C3h-symmetrical star-shaped compounds with pyridine end groups are reported. In all cases, the acid-mediated cyclocondensations of the corresponding aryl methyl ketone provided the central benzene ring. For the preceding preparation of the functionalized compound arms, Suzuki couplings were applied. The crucial introduction of the pyridine C-2 and C-6 substituents occurred by Fe(acac)3-catalyzed alkylations (acac = acetylacetonate). The preparation of the C3-symmetrical compound involved an alternating sequence of halogenations and coupling reactions. The self-assembly behavior of the four resulting star-shaped compounds at the interface between 1-phenyloctane and the basal plane of highly oriented pyrolytic graphite (HOPG) was studied by scanning tunnelling microscopy (STM). We found self-assembled monolayers with structures strongly dependent on the substitution patterns of the investigated compounds. The reduction of the symmetry from a D3h- to a C3h-symmetrical compound led to an entirely different self-assembly behavior with the change from a hexagonal to a lamellar arrangement.

Unprecedented Strong Lewis Bases – Synthesis and Mehtyl Cation Affinities of Dimethylamino-Substituted Terpyridines

P. Hommes, C. Fischer, C. Lindner, H. Zipse, H.-U. Reissig

Angew. Chem. Int. Ed. 2014, 53, 7647-7651


A versatile method for the synthesis of functionalized 2,2′:6′,2′′-terpyridines by assembly of the terminal pyridine rings is presented. The cyclization precursors—bis-β-ketoenamides—are prepared from 4-substituted 2,6-pyridinedicarboxylic acids and acetylacetone or its corresponding enamino ketone. Treatment with trimethylsilyl trifluoromethanesulfonate induces a twofold intramolecular condensation providing an efficient access to 4,4′′-di- and 4,4′,4′′-trifunctionalized 6,6′′-dimethyl-2,2′:6′,2′′-terpyridines. Using this method, hitherto unknown 4,4′′-bis(dimethylamino)- and 4,4′,4′′-tris(dimethylamino)terpyridines have been prepared that show remarkably high calculated Lewis basicities.

Extrem starke Lewis-Basen – Synthese und Methylkationaffinitäten von Dimethylamino-substituierten Terpyridinen

P. Hommes, C. Fischer, C. Lindner, H. Zipse, H.-U. Reissig

Angew. Chem. 2014, 126, 7778-7782


Es wird eine vielseitig einsetzbare Methode zur Herstellung von funktionalisierten 2,2′:6′,2′′-Terpyridinen vorgestellt, bei der die äußeren Pyridinringe über Cyclisierungsreaktionen aufgebaut werden. Die Vorstufen – Bis-β-ketoenamide – werden aus in 4-Position substituierten 2,6-Pyridindicarbonsäuren und Acetylaceton oder dem entsprechenden Enaminoketon hergestellt. Ihre Cyclisierung erfolgt mithilfe von Trifluormethansulfonsäuretrimethylsilylester durch eine zweifache intramolekulare Kondensation. Die Methode bietet einen effizienten Zugang zu 4,4′′-di- und 4,4′,4′′-trifunktionalisierten 6,6′′-Dimethyl-2,2′:6′,2′′-terpyridinen und ermöglicht die Herstellung bislang unbekannter 4,4′′-Bis(dimethylamino)- und 4,4′,4′′-Tris(dimethylamino)terpyridine, für die extrem hohe Lewis-Basizitäten berechnet wurden.

Sense codon emancipation for proteome-wide incorporation of noncanonical amino acids: rare isoleucine codon AUA as a target for genetic code expansion

Bohlke N, Budisa N.

FEMS Microbiol Lett., 2014, 351, 133-144


One of the major challenges in contemporary synthetic biology is to find a route to engineer synthetic organisms with altered chemical constitution. In terms of core reaction types, nature uses an astonishingly limited repertoire of chemistries when compared with the exceptionally rich and diverse methods of organic chemistry. In this context, the most promising route to change and expand the fundamental chemistry of life is the inclusion of amino acid building blocks beyond the canonical 20 (i.e. expanding the genetic code). This strategy would allow the transfer of numerous chemical functionalities and reactions from the synthetic laboratory into the cellular environment. Due to limitations in terms of both efficiency and practical applicability, state-of-the-art nonsense suppression- or frameshift suppression-based methods are less suitable for such engineering. Consequently, we set out to achieve this goal by sense codon emancipation, that is, liberation from its natural decoding function – a prerequisite for the reassignment of degenerate sense codons to a new 21st amino acid. We have achieved this by redesigning of several features of the post-transcriptional modification machinery which are directly involved in the decoding process. In particular, we report first steps towards the reassignment of 5797 AUA isoleucine codons in Escherichia coli using efficient tools for tRNA nucleotide modification pathway engineering.

Coupling Bioorthogonal Chemistries with Artificial Metabolism: Intracellular Biosynthesis of Azidohomoalanine and its Incorporation into Recombinant Proteins

Ma, Y., Biava, H., Contestabile, R., Budisa, N. & M.L. di Salvo

Molecules, 2014, 19(1), 1004-1022


In this paper, we present a novel, “single experiment” methodology based on genetic engineering of metabolic pathways for direct intracellular production of non-canonical amino acids from simple precursors, coupled with expanded genetic code. In particular, we engineered the intracellular biosynthesis of L-azidohomoalanine from O-acetyl-L-homoserine and NaN3, and achieved its direct incorporation into recombinant target proteins by AUG codon reassignment in a methionine-auxotroph E. coli strain. In our system, the host’s methionine biosynthetic pathway was first diverted towards the production of the desired non-canonical amino acid by exploiting the broad reaction specificity of recombinant pyridoxal phosphate-dependent O-acetylhomoserine sulfhydrylase from Corynebacterium glutamicum. Then, the expression of the target protein barstar, accompanied with efficient L-azidohomoalanine incorporation in place of L-methionine, was accomplished. This work stands as proof-of-principle and paves the way for additional work towards intracellular production and site-specific incorporation of biotechnologically relevant non-canonical amino acids directly from common fermentable sources.

Receptor binding and pH stability — How influenza A virus hemagglutinin affects host-specific virus infection

C. M. Mair, K. Ludwig, A. Herrmann, C. Sieben

Biochim. Biophys. Acta-Biomembranes, 2014, 1838 (4), 1153–1168 ...

Influenza A virus strains adopt different host specificities mainly depending on their hemagglutinin (HA) protein. Via HA, the virus binds sialic acid receptors of the host cell and, upon endocytic uptake, HA triggers fusion between the viral envelope bilayer and the endosomal membrane by a low pH-induced conformational change leading to the release of the viral genome into the host cell cytoplasm. Both functions are crucial for viral infection enabling the genesis of new progeny virus. Adaptation to different hosts in vitro was shown to require mutations within HA altering the receptor binding and/or fusion behavior of the respective virus strain. Human adapted influenza virus strains (H1N1, H3N2, H2N2) as well as recent avian influenza virus strains (H5, H7 and H9 subtypes) which gained the ability to infect humans mostly contained mutations in the receptor binding site (RBS) of HA enabling increased binding affinity of these viruses to human type (α-2,6 linked sialic acid) receptors. Thus, the receptor binding specificity seems to be the major requirement for successful adaptation to the human host; however, the RBS is not the only determinant of host specificity. Increased binding to a certain cell type does not always correlate with infection efficiency. Furthermore, viruses carrying mutations in the RBS often resulted in reduced viral fitness and were still unable to transmit between mammals. Recently, the pH stability of HA was reported to affect the transmissibility of influenza viruses. This review summarizes recent findings on the adaptation of influenza A viruses to the human host and related amino acid substitutions resulting in altered receptor binding specificity and/or modulated fusion pH of HA. Furthermore, the role of these properties (receptor specificity and pH stability of HA) for adaptation to and transmissibility in the human host is discussed. This article is part of a Special Issue entitled: Viral Membrane Protiens -- Channels for Cellular Networking.

A Histidine Residue of the Influenza Virus Hemagglutinin Controls the pH Dependence of the Conformational Change Mediating Membrane Fusion

C. M. Mair, T. Meyer, K. Schneider, Q. Huang, M. Veit, A. Herrmann

J. Virol., 2014, 88, 13189-13200 ...

The conformational change of the influenza virus hemagglutinin (HA) protein mediating the fusion between the virus envelope and the endosomal membrane was hypothesized to be induced by protonation of specific histidine residues since their pKas match the pHs of late endosomes (pKa of ∼6.0). However, such critical key histidine residues remain to be identified. We investigated the highly conserved His184 at the HA1-HA1 interface and His110 at the HA1-HA2 interface of highly pathogenic H5N1 HA as potential pH sensors. By replacing both histidines with different amino acids and analyzing the effect of these mutations on conformational change and fusion, we found that His184, but not His110, plays an essential role in the pH dependence of the conformational change of HA. Computational modeling of the protonated His184 revealed that His184 is central in a conserved interaction network possibly regulating the pH dependence of conformational change via its pKa. As the propensity of histidine to get protonated largely depends on its local environment, mutation of residues in the vicinity of histidine may affect its pKa. The HA of highly pathogenic H5N1 viruses carries a Glu-to-Arg mutation at position 216 close to His184. By mutation of residue 216 in the highly pathogenic as well as the low pathogenic H5 HA, we observed a significant influence on the pH dependence of conformational change and fusion. These results are in support of a pKa-modulating effect of neighboring residues.

pH-controlled two-step uncoating of influenza virus

S. Li, C. Sieben, K. Ludwig, A. Herrmann, F. Eghiaian, I. A. T. Schaap

Biophys J., 2014, 106, 1447-1456 ...

Upon endocytosis in its cellular host, influenza A virus transits via early to late endosomes. To efficiently release its genome, the composite viral shell must undergo significant structural rearrangement, but the exact sequence of events leading to viral uncoating remains largely speculative. In addition, no change in viral structure has ever been identified at the level of early endosomes, raising a question about their role. We performed AFM indentation on single viruses in conjunction with cellular assays under conditions that mimicked gradual acidification from early to late endosomes. We found that the release of the influenza genome requires sequential exposure to the pH of both early and late endosomes, with each step corresponding to changes in the virus mechanical response. Step 1 (pH 7.5–6) involves a modification of both hemagglutinin and the viral lumen and is reversible, whereas Step 2 (pH <6.0) involves M1 dissociation and major hemagglutinin conformational changes and is irreversible. Bypassing the early-endosomal pH step or blocking the envelope proton channel M2 precludes proper genome release and efficient infection, illustrating the importance of viral lumen acidification during the early endosomal residence for influenza virus infection.

Fluoreszente Mimetika von CMP-Neu5Ac sind hochaffine, zellgängige Polarisationssonden eukaryotischer und bakterieller Sialyltransferasen und inhibieren die zelluläre Sialylierung

J. J. Preidl, V. S. Gnanapragassam, M. Lisurek, J. Saupe, R. Horstkorte, J. Rademann

Angew. Chem. 2014, 126, 5808-5813


Oligosaccharide der Glycolipide und Glycoproteine auf der äußeren Membran humaner Zellen tragen terminale Neuraminsäuren, die für die Zell-Zell-Erkennung und die Adhäsion von Zellen, Bakterien und Viruspartikeln verantwortlich sind. Die Synthese der Neuraminsäure-tragenden Glycoside wird von intrazellulären Sialyltransferasen vollzogen. Daher könnte die chemische Manipulation der zellulären Sialylierung ein Weg zur Hemmung der Krebsentwicklung sowie von Entzündungen und Infektionen sein. Hier wird die Entwicklung und Anwendung der ersten nanomolaren fluoreszenten Inhibitoren von Sialyltransferasen beschrieben. Die erhaltenen Mimetika von Kohlenhydratnukleotiden erwiesen sich als Liganden der vier kommerziell erhältlichen und in Fluoreszenzpolarisations-Assays getesteten eukaryotischen wie bakteriellen Sialyltransferasen. Darüber hinaus wurde beobachtet, dass die anionischen Mimetika schnell und effizient in Vesikeln in Zellen eindrangen und zu den kernnahen Organellen von CHO-Zellen wanderten. Die neuen Verbindungen inhibieren die zelluläre Sialylierung in zwei Zelllinien und eröffnen neue Perspektiven zur Untersuchung zellulärer Sialylierung.

Fluorescent Mimetics of CMP-Neu5Ac Are Highly Potent, Cell-Permeable Polarization Probes of Eukaryotic and Bacterial Sialyltransferases and Inhibit Cellular Sialylation

J. J. Preidl, V. S. Gnanapragassam, M. Lisurek, J. Saupe, R. Horstkorte, J. Rademann

Angew. Chem. Int. 2014, 53, 5700-5705


Oligosaccharides of the glycolipids and glycoproteins at the outer membranes of human cells carry terminal neuraminic acids, which are responsible for recognition events and adhesion of cells, bacteria, and virus particles. The synthesis of neuraminic acid containing glycosides is accomplished by intracellular sialyl transferases. Therefore, the chemical manipulation of cellular sialylation could be very important to interfere with cancer development, inflammations, and infections. The development and applications of the first nanomolar fluorescent inhibitors of sialyl transferases are described herein. The obtained carbohydrate-nucleotide mimetics were found to bind all four commercially available and tested eukaryotic and bacterial sialyl transferases in a fluorescence polarization assay. Moreover, it was observed that the anionic mimetics intruded rapidly and efficiently into cells in vesicles and translocated to cellular organelles surrounding the nucleus of CHO cells. The new compounds inhibit cellular sialylation in two cell lines and open new perspectives for investigations of cellular sialylation.

Multivalent presentation of the cell-penetrating peptide nona-arginine on a linear scaffold strongly increases its membrane-perturbing capacity

A. Chakrabarti, J. J. Witsenburg, M. D. Sinzinger, M. Richter, R. Wallbrecher, J. C. Cluitmans, W. P.R. Verdurmen, S. Tanis, M. J. W. Adjobo-Hermans, J. Rademann, R. Brock

Biochimica et Biophysica Acta, 2014, 1838, 3097–3106


Arginine-rich cell-penetrating peptides (CPP) are widely employed as delivery vehicles for a large variety of macromolecular cargos. As a mechanism-of-action for induction of uptake cross-linking of heparan sulfates and interaction with lipid head groups have been proposed. Here, we employed a multivalent display of the CPP nona-arginine (R9) on a linear dextran scaffold to assess the impact of heparan sulfate and lipid interactions on uptake and membrane perturbation. Increased avidity through multivalency should potentiate molecular phenomena that may only play a minor role if only individual peptides are used. To this point, the impact of multivalency has only been explored for dendrimers, CPP-decorated proteins and nanoparticles. We reasoned that multivalency on a linear scaffold would more faithfully mimic the arrangement of peptides at the membrane at high local peptide concentrations. On average, five R9 were coupled to a linear dextran backbone. The conjugate displayed a direct cytoplasmic uptake similar to free R9 at concentrations higher than 10 μM. However, this uptake was accompanied by an increased membrane disturbance and cellular toxicity that was independent of the presence of heparan sulfates. In contrast, for erythrocytes, the multivalent conjugate induced aggregation, however, showed only limited membrane perturbation. Overall, the results demonstrate that multivalency of R9 on a linear scaffold strongly increases the capacity to interact with the plasma membrane. However, the induction of membrane perturbation is a function of the cellular response to peptide binding.

Chemoselective Wittig and Michael ligations of unprotected peptidyl phosphoranes in water furnish potent inhibitors of caspase-3

K. Holland-Nell, M. I. Fernández-Bachiller, Ahsanullah, J. Rademann

Org. Lett. 2014, 16, 4428-4431


Unprotected peptidyl phosphoranes 1 with sequence Ac-l-aspartyl-l-glutamyl-l-valinyl-l-aspartyl are released from polymer support and react with aliphatic and aromatic aldehydes in aqueous medium in a Wittig ligation. Obtained vinyl ketones 6–12 are potent inhibitors of caspase-3. Vinyl ketone 6, derived from formaldehyde, undergoes Michael ligations with thiol nucleophiles furnishing products 14–16, also in aqueous medium. The demonstrated ligation reactions enable the modification of complex functionalized peptides in water providing bioactive protein ligands without side-chain protection.

Computational and experimental prediction of human C-type lectin receptor druggability

Jonas Aretz, Eike-Christian Wamhoff, Jonas Hanske, Dario Heymann and Christoph Rademacher

Front. Immunol., 2014, 5, 323


Mammalian C-type lectin receptors (CTLRS) are involved in many aspects of immune cell regulation such as pathogen recognition, clearance of apoptotic bodies, and lymphocyte homing. Despite a great interest in modulating CTLR recognition of carbohydrates, the number of specific molecular probes is limited. To this end, we predicted the druggability of a panel of 22 CTLRs using DoGSiteScorer. The computed druggability scores of most structures were low, characterizing this family as either challenging or even undruggable. To further explore these findings, we employed a fluorine-based nuclear magnetic resonance screening of fragment mixtures against DC-SIGN, a receptor of pharmacological interest. To our surprise, we found many fragment hits associated with the carbohydrate recognition site (hit rate = 13.5%). A surface plasmon resonance-based follow-up assay confirmed 18 of these fragments (47%) and equilibrium dissociation constants were determined. Encouraged by these findings we expanded our experimental druggability prediction to Langerin and MCL and found medium to high hit rates as well, being 15.7 and 10.0%, respectively. Our results highlight limitations of current in silico approaches to druggability assessment, in particular, with regard to carbohydrate-binding proteins. In sum, our data indicate that small molecule ligands for a larger panel of CTLRs can be developed.

Carbohydrate-Lectin Recognition of Sequence-Defined Heteromultivalent Glycooligomers

Daniela Ponader, Pauline Maffre, Jonas Aretz, Daniel Pussak, Nina M. Ninnemann, Stephan Schmidt, Peter H. Seeberger, Christoph Rademacher, G. Ulrich Nienhaus and Laura Hartmann

J. Am. Chem. Soc., 2014, 136, 2008–2016


Multivalency as a key principle in nature has been successfully adopted for the design and synthesis of artificial glycoligands by attaching multiple copies of monosaccharides to a synthetic scaffold. Besides their potential in various applied areas, e.g. as antiviral drugs, for the vaccine development and as novel biosensors, such glycomimetics also allow for a deeper understanding of the fundamental aspects of multivalent binding of both artificial and natural ligands. However, most glycomimetics so far neglect the purposeful arranged heterogeneity of their natural counterparts, thus limiting more detailed insights into the design and synthesis of novel glycomimetics. Therefore, this work presents the synthesis of monodisperse glycooligomers carrying different sugar ligands at well-defined positions along the backbone using for the first time sequential click chemistry and stepwise assembly of functional building blocks on solid support. This approach allows for straightforward access to sequence-defined, multivalent glycooligomers with full control over number, spacing, position, and type of sugar ligand. We demonstrate the synthesis of a set of heteromultivalent oligomers presenting mannose, galactose, and glucose residues. All heteromultivalent structures show surprisingly high affinities toward Concanavalin A lectin receptor in comparison to their homomultivalent analogues presenting the same number of binding ligands. Detailed studies of the ligand/receptor interaction using STD-NMR and 2fFCS indeed indicate a change in binding mechanism for trivalent glycooligomers presenting mannose or combinations of mannose and galactose residues. We find that galactose residues do not participate in the binding to the receptor, but they promote steric shielding of the heteromultivalent glycoligands and thus result in an overall increase in affinity. Furthermore, the introduction of nonbinding ligands seems to suppress receptor clustering of multivalent ligands. Overall these results support the importance of heteromultivalency specifically for the design of novel glycoligands and help to promote a fundamental understanding of multivalent binding modes.

Env decorated Phosphatidylserine Liposomes Trigger Phagocytosis of HIV-virus-like Particles in Macrophages.

A. Gramatica, R. A., Petazzi, M. Lehmann, J. Ziomkowska, A. Herrmann, S. Chiantia,

Nanomedicine, 2014,10 (5), 981-989 ...

Macrophages represent an important cellular target of HIV-1. Interestingly, they are also believed to play a potential role counteracting its infection. However, HIV-1 is known to impair macrophage immune functions such as antibody-mediated phagocytosis. Here, we present immunoliposomes that can bind HIV-1 virus-like particles (HIV-VLPs) while being specifically phagocytosed by macrophages, thus allowing the co-internalization of HIV-VLPs. These liposomes are decorated with anti-Env antibodies and contain phosphatidylserine (PS). PS mediates liposome internalization by macrophages via a mechanism not affected by HIV-1. Hence, PS-liposomes mimic apoptotic cells and are internalized into the macrophages due to specific recognition, carrying the previously bound HIV-VLPs. With a combination of flow cytometry, confocal live-cell imaging and electron microscopy we demonstrate that the PS-immunoliposomes presented here are able to elicit efficient HIV-VLPs phagocytosis by macrophages and might represent a new nanotechnological approach to enhance HIV-1 antigen presentation and reduce the ongoing inflammation processes.

Double-Clicking Peptides onto Phosphorothioate Oligonucleotides: Combining Two Proapoptotic Agents in One Molecule

Frank Abendroth and Oliver Seitz

Angew. Chem. Int. Ed. 2014, 53, 10504 –10509


Described here is a method for the conjugation of phosphorothioate oligonucleotides (PSOs) with peptides. PSOs are key to antisense technology. Peptide–PSO conjugates may improve target specificity, tissue distribution, and cellular uptake of PSOs. However, the highly nucleophilic phosphorothioate structure poses a challenge to conjugation chemistry. Herein, we introduce a new method which involves a sequence of oxime ligation and strain-promoted [2+3] cycloaddition. The usefulness of the method was demonstrated in the synthesis of peptide–PSO conjugates that targeted two suppressors of both the intrinsic and the extrinsic pathway of apoptosis. It is shown that the activity of a PSO sequence targeted against mRNA from c-Flip can be enhanced by conjugation with a peptide mimetic designed to inhibit the X-linked inhibitor of apoptosis protein (XIAP).

Progress in the direct structural characterization of fibrous amphiphilic supramolecular assemblies in solution by transmission electron microscopic techniques

H. von Berlepsch, K. Ludwig, B. Schade, R. Haag and C. Böttcher

Adv. Colloid Interface Sci. 2014, 208C, 279-292 ...

The self-assembly of amphiphilic molecules into fibrous structures has been the subject of numerous studies over past decades due to various current and promising technical applications. Although very different in their head group chemistry many natural as well as synthetic amphiphilic compounds derived from carbohydrates, carbocyanine dyes, or amino acids tend to form fibrous structures by molecular self-assembly in water predominantly twisted ribbons or tubes. Often a transition between these assembly structures is observed, which is a phenomenon already theoretically approached by Wolfgang Helfrich and still focus point in current research. With the development of suitable sample preparation and electron optical imaging techniques, cryogenic transmission electron microscopy (cryo-TEM) in combination with three-dimensional (3D) reconstruction techniques has become a particular popular direct characterization technique for supramolecular assemblies in general. Here we review the recent progress in deriving precise structural information from cryo-TEM data of particularly fibrous structures preferably in three dimensions.

A crucial role of L-selectin in C protein-induced experimental polymyositis in mice.

K. Oishi, Y. Hamaguchi, T. Matsushita, M. Hasegawa, N. Okiyama, J. Dernedde, M. Weinhart, R. Haag, T.F. Tedder, K. Takehara, H. Kohsaka, M. Fujimoto

Arthritis Rheumatol. 2014, 66, 1864-1871


To investigate the role of adhesion molecules in C protein–induced myositis (CIM), a murine model of polymyositis (PM). CIM was induced in wild-type mice, L-selectin–deficient (L-selectin−/−) mice, intercellular adhesion molecule 1 (ICAM-1)–deficient (ICAM-1−/−) mice, and mice deficient in both L-selectin and ICAM-1 (L-selectin−/−ICAM-1−/− mice). Myositis severity, inflammatory cell infiltration, and messenger RNA expression in the inflamed muscles were analyzed. The effect of dendritic polyglycerol sulfate, a synthetic inhibitor that suppresses the function of L-selectin and endothelial P-selectin, was also examined. L-selectin−/− mice and L-selectin−/−ICAM-1−/− mice developed significantly less severe myositis compared to wild-type mice, while ICAM-1 deficiency did not inhibit the development of myositis. L-selectin−/− mice that received wild-type T cells developed myositis. Treatment with dendritic polyglycerol sulfate significantly diminished the severity of myositis in wild-type mice compared to treatment with control. These data indicate that L-selectin plays a major role in the development of CIM, whereas ICAM-1 plays a lesser role, if any, in the development of CIM. L-selectin–targeted therapy may be a candidate for the treatment of PM.

Self-recovering stimuli-responsive macrocycle-equipped supramolecular ionogels with unusual mechanical properties

Z. Qi, N. L. Traulsen , P. Malo de Molina , C. Schlaich, M. Gradzielski, C. A. Schalley

Org. Biomol. Chem. 2014, 12, 503-510

Full TextSupporting Information

A chiral, crown-ether-functionalized bisurea gelator forms supramolecular gels in ionic liquids. The resulting ionogels show a remarkably high thermal stability with gel-sol transition temperatures (Tgs) reaching more than 100 °C. The mechanical strength of these ionogels is surprisingly high and even comparable to that of cross-linked protein fibres. Furthermore, the ionogels exhibit rapid self-recovery properties after structural damage caused by deformation. Pseudorotaxanes form from the gelators’ benzo[21]crown-7 ethers as the wheels and secondary ammonium ions as the axles despite of the competition between that cation and the imidazolium ions of the ionic liquid for crown ether binding. Pseudorotaxane formation as an external chemical stimulus triggers the gel-sol transition of the ionogels.

Multivalency in the Gas Phase: H/D-Exchange Reactions Unravel the Dynamic 'Rock 'n Roll' Motion in Dendrimer-Dendrimer Complexes

Z. Qi, C. Schlaich, C. A. Schalley

Chem. Eur. J. 2013, 19, 14867-14875

Full TextSupporting Information

Non-covalent dendrimer-dendrimer complexes were successfully ionized by electrospray ionization (ESI) of partly protonated amino-terminated polypropylene amin (POPAM) and POPAM dendrimers fully functionalized with benzo[21]crown-7 on all branches. Hydrogen/deuterium exchange (HDX) experiments conducted on dendrimer-dendrimer complexes in the high vacuum of a mass spectrometer give rise to a complete exchange of all labile N-H hydrogen atoms. As crown ethers represent non-covalent protective groups against HDX reactions on the ammonium group they coordinate to, this result provides evidence for a very dynamic binding situation: Each crown is mobile enough to move from one ammonium binding site to another one. Schematically, one might compare this motion with two rock ‘n roll dancers that swirl around each other without completely losing all contact at any time. Although the multivalent attachment certainly increases the overall affinity, the “microdynamics” of individual site binding and dissociation remains fast.

Fibrous Networks with Incorporated Macrocycles: A Chiral Stimuli-Responsive Supramolecular Supergelator and its Application to Biocatalysis in Organic Media

Z. Qi, C. Wu, P. Malo de Molina, H. Sun, A. Schulz, C. Griesinger, M. Gradzielski, R. Haag, M. B. Ansorge-Schumacher, C. A. Schalley

Chem. Eur. J. 2013, 19, 10150-10159

Full TextSupporting Information

A new and versatile, crown ether appended, chiral supergelator has been designed and synthesized based on the bis-urea motif. The introduction of a stereogenic center improved its gelation ability significantly relative to its achiral analogue. This low-molecular-weight gelator forms supramolecular gels in a variety of organic solvents. It is sensitive to multiple chemical stimuli and the sol–gel phase transitions can be reversibly triggered by host–guest interactions. The gel can be used to trap enzymes and release them on demand by chemical stimuli. It stabilizes the microparticles in Pickering emulsions so that enzyme-catalyzed organic reactions can take place in the polar phase inside the microparticles, the organic reactants diffusing through the biphasic interface from the surrounding organic phase. Because of the higher interface area between the organic and polar phases, enzyme activity is enhanced in comparison with simple biphasic systems.

Pseudorotaxanes with Self-Sorted Sequence and Stereochemistry

C. Talotta, C. Gaeta, Z. Qi, C. A. Schalley, P. Neri

Angew. Chem. Int. Ed. 2013, 52, 7437-7441; Angew. Chem. 2013, 125, 7585-7589

Full Text (International Edition)Full Text (German Edition)Supporting Information

Partner preferences in pseudorotaxane formation were exploited to establish an integrative self-sorting system able to discriminate simultaneously at the sequence and stereochemical level (see picture). It was found that calix[6]arenes were threaded selectively with a preferred orientation onto bisammonium axles, even when the structural differences between the possible building blocks were small and located remote from the binding sites.

Generation of a Dynamic System of Three-Dimensional Tetrahedral Polycatenanes

S. P. Black, A. R. Stefankiewicz, M. M. J. Smulders, D. Sattler, C. A. Schalley, J. R. Nitschke, J. K. M. Sanders

Angew. Chem. Int. Ed. 2013, 52, 5749-5752; Angew. Chem. 2013, 125, 5861-5864

Full Text (International Edition)Full Text (German Edition)Supporting InformationHighlighted in Nature Chemistry

Seven of the best: A dynamic combinatorial library of polycatenated tetrahedra was prepared by complexation between a dynamic Fe4L6 tetrahedral cage, constructed from ligands containing an electron-deficient naphthalenediimide core, and an electron-rich aromatic crown ether, 1,5-dinaphtho[38]crown-10. The highest order species in the library is the tetrahedral [7]catenane.

Stabilization of Peptides for Intracellular Applications by branched Phosphoramidate-Linked PEG Chains

N. Nischan, A. Chakrabarti, R.A. Serwa, P.H.M. Bovée-Geurts, R. Brock, C.P.R. Hackenberger

Angew. Chem. Int. Ed., 2013, 52, 11920 - 11924 ...

PEG intracellularly! Although long known to enhance residence half-life of peptides in serum and lysates, the effect of PEGylation on biological probes in cells has received only limited attention. Here it is shown that phosphoramidate-linked PEGylated proapoptotic peptides display a dramatically increased stability in Jurkat cell lysate and a homogenous intracellular distribution as well as high apoptotic activity after introduction into cells.

Solvation thermodynamics and heat capacity of polar and charged solutes in water

Felix Sedlmeier and Roland R. Netz

J. Chem. Phys., 2013, 138, 115101 ...

The solvation thermodynamics and in particular the solvation heat capacity of polar and charged solutes in water is studied using atomistic molecular dynamics simulations. As ionic solutes we consider a F− and a Na + ion, as an example for a polar molecule with vanishing net charge we take a SPC/E water molecule. The partial charges of all three solutes are varied in a wide range by a scaling factor. Using a recently introduced method for the accurate determination of the solvation free energy of polar solutes, we determine the free energy, entropy, enthalpy, and heat capacity of the three different solutes as a function of temperature and partial solute charge. We find that the sum of the solvation heat capacities of the Na + and F− ions is negative, in agreement with experimental observations, but our results uncover a pronounced difference in the heat capacity between positively and negatively charged groups. While the solvation heat capacity ΔC p stays positive and even increases slightly upon charging the Na + ion, it decreases upon charging the F− ion and becomes negative beyond an ion charge of q = −0.3e. On the other hand, the heat capacity of the overall charge-neutral polar solute derived from a SPC/E water molecule is positive for all charge scaling factors considered by us. This means that the heat capacity of a wide class of polar solutes with vanishing net charge is positive. The common ascription of negative heat capacities to polar chemical groups might arise from the neglect of non-additive interaction effects between polar and apolar groups. The reason behind this non-additivity is suggested to be related to the second solvation shell that significantly affects the solvation thermodynamics and due to its large spatial extent induces quite long-ranged interactions between solvated molecular parts and groups.

Adsorption of multivalent alkylthiols on Au(111) surface: Insights from DFT

E. Fertitta, E. Voloshina, B. Paulus

J. Comp. Chem., 2013, 35, 204 - 213

" ...

The adsorption of multivalent thiols on gold (111) surface was investigated using density functional theory applying the Perdew–Burke–Ernzerhof functional. Through the comparison of differences in energetics, structure and charge density distribution of a set of monodentate and polydentate thiols, we have described in detail the factors affecting the adsorption energy and the role played by the multivalence, which causes a decreasing of adsorption energy because of both electronic and steric hindrance effects. Finally, the comparison between the adsorption of 1,2- and 1,3-disulfides revealed how the chain length may affect the cleavage of the S[BOND]S bond when they adsorb on Au(111) surface.

Interaction of Pyridine Derivatives with Gold (111) Surface as a Model for Adsorption to Large Nanoparticles

D. Mollenhauer, N. Gaston, E. Voloshina and B. Paulus

J. Phys. Chem. C, 2013 117, 4470 ...

In recent years pyridine derivatives, namely 4-(N,N-dimethylamino)pyridine and 4-methoxypyridine, were found to be excellent agents for the stabilization of gold nanoparticles. In order to gain a better understanding of these systems we have simulated the interaction of gold nanoparticles with pyridine and pyridine derivatives with donor substituents in position 4 by quantum-chemical calculations. The relatively large nanoparticles were modeled by the most stable and relevant surface, Au(111). To account for dispersion effects within the density functional theory approach a dispersion correction in the D3 scheme was applied. Due to the dispersion effects the preferred orientation on Au(111) changed from perpendicular for pyridine to parallel for pyridine derivatives. In addition, the adsorption at edges and corner sites of nanoparticles was considered through the use of gold ad-atoms on the surface. For the pyridine–gold interaction a change of the nature of binding was observed.

Inhibition of clathrin by pitstop 2 activates the spindle assembly checkpoint and induces cell death in dividing HeLa cancer cells

Smith CM, Haucke V, McCluskey A, Robinson PJ, Chircop M

Molecular Cancer 2013, 12:4 ...

During metaphase clathrin stabilises the mitotic spindle kinetochore(K)-fibres. Many anti-mitotic compounds target microtubule dynamics. Pitstop 2™ is the first small molecule inhibitor of clathrin terminal domain and inhibits clathrin-mediated endocytosis. We investigated its effects on a second function for clathrin in mitosis.

Stabilisierung von Peptiden für intrazelluläre Anwendungen mit Phosphoramidat-verzweigten Polyethylenglycol-Ketten

N. Nischan, A. Chakrabarti, R.A. Serwa, P.H.M. Bovée-Geurts, R. Brock, C.P.R. Hackenberger

Angew. Chem., 2013, 125, 12138 - 12142 ...

PEG intrazellulär! Obwohl PEGylierungen die biologische Halbwertszeit von Peptiden in Serum und Lysat verlängern, wurde der Einfluss der PEGylierung auf das Verhalten biologischer Wirkstoffe in Zellen bis jetzt kaum untersucht. Hier wird gezeigt, dass Phosphoramidat-verzweigte PEGylierte proapoptotische Peptide eine erhöhte Stabilität in Jurkat-Zelllysat aufweisen, homogen in der Zelle verteilt sind und eine hohe apoptotische Aktivität in Zellen zeigen.

Alkinphosphonite für sequenzielle Azid-Azid-Kupplungen

M.R.J. Vallée, L.M. Artner, J. Dernedde, C.P.R. Hackenberger

Angew. Chem., 2013, 125, 9682–9686 ...

Wenn Staudinger auf Huisgen trifft! Eine Kombination der Kupfer-katalysierten Variante der Huisgen-Azid-Alkin-Cycloaddition (CuAAC) mit der Staudinger-Reaktion führt zu einer chemischen Methode, die die sequenzielle Kupplung zweier unterschiedlicher Azidbausteine in hohen Ausbeuten ermöglicht. Mithilfe dieses modularen Protokolls gelingt eine finale metallfreie Konjugation von funktionellen Bausteinen mit Aziden.

Alkyne Phosphonites for Sequential Azide–Azide Couplings

M.R.J. Vallée, L.M. Artner, J. Dernedde, C.P.R. Hackenberger

Angew. Chem. Int. Ed., 2013, 52, 9504 - 9508 ...

When Staudinger meets Huisgen! A combination of the copper-catalyzed variant of the Huisgen azide–alkyne cycloaddition (CuAAC) and the Staudinger reaction, the two most successful chemoselective reactions for the transformation of azides, leads to a chemical method that allows the sequential coupling of two different azido building blocks in high yields. This modular procedure enables a final metal-free conjugation of functional building blocks to azides.

Completion of Proteomic Data Sets by K(d) Measurement Using Cell-Free Synthesis of Site-Specifically Labeled Proteins

Paul Majkut, Iris Claußnitzer, Helmut Merk, Christian Freund, Christian P. R. Hackenberger, Michael Gerrits

PLoS ONE, 2013, 8(12): e82352 ...

The characterization of phosphotyrosine mediated protein-protein interactions is vital for the interpretation of downstream pathways of transmembrane signaling processes. Currently however, there is a gap between the initial identification and characterization of cellular binding events by proteomic methods and the in vitro generation of quantitative binding information in the form of equilibrium rate constants (Kd values). In this work we present a systematic, accelerated and simplified approach to fill this gap: using cell-free protein synthesis with site-specific labeling for pull-down and microscale thermophoresis (MST) we were able to validate interactions and to establish a binding hierarchy based on Kd values as a completion of existing proteomic data sets. As a model system we analyzed SH2-mediated interactions of the human T-cell phosphoprotein ADAP. Putative SH2 domain-containing binding partners were synthesized from a cDNA library using Expression-PCR with site-specific biotinylation in order to analyze their interaction with fluorescently labeled and in vitro phosphorylated ADAP by pull-down. On the basis of the pull-down results, selected SH2’s were subjected to MST to determine Kd values. In particular, we could identify an unexpectedly strong binding of ADAP to the previously found binding partner Rasa1 of about 100 nM, while no evidence of interaction was found for the also predicted SH2D1A. Moreover, Kd values between ADAP and its known binding partners SLP-76 and Fyn were determined. Next to expanding data on ADAP suggesting promising candidates for further analysis in vivo, this work marks the first Kd values for phosphotyrosine/SH2 interactions on a phosphoprotein level.

Understanding selectin counter-receptor binding from electrostatic energy computations and experimental binding studies.

A.L. Woelke, C. Kuehne, T. Meyer, G. Galstyan, J. Dernedde, E.W. Knapp

J. Phys. Chem. B, 2013, 117, 16443-16454


Higher organisms defend themselves against invading micro-organisms and harmful substances with their immune system. Key players of the immune system are the white blood cells (WBC), which in case of infection move in an extravasation process from blood vessels toward infected tissue promoting inflammation. This process starts with the attachment of the WBC to the blood vessel wall, mediated by protein pair interactions of selectins and counter-receptors (C-R). Individual selectin C-R binding is weak and varies only moderately between the three selectin types. Multivalency enhances such small differences, rendering selectin-binding type specific. In this work, we study selectin C-R binding, the initial step of extravasation. We performed electrostatic energy computations based on the crystal structure of one selectin type co-crystallized with the ligating part of the C-R. The agreement with measured free energies of binding is satisfactory. Additionally, we modeled selectin mutant structures in order to explain differences in binding of the different selectin types. To verify our modeling procedures, surface plasmon resonance data were measured for several mutants and compared with computed binding affinities. Binding affinities computed with soaked rather than co-crystallized selectin C-R structures do not agree with measured data. Hence, these structures are inappropriate to describe the binding mode. The analysis of selectin/C-R binding unravels the role played by individual molecular components in the binding event. This opens new avenues to prevent immune system malfunction, designing drugs that can control inflammatory processes by moderating selectin C-R binding.

Toward animal cell culture-based influenza vaccine design: viral hemagglutinin N-glycosylation markedly impacts immunogenicity

J. Hütter, J.V. Rödig, D. Höper, U. Reichl, P.H. Seeberger, E. Rapp, B. Lepenies

J. Immunol., 2013, 190(1), 220-30 ...

The glycoproteins hemagglutinin (HA) and neuraminidase are the major determinants of host range and tissue tropism of the influenza virus. HA is the most abundant protein in the virus particle membrane and represents the basis of most influenza vaccines. It has been reported that influenza virus HA N-glycosylation markedly depends on the host cell line used for virus production. However, little is known about how differential glycosylation affects immunogenicity of the viral proteins. This is of importance for virus propagation in chicken eggs as well as for innovative influenza vaccine production in mammalian cell lines. In this study, we investigated the impact of the differential N-glycosylation patterns of two influenza A virus PR/8/34 (H1N1) variants on immunogenicity. Madin–Darby canine kidney cell–derived and Vero cell–derived glycovariants were analyzed for immunogenicity in a TCR-HA transgenic mouse model. Next-generation pyrosequencing validated the congruence of the potential HA N-glycosylation sites as well as the presence of the HA peptide recognized by the TCR-HA transgenic T cells. We show that differential HA N-glycosylation markedly affected T cell activation and cytokine production in vitro and moderately influenced IL-2 production in vivo. Cocultivation assays indicated that the difference in immunogenicity was mediated by CD11c+ dendritic cells. Native virus deglycosylation by endo- and exoglycosidases dramatically reduced cytokine production by splenocytes in vitro and markedly decreased HA-specific Ab production in vivo. In conclusion, this study indicates a crucial importance of HA N-glycosylation for immunogenicity. Our findings have implications for cell line–based influenza vaccine design.

Targeting C-type lectin receptors with multivalent carbohydrate ligands

B. Lepenies, J. Lee, S. Sonkaria

Adv. Drug. Deliv. Rev., 2013, 65(9), 1271-81 ...

C-type lectin receptors (CLRs) represent a large receptor family including collectins, selectins, lymphocyte lectins, and proteoglycans. CLRs share a structurally homologous carbohydrate-recognition domain (CRD) and often bind carbohydrates in a Ca2 +-dependent manner. In innate immunity, CLRs serve as pattern recognition receptors (PRRs) and bind to the glycan structures of pathogens and also to self-antigens. In nature, the low affinity of CLR/carbohydrate interactions is overcome by multivalent ligand presentation at the surface of cells or pathogens. Thus, multivalency is a promising strategy for targeting CLR-expressing cells and, indeed, carbohydrate-based targeting approaches have been employed for a number of CLRs, including asialoglycoprotein receptor (ASGPR) in the liver, or DC-SIGN expressed by dendritic cells. Since CLR engagement not only mediates endocytosis but also influences intracellular signaling pathways, CLR targeting may allow for cell-specific drug delivery and also the modulation of cellular functions. Glyconanoparticles, glycodendrimers, and glycoliposomes were successfully used as tools for CLR-specific targeting. This review will discuss different approaches for multivalent CLR ligand presentation and aims to highlight how CLR targeting has been employed for cell specific drug delivery. Major emphasis is directed towards targeting of CLRs expressed by antigen-presenting cells to modulate immune responses.

The C-type lectin receptor SIGNR3 binds to fungi present in commensal microbiota and influences immune regulation in experimental colitis

M. Eriksson, T. Johannssen, D. von Smolinski, A.D. Gruber, P.H. Seeberger, B. Lepenies

Front. Immunol., 2013, 4, 196 ...

Inflammatory bowel disease is a condition of acute and chronic inflammation of the gut. An important factor contributing to pathogenesis is a dysregulated mucosal immunity against commensal bacteria and fungi. Host pattern-recognition receptors (PRRs) sense commensals in the gut and are involved in maintaining the balance between controlled responses to pathogens and overwhelming innate immune activation. C-type lectin receptors (CLRs) are PRRs recognizing glycan structures on pathogens and self-antigens. Here we examined the role of the murine CLR specific intracellular adhesion molecule-3 grabbing non-integrin homolog-related 3 (SIGNR3) in the recognition of commensals and its involvement in intestinal immunity. SIGNR3 is the closest murine homolog of the human dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) receptor recognizing similar carbohydrate ligands such as terminal fucose or high-mannose glycans. We discovered that SIGNR3 recognizes fungi present in the commensal microbiota. To analyze whether this interaction impacts the intestinal immunity against microbiota, the dextran sulfate sodium-induced colitis model was employed. SIGNR3−/− mice exhibited an increased weight loss associated with more severe colitis symptoms compared to wild-type control mice. The increased inflammation in SIGNR3−/− mice was accompanied by a higher level of TNF-α in colon. Our findings demonstrate for the first time that SIGNR3 recognizes intestinal fungi and has an immune regulatory role in colitis.

Selektive katalytische Hydrodefluorierung als Schlüsselschritt zur Synthese bisher unzugänglicher Aminopyridinderivate

G. Podolan, D. Lentz, H.-U. Reissig

Angew. Chem. 2013, 125, 9669 –9672


Fluor macht's möglich! Die regioselektive nucleophile Substitution von (Oligo)fluorpyridinen mit geeigneten Aminen und nachfolgende katalytische Hydrodefluorierung ebnen den Weg zu bisher unzugänglichen Aminopyridinen, die als neue Liganden von Interesse sind. Bis zu vier Fluoratome werden in einem Schritt mithilfe eines kostengünstigen Titankatalysators regioselektiv entfernt.

Investigations of Host-Guest Interactions with Shape-persistent Nonionic Dendritic Micelles

R. Tyagi, S. Malhotra, A. Thünemann, A. Sedighi, M. Weber, A. Schäfer, R. Haag

J. Phys. Chem. C, 2013, 117, 12307–12317


The interaction of self-assembled dendritic amphiphiles with drugs and dyes in aqueous solutions is of great significance for designing and optimizing shape-persistent delivery systems. Here we present deeper insight for two examples of low molecular weight (LMW) nonionic dendritic amphiphiles as host molecules and a series of selected aromatic guest model molecules (benzene, naphthalene, biphenyl, terphenyl, anthracene, and pyrene). Aromatic guest molecules were incorporated into the self-assemblies of dendritic nanocarriers, and the resultant complexes were studied by a combination of UV, NMR, computational simulation, and small-angle X-ray-scattering (SAXS) techniques in order to determine the loading capacity, localization, and specific interactions in dendritic amphiphiles with guest molecules. Our findings revealed that the localization of guest molecules in the hydrophobic region and the loading capacity of guest molecules are dependent on their size and the arrangement of aromatic rings instead of the loading amount. Furthermore, the shape of self-assembled host molecules was found to be ellipsoidal and highly persistent even after loading the guest molecules. To the best of our knowledge, this is the first systematic host–guest study, particularly with low molecular weight nonionic dendritic amphiphilies and aromatic guest molecules. Thus, this study opens new possibilities and ways to explore the transport behavior of aromatic drugs with such nanocarriers.

Synthesis and Properties of Branched Oligo(2-thienyl)- and Oligo(2,2’-bithien-5-yl)-Substituted Pyridine Derivatives

M. K. Bera, S. L. Gholap, P. Hommes, K. Neuthe, D. Trawny, J. P. Rabe, D. Lentz, R. Zimmer, H.-U. Reissig

Adv. Synth. Catal. 2013, 355, 3463 - 3474


Starting from easily accessible precursors we describe the preparation of a series of branched oligo(2-thienyl)- and oligo(2,2′-bithienyl)-substituted pyridine derivatives. With palladium-catalyzed cross-coupling reactions of pyridyl nonaflates/triflates as key steps we synthesized 2,6-di(2-thienyl)pyridines bridged by thiophene or benzene rings. By selective bromination of 2,6-di(2-thienyl)pyridine and 2,4,6-tri(2-thienyl)pyridine and subsequent coupling reactions an access to oligo(2,2′-bithien-5-yl)-substituted pyridine derivatives was gained. The constitution and solid state conformation of 2,6-bis(2,2’-bithien-5-yl)pyridine was determined by X-ray analysis. This series of new pyridine-thiophene conjugates was systematically investigated by UV/vis spectroscopy. Large Stokes shifts in the neutral and protonated form were observed. The electrochemical oxidation of two typical compounds was studied, however, these oxidations were irreversible forming a polymeric film at the anode. As a selected example, a thiophene-bridged 2,6-di(2-thienyl)pyridine derivative was also investigated by scanning tunneling microscopy showing an interesting self-assembly into a highly ordered monolayer on highly oriented pyrolytic graphite.

Carbohydrate-PNA and Aptamer-PNA Conjugates for the Spatial Screening of lectins and Lectin Assemblies

C. Scheibe, S. Wedepohl, S.B. Riese, J. Dernedde, O. Seitz

ChemBioChem, 2013, 14, 236-250 ...

Nucleic acid architectures offer intriguing opportunities for the interrogation of structural properties of protein receptors. In this study, we performed a DNA-programmed spatial screening to characterize two functionally distinct receptor systems: 1) structurally well-defined Ricinus communis agglutinin (RCA120), and 2) rather ill-defined assemblies of L-selectin on nanoparticles and leukocytes. A robust synthesis route that allowed the attachment both of carbohydrate ligands—such as N-acetyllactosamine (LacNAc), sialyl-Lewis-X (sLeX), and mannose—and of a DNA aptamer to PNAs was developed. A systematically assembled series of different PNA–DNA complexes served as multivalent scaffolds to control the spatial alignments of appended lectin ligands. The spatial screening of the binding sites of RCA120 was in agreement with the crystal structure analysis. The study revealed that two appropriately presented LacNAc ligands suffice to provide unprecedented RCA120 affinity (KD=4 μM). In addition, a potential secondary binding site was identified. Less dramatic binding enhancements were obtained when the more flexible L-selectin assemblies were probed. This study involved the bivalent display both of the weak-affinity sLeX ligand and of a high-affinity DNA aptamer. Bivalent presentation led to rather modest (sixfold or less) enhancements of binding when the self-assemblies were targeted against L-selectin on gold nanoparticles. Spatial screening of L-selectin on the surfaces of leukocytes showed higher affinity enhancements (25-fold). This and the distance–activity relationships indicated that leukocytes permit dense clustering of L-selectin.

Insight into the Molecular Mechanisms of Protein Stabilizing Osmolytes from Global Force-Field Variations

Emanuel Schneck, Dominik Horinek and Roland R. Netz

J. Phys. Chem. B, 2013, 117, 8310-8321

A prominent class of osmolytes that are able to stabilize proteins in their native fold consist of small highly water-soluble molecules with a large dipole moment and hydrophobic groups attached to the positively charged end of the molecule, for which we coin the term dipolar/hydrophobic osmolytes. For TMAO, which is a prime member of this class, we perform large-scale water-explicit MD simulations and determine the bulk solution activity coefficient as well as the affinity to a stretched polyglycine chain for varying TMAO dipolar strength and hydrophobicity. Double optimization with respect to experimental values for the activity coefficient and the polyglycine transfer free energy is achieved. The resulting optimal TMAO force field shows excellent transferability to different concentrations and also reproduces transfer free energies of various amino acids, including the tryptophan anomaly, for which TMAO acts as a denaturant. By globally analyzing the thermodynamic and structural properties of suboptimal TMAO force fields, we identify the frustration between dipolar and hydrophobic interactions as the working mechanism and the design principle of dipolar/hydrophobic osmolytes.

Hydration Interaction between Phospholipid Membranes: Insight into Different Measurement Ensembles from Atomistic Molecular Dynamics Simulations

Matej Kanduc, Emanuel Schneck, Roland R. Netz

Langmuir 2013, 29, 9126-9137

Using the novel thermodynamic extrapolation technique in molecular dynamics simulations, we investigate the interaction between phospholipid bilayers subject to various boundary conditions that correspond to established experimental methods for the determination of pressure–distance curves: the osmotic stress method, the hydrostatic method, and the surface force apparatus method. We discuss the roles of van der Waals and Helfrich undulation pressures in the force balance and find that they do not play a major role in the distance range below 28 water molecules per lipid as considered by us. We address the influence of experimental boundary conditions on bilayer structural changes as well as the consequences on interaction pressures. Significant discrepancies are observed between pressures obtained in osmotic stress and hydration methods on one hand and the surface force apparatus method on the other hand. We quantify the contribution of lipid volume compressibility to the total work of dehydration and find it to be substantial for high pressures. In a wide hydration range, the interaction pressure is mostly determined by the area per lipid molecule. This means that the influence of fatty acid chemistry on experimental pressure–distance curves is indirect and mediated by the area per lipid.

Free nanoparticles studied by soft X-rays

E. Antonsson, H. Bresch, R. Lewinski, B. Wassermann, T. Leisner, C. Graf, B. Langer und E. Rühl

Chem. Phys. Lett., Frontiers Article 559, 1-11 (2013).


The use of nanoparticle beams for the preparation and characterization of isolated nanoscopic matter is reviewed. It is a general preparation scheme which is shown to be of broad use, whenever the intrinsic properties of nanoscopic matter without any interactions with other particles or substrates are of interest. The size regime spans a few nanometers up to the micron sizes, wherein a large variety of substances and materials can be studied. Similarly broad are the detection schemes and properties, which can be investigated by this approach. This review covers various properties of isolated nanoparticles, including their size, surface properties, optical constants, as well as their local electronic structure.

Size-Effects in Free Nanoparticles Studied by Soft X-Rays

E. Antonsson, H. Bresch, R. Lewinski, B. Wassermann, T. Leisner, C. Graf, B. Langer und E. Rühl

Chem. Phys. Lett., 2013 Frontiers Article 559, 1-11 ...

The use of nanoparticle beams for the preparation and characterization of isolated nanoscopic matter is reviewed. It is a general preparation scheme which is shown to be of broad use, whenever the intrinsic properties of nanoscopic matter without any interactions with other particles or substrates are of interest. The size regime spans a few nanometers up to the micron sizes, wherein a large variety of substances and materials can be studied. Similarly broad are the detection schemes and properties, which can be investigated by this approach. This review covers various properties of isolated nanoparticles, including their size, surface properties, optical constants, as well as their local electronic structure.

Control over Unfolding Pathways by Localizing Photoisomerization Events within Heterosequence Oligoazobenzene Foldamers

Z. Yu, S. Hecht

Angew. Chem. 2013, 125, 13985-13989


Zwei photoschaltbare Foldamere, die Azobenzol-Einheiten als Energie-Akzeptoren beinhalten, wurden entwickelt. Ihr Entfaltungsmechanismus kann durch den kontrollierten Einbau dieser photoinduzierten Auslöser in der Mitte oder an den Enden der Helix gesteuert werden.

Control over Unfolding Pathways by Localizing Photoisomerization Events within Heterosequence Oligoazobenzene Foldamers

Z. Yu, S. Hecht

Angew. Chem. Int. Ed. 2013, 52, 13740-13744


From the inside out or from the outside in? Two photoswitchable foldamers that incorporate azobenzene moieties as the energy-acceptor units have been designed. The pathway of helix unfolding can be controlled by localizing these photoinduced triggers either at the core or at the termini of the helix.

Elucidating the backbone conformation of photoswitchable foldamers using vibrational circular dichroism

S. R. Domingos, S. J. Roeters, S. Amirjalayer, Z. Yu, S. Hecht, S. Woutersen

Phys. Chem. Chem. Phys. 2013, 15, 17263-17267


The backbone conformation of amphiphilic oligo(azobenzene) foldamers is investigated using vibrational circular dichroism (VCD) spectroscopy on a mode involving the stretching of the N[double bond, length as m-dash]N bonds in the backbone. From denaturation experiments, we find that the VCD response in the helical conformation arises mainly from through-space interaction between the N[double bond, length as m-dash]N-stretch transition-dipole moments, so that the coupled-oscillator model can be used to predict the VCD spectrum associated with a particular conformation. Using this approach, we elucidate the origin of the VCD signals in the folded conformation, and can assign the observed partial loss of VCD signals upon photo-induced unfolding to specific conformational changes. Our results show that the N[double bond, length as m-dash]N-stretch VCD response provides an excellent probe of the helical conformation of the N[double bond, length as m-dash]N bonds in this type of switchable molecular system.

The role of statistics and microenvironment for the photoresponse in multi-switch architectures: The case of photoswitchable oligoazobenzene foldamers

Z. Yu, S. Weidner, T. Risse, S. Hecht

Chem. Sci. 2013, 4, 4156-4167


Systems containing multiple photochromic units possibly display a synergistic interplay of individual switching events and hence potentially give rise to unprecedented photoresponsive behavior. Among such systems photoswitchable foldamers are attractive as the photoisomerization events are coupled to the helix–coil conformational transition. To gain comprehensive insight into the role of the number of switching units (statistics) as well as their specific location and relative orientation in the helix backbone, several series of foldamers have been synthesized and characterized. In these series of foldamers, the local environment of the photoswitchable units was precisely tuned as π,π-stacking interactions were enforced to occur between specific pairs, i.e. azobenzene–azobenzene, azobenzene–tolane, or phenylene–phenylene units. These particular arrangements are reflected not only in the stability of the helical conformation, but also affect the photoresponsive behavior, i.e. the rate of photoisomerization and extent of denaturation. Furthermore, determining the intramolecular spin–spin distance in a series of TEMPO-labeled foldamers with variable chain lengths and different spatial locations of the spin-labels deduced an independent verification of the photoinduced helix–coil transition by ESR spectroscopy. Quantitative analysis of the corresponding ESR spectra shows an excellent correlation of the extent of intramolecular spin–spin coupling and the intensity of the Cotton effect in CD spectroscopy. From all of these results an unusual relationship between the rate of photoisomerization and the extent of photoinduced denaturation could be unraveled, as they are not going hand-in-hand but compete with each other, i.e. the easier the individual switching event is, the harder it becomes to achieve a high degree of unfolding. This insight into the effect of microenvironment on the ease of individual switching events and the role of statistics on the resulting degree of the overall conformational transition is of general interest for the design of multi-switch architectures with improved photoresponse.

Nonsteroidal Bivalent Estrogen Ligands - An Application of the Bivalent Concept to the Estrogen Receptor

M. Shan, K. E. Carlson, A. Bujotzek, A. Wellner, R. Gust, M. Weber, J. A. Katzenellenbogen, R. Haag

ACS Chem. Biol., 2013, 8 (4), pp 707–715 ...

The estrogen receptor (ER) is a hormone-regulated transcription factor that binds, as a dimer, to estrogens and to specific DNA sequences. To explore at a fundamental level the geometric and topological features of bivalent-ligand binding to the ER dimer, dimeric ER crystal structures were used to rationally design nonsteroidal bivalent estrogen ligands. Guided by this structure-based ligand design, we prepared two series of bivalent ligands (agonists and antagonists) tethered by flexible spacers of varying lengths (7–47 Å) and evaluated their ER-binding affinities for the two ER subtypes and their biological activities in cell lines. Bivalent ligands based on the agonist diethylstilbestrol (DES) proved to be poor candidates, but bivalent ligands based on the antagonist hydroxytamoxifen (OHT) were well suited for intensive study. Binding affinities of the OHT-based bivalent ligands were related to spacer length in a distinctive fashion, reaching two maximum values at 14 and 29 Å in both ER subtypes. These results demonstrate that the bivalent concept can operate in determining ER-ligand binding affinity and suggest that two distinct modes operate for the binding of bivalent estrogen ligands to the ER dimers, an intermolecular as well as an intramolecular mode. Our insights, particularly the possibility of intramolecular bivalent binding on a single ER monomer, may provide an alternative strategy for preparing more selective and active ER antagonists for endocrine therapy of breast cancer.

A meshless discretization method for Markov State Models applied to explicit water peptide folding simulations

K. Fackeldey, A. Bujotzek, M. Weber

In: Lecture Notes in Computational Science and Engineering, Vol 89, Meshfree Methods for Partial Differential Equations VI, M. Griebel, M.A. Schweitzer (Eds.), 2013 Springer, 141-154


Markov State Models (MSMs) are widely used to represent molecular conformational changes as jump-like transitions between subsets of the conformational state space. However, the simulation of peptide folding in explicit water is usually said to be unsuitable for the MSM framework. In this article, we summarize the theoretical background of MSMs and indicate that explicit water simulations do not contradict these principles. The algorithmic framework of a meshless conformational space discretization is applied to an explicit water system and the sampling results are compared to a long-term molecular dynamics trajectory. The meshless discretization approach is based on spectral clustering of stochastic matrices (MSMs) and allows for a parallelization of MD simulations. In our example of Trialanine we were able to compute the same distribution of a long term simulation in less computing time.

Selective Catalytic Hydrodefluorination as a Key Step for the Synthesis of Hitherto Inaccessible Aminopyridine Derivatives

G. Podolan, D. Lentz, H.-U. Reissig

Angew. Chem. Int. Ed. 2013, 52, 9491 –9494


Fluorine makes it possible! The regioselective nucleophilic substitution of (oligo)fluoropyridines with the appropriate amines and the subsequent catalytic hydrodefluorination paves the way to hitherto inaccessible aminopyridine derivatives, which are of interest as new ligands. Up to four fluorine atoms can be removed regioselectively in one step in a reaction employing an inexpensive titanium precatalyst.

Systems Chemistry: Logic gates based on the stimuli-responsive gel-sol transition of a crown-ether functionalized bis-urea-gelator

Z. Qi, P. Malo de Molina, W. Jiang, Q. Wang, K. Nowosinski, A. Schulz, M. Gradzielski, C. A. Schalley

Chem. Sci. 2012, 3, 2073-2082

Full TextSupporting InformationHighlighted on the Chemical Science Blog

A quite simple, achiral benzo-21-crown-7-substituted bis(urea) low-molecular weight gelator hierarchically assembles into helical fibrils, which further develop into bundles and finally form a stable gel in acetonitrile. The gel–sol transition can be controlled by three different molecular recognition events: K+ binding to the crown ethers, pseudorotaxane formation with secondary ammonium ions and Cl− binding to the urea units. Addition of a cryptand that scavenges the K+ ions and Ag+ addition to remove the chloride and bases/acids, which mediate pseudorotaxane formation, can reverse this process. With the gelator, and these chemical stimuli, a number of different systems can be designed that behave as logic gates. Depending on the choice of components, OR, AND, XOR, NOT, NOR, XNOR and INHIBIT gates have been realized. Thus, the gel–sol transition as a property of the system as a whole is influenced in a complex manner. For some cases, the type of logic gate is defined by input signal concentration so that an even more complex reaction of the gel towards the two input signals is achieved.

Wavefunction-based electron correlation methods for solids

C. Müller, B. Paulus

Phys. Chem. Chem. Phys., 2012, 14, 7605-7614 ...

In this article we provide an overview of the most common ways of treating electron correlation effects in 3D-periodic systems with some emphasize on wavefunction-based correlation methods such as the method of increments and the local MP2 method implemented in the CRYSCOR program. We discuss strengths and weaknesses of the different approaches and give examples for their application. Additionally, for the method of increments we discuss recent developments for its application to open shell systems and problems related to the treatment of graphene sheets.

Surface Functionalization of Silica Nanoparticles Supports Colloidal Stability in Physiological Media and Facilitates Internalization in Cells

C. Graf, Q. Gao, I. Schütz, C. Niki Noufele, W. Ruan, U. Posselt, E. Korotianskiy, D. Nordmeyer, F. Rancan, S. Hadam, A. Vogt, J. Lademann, V. Haucke und E. Rühl

Langmuir 18, 2012 7598


The influence of the surface functionalization of silica particles on their colloidal stability in physiological media is studied and correlated with their uptake in cells. The surface of 55 ± 2 nm diameter silica particles is functionalized by amino acids or amino- or poly(ethylene glycol) (PEG)-terminated alkoxysilanes to adjust the zeta potential from highly negative to positive values in ethanol. A transfer of the particles into water, physiological buffers, and cell culture media reduces the absolute value of the zeta potential and changes the colloidal stability. Particles stabilized by l-arginine, l-lysine, and amino silanes with short alkyl chains are only moderately stable in water and partially in PBS or TRIS buffer, but aggregate in cell culture media. Nonfunctionalized, N-(6-aminohexyl)-3-aminopropyltrimethoxy silane (AHAPS), and PEG-functionalized particles are stable in all media under study. The high colloidal stability of positively charged AHAPS-functionalized particles scales with the ionic strength of the media, indicating a mainly electrostatical stabilization. PEG-functionalized particles show, independently from the ionic strength, no or only minor aggregation due to additional steric stabilization. AHAPS stabilized particles are readily taken up by HeLa cells, likely as the positive zeta potential enhances the association with the negatively charged cell membrane. Positively charged particles stabilized by short alkyl chain aminosilanes adsorb on the cell membrane, but are weakly taken up, since aggregation inhibits their transport. Nonfunctionalized particles are barely taken up and PEG-stabilized particles are not taken up at all into HeLa cells, despite their high colloidal stability. The results indicate that a high colloidal stability of nanoparticles combined with an initial charge-driven adsorption on the cell membrane is essential for efficient cellular uptake.

A novel hSH3 domain scaffold engineered to bind folded domains in CD2BP2 and HIV capsid protein.

K. Piotukh, C. Freund

Protein Eng. Des. Sel. 2012, 25,649-56 ...

Engineered protein scaffolds destined to target and inhibit molecular interactions in the context of disease bear great diagnostic and therapeutic potential. Apart from antibodies several alternative scaffolds have been exploited over the years making use of the fact that individual domain families are best suited for certain target families. Here we capitalize on the helically extended SH3 domain hSH3N of the ADAP protein as loop-randomized template that was tested against HIV capsid (CA) protein and the GYF domain of human CD2BP2 as molecular targets. Phage display of mutant proteins resulted in the isolation of variants with changes in all randomized positions compared with wild-type hSH3N. The soluble scaffolds bind with 340 and 600 nM affinity to CA and CD2BP2, respectively, and employ large molecular surfaces to pull out these targets from complex mixtures.

Multivalency as a Chemical Organization and Action Principle

C. Fasting, C.A. Schalley, M. Weber, O. Seitz, S. Hecht, B. Koksch, J. Dernedde, C. Graf, E.-W. Knapp, R. Haag

Angew. Chem. Int Ed., 2012, 51, 10472-10498


Multivalent interactions can be applied universally for a targeted strengthening of an interaction between different interfaces or molecules. The binding partners form cooperative, multiple receptor–ligand interactions that are based on individually weak, noncovalent bonds and are thus generally reversible. Hence, multi- and polyvalent interactions play a decisive role in biological systems for recognition, adhesion, and signal processes. The scientific and practical realization of this principle will be demonstrated by the development of simple artificial and theoretical models, from natural systems to functional, application-oriented systems. In a systematic review of scaffold architectures, the underlying effects and control options will be demonstrated, and suggestions will be given for designing effective multivalent binding systems, as well as for polyvalent therapeutics.

Multivalenz als chemisches Organisations- und Wirkprinzip

C. Fasting, C.A. Schalley, M. Weber, O. Seitz, S. Hecht, B. Koksch, J. Dernedde, C. Graf, E.-W. Knapp, R. Haag

Angew. Chem., 2012, 124, 10622-10650


Multivalente Wechselwirkungen können universell zur gezielten Bindungsverstärkung zwischen verschiedenen Grenzflächen oder Molekülen genutzt werden. Die Bindungspartner bilden dabei kooperativ multiple Rezeptor-Ligand-Wechselwirkungen, die auf einzelnen schwachen, nichtkovalenten Bindungen basieren und daher prinzipiell reversibel sind. Daher spielen multi- und polyvalente Wechselwirkungen in biologischen Systemen eine entscheidende Rolle für Erkennungs-, Adhäsions- und Signalprozesse. Die wissenschaftliche und praktische Etablierung dieses Prinzips wird anhand der Entwicklung von natürlichen Systemen über einfache artifizielle und theoretische Modelle hin zu anwendungsorientierten funktionalen Systemen demonstriert. Anhand eines systematischen Überblicks über Gerüstarchitekturen werden die zugrunde liegenden Wirk- und Steuerungsmöglichkeiten aufgezeigt und Designvorschläge für möglichst effektive multivalente Bindungspartner bis hin zu polyvalenten Therapeutika aufgezeigt.

Unexpected One-Step Formation of Iodo[1,3]dioxolo[4,5-c]pyridine Derivatives by a Hofmann-Löffler-Freytag Reaction – Studies towards the Synthesis of a Pyridine Containing Macrocycle

T. Lechel, G. Podolan, B. Brusilowskij, C.A. Schalley, H.-U. Reissig

Eur. J. Org. Chem. 2012, 5685-5692


During attempts to prepare functionalized 5-iodopyridine derivatives the unexpected formation of iodo[1,3]dioxolo[4,5-c]pyridines was discovered. The conversion of 3-alkoxypyridin-4-ols into the corresponding 5-iodo compounds was achieved by reaction with one equivalent of iodine or tetramethylammonium dichloroiodate under basic conditions. When three equivalents of iodine were used in chlorinated solvents, after 5-iodination, subsequent reaction of the 3-alkoxy group took place to form a 1,3-dioxolane ring with the 4-hydroxyl group. Generation of the resulting iodo[1,3]dioxolo[4,5-c]pyridines is explained by a radical process known as the Hofmann–Löffler–Freytag reaction. Two 6-ethynylpyridine derivatives were examined in the iodination process to establish a route to pyridine-containing macrocycles. The pentasubstituted 5-iodopyridine derivative 21 could be prepared; however, attempts to achieve cyclotrimerization of this building block under different conditions were not successful. Reaction of 21 with copper chloride allowed isolation of a copper acetylide 22, which aggregates to a triangular trimeric complex containing four copper(I) ions such as [23·Cu]+ as monitored by ESI mass spectrometry.

Synthesis of Novel Carbohydrate Mimetics via 1,2-Oxazines

Léa Bouché, H.-U. Reissig

Pure Appl. Chem. 2012, 84, 23-36 ...

The combination of lithiated alkoxyallenes with carbohydrate-derived nitrones constitutes a flexible entry to highly functionalized enantiopure 1,2-oxazine derivatives. They can be used as precursors for acyclic and cyclic carbohydrate-like products such as amino sugar alcohols, azetidine and pyrrolidine derivatives. The Lewis acid-promoted rearrangement of 1,3-dioxolanyl-substituted 1,2-oxazines to bicyclic compounds allows an efficient route to novel amino pyran and oxepane derivatives. After subsequent transformations, new carbohydrate mimetics or “real” carbohydrates were obtained in good yield and often in a stereodivergent fashion. These compounds have already been employed for the preparation of unusual di- and trisaccharide derivatives. Several of the products prepared showed interesting biological activities, e.g., as L- and P-selectin inhibitors with IC50 values in the subnanomolar range.

Substituent Effects on Axle Binding in Amide Pseudorotaxanes: Comparison of NMR Titration and ITC Data with DFT Calculations

L. Kaufmann, E.V. Dzyuba, F. Malberg, N.L. Löw, M. Groschke, B. Brusilowskij, J. Huuskonen, K. Rissanen, B. Kirchner, C.A. Schalley

Org. Biomol. Chem. 2012, 10, 5954-5964


The binding behaviour of differently substituted diamide axle molecules to Hunter/Vögtle tetralactam macrocycles was studied with a combination of NMR titration, isothermal titration calorimetry (ITC) experiments and calculations employing density functional theory (DFT), along with dispersion-corrected exchange-correlation functionals. Guests with alkyl or alkenyl chains attached to the diamide carbonyl groups have a significantly higher binding affinity to the macrocycle than guests with benzoyl amides and their substituted analogues. While the binding of the benzoyl and alkenyl substituted axles is enthalpically driven, the alkyl-substituted guest binds mainly because of a positive binding entropy. The electronic effects of para-substituents at the benzoyl moieties have an influence on the binding affinities. Electron donating substituents increase, while electron-withdrawing substituents decrease the binding energies. The binding affinities obtained from both NMR titration and ITC experiments correlate well with each other. The substituent effects observed in the experimental data are reflected in adiabatic interaction energies calculated with density functional methods. The calculated structures also agree well with pseudorotaxane crystal structures.

Synthesis of Multivalent Host and Guest Molecules for the Construction of Multithreaded Diamide Pseudorotaxanes.

N.L. Löw, E.V. Dzyuba, B. Brusilowskij, L. Kaufmann, E. Franzmann, W. Maison, E. Brandt, D. Aicher, A. Wiehe, C.A. Schalley

Beilstein J. Org. Chem. 2012, 8, 234–245


A series of di-, tri- and tetravalent axles and wheels for the synthesis of pseudorotaxanes bearing the tetralactam macrocycle/diamide axle binding motif was prepared. Starting from iodinated monovalent precursors, Sonogashira cross-coupling reactions were utilized to couple the binding sites to appropriate spacer groups. Through this “Lego” or “toolbox” approach, the convergent synthesis of host and guests with a well-defined number of the binding sites is possible. In addition, the spatial arrangement of the binding sites can be controlled through the quite rigid connections between linker and binding sites. Although a quantitative assessment of binding strengths was not possible by NMR titration experiments, typical and significant shifts of the signals of the diamide moiety indicate qualitatively the formation of pseudorotaxanes from the axle and wheel precursors.

Cooperative Switching Events in Azobenzene Foldamer Denaturation.

Z. Yu, S. Hecht

Chem. Eur. J., 2012, 18, 10519–10524 ...

Insight into the mechanism of photoswitchable foldamer denaturation was gained by incorporating two azobenzene switching units (see figure; orange) into the helix backbone. Optimizing the location of the units leads to cooperative photoisomerization events, causing complete unfolding. The switching cooperativity coupled with the cooperative unfolding transition enables the design of highly photosensitive systems.

Synthesis of a New Class of Bis(thiourea)hydrazide Pseudopeptides as Potential Inhibitors of β–Sheet Aggregation

J. J. Klein, S. Hecht

Org. Lett., 2012, 14(1), 330–333 ...

The modular synthesis of a novel pseudopeptide scaffold based on a bis(thiourea)hydrazide motif is reported. This compound class is designed to display “amphifinity”, i.e. association with a peptide strand on one but not the other face of the scaffold, and hence could potentially inhibit β-sheet aggregation.

Quantifying the rebinding effect in multivalent chemical ligand-receptor systems

M. Weber, A. Bujotzek, R. Haag

J. Chem. Phys., 2012, 137(5), 054111, ...

Multivalent ligand-receptor systems often show an enhancement in binding compared to the constituent monovalent systems. This “cooperativity effect” is often attributed to the favorable spatial preorganisation of the ligands by the connecting spacer that leads to a reduction of entropy loss at ligand binding. A different factor that has been proposed to contribute to the cooperativity effect is “rebinding”: As soon as a single ligand-receptor complex dissociates, the presence of another ligand “on coat-tails” will increase the probability of another binding event, which in turn will drive the system to a state where all ligands are bound. In this article, we derive a first quantitative description of the rebinding effect. In order to model the inherent memory effect of a spacer-connected system, we pursue a mathematical approach based on Markov state models and conformation dynamics. The theoretical investigations are illustrated by studying different prototypic ligand-receptor systems.

Chelate Cooperativity and Spacer Length Effects on the Assembly Thermodynamics and Kinetics of Divalent Pseudorotaxanes.

W. Jiang, K. Nowosinski, N.L. Löw, E.V. Dzyuba, F. Klautzsch, A. Schäfer, J. Huuskonen, K. Rissanen, C.A. Schalley

J. Am. Chem. Soc. 2012, 134, 1860-1868


Homo- and heterodivalent crown-ammonium pseudorotaxanes with different spacers connecting the two axle ammonium binding sites have been synthesized and characterized by NMR spectroscopy and ESI mass spectrometry. The homodivalent pseudorotaxanes are investigated with respect to the thermodynamics of divalent binding and to chelate cooperativity. The shortest spacer exhibits a chelate cooperativity much stronger than that of the longer spacers. On the basis of crystal structure, this can be explained by a noninnocent spacer, which contributes to the binding strength in addition to the two binding sites. Already very subtle changes in the spacer length, i.e., the introduction of an additional methylene group, cause substantial changes in the magnitude of cooperative binding as expressed in the large differences in effective molarity. With a similar series of heterodivalent pseudorotaxanes, the spacer effects on the barrier for the intramolecular threading step has been examined with the result that the shortest spacer causes a strained transition structure and thus the second binding event occurs slower than that of the longer spacers. The activation enthalpies and entropies show clear trends. While the longer spacers reduce the enthalpic strain that is present in the transition state for the shortest member of the series, the longer spacers become entropically slightly more unfavorable because of conformational fixation of the spacer chain during the second binding event. These results clearly show the noninnocent spacers to complicate the analysis of multivalent binding. An approximate description which considers the binding sites to be connected just by a flexible chain turns out to be more a rough approximation than a good model. The second conclusion from the results presented here is that multivalency is expressed in both the thermodynamics and the kinetics in different ways. A spacer optimized for strong binding is suboptimal for fast pseudorotaxane formation.

Exploring the palladium and platinum bis(pyridyl) complex motif – do substituents affect NMR spectroscopic, X-ray crystallographic, (tandem) mass spectrometric, and isothermal titration calorimetry data following chemical intuition?

T. Weilandt, N.L. Löw, G. Schnakenburg, J. Daniels, M. Nieger, C.A. Schalley, A. Lützen

Chem. Eur. J., 2012, 18, 16665-16676


A series of ten palladium[BOND]bis(pyridine) complexes, as well as their corresponding platinum complexes, have been synthesized. The pyridine ligands in each series carried different σ-donor and/or π-acceptor/donor substituents at the para-position of their pyridine rings. These complexes were analysed by NMR spectroscopy, X-ray crystallography, (tandem) MS, and isothermal titration calorimetry (ITC) to validate whether these methods allowed us to obtain a concise and systematic picture of the relative and absolute thermodynamic stabilities of the complexes, as determined by the electronic effects of the substituents. Interestingly, the NMR spectroscopic data hardly correlated with the expected substituent effects but the heteronuclear platinum[BOND]phosphorus coupling constants did. Crystallographic data were found to be blurred by packing effects. Instead, tandem MS and ITC data were in line with each other and followed the expected trends.

PNA-sugar conjugates as tools for the spatial screening of carbohydrate–lectin interactions

C. Scheibe, O. Seitz

Pure Appl. Chem. 2012, 84 (1), 77-85 ...

Multivalent carbohydrate–lectin interactions are essential for a multitude of biological recognition events. Much effort has been spent in the synthesis of potent multivalent scaffolds in order to mimic or inhibit biological carbohydrate–protein interactions. However, the defined spatial presentation of carbohydrates remained a challenging task. Peptide nucleic acid (PNA)- and DNA-based double helices are useful scaffolds that enable the controlled display of carbohydrate ligands in a modular approach. The hybridization of PNA‑sugar conjugates with complementary DNA strands provides multivalent complexes with defined spatial presentation of carbohydrates, which facilitates the spatial screening of carbohydrate–lectin interactions with Ångström-scale precision

Light-Harvesting in Multichromophoric Rotaxanes

M.E. Gallina, B. Baytekin, C.A. Schalley, P. Ceroni

Chem. Eur. J. 2012, 18, 1528-1535


Two rotaxanes with benzyl ether axles and tetralactam wheels were synthesized through an anion template effect. They carry naphthalene chromophores attached to the stopper groups and a pyrene chromophore attached to the wheel. The difference between the two rotaxanes is represented by the connecting unit of the naphthyl chromophore to the rotaxane axle: a triazole or an alkynyl group. Both rotaxanes exhibit excellent light-harvesting properties: excitation of the naphthalene chromophores is followed by energy transfer to the pyrene unit with efficiency higher than 90 % in both cases. This represents an example of light-harvesting function among chromophores belonging to mechanically interlocked components, that is, the axle and the wheel of the rotaxanes.

Phenanthroline- and Terpyridine-Substituted Tetralactam Macrocycles: A Facile Route to Rigid Di- and Trivalent Receptors and Interlocked Moleculesy

E.V. Dzyuba, B. Baytekin, D. Sattler, C.A. Schalley

Eur. J. Org. Chem. 2012, 6, 1171-1178


Bromo-substituted Hunter/Vögtle-type tetralactam macrocycles (TLMs) represent key intermediates for the attachment of terpyridyl and phenanthroline metal binding sites through cross-coupling reactions. From these monovalent precursors, metal complexes can easily be obtained that present the macrocycles in a multivalent fashion. Depending on the nature of the metal ion, the properties of the complexes can be tuned with respect to valency (e.g., phen-TLM + CuI: divalent, phen-TLM + FeII: trivalent) and lability against TLM ligand exchange (e.g., CuI: slow, but reversible exchange, RuIICl2: kinetically inert).

On the Relationship between Peptide Adsorption Resistance and Surface Contact Angle: A Combined Experimental and Simulation Single-Molecule Study

N. Schwierz, D. Horinek, S. Liese, T. Pirzer, B.N. Balzer, T. Hugel, R. R. Netz

J. Am. Chem. Soc., 2012, 134, 19628 - 19638 ...

The force-induced desorption of single peptide chains from mixed OH/CH3-terminated self-assembled monolayers is studied in closely matched molecular dynamics simulations and atomic force microscopy experiments with the goal to gain microscopic understanding of the transition between peptide adsorption and adsorption resistance as the surface contact angle is varied. In both simulations and experiments, the surfaces become adsorption resistant against hydrophilic as well as hydrophobic peptides when their contact angle decreases below θ ≈ 50°–60°, thus confirming the so-called Berg limit established in the context of protein and cell adsorption. Entropy/enthalpy decomposition of the simulation results reveals that the key discriminator between the adsorption of different residues on a hydrophobic monolayer is of entropic nature and thus is suggested to be linked to the hydrophobic effect. By pushing a polyalanine peptide onto a polar surface, simulations reveal that the peptide adsorption resistance is caused by the strongly bound water hydration layer and characterized by the simultaneous gain of both total entropy in the system and total number of hydrogen bonds between water, peptide, and surface. This mechanistic insight into peptide adsorption resistance might help to refine design principles for anti-fouling surfaces.

Hydration repulsion between biomembranes results from an interplay of dehydration and depolarization

Emanuel Schneck, Felix Sedlmeier, and Roland R. Netz

Proc. Natl. Acad. Sci. USA, 2012, 109, 14405 - 14409 ...

Hydration repulsion dominates the interaction between polar surfaces in water at nanometer separations and ultimately prevents the sticking together of biological matter. Although confirmed by a multitude of experimental methods for various systems, its mechanism remained unclear. A simulation technique is introduced that yields accurate pressures between solvated surfaces at prescribed water chemical potential and is applied to a stack of phospholipid bilayers. Experimental pressure data are quantitatively reproduced and the simulations unveil a rich microscopic picture: Direct membrane–membrane interactions are attractive but overwhelmed by repulsive indirect water contributions. Below about 17 water molecules per lipid, this indirect repulsion is of an energetic nature and due to desorption of hydration water; for larger hydration it is entropic and suggested to involve water depolarization. This antagonistic nature and the presence of various compensating contributions indicate that the hydration repulsion is less universal than previously assumed and rather involves finely tuned surface-water interactions.

Influenza virus binds its host cell using multiple dynamic interactions

C. Sieben, C. Kappel, R. Zhu, A. Wozniak, C. Rankl, P. Hinterdorfer, H. Grubmüller, A. Herrmann

Proc Natl Acad Sci U S A. 2012, 109(34), 13626-13631

Influenza virus belongs to a wide range of enveloped viruses. The major spike protein hemagglutinin binds sialic acid residues of glycoproteins and glycolipids with dissociation constants in the millimolar range [Sauter NK, et al. (1992) Biochemistry 31:9609–9621], indicating a multivalent binding mode. Here, we characterized the attachment of influenza virus to host cell receptors using three independent approaches. Optical tweezers and atomic force microscopy-based single-molecule force spectroscopy revealed very low interaction forces. Further, the observation of sequential unbinding events strongly suggests a multivalent binding mode between virus and cell membrane. Molecular dynamics simulations reveal a variety of unbinding pathways that indicate a highly dynamic interaction between HA and its receptor, allowing rationalization of influenza virus–cell binding quantitatively at the molecular level.

Chemoselective Staudinger-phosphite reaction of symmetrical glycosyl-phosphites with azido-peptides and polygycerols

V. Böhrsch, T. Mathew, M. Zieringer, M.R. Vallée, L.M. Artner, J. Dernedde, R. Haag, C.P. Hackenberger

Org. Biomol. Chem. 2012, 10, 6211-6216 ...

In this paper we present the synthesis of glyco-phosphoramidate conjugates as easily accessible analogs of glyco-phosphorous esters via the Staudinger-phosphite reaction. This protocol takes advantage of synthetically accessible symmetrical carbohydrate phosphites in good overall yields, in which ethylene or propylene linkers can be introduced between phosphorous and galactose or lactose moieties. The phosphites were finally applied for the chemoselective reaction with azido-peptides and polyazido(poly)glycerols.

Remote control of lipophilic nucleic acids domain partitioning by DNA hybridization and enzymatic cleavage

M. Schade, A. Knoll, A. Vogel, O. Seitz, J. Liebscher, D. Huster, A. Herrmann, A. Arbuzova

J. Am. Chem. Soc., 2012, 134 (50), 20490–20497 ...

Lateral partitioning of lipid-modified molecules between liquid-disordered (ld) and liquid-ordered (lo) domains depends on the type of lipid modification, presence of a spacer, membrane composition, and temperature. Here, we show that the lo domain partitioning of the palmitoylated peptide nucleic acid (PNA) can be influenced by formation of a four-component complex with the ld domain partitioning tocopherol-modified DNA: the PNA–DNA complex partitioned into the ld domains. Enzymatic cleavage of the DNA linker led to the disruption of the complex and restored the initial distribution of the lipophilic nucleic acids into the respective domains. This modular system offers strategies for dynamic functionalization of biomimetic surfaces, for example, in nanostructuring and regulation of enzyme catalysis, and it provides a tool to study the molecular basis of controlled reorganization of lipid-modified proteins in membranes, for example, during signal transduction.

Sequence-defined glycopolymer segments presenting mannose: synthesis and lectin binding affinity

D. Ponader, F. Wojcik, F. Beceren-Braun, J. Dernedde, L. Hartmann

Biomacromolecules 2012, 13, 1845-52 ...

We present for the first time the synthesis of sequence-defined monodisperse glycopolymer segments via solid-phase polymer synthesis. Functional building blocks displaying alkyne moieties and hydrophilic ethylenedioxy units were assembled stepwise on solid phase. The resulting polymer segments were conjugated with mannose sugars via 1,3-dipolar cycloaddition. The obtained mono-, di-, and trivalent mannose structures were then subject to Con A lectin binding. Surface plasmon resonance studies showed a nonlinear increase in binding regarding the number and spacing of sugar ligands. The results of Con A lectin binding assays indicate that the chemical composition of the polymeric scaffold strongly contributes to the binding activities as well as the spacing between the ligands and the number of presented mannose units. Our approach now allows for the synthesis of highly defined glycooligomers and glycopolymers with a diversity of properties to investigate systematically multivalent effects of polymeric ligands.

Probes for the dual color imaging of two viral mRNA targets in Influenza H1N1 infected live cells

S. Kummer, A. Knoll, E. Socher, L. Bethge, A. Herrmann, O. Seitz

Bioconjugate Chem. 2012, 23, 2051−2060 ...

Fluorogenic hybridization probes that allow RNA imaging provide information as to how the synthesis and transport of particular RNA molecules is orchestrated in living cells. In this study, we explored the peptide nucleic acid (PNA)-based FIT-probes in the simultaneous imaging of two different viral mRNA molecules expressed during the replication cycle of the H1N1 influenza A virus. PNA FIT-probes are non-nucleotidic, nonstructured probes and contain a single asymmetric cyanine dye which serves as a fluorescent base surrogate. The fluorochrome acts as a local intercalator probe and reports hybridization of target DNA/RNA by enhancement of fluorescence. Though multiplexed hybridization probes are expected to facilitate the analysis of RNA expression, there are no previous reports on the dual color imaging of two different viral mRNA targets. In this work, we developed a set of two differently colored PNA FIT-probes that allow the spectrally resolved imaging of mRNA coding for neuraminidase (NA) and matrix protein 1 (M1); proteins which execute distinct functions during the replication of the influenza A virus. The probes are characterized by a wide range of applicable hybridization temperatures. The same probe sequence enabled live-cell RNA imaging (at 37 °C) as well as real-time PCR measurements (at 60 °C annealing temperature). This facilitated a comprehensive analysis of RNA expression by quantitative (qPCR) and qualitative (imaging) means. Confocal laser scanning microscopy showed that the viral-RNA specific PNA FIT-probes neither stained noninfected cells nor cells infected by a control virus. The joint use of differently colored PNA FIT-probes in this feasibility study revealed significant differences in the expression pattern of influenza H1N1 mRNAs coding for NA or M1. These experiments provide evidence for the usefulness of PNA FIT-probes in investigations on the temporal and spatial progression of mRNA synthesis in living cells for two mRNA species.

Graphene on Rh(111): Scanning tunneling and atomic force microscopies studies

E. N. Voloshina, Yu. S. Dedkov, S. Torbrügge, A. Thissen, and M. Fonin

Appl. Phys. Lett., 2012 ,100, 241606 ...

The electronic and crystallographic structure of the graphene/Rh(111) moiré lattice is studied via combination of density-functional theory calculations and scanning tunneling and atomic force microscopy(STM and AFM). Whereas the principal contrast between hills and valleys observed in STM does not depend on the sign of applied bias voltage, the contrast in atomically resolved AFM images strongly depends on the frequency shift of the oscillating AFM tip. The obtained results demonstrate the perspectives of application atomic force microscopy/spectroscopy for the probing of the chemical contrast at the surface.

Graphene on metallic surfaces: problems and perspectives

E. Voloshina and Yu. S. Dedkov

Phys. Chem. Chem. Phys., 2012, 14, 13502 ...

The present manuscript summarizes the modern view on the problem of the graphene–metal interaction. Presently, the close-packed surfaces of d metals are used as templates for the preparation of highly-ordered graphene layers. Different classifications can be introduced for these systems: graphene on lattice-matched and graphene on lattice-mismatched surfaces where the interaction with the metallic substrate can be either “strong” or “weak”. Here these classifications, with the focus on the specific features in the electronic structure in all cases, are considered on the basis of large amount of experimental and theoretical data, summarized and discussed. The perspectives of the graphene–metal interfaces in fundamental and applied physics and chemistry are pointed out.

Multivalent Design of Apoptosis-Inducing Bid-BH3 Peptide–Oligosaccharides Boosts the Intracellular Activity at Identical Overall Peptide Concentrations

M. Richter, A. Chakrabarti, I. R. Ruttekolk, B. Wiesner, M. Beyermann, R. Brock, J. Rademann

Chem. Eur. J. 2012, 18, 16708-16715


Multivalent peptide–oligosaccharide conjugates were prepared and used to investigate the multivalency effect concerning the activity of Bid-BH3 peptides in live cells. Dextran oligosaccharides were carboxyethylated selectively in the 2-position of the carbohydrate units and activated for the ligation of N-terminally cysteinylated peptides. Ligation through maleimide coupling was found to be superior to the native chemical ligation protocol. Monomeric Bid-BH3 peptides were virtually inactive, whereas pentameric peptide conjugates induced apoptosis up to 20-fold stronger at identical peptide concentrations. Comparison of lowly multivalent and highly multivalent peptide dextrans proved a multivalency effect in life cells which was specific for the BH3 peptide sequence.

Benzoylphosphonat-basierte, photoaktive Phosphotyrosinpeptidmimetika zur funktionellen Modulierung von Proteintyrosinphosphatasen und hochspezifischen Markierung von SH2-Domänen

A. Horatscheck, S. Wagner, J. Ortwein, M. Lisurek, B. G. Kim, S. Beligny, A. Schütz, J. Rademann

Angew. Chem. 2012, 124, 9577-9583


Ein Lichtschalter für Phosphotyrosin erkennende Domänen: Die Bestrahlung des bioisosteren Benzoylphosphonats genügt, um die biologische Aktivität von Zielproteinen „auszuknipsen“ oder gezielt eine kovalente Markierung anzubringen (siehe Schema). Mögliche Anwendungen für photoaktive Bioisostere finden sich in der funktionellen Zellbiologie, der Bioanalytik und der Proteomforschung.

Site-selective modification of proteins for the synthesis of structurally defined multivalent scaffolds

L.M. Artner, L. Merkel, N. Bohlke, F. Beceren-Braun, C. Weise, J. Dernedde, N. Budisa, C.P.R. Hackenberger

Chem. Commun. 2012, 48, 522 - 524 ...

A combination of classical site-directed mutagenesis, genetic code engineering and bioorthogonal reactions delivered a chemically modified barstar protein with one or four carbohydrates installed at specific residues. These protein conjugates were employed in multivalent binding studies, which support the use of proteins as structurally defined scaffolds for the presentation of multivalent ligands.

The Alzheimer’s disease-related tau protein as a new target for chemical protein engineering

M. Broncel, E. Krause, D. Schwarzer, C. P. R. Hackenberger

Chem. Eur. J. 2012, 18, 2488 - 2492 ...

Site-specifically phosphorylated tau: A semisynthetic strategy has been applied for the first time to generate a phosphorylated and biotinylated fully functional tau protein (see figure). The presented methodology allows for an unambiguous verification of individual phosphorylation sites on tau and significantly improves its purification.

Glycan-spezifisches metabolisches Oligosaccharid-Engineering von C7-substituierten Sialinsäuren

H. Möller, V. Böhrsch, J. Bentrop, J. Bender, S. Hinderlich, C. P. R. Hackenberger

Angew. Chem. 2012, 124, 6088 - 6092 ...

Eine neue Sialinsäure in der Familie! Ein biosynthetischer Ansatz eröffnet den Weg zu früher nicht zugänglichen C7-Modifikationen von Sialinsäuren in lebenden Zellen. Dies wurde durch metabolischen Einbau von synthetischem N-Acetyl-4-azido-4-desoxymannosamin in Glykane von Säugerzellen und Zebrafischlarven unter Erhaltung der bioorthogonalen Funktionalität der Azidogruppe für die Markierung mit biophysikalischen Sonden verwirklicht.

Glycan-Specific Metabolic Oligosaccharide Engineering of C7-Substituted Sialic Acids

H. Möller, V. Böhrsch, J. Bentrop, J. Bender, S. Hinderlich, C. P. R. Hackenberger

Angew. Chem. Int. Ed. 2012, 51, 5986 - 5990 ...

A new sialic acid in the house! A biosynthetic approach gives access to previously inaccessible C7 modifications of sialic acids in living cells. Metabolic incorporation of synthetically derived N-acetyl-4-azido-4-deoxymannosamine (see scheme) into glycans of mammalian cells and zebrafish larvae preserves the bioorthogonal functionality of its azido group for subsequent labeling with biophysical probes.

Local correlation method for metals: Benchmarks for surface and adsorption energies

E. Voloshina

Phys. Rev. B, 2012 83, 045444 ...

Highly accurate methods such as coupled cluster (CC) techniques can be used for periodic systems within the framework of the method of increments. Its extension to a low-dimensional conducting system is considered. To demonstrate the presented approach, a clean Mg(0001) surface is selected, where the CC treatment with single and double excitations and perturbative triples is used for calculation of the surface energy. A further example concerns the adsorption energy of Xe on the metal surface. The obtained results can be used to verify the performance of the approximate methods. Along with the computational speed-up at the high level of accuracy, application of the method of increments provides for a possibility to analyze the influence of individual correlation energy increments on the studied property.

Benzoylphosphonate-based photoactive phosphopeptide mimetics for functional modulation of protein tyrosine phosphatases and highly specific covalent labeling of SH2-domains

A. Horatscheck, S. Wagner, J. Ortwein, M. Lisurek, B. G. Kim, S. Beligny, A. Schütz, J. Rademann

Angew. Chem. Int. Ed. 2012, 51, 9441-9447


A light switch for phosphotyrosine- recognizing proteins: Irradiation of the bioisosteric benzoylphosphonate suffices to “turn off” the activity of target proteins and to label them covalently (see scheme). Photoactive bioisosters may find applications in functional cell biology, bioanalytics, and proteome research.

Fmoc-Based Synthesis of Peptide Thioacids for Azide Ligations via 2-Cyanoethyl Thioesters

R. Raz, J. Rademann

Org. Lett. 2012, 14, 5038–5041


Rapid and efficient preparation of peptide thioacids from 2-cyanoethyl peptide thioesters has been accomplished. S-2-Cyanoethyl peptide thioesters were obtained cleanly without the need for purification from resin-bound tert-butyl peptide thioesters using 3-mercaptopropionitrile as a nucleophile. Elimination of the 2-cyanoethyl group proceeded rapidly (t1/2 < 8 min) under mild conditions and furnished peptide thioacids up to the size of a 16-mer. Peptide thioacids could be isolated or formed in situ and reacted smoothly with electron-deficient azides yielding an amide as the ligation product.

Soluble peptidyl-phosphoranes for metal-free, stereoselective ligations in organic and aqueous solution

Ahsanullah, S. I. Al-Gharabli, J. Rademann

Org. Lett, 2012, 14, 14-17


Protocols for solid-phase syntheses of soluble peptidyl phosphoranes are presented. Various supported phosphoranylidene acetates were prepared on Rink amide or via alkylation of trialkyl- and triarylphosphines with bromoacetyl Wang ester. C-Acylation was conducted racemization-free with activated Fmoc-amino acids, followed by SPPS (solid-phase peptide synthesis). Acidic conditions released decarboxylated peptidyl phosphoranes into solution. The protocol allowed for the electronic variation of peptidyl phosphoranes which were investigated in ligation reactions with azides in organic and aqueous solvents.

DNA as Molecular Ruler: Interrogation of a Tandem SH2 Domain with Self-Assembled, Bivalent DNA–Peptide Complexes

H. Eberhard, F. Diezmann, O. Seitz

Angew. Chem. Int. Ed. 2011, 50, 1-6. ...

Two peptides on display: The self-assembly of DNA complexes enables the bivalent presentation of phosphopeptides. Flexibility and distance in the ligand arrangement can be adjusted through the choice of appropriate DNA templates. Spatial screening of the tandem SH2 domain of Syk kinase with these probes indicated the accessible arrangements of the two homologous binding pockets and the flexibility of the connecting protein linker.

Theoretical study on the adsorption of pyridine derivatives on graphene

E. Voloshina, D. Mollenhauer, L. Chiappisi, B. Paulus

Chem. Phys. Lett. 2011, 510 (4-6), 220-223 ...

The adsorption of pyridine and its derivatives on the graphene surface has been studied using density functional theory (DFT). Adsorption geometries and energies as well as nature of binding have been analyzed. Dispersion effects have been taken into account via a semiempirical DFT-D2 method. Influence of electron-donor and electron-acceptor substituents in 4-position of the heterocyclic ring, effect of substrate and adsorbate’s concentration on the interaction energy have been investigated. Impact of the pyridine adsorption on the electronic band structure of graphene has been studied.

[4]Pseudorotaxanes with Remarkable Self-Sorting Selectivities

W. Jiang, D. Sattler, K. Rissanen, C.A. Schalley

Org. Lett. 2011, 13, 4502-4505


The synthesis and characterization of several self-assembled [4]pseudorotaxanes is reported, some of which form in a programmed way based on two similar yet orthogonal crown ether/secondary ammonium ion binding motifs. A preference for the formation of a [4]pseudorotaxane with an antiparallel rather than parallel alignment of crown ether building blocks is observed even in the absence of such orthogonal binding sites, when a homodivalent axle is used.

Adsorption Behavior of 4-Methoxypyridine on Gold Nanoparticles

H. Lange, J. Maultzsch, W. Meng, D. Mollenhauer, B. Paulus, N. Peica, S. Schlecht, C. Thomsen

Langmuir 2011, 27, 7258 ...

We demonstrate a phase transfer method to create stable colloidal solutions of Au nanoparticles with 4-methoxypyridine ligands. We then investigate the adsorption behavior of 4-methoxypyridine onto gold surfaces by Raman spectroscopy, DFT calculations, and 1H NMR. In contrast to unsubstituted pyridine and the frequently used (N,N-dimethylamino)pyridine (DMAP), a flat adsorption of 4-methoxypyridine on gold was found.

Role of the clathrin terminal domain in regulating coated pit dynamics revealed by small molecule inhibition

von Kleist, L., Stahlschmidt, W., Bulut, H., Gromova, K., Puchkov, D., Robertson, M., MacGregor, K.A., Tomlin, N., Pechstein, A., Chau, N., Chircop, M., Sakoff, J., von Kries, J., Saenger, W., Kräusslich, H.-G., Shupliakov, O., Robinson, P., McCluskey, A., Haucke, V.

Cell, 2011, 146, 471-484 ...

Clathrin-mediated endocytosis (CME) regulates many cell physiological processes such as the internalization of growth factors and receptors, entry of pathogens, and synaptic transmission. Within the endocytic network, clathrin functions as a central organizing platform for coated pit assembly and dissociation via its terminal domain (TD). We report the design and synthesis of two compounds named pitstops that selectively block endocytic ligand association with the clathrin TD as confirmed by X-ray crystallography. Pitstop-induced inhibition of clathrin TD function acutely interferes with receptor-mediated endocytosis, entry of HIV, and synaptic vesicle recycling. Endocytosis inhibition is caused by a dramatic increase in the lifetimes of clathrin coat components, including FCHo, clathrin, and dynamin, suggesting that the clathrin TD regulates coated pit dynamics. Pitstops provide new tools to address clathrin function in cell physiology with potential applications as inhibitors of virus and pathogen entry and as modulators of cell signaling.

Coupling to polymeric scaffolds stabilizes biofunctional peptides for intracellular applications

I. Ruttekolk, A. Chakrabarti, M. Richter, F. Duchardt, H. Glauner, W.P.R. Verdurmen, J. Rademann, R. Brock

Mol. Pharmac. 2011, 79(4), 692-700 ...

Here, we demonstrate that coupling to N-hydroxypropyl methacrylamide (HPMA) copolymer greatly enhances the activity of apoptosis-inducing peptides inside cells. Peptides corresponding to the BH3 domain of Bid were coupled to a thioester-activated HPMA (28.5 kDa) via native chemical ligation in a simple one-pot synthesis. Peptides and polymer conjugates were introduced into cells either by electroporation or by conjugation to the cell-penetrating peptide nona-arginine. The molecular basis of the increased activity is elucidated in detail. Loading efficiency and intracellular residence time were assessed by confocal microscopy. Fluorescence correlation spectroscopy was used as a separation-free analytical technique to determine proteolytic degradation in crude cell lysates. HPMA conjugation strongly increased the half-life of the peptides in crude cell lysates and inside cells, revealing proteolytic protection as the basis for higher activity.

Graphene on ferromagnetic surfaces and its functionalization with water and ammonia.

S. Böttcher, M. Weser, Yu. S. Dedkov, K. Horn, E.N. Voloshina, B. Paulus

Nanoscale Research Letters 2011, 6:214 ...

Nanoscale Res. Lett.

In this article, an angle-resolved photoelectron spectroscopy (ARPES), X-ray absorption spectroscopy (XAS), and density-functional theory (DFT) investigations of water and ammonia adsorption on graphene/Ni(111) are presented. The results of adsorption on graphene/Ni(111) obtained in this study reveal the existence of interface states, originating from the strong hybridization of the graphene π and spin-polarized Ni 3d valence band states. ARPES and XAS data of the H2O (NH3)/graphene/Ni(111) system give an information regarding the kind of interaction between the adsorbed molecules and the graphene on Ni(111). The presented experimental data are compared with the results obtained in the framework of the DFT approach.

Multivalent presentation of mannose on hyperbranched polyglycerol and their interaction with concanavalin A lectin

I. Papp, J. Dernedde, S. Enders, S.B. Riese, T.C. Shiao, R. Roy, R. Haag

ChemBioChem, 2011, 12, 7,1075-1083

" ...

We describe the synthesis of multivalent mannose derivatives by using hyperbranched polyglycerols (hPG) as a scaffold with different linker structures. Grafting of protected mannose (Man) units is achieved by using CuI-catalyzed Huisgen click chemistry with either an anomeric azide or propargyl ether onto complementarily functionalized alkyne or azido polymer surfaces. NMR spectroscopy, dynamic light scattering (DLS), IR spectroscopy, size-exclusion chromatography (SEC), and elemental analysis have been used to characterize the hPG–Man compounds. The surface availability and bioactivity of Man-modified polymers were evaluated by using a competitive surface plasmon resonance (SPR)-based binding assay by interactions of the glycopolymers with concanavalin A (Con A), a lectin that binds mannose containing molecules. The results indicated that the novel glycoarchitectures presented in this work are efficient inhibitors of Con A–mannose recognition and resulted in inhibitor concentrations (mean IC50) from the micro- to the nanomolar range, whereas the corresponding monovalent mannoside (methyl-Man) requires millimolar concentrations. The results provide an interesting structure–activity relationship for libraries of materials that differ in the linkage of the sugar moiety presented on a biocompatible polyglycerol scaffold.

Synthesis of dendritic polyglycerol anions and their efficiency toward L-selectin inhibition

M. Weinhart, D. Gröger, S. Enders, J. Dernedde, R. Haag

Biomacromolecules 2011, 12, 2502-2511 ...

A versatile route for the synthesis of highly functionalized, polyanionic macromolecules based on dendritic polyglycerol was applied by means of the Huisgen–Sharpless–Meldal 1,3-dipolar cycloaddition (“click-reaction”) of polyglycerolazide precursors and alkyne-functionalized anions such as sulfonates, carboxylates, phosphonates, and bisphosphonates. In addition, the corresponding polyglycerol phosphate has been synthesized via direct hydroxyl interconversion of polyglycerol to the corresponding phosphate with a degree of functionalization >80% by analogy to the synthesis of previously reported polyglycerol sulfates (dPGS). On the basis of the finding that dPGS exhibits high affinity for L- and P-selectin, the potential of these novel polyanionic, multivalent macromolecules of varying anionic nature as L-selectin inhibitors has been evaluated in vitro by means of a competitive concentration dependent binding assay. Affinity of all polyanions toward L-selectin was demonstrated with distinct IC50 values ranging from the low nanomolar to the high micromolar range. The efficiency of L-selectin inhibition increases in the order carboxylate < phosphate < phosphonate ≈ sulfonate < bisphosphonate < sulfate. Additional DLS and ζ-potential measurements of these polyanions were performed to correlate their binding affinity toward L-selectin with their anionic nature. However, a direct correlation of effective charge and particle size with the determined IC50 values turned out to require further in-depth studies on the microstructure of the polyanions but clearly indicate an exceptional position of dPGS among the studied dendritic polyelectrolytes.

Synthesis and evaluation of nonsulfated and sulfated glycopolymers as L- and P-selectin inhibitors

J. Dernedde, I. Papp, S. Enders, S. Wedepohl, F. Paulus, R. Haag

J. Carbohydr. Chem. 2011, 30, 347–360 ...

Selectins are carbohydrate-binding proteins and responsible for leukocyte extravasation in inflammation. Here we demonstrate the potential of synthetic glycocompounds as inhibitors for the selectin-ligand interaction. Pentaerythritol derivatives showed distinct selectin-binding properties with IC50 values up to 1.5 μM. Multivalent hyperbranched polyglycerol (hPG) derivatives did not lead to a substantial increase in inhibition, but a more than 1000-fold enhancement was realized when sulfated glyco-hPGs were tested. IC50 values in the high picomolar to low nanomolar range were obtained for selectin inhibition, which highlights the relevance of sulfate groups that seem to dominate the binding mode.

Bending and puncturing the influenza lipid envelope

S. Li, F. Eghiaian, C. Sieben, A. Herrmann, I.A.T. Schaap

Biophysi. J. 2011, 100(3), 637-645 ...

Lysosomes, enveloped viruses, as well as synaptic and secretory vesicles are all examples of natural nanocontainers (diameter ≈ 100 nm) which specifically rely on their lipid bilayer to protect and exchange their contents with the cell. We have applied methods primarily based on atomic force microscopy and finite element modeling that allow precise investigation of the mechanical properties of the influenza virus lipid envelope. The mechanical properties of small, spherical vesicles made from PR8 influenza lipids were probed by an atomic force microscopy tip applying forces up to 0.2 nN, which led to an elastic deformation up to 20%, on average. The liposome deformation was modeled using finite element methods to extract the lipid bilayer elastic properties. We found that influenza liposomes were softer than what would be expected for a gel phase bilayer and highly deformable: Consistent with previous suggestion that influenza lipids do not undergo a major phase transition, we observe that the stiffness of influenza liposomes increases gradually and weakly (within one order of magnitude) with temperature. Surprisingly, influenza liposomes were, in most cases, able to withstand wall-to-wall deformation, and forces >1 nN were generally required to puncture the influenza envelope, which is similar to viral protein shells. Hence, the choice of a highly flexible lipid envelope may provide as efficient a protection for a viral genome as a stiff protein shell.

DNA-programmed spatial screening of carbohydrate–lectin interactions.

C. Scheibe, A. Bujotzek, J. Dernedde, M. Weber, O. Seitz

Chem. Sci. 2011, 2, 770-775. ...

A wide range of multivalent scaffolds was assembled by using only five different PNA oligomers and various DNA templates. The flexibility of the PNA–DNA duplexes could be increased by introducing nick-sites and partially unpaired regions, as confirmed by MD simulations. The self-organized glyco-assemblies were used in a spatial screening of accessible carbohydrate binding sites in the Erythrina cristagalli lectin (ECL). This systematic investigation revealed a distance dependence which is in agreement with the crystal structure analysis.

DNA-Triggered Synthesis and Bioactivity of Proapoptotic Peptides

A. Erben, T.N. Grossmann, O. Seitz

Angew. Chem. Int. Ed. 2011, 50, 2828 - 2832

" ...

A great leap for an amino acid is enabled by a DNA-triggered reaction which involves the transfer of an aminoacyl group from a donating thioester-linked PNA–peptide hybrid to a peptide–PNA acceptor (see scheme). The formed peptide conjugates acted as antagonists to the X-linked inhibitor of apoptosis protein (XIAP) and allowed reactivation of initiator caspase-9 as well as the executioner

CH•••O Hydrogen Bonds in “Clicked” Diketopiperazine-Based Amide Rotaxanes

E.V. Dzyuba, L. Kaufmann, N.L. Löw, A.K. Meyer, H.D.F. Winkler, K. Rissanen, C.A. Schalley

Org. Lett. 2011, 13, 4838-4841


Two amide [2]rotaxanes were synthesized in high yields using a novel N,N′-dipropargyl diketopiperazine axle centerpiece as the template to which the stoppers are attached through “click chemistry”. 1H and 2D NMR spectra provide evidence for two different H-bonding motifs, in one of which the triazole CH groups form C–H···O═C bonds with the wheel carbonyl O atoms. This motif can be controlled and switched reversibly by competitive anion binding.

DBA-guided display of proteins and protein ligands for the interrogation of biology.

F. Diezmann, O. Seitz

Chem. Soc. Rev. 2011, 40, 5789 - 5801 ...

The self-assembly of nanosized DNA templates—based on formation of duplex, triplex, quadruplex or even pentaplex structures—provides unique opportunities for the controlled presentation of appended functional units. Recently, researchers have recognized the potential of such DNA scaffolds to address questions in the life sciences. In this critical review the focus is on the exploration of proteins. It is shown how different scaffolds can be used to control localization, structure and bioactivity of proteins and protein ligands. Further examples demonstrate that DNA-based recognition can even be used to trigger the formation of protein targeted molecules. Potential and existing applications in protein detection, drug discovery, structural characterization of protein targets as well as in the design of nucleic acid responsive pharmacophores are discussed

Ascorbate as an alternative to thiol additives in native chemical ligation.

H. Rohde, J. Schmalisch, Z. Harpaz, F. Diezmann, O. Seitz

Chem.Bio.Chem. 2011, 12, 1396 - 1400 ...

Vitamin C for peptide ligation: Phosphine and thiol additives maintain reducing environments and increase the reactivities of peptide thioesters in native chemical ligation. Thiol additives such as thiophenol also act as radical scavengers that inhibit phosphine-induced desulfurization of cysteine. This role can be assumed by the odourless, nontoxic, highly water-soluble and inexpensive ascorbate, which can replace the usually added thiols.

9-Fluorenylmethoxycarbonyl-Based Solid-Phase Synthesis of Peptide alpha-Thioesters

F. Mende, O. Seitz

Angew. Chem. Int. Ed. 2011, 50, 1232-1240. ...

Peptide thioesters play a key role in convergent protein synthesis strategies such as native chemical ligation, traceless Staudinger ligation, and Ag+-mediated thioester ligation. The Boc-based solid-phase synthesis provides a very reliable access to peptide thioesters. However, the acid lability of many peptide modifications and the requirements of most parallel peptide synthesizers call for the milder Fmoc-based solid-phase synthesis. The Fmoc-based synthesis of peptide thioesters is more cumbersome and typically proceeds with lower yields than the synthesis of peptide acids and peptide amides. The success of native chemical ligation and related technologies has sparked intensive research effort devoted to the development of new methods. The recent progress in this rapidly expanding field is reviewed.

Conformational Analysis of Bivalent Estrogen Receptor Ligands: From Intramolecular to Intermolecular Binding

M. Shan, A. Bujotzek, F. Abendroth, A. Wellner, R. Gust, O. Seitz, M. Weber, R. Haag

ChemBioChem 2011, 12 (17), 2587-2598 ...

The estrogen receptor binding affinities of bivalent raloxifene ligands tethered by flexible spacers of different lengths have been evaluated in vitro. Two bivalent binding modes, intra- and intermolecular, were hypothesized to explain their different binding properties. The binding affinities of these bivalent ligands in an aqueous environment are influenced by their conformations, which can be determined by 2D NMR and UV spectral methods. Moreover, computer modeling and simulations were performed to explain the binding modes of these bivalent ligands and to estimate the conformational entropy difference between their unbound and bound states. It was found that bivalent ligands tethered by long spacers had weaker binding affinities because of the shielding of the binding moieties that results from their folded conformations; those tethered by short spacers had stronger affinities because they exposed their ligands to the receptor.

Fluorescence Imaging of Influenza H1N1 mRNA in Living Infected Cells Using Single-Chromophore FIT-PNA

S. Kummer, A. Knoll, E. Socher, L. Bethge, A. Herrmann, O. Seitz

Angew. Chem. Int. Ed. 2011, 50, 1931-1934 ...

A message from the virus was detected by a peptide nucleic acid probe that contains thiazole orange as a fluorescent base surrogate (see picture). The high specificity and biostability of the probes lead to significant improvements to the signal-to-background ratio in imaging the mRNA from the influenza H1N1 virus in living infected cells.

Membrane-Mediated Induction and Sorting of K-Ras Microdomain Signaling Platforms

K. Weise, S. Kapor, C. Denter, J. Nikolaus, N. Opitz, S. Koch, G. Triola, A. Herrmann, H. Waldmann, R. Winter

JACS, 2011, 133, 880-887 ...

The K-Ras4B GTPase is a major oncoprotein whose sig-naling activity depends on its correct localization to negatively charged subcellular membranes and nanoclustering in membrane microdomains. Selective localization and clustering are mediated by the polybasic farnesylated C-terminus of K-Ras4B, but the mechanisms and molecular determinants involved are largely unknown. In a combined chemical biological and biophysical approach we investigated the partitioning of semisynthetic fully functional lipidated K-Ras4B proteins into heterogeneous anionic model membranes and membranes composed of viral lipid extracts. Independent of GDP/GTP-loading, K-Ras4B is preferentially localized in liquid-disordered (ld) lipid domains and forms new protein-containing fluid domains that are recruiting multivalent acidic lipids by an effective, electrostatic lipid sorting mechanism. In addition, GDP-GTP exchange and, thereby, Ras activation results in a higher concentration of activated K-Ras4B in the nanoscale signaling platforms. Conversely, palmitoylated and farnesylated N-Ras proteins partition into the ld phase and concentrate at the ld/lo phase boundary of heterogeneous membranes. Next to the lipid anchor system, the results reveal an involvement of the G-domain in the membrane interaction process by determining minor but yet significant structural reorientations of the GDP/GTP-K-Ras4B proteins at lipid interfaces. A molecular mechanism for isoform-specific Ras signaling from separate membrane microdomains is postulated from the results of this study.

A Chiral Pool Strategy for the Synthesis of Enantiopure Hydroxymethyl-Substituted Pyridine Derivatives.

C. Eidamshaus, H.-U. Reissig

Eur. J. Chem. 2011, 1162-1166.


A simple procedure for the synthesis of enantiopure hydroxymethyl-substituted pyridine derivatives is presented. The developed method is based on TMSOTf-promoted cyclocondensations of β-ketoenamides, leading to differently substituted 4-hydroxypyridine/4-pyridone derivatives. The required β-ketoenamides were prepared by acylation ofeasily available enamino ketones with suitably protected enantiopure carboxylic chlorides. Most of the experiments were performed with D-mandelic acid as starting material. It has been shown that all steps occur essentially without racemisation. Several of the prepared 4-pyridone derivatives were transformed into the corresponding pyrid-4-yl nonaflates and subjected to a series of palladium-catalysed transformations, such as Suzuki, Heck or Sonogashira reactions. In addition, regioselective side-chain functionalisation of unsymmetrically 2,6-disubstituted pyridine derivatives was accomplished by application of Boekelheide rearrangements of the corresponding pyridine N-oxides. The presented methods allow a flexible, rapid and scalable approach to highly substituted, enantiopure pyridine derivatives.

A Practical Two-Step Procedure for the Preparation of Enantiopure Pyridines: Multicomponent Reactions of Alkoxyallenes, Nitriles and Carboxylic Acids followed by a Cyclocondensation Reaction.

C. Eidamshaus, R. Kumar, M.K. Bera, H.-U. Reissig

Beilstein J. Org. Chem. 2011, 7, 962-975.


A practical approach to highly functionalized 4-hydroxypyridine derivatives with stereogenic side chains in the 2- and 6-positions is described. The presented two-step process utilizes a multicomponent reaction of alkoxyallenes, nitriles and carboxylic acids to provide β-methoxy-β-ketoenamides which are transformed into 4-hydroxypyridines in a subsequent cyclocondensation. The process shows broad substrate scope and leads to differentially substituted enantiopure pyridines in good to moderate yields. The preparation of diverse substituted lactic acid derived pyrid-4-yl nonaflates is described. Additional evidence for the postulated mechanism of the multicomponent reaction is presented.

Direct Reweighting Strategies in Conformation Dynamics

M. Klimm, A. Bujotzek, M. Weber

MATCH Commun. Math. Comput. Chem., 2011, 65 (2), 333-346 ...

The aim of conformation dynamics is the identification of metastable sets in molecular conformation space and the computation of their statistical weights. In this article, we present two novel strategies that permit a direct calculation of the statistical weights, both based on the estimation of free energy differences. The first approach requires an inverse balance condition that leads to a direct calculation of the statistical weights as eigenvalues of a transition matrix. The second approach approximates entropy differences between metastable sets, which is again used to derive according free energy differences and, hence, the statistical weights. In conclusion, we present two examples that illustrate the applicability of these strategies.

A rapidly-mixing Monte Carlo method for the simulation of slow molecular processes.

V. Durmaz, K. Fackelday, M. Weber

Applications of Monte Carlo Methods in Biology, Medicine and Other Fields of Science, C. J. Mode (ed.), InTech, chapter 22, 2011 ...

Since the middle of the last century, the continously increasing computational power has been adopted to molecular modeling and the simulation of molecular dynamics as well. In this field of research, one is interested in the dynamical behaviour of molecular systems. In contrast to the beginnings when only single or very few atoms could be simulated, the systems under consideration have grown to the size of macromolecules like proteins, DNA, or membrane structures nowadays resulting in high-dimensional conformational spaces. This development is triggered by permanently increasing computational power, the utilization of massively parallel hardware as well as improved algorithms and enhanced molecular force fields, covering chemical and especially biological molecular systems at a progressive rate. Applications basing on molecular modeling help to understand and predict molecular phenomena in various fields of applications providing information on e. g. molecular conformations and recognition, protein folding, drug-design, or binding affinities. Typical fields benefiting from their usage are pharmacy, medicine, chemistry and materials research. Unfortunately, often the atomistic structure is so complex that a satisfactory mapping of the processes can hardly be realized, due to the large number of atoms and in particular, the difference in time scales. More precisely, for the molecular function of a protein for example, its folding is a key issue. In contrast to this folding event that may last up to several seconds or even minutes, the time step of an ordinary trajectory based molecular simulation is linked to the fastest molecular oscillation which occurs in case of the chemical H − C bond with a time period around few 10−15 seconds. Even today, exorbitant computational effort and time need to be invested in order to capture such interesting processes.

Towards a rational spacer design for bivalent inhibition of estrogen receptor

A. Bujotzek, M. Shan, R. Haag, M. Weber

J. Comput Aided Mol Des 2011, 25, 253-262 ...

Estrogen receptors are known drug targets that have been linked to several kinds of cancer. The structure of the estrogen receptor ligand binding domain is available and reveals a homodimeric layout. In order to improve the binding affinity of known estrogen receptor inhibitors, bivalent compounds have been developed that consist of two individual ligands linked by flexible tethers serving as spacers. So far, binding affinities of the bivalent compounds do not surpass their monovalent counterparts. In this article, we focus our attention on the molecular spacers that are used to connect the individual ligands to form bivalent compounds, and describe their thermodynamic contribution during the ligand binding process. We use computational methods to predict structural and entropic parameters of different spacer structures. We find that flexible spacers introduce a number of effects that may interfere with ligand binding and possibly can be connected to the low binding affinities that have been reported in binding assays. Based on these findings, we try to provide guidelines for the design of novel molecular spacers.

Synthesis of 5-Acetyloxazoles and 1,2-Diketones from β-Alkoxy-β-ketoenamides and Their Subsequent Transformations.

T. Lechel, M. Gerhard, D. Trawny, B. Brusilowskij, L. Schefzig, R. Zimmer, J.P. Rabe, D. Lentz, C.A. Schalley, H.-U. Reissig

Chem. Eur. J. 2011, 17, 7480-7491.


Lithiated alkoxyallenes, nitriles, and carboxylic acids have been employed as precursors in a three-component reaction leading to highly substituted β-alkoxy-β-ketoenamides. Upon treatment with trifluoroacetic acid, these enamides could be easily cyclized to 5-acetyloxazole derivatives. The synthesis is very flexible with respect to the substitution pattern at C-2 and C-4 of the oxazole core. A mechanistic suggestion for the oxazole formation is presented on the basis of 18O-labeled compounds and their mass spectrometric analysis. In several cases, 1,2-diketones are formed as side products or even as major components. The acetyl moiety at C-5 of the oxazole derivatives can efficiently be converted into alkenyl or alkynyl moieties, which allows a multitude of subsequent reactions. Condensation reactions of the acetyl group provided the expected oxime or hydrazone. By applying a Fischer reaction, the phenylhydrazone could be transferred into an indole, which emphasizes the potential of 5-acetyloxazoles for the preparation of highly substituted (poly)heterocyclic systems. The alkynyl group at C-2 is prone to addition reactions, providing an enamine with interesting photophysical properties. Sonogashira couplings were performed with 5-alkynyl-substituted oxazoles, furnishing the expected aryl-substituted products. This alkynyl unit was employed for the preparation of a new, star-shaped trisoxazole derivative. The ability of this multivalent compound to form self-assembled monolayers between the basal plane of highly oriented pyrolytic graphite and 1-phenyloctane was demonstrated by scanning tunneling microscopy (STM). The star-shaped compound seems to prefer the C3-symmetric arrangement in this two-dimensional crystal. Two 1,2-diketones were smoothly converted into functionalized quinoxaline derivatives.

Accurate Quantum-Chemical Description of Gold Complexes with Pyridine and its Derivatives.

D. Mollenhauer, J. Floß, H.-U. Reissig, E. Voloshina, B. Paulus

J. Comput. Chem. 2011, 32, 1839-1845.


Interaction of gold with pyridine and its derivatives was studied by means of different wavefunction-based correlation methods and standar DFT functionals as well as accounting for dispersion correction. Comparison of the calculated binding energies with benchmark CCSD(T)results allows us to find an appropriate computational method, when considering the two structures reflecting the interaction of gold with the lone pair at nitrogen, on the one hand, and with the π-system of pyridine, on the other hand. Additional binding sites were evaluated, when performing potential energy surface calculations and structure optimizations. The enhancement of the interaction energy due to donor substituents in the 4-position of the pyridine molecule has been investigated.

Stereoselective Synthesis of Aza, Amino and Imion Sugar Derivatives by Hydroboration of 3,6-Dihydro-2H-1,2-oxazines as Key Reaction

V. Dekaris, R. Pulz, A. Al-Harrasi, D. Lentz, H.-U. Reissig

Eur. J. Org. Chem. 2011, 3210 - 3219


Starting from enantiopure 3,6-dihydro-2H-1,2-oxazinessyn-1 we introduced an additional hydroxy group in a stereoselective fashion by a standard hydroboration/oxidation protocol. Under “regular“ conditions substrate control was sufficient to achieve a very high degree of stereoselectivity. However, a diastereomeric product was isolated when a partially “degraded” borane reagent was used. We could synthesise this new diastereomer on purpose by addition of alcohols to the “fresh“ hydroboration reagent. The level of stereoinduction increased with the steric bulk of the added alcohol: MeOH < nBuOH < iPrOH < tBuOH. After a two-step oxidation/reduction sequence, another 5-hydroxy-1,2-oxazine epimer was accessible. The obtained 5-hydroxy-1,2-oxazine diastereomers 2 were used as versatile intermediates in a series of transformations leading to several amino polyol derivatives. Complete deprotection of both diastereomers without cleavage of the N–O bond led to the novel polyhydroxylated tetrahydro-2H-1,2-oxazines 3 and epi-3. By change of the deprotection conditions the open-chain amino polyol 4 with D-iditol configuration became accessible. In an alternative sequence 5-hydroxy-1,2-oxazines were utilised to synthesise imino sugars (polyhydroxylated pyrrolidines). Samarium diiodide induced cleavage of the 1,2-oxazine N–O bond furnished 1,4-amino alcohols, which were cyclised to give the corresponding pyrrolidine derivatives after activation by mesyl chloride. This sequence either led to a 3-methoxy-substituted trihydroxylated pyrrolidine derivative or to the related fully deprotected compound.

The Role of Dimension in Multivalent Binding Events: Structure-Activity Relationship of Dendritic Polyglycerol Sulfate Binding to L-selectin in Correlation with Size and Surface Charge Density.

M. Weinhart, D. Gröger, S. Enders, S.B. Riese, J. Dernedde, R.K. Kainthan, D.E. Brooks, R. Haag

Macromol. Biosci., 2011. 11, 8, 1088-1098


L-, P-, and E-Selectin are cell adhesion molecules that play a crucial role in leukocyte recruitment from the blood stream to the afflicted tissue in an acute and chronic inflammatory setting. Since selectins mediate the initial contact of leukocytes to the vascular endothelium, they have evolved as a valuable therapeutic target in diseases related to inflammation by inhibition of the physiological selectin–ligand interactions. In a previous study, it was demonstrated that dPGS, a fully synthetic heparin analogue, works as an efficient inhibitor towards L- and P-selectin in vitro as well as in vivo. Herein, the focus is directed towards the effect of size and charge density of the polyanion. The efficiency of L-selectin inhibition via an SPR-based in vitro assay and a cell-based flow chamber assay is investigated with dPGS ranging from approximately 4 to 2000 kDa. SPR measurements show that the inhibitory potential of highly sulfated dPGS increases with size and charge density. Thereby, IC50 values from the micromolar to the low picomolar range are determined. The same tendency could be observed in a cell-based flow chamber assay with three representative dPGS samples. This structure–affinity relationship of dPGS suggests that the strong inhibitory potential of dPGS is not only based on the strong electrostatic interaction with areas of cationic surface potential on L-selectin but is also due to a steric shielding of the carbohydrate binding site by large, flexible dPGS particles.

Synthesis of Dendritic Polyglycerol Anions and Their Efficiency Toward L-Selectin Inhibition.

M. Weinhart, D. Gröger, S. Enders, J. Dernedde, R. Haag

Biomacromolecules 2011 12 (7), 2502-2511


A versatile route for the synthesis of highly functionalized, polyanionic macromolecules based on dendritic polyglycerol was applied by means of the Huisgen–Sharpless–Meldal 1,3-dipolar cycloaddition (“click-reaction”) of polyglycerolazide precursors and alkyne-functionalized anions such as sulfonates, carboxylates, phosphonates, and bisphosphonates. In addition, the corresponding polyglycerol phosphate has been synthesized via direct hydroxyl interconversion of polyglycerol to the corresponding phosphate with a degree of functionalization >80% by analogy to the synthesis of previously reported polyglycerol sulfates (dPGS). On the basis of the finding that dPGS exhibits high affinity for L- and P-selectin, the potential of these novel polyanionic, multivalent macromolecules of varying anionic nature as L-selectin inhibitors has been evaluated in vitro by means of a competitive concentration dependent binding assay. Affinity of all polyanions toward L-selectin was demonstrated with distinct IC50 values ranging from the low nanomolar to the high micromolar range. The efficiency of L-selectin inhibition increases in the order carboxylate < phosphate < phosphonate ≈ sulfonate < bisphosphonate < sulfate. Additional DLS and ζ-potential measurements of these polyanions were performed to correlate their binding affinity toward L-selectin with their anionic nature. However, a direct correlation of effective charge and particle size with the determined IC50 values turned out to require further in-depth studies on the microstructure of the polyanions but clearly indicate an exceptional position of dPGS among the studied dendritic polyelectrolytes.

Inhibition of Influenza Virus Activity by Multivalent Glycoarchitectures with Matched Sizes

I. Papp, C. Sieben, A.L. Sisson, J. Kostka, C. Böttcher, K. Ludwig, A. Herrmann, R. Haag

ChemBioChem, 2011, 12, 6, 887-895


We describe the synthesis of a series of sialic acid-conjugated, polyglycerol-based nanoparticles with diameters in the range of 1–100 nm. Particle sizes were varied along with the degree of functionalization to match the corresponding virus size and receptor multiplicity in order to achieve maximum efficiency. To build up these architectures, we used biocompatible, hyperbranched polyglycerols as scaffolds and recently developed polyglycerol-based nanogels, the sizes of which can be varied between 2–4 nm and 40–100 nm, respectively. We demonstrate here that such multivalent nanoparticles inhibit influenza A virus cell binding and fusion and consequently infectivity. The potential of multivalency is evident from larger particles showing very efficient inhibition of viral infection up to 80 %. Indeed, both the size of the nanoparticle and the amount of ligand density are important determinants of inhibition efficiency. The inhibitory activity of the tested polymeric nanoparticles drastically increased with size. Particles with similar dimensions to the virus (50–100 nm) are exceedingly effective. We also observed a saturation point in degree of surface functionalization (i.e. ligand density), above which inhibition was not significantly improved. Our study emphasizes the importance of matching particle sizes and ligand densities to mimic biological surfaces and improve interactions; this is a vital concept underlying multivalent interactions.

Hyperbranched polyglycerols on the nanometer and micrometer scale.

D. Steinhilber, S. Seiffert, J.A. Heyman, F. Paulus, D.A. Weitz, R. Haag

Biomaterials 2011, 32, 1311-1316.


We report the preparation of polyglycerol particles on different length scales by extending the size of hyperbranched polyglycerols (3 nm) to nanogels (32 nm) and microgels (140 and 220 μm). We use miniemulsion templating for the preparation of nanogels and microfluidic templating for the preparation of microgels, which we obtain through a free-radical polymerization of hyperbranched polyglycerol decaacrylate and polyethylene glycol-diacrylate. The use of mild polymerization conditions allows yeast cells to be encapsulated into the resultant microgels with cell viabilities of approximately 30%.

Designing Structural Motifs for Clickamers: Exploiting the 1,2,3-Triazole Moiety to Generate Conformationally Restricted Molecular Architectures.

D. Zornik, R. M. Meudtner, T. El Malah, C. M. Thiele, S. Hecht

Chem. Eur. J. 2011, 17, 1473-1484.


Noncovalent interactions, especially hydrogen-bonding interactions as well as electrostatic forces, confined within one macromolecule are the key to designing foldamers that adopt well-defined conformations in solution. In the context of significant recent activities in the area of triazole-connected foldamers, so-called clickamers, we present a fundamental study that compares various model compounds that bear adjacent N-, O-, or F-heteroatom substituents. The interplay of attractive and repulsive interactions leads to rotational constraints around the single bonds attached to both the 1- and 4-positions of the 1,2,3-triazole moiety and should therefore be able to induce well-defined conformational preferences in higher oligomers and polymers, that is, foldamers. Various compounds were synthesized and characterized with regard to their preferred conformations in all three aggregation states—that is, in the gas phase, in solution as well as in the solid state—by employing DFT calculations, NMR spectroscopic experiments, and X-ray crystallography, respectively. On the basis of the thus-obtained general understanding of the conformational behavior of the individual connection motifs, heterostructures were prepared from different motifs without affecting their distinct folding characteristics. Therefore, this work provides a kind of foldamer construction kit, which should enable the design of various clickamers with specific shape and incorporated functionality.

Reversible and Quantitative Denaturation of Amphiphilic Oligo(azobenzene) Foldamers.

Z. Yu, S. Hecht

Angew. Chem. 2011, 123, 1678-1681.


Intelligente photoschaltbare Foldamere, die ausschließlich aus Azobenzol-Einheiten bestehen, nehmen im Dunkeln stabile helicale Formen an. Sie lassen sich durch UV-Strahlung leicht und reversibel entfalten, erkennbar durch verschwindende Circulardichroismus-Signale (siehe Bild). Die Variation der Oligomerlänge führt zur Optimierung der Effizienz und damit zu quantitativem lichtinduziertem Entfalten und erneutem Falten der Foldamere.

β-Alkoxy-γ-amino Aldehydes by Internal Redox Ring Cleavages of Carbohydrate-Derived Enantiopure 1,2-Oxazines and Preparation of Heterocycles with Aminopolyol Side Chain

A. Al-Harrasi, L. Bouché, R. Zimmer, H.-U. Reissig

Synthesis 2011, No. 1, pp 0109-0118


N-Methylation of syn- or anti-configured 3,6-dihydro-2H-1,2-oxazines and subsequent treatment with triethylamine smoothly provided enantiopure α,β-unsaturated β-alkoxy-γ-amino aldehydes bearing different protected diol, triol, or tetrol side chains in good to excellent yields. The N-O bond cleavage occurs under mild conditions and involves an internal redox process. The method is also applicable to tetrahydro-2H-1,2-oxazines, which either lead to 4-amino ketose or aldose derivatives (d-sorbose or d-idose configuration). The equivalency of the generated β-alkoxyenal moiety with 1,3-dicarbonyl compounds could be demonstrated by condensation reactions with hydrazine or 2-aminoimidazole derivatives providing a series of new pyrazole or imidazo[1,2-a]pyrimidine derivatives with stereodefined and protected aminopolyol side chains.

Multivalent Interaction and Selectivities in Selectin Binding of Functionalized Gold Colloids Decorated with Carbohydrate Mimetics.

M. Roskamp, S. Enders, F. Pfrengle, S. Yekta, V. Dekaris, J. Dernedde, H.-U. Reissig, S. Schlecht

Org. Biomol. Chem. 2011, 9, 7448-7456. 


Colloidal gold particles with functionalized organic shells were applied as novel selectin binders. The ligand shell was terminated with different monocyclic carbohydrate mimetics as simplified analogs of the sLex unit found in biological selectin ligands. The multivalent presentation of the sulfated selectin binding epitopes on the gold particles led to extremely high binding affinities towards L- and P-selectin and IC50 values in the subnanomolar range. Depending on the ring size of the sulfated carbohydrate mimetic, its substitution pattern and its configuration, different selectivities for either L-selectin or P-selectin were obtained. These selectivities were not found for gold particles with simple acyclic sulfated alcohols, diols and triols in the ligand shell. In addition, the influence of the particle size and the thickness of the hydrophobic organic shell were systematically investigated.

Inhibition of Influenza Virus Infection by Multivalent Sialic-Acid-Functionalized Gold Nanoparticles.

I. Papp, C. Sieben, K. Ludwig, M. Roskamp, C. Böttcher, S. Schlecht, A. Herrmann, R. Haag

Small 2010, 6, No. 24, 2900-2906.


An efficient synthesis of sialic-acid-terminated glycerol dendron to chemically functionalize 2 nm and 14 nm gold nanoparticles (AuNPs) is described. These nanoparticles are highly stable and show high activity towards the inhibition of influenza virus infection. As the binding of the viral fusion protein hemagglutinin to the host cell surface is mediated by sialic acid receptors, a multivalent interaction with sialic-acid-functionalized AuNPs is expected to competitively inhibit viral infection. Electron microscopy techniques and biochemical analysis show a high binding affinity of the 14 nm AuNPs to hemagglutinin on the virus surface and, less efficiently, to isolated hemagglutinin. The functionalized AuNPs are nontoxic to the cells under the conditions studied. This approach allows a new type of molecular-imaging activity-correlation and is of particular relevance for further application in alternative antiviral therapy.

Biocatalytic approach for the synthesis of glycerol-based macroamphiphiles and their self-assembly to micellar nanotransporters.

S. Gupta, M. K. Pandey, K. Levon, R. Haag, A. C. Watterson, V. S. Parmar, S. Sunil

Macromol. Chem. Phys., 2010, 211, 239-244.


Glycerol is one of the most versatile and valuable chemical substances and is utilized in a variety of commercial products with no known adverse pharmacological or environmental effects. Moreover, it exhibits good chemical stability and inertness under biological conditions. We have designed and developed Candida antarctica lipase (CAL B) catalyzed biocatalytic method to synthesize novel polymeric systems using glycerol and poly[ethylene glycol bis(carboxymethyl) ether] dimethylester. Both synthons are biocompatible, non-toxic, and are readily available. The polymerization occurs regioselectively through the primary hydroxyl groups of glycerol leaving the secondary hydroxyls for post-polymerization chemical modifications and also for attaching drugs/bioactive molecules. We have utilized these sites for the synthesis of amphiphilic polymers by attaching alkyl chains at the secondary hydroxyl groups simply by acylation. The drug encapsulation abilities of the resulting polymers have been evaluated for potential biomedical applications.

Supramolecular aggregates of water soluble dendritic polyglycerol architectures with hydrophobic cores.

I. N. Kurniasih, H. Liang, J. P. Rabe, R. Haag

Rapid. Macromol. Commun., 2010, 31(17), 1516-1520.


Dendritic core–shell architectures which are based on hyperbranched polyglycerol for the solubilization of hydrophobic drugs have been synthesized and characterized. The core of hyperbranched polyglycerol has been modified with hydrophobic biphenyl groups or perfluorinated chains to increase the core hydrophobicity of the macromolecules. These amphiphilic core–shell type architectures were then used to solubilize pyrene, nile red, and a perfluoro tagged diazo dye, as well as the drug nimodipine in water. Specific host–guest interactions such as fluorous–fluorous interactions could be tailored by this flexible core design and determined by UV spectroscopy. The transport capacity increased 450-fold for nile red, 47-fold for nimodipine, and 37-fold for pyrene at a polymer concentration of only 0.1 wt.-%. Surface tension measurements and scanning force microscopy (SFM) were used to reveal the aggregation properties of these complexes. The formation of supramolecular aggregates with diameters of ≈20 nm and critical aggregate concentrations of 2 × 10−6 mol · L−1 have been observed. This indicates the controlled self-assembly of the presented amphiphilic dendritic core–shell type architectures.

In vivo delivery of siRNA to tumors and their vasculature by novel dendritic nanocarriers.

P. Ofek, W. Fischer, M. Calderon, R. Haag, R. Satchi-Fainaro

FASEB Journal, 2010, 24(9), 3122-3134.


New targets for RNA interference (RNAi)-based cancer therapy are constantly emerging from the increasing knowledge on key molecular pathways that are paramount for carcinogenesis. Nevertheless, in vivo delivery of small interfering RNA (siRNA) remains a crucial challenge for therapeutic success. siRNAs on their own are not taken up by most mammalian cells in a way that preserves their activity. Moreover, when applied in vivo, siRNA-based approaches are all limited by poor penetration into the target tissue and low silencing efficiency. To circumvent these limitations, we have developed novel polymerized polyglycerol-based dendrimer core shell structures to deliver siRNA to tumors in vivo. These cationic dendrimers can strongly improve the stability of the siRNA, its intracellular trafficking, its silencing efficacy, and its accumulation in the tumor environment owing to the enhanced permeability and retention effect. Here, we show that our dendritic nanocarriers exhibited low cytotoxicity and high efficacy in delivering active siRNA into cells. With use of human glioblastoma and murine mammary adenocarcinoma cell lines as model systems, these siRNA-dendrimer polyplexes silenced the luciferase gene, ectopically overexpressed in these cells. Importantly, significant gene silencing was accomplished in vivo within 24 h of treatment with our luciferase siRNA-nanocarrier polyplexes, as measured by noninvasive intravital bioluminescence imaging. Moreover, our siRNA-nanocarriers show very low levels of toxicity as no significant weight loss was observed after intravenous administration of the polyplexes. We show a proof of concept for siRNA delivery in vivo using a luciferase-based model. We predict that in vivo silencing of important cell growth and angiogenesis regulator genes in a selective manner will justify this approach as a successful anticancer therapy.

Novel chemoenzymatic methodology for the regioselective glycine loading on polyhydroxy compounds.

S. Malhotra, M. Calderon, A. K. Prasad, V. S. Parmar, R. Haag

Org. Biomol. Chem., 2010, 8, 2228-2237.


In the present work, we have developed a highly efficient temperature-dependent chemo-enzymatic methodology for the regioselective synthesis of novel esters of glycerol, G1 tri-glycerol dendrons and related esters for the first time using 4-nitrophenyl 2-(tert-butoxycarbonyl)acetate (Boc-gly-Ph-pNO2) (2) as the acylating agent. This methodology offers efficient and controlled loading of amino acid (glycine) on polyhydroxy compounds.

Structure-biocompatibility relationship of dendritic polyglycerol derivatives.

J. Khandare, A. Mohr, M. Calderon, P. Welker, K. Licha, R. Haag

Biomaterials, 2010, 31, 4268–4277


Nanocarriers possess advanced physicochemical properties that improve bioavailability, enhance cellular dynamics, and control targetability in drug delivery. In particular, dendritic polyglycerol is a promising new biocompatible scaffold for drug delivery. The present explores the structure-biocompatibility relationship of dendritic polyglycerol (dPG) derivatives possessing neutral, cationic, and anionic charges. The effect of solution pH on the surface charge was studied in buffered aqueous solution between pH 4.8 and 7.4. Surface charge properties of dPG derivatives are discussed in terms of surface functionalities and compared with amine and hydroxyl terminated polyamidoamine (PAMAM) dendrimers. Zeta potential measurements and fluorescence quenching studies address the binding interactions of dPGs to bovine serum albumin in order to explore the applicability of dPG derivatives for systemic delivery. Cellular entry of dPG-dye conjugate was evaluated using A549 lung epithelial cells, while in vitro toxicity was studied for various dPGs and compared to PAMAM dendrimers, polyethyleneimine (PEI), dextran, and linear polyethylene glycol (PEG) using human hematopoietic cell line U-937. Cellular uptake studies of dye labelled dPGs inferred that the charged derivatives (dPG-sulfate and dPG-amine) are more rapidly internalized primarily inside the cytosol of A549 cells compared to the neutral dPG. The cell compatibility results show that the dendritic polyglycerols are as safe as linear PEG polymer or dextran, which indicates the suitability of dPG derivatives in delivering therapeutic agents systemically.

Functional dendritic polymer architectures as stimuli responsive nanocarriers.

M. Calderon, M. A. Quadir, M. Strumia, R. Haag

Biochimie, 2010, 92(9), 1242-1251.


Stimuli-responsive polymer architectures are molecular systems which evolve with an external signal. The observed changes are mainly decomposition, isomerization, polymerization, activation, supramolecular aggregation, and structural modifications of these molecules. The external stimuli, which can be combined in order to provoke these molecular changes, are numerous. In this review, we have chosen to present an overview on different mechanisms to impart responsiveness to dendritic polymers, with the particular aim of delivery and release of bioactive molecules.

Dendritic Polyglycerols for Biomedical Applications.

M. Calderon, M. A. Quadir, S. K. Sharma, R. Haag

Adv. Mater., 2010, 22, 190–218.


The application of nanotechnology in medicine and pharmaceuticals is a rapidly advancing field that is quickly gaining acceptance and recognition as an independent area of research called “nanomedicine”. Urgent needs in this field, however, are biocompatible and bioactive materials for antifouling surfaces and nanoparticles for drug delivery. Therefore, extensive attention has been given to the design and development of new macromolecular structures. Among the various polymeric architectures, dendritic (“treelike”) polymers have experienced an exponential development due to their highly branched, multifunctional, and well-defined structures. This Review describes the diverse syntheses and biomedical applications of dendritic polyglycerols (PGs). These polymers exhibit good chemical stability and inertness under biological conditions and are highly biocompatible. Oligoglycerols and their fatty acid esters are FDA-approved and are already being used in a variety of consumer applications, e.g., cosmetics and toiletries, food industries, cleaning and softening agents, pharmaceuticals, polymers and polymer additives, printing photographing materials, and electronics. Herein, we present the current status of dendritic PGs as functional dendritic architectures with particular focus on their application in nanomedicine, in drug, dye, and gene delivery, as well as in regenerative medicine in the form of non-fouling surfaces and matrix materials.

Site-specific Functionalisation of Proteins by a Staudinger-type reaction using unsymmetrical phosphites

V. Böhrsch, R. Serwa, P. Majkut, E. Krause, C.P.R. Hackenberger

Chem. Commun. 2010, 46, 3176-3178 ...

Unsymmetrical phosphites react efficiently in a Staudinger reaction with p-azido-phenylalanine, which can be applied for the biotinylation of proteins, thereby expanding the scope of the chemoselective Staudinger-phosphite reaction of aryl azides with symmetrical phosphites to the corresponding phosphoramidates.

Tandem Mass Spectrometry for the Analysis of Self-Sorted Pseudorotaxanes: The Effects of Coulomb Interactions.

W. Jiang, C.A. Schalley

J. Mass Spectrom. 2010, 45, 788-798.


The increasing complexity of self-assembled supramolecules generates the need for analytical techniques that can accurately elucidate their structures. Here, we explore the ability of tandem mass spectrometry to deliver structural information on a series of self-sorted crown ether/ammonium pseudorotaxanes. Of these intertwined molecules, different charge states are accessible and the effects of Coulomb interactions on the fragmentation pattern can be examined. Three different cases can be distinguished: (1) one or more counterions are present in the complex and compete with the crown for binding to the ammonium ion. This destabilizes the supramolecular bond. (2) In multiply charged complexes, charge repulsion significantly alters the fragmentation behavior as compared with singly charged ions. (3) If guest and host are both charged, the supramolecular bond becomes very weak. The different charge states provide different pieces of information about the supramolecules under study. Although singly charged complexes provide data on the building block connectivity, the doubly charged analogs are more reliable with respect to complex stoichiometry. As there are several factors which may cause differences in the gas phase and solution behavior of supramolecules (the presence and absence of solvation, changes in the strength of non-covalent interactions upon ionization), it is important to establish well understood correlations between the complexes' gas-phase behavior and their solution structures. A more detailed understanding will help to characterize the structures of even more complex supramolecular architectures by mass spectrometry.

Internally Protected Amino Sugar Equivalents from Enantiopure1,2-Oxazines: Synthesis of Variably Configured Carbohydrates with C-Branched Amino Sugar Units

F. Pfrengle, H.-U. Reissig

Chem. Eur. J. 2010, 16, 11915-11925 ...

A stereodivergent synthesis of differently configured C2-branched 4-amino sugar derivatives was accomplished. The Lewis acid mediated rearrangement of phenylthio-substituted 1,2-oxazines delivered glycosyl donor equivalents that can directly be employed in glycosidation reactions. Treatment with methanol provided internally protected amino sugar equivalents that have been transformed into the stereoisomeric methyl glycosides 28, ent-28, 29, ent-29 and 34 in two simple reductive steps. Reaction with natural carbohydrates or bicyclic amino sugar precursors allowed the synthesis of homo-oligomeric di- and trisaccharides 44, 46 and 47 or a hybrid trisaccharide 51 with natural carbohydrates. Access to a bivalent amino sugar derivative 54 was accomplished by reaction of rearrangement product 10 with 1,5-pentanediol. Alternatively, when a protected L-serine derivative was employed as glycosyl acceptor, the glycosylated amino acid 60 was efficiently prepared in few steps. In this report we describe the synthesis of unusual amino sugar building blocks from enantiopure 1,2-oxazines that can be attached to natural carbohydrates or natural product aglycons to produce new natural product analogues with potential applications in medicinal chemistry.

Monitoring Self-Sorting by Electrospray Ionization Mass Spectrometry: Formation Intermediates and Error-Correction during the Self-Assembly of Multiply Threaded Pseudorotaxanes.

W. Jiang, P.C. Mohr, A. Schäfer, C.A. Schalley

J. Am. Chem. Soc. 2010, 132, 2309-2320.


Three binary pseudorotaxanes, which are based on two different secondary ammonium/crown ether binding motifs, have been studied by 1H NMR and 1H,1H EXSY NMR experiments with respect to their thermodynamic stabilities and their axle exchange kinetics. The stability ranking does not follow the order of axle exchange rates, and the thermodynamically most stable axle−wheel combinations assemble only slowly. On the basis of these binding motifs, a series of self-sorting systems have been studied ranging from simple four-component mixtures through sequence-specific pseudorotaxanes to multiply threaded complexes. Because of the mismatch of kinetic and thermodynamic order, wrongly assembled structures are unavoidable, which require error-correction steps to yield the final thermodynamically controlled self-sorted products. These error-correction steps can easily be monitored by electrospray mass spectrometry, when a mixed-flow microreactor is coupled to the ion source to cover second time scales. Self-assembly intermediates, wrongly assembled structures, and the final thermodynamic products can be simultaneously identified. The determination of preferred assembly pathways as well as the formation of dead-end structures provides a clear picture of a rich kinetic behavior of the self-sorting systems under study.

Adaptin' endosomes for synaptic vesicle recycling, learning and memory

M. Krauss, V. Haucke

EMBO J. 2010, 29(8), 1313-1315 ...

The pathways by which neurotransmitter-filled presynaptic vesicles (SVs) are generated and recycled have been debated for a long time. Glyvuk et al (2010) in this issue of The EMBO Journal describe an unanticipated role for the clathrin adaptor AP-1 and in particular its σ1B subunit in SV recycling. SV reformation is defective in σ1B-deficient mice, which instead accumulate large endosomelike vacuoles. These defects are paired with reduced motor coordination and long-term spatial memory. This work thus not only provides novel insights into the role of clathrin/AP-1 coats in SV recycling from endosomes, but also unravels a molecular mechanism that may contribute to some forms of X-linked mental retardation.

Binding activity of recombinant human L-selectin-Fc(γ) is modified by sialylation

S. Enders, S.B. Riese, G. Bernhard, J. Dernedde, W. Reutter, R. Tauber

Biochem. E. J. 2010, 48, 253-259. ...

The adhesion molecule L-selectin expressed on most leukocytes mediates tethering and rolling of leukocytes on activated endothelia and initiates the extravasation of leukocytes into inflamed tissues. Recombinant L-selectin-Fcγ is widely used both as a tool to study this key step of inflammation and as an anti-inflammatory compound in animal models of inflammation. Since previous studies on cellular L-selectin have indicated that glycosylation influences adhesive interactions of the adhesion molecule, we have examined whether the binding activity of L-selectin-Fcγ is affected by sialylation. Different forms of recombinant human L-selectin-Fcγ were expressed in CHO and K-562 cells and were purified by affinity chromatography using Protein A-Sepharose. A hypersialylated form of L-selectin-Fcγ was generated by culturing cells in the presence of 5 mM N-acetyl-beta-d-mannosamine, while a desialylated variant was obtained by treatment of purified L-selectin-Fcγ with neuraminidase. Binding activity to the synthetic biligand SiaLex-PAA-sTyr was measured by surface plasmon resonance (SPR) technology. While hypersialylated L-selectin-Fcγ showed decreased binding activity, desialylation elevated L-selectin-Fcγ binding to SiaLex-PAA-sTyr. The data show that sialylation of L-selectin-Fcγ reduces binding activity to ligand epitopes containing sialyl Lewis x and sulfated tyrosine residues. For the production of biologically active L-selectin-Fcγ conditions should be chosen that favor the generation of non-sialylated or of scarcely sialylated forms of the recombinant glycoprotein.

Cyclative cleavage of azido-peptidyl-phosphoranes delivers locked cis-triazolyl-cyclopeptides as privileged protein binders

Ahsanullah, J. Rademann

Angew. Chem. Int. Ed. 2010, 49, 5378 –5382 ...

Support and guidance: Azidopeptidyl phosphoranes on a solid support react very efficiently through cyclative cleavage to yield cyclopeptides with an incorporated triazole ring. The solid support is advantageous as cyclization is favored strongly over oligomerization reactions and thus only cyclized products are released.

Multiple glycosylation of de novo designed α-helical coiled coil peptides.

J.A. Falenski, U.I.M. Gerling, B. Koksch

Bioorg. Med. Chem. 2010, 18, 3703–3706.


The aim of this study was to investigate the influence of multiple O-glycosylation in α-helical coiled coil peptides on the folding and stability. For this purpose we systematically incorporated one to six β-galactose residues into the solvent exposed positions of a 26 amino acid long coiled coil helix. Surprisingly, circular dichroism spectroscopy showed no unfolding of the coiled coil structure for all glycopeptides. Thermally induced denaturations reveal a successive but relative low destabilization of the coiled coil structure upon introduction of β-galactose residues. These first results indicate that O-glycosylation of the glycosylated variants is easily tolerated by this structural motif and pave the way for further functional studies.

How Post-Translational Modifications Influence Amyloid Formation: A Systematic Study of Phosphorylation and Glycosylation in Model Peptides.

M. Broncel, J.A. Falenski, S.C. Wagner, C.P.R. Hackenberger, B. Koksch

Chem. Eur. J. 2010, 16(26), 7881-88.


A reciprocal relationship between phosphorylation and O-glycosylation has been reported for many cellular processes and human diseases. The accumulated evidence points to the significant role these post-translational modifications play in aggregation and fibril formation. Simplified peptide model systems provide a means for investigating the molecular changes associated with protein aggregation. In this study, by using an amyloid-forming model peptide, we show that phosphorylation and glycosylation can affect folding and aggregation kinetics differently. Incorporation of phosphoserines, regardless of their quantity and position, turned out to be most efficient in preventing amyloid formation, whereas O-glycosylation has a more subtle effect. The introduction of a single β-galactose does not change the folding behavior of the model peptide, but does alter the aggregation kinetics in a site-specific manner. The presence of multiple galactose residues has an effect similar to that of phosphorylation.

Towards understanding secondary structure transitions: phosphorylation and metal coordination in model peptides.

M. Broncel, S.C. Wagner, K. Paul, C.P.R. Hackenberger and B. Koksch

Org. Biomol. Chem. 2010, 8, 2575-2579.


Secondary structure transitions are important modulators of signal transduction and protein aggregation. Phosphorylation is a well known post-translational modification capable of dramatic alteration of protein secondary structure. Additionally, phosphorylated residues can induce structural changes through metal binding. Data derived from the Protein Data Bank demonstrate that magnesium and manganese are metal ions most favored by phosphate. Due to the complexity of molecular interactions as well as the challenging physicochemical properties of natural systems, simplified peptide models have emerged as a useful tool for investigating the molecular switching phenomenon. In this study using a coiled coil model peptide, we show structural consequences of phosphorylation and subsequent magnesium and manganese ions coordination. In the course of our experiment we obtained a switch cascade starting from a stable helical conformation of the control peptide, continuing through the phosphorylation-induced unfolded structure, and ending with a metal-stabilized α-helix (Mg2+) or helical fibers (Mn2+), each of which could be transferred back to the unfolded form upon EDTA chelation. This study demonstrates how small peptide models can aid in the evaluation and a better understanding of protein secondary structure transitions.

Enzymatically trigerred amyloid formation: an approach for studying peptide aggregation.

M. Broncel, S.C. Wagner, C.P.R. Hackenberger, B. Koksch

Chem. Commun. 2010, 46, 3080-3082.


A strategy has been demonstrated that utilizes a phosphatase as a natural tool for the triggering and control of amyloid formation in a coiled coil peptide model under conditions that closely approximate a physiological environment.

Nanoparticle-Induced Folding and Fibril Formation of Coiled-Coil-Based Model Peptides.

S.C. Wagner, M. Roskamp, M. Pallerla, R.R. Araghi, S. Schlecht and B. Koksch

Small 2010, 6 (12), 1321-1328.


Nanomedicine is a rapidly growing field that has the potential to deliver treatments for many illnesses. However, relatively little is known about the biological risks of nanoparticles. Some studies have shown that nanoparticles can have an impact on the aggregation properties of proteins, including fibril formation. Moreover, these studies also show that the capacity of nanoscale objects to induce or prevent misfolding of the proteins strongly depends on the primary structure of the protein. Herein, light is shed on the role of the peptide primary structure in directing nanoparticle-induced misfolding by means of two model peptides. The design of these peptides is based on the α-helical coiled-coil folding motif, but also includes features that enable them to respond to pH changes, thus allowing pH-dependent β-sheet formation. Previous studies showed that the two peptides differ in the pH range required for β-sheet folding. Time-dependent circular dichroism spectroscopy and transmission electron microscopy are used to characterize peptide folding and aggregate morphology in the presence of negatively charged gold nanoparticles (AuNPs). Both peptides are found to undergo nanoparticle-induced fibril formation. The determination of binding parameters by isothermal titration calorimetry further reveals that the different propensities of both peptides to form amyloid-like structures in the presence of AuNPs is primarily due to the binding stoichiometry to the AuNPs. Modification of one of the peptide sequences shows that AuNP-induced β-sheet formation is related to the structural propensity of the primary structure and is not a generic feature of peptide sequences with a sufficiently high binding stoichiometry to the nanoparticles.

Cyclisierende Abspaltungen über dipolare Cycloadditionen: Polymergebundene Azidopeptidylphosphorane liefern konformativ fixierte cis-Triazolylcyclopeptide als privilegierte Proteinbinder

Ahsanullah, J. Rademann

Angew. Chem. 2010, 122, 5506 –5510 ...

Hilfreiche Stütze: Cyclopeptide mit eingebautem Triazolring lassen sich sehr effizient erhalten, wenn man Azidopeptidylphosphorane in einer cyclisierenden Abspaltung an einer Festphase umsetzt (siehe Schema). Die Festphase ist besonders hilfreich, indem sie Cyclisierungen gegenüber Oligomerisierungen begünstigt, sodass nur cyclische Produkte freigesetzt werden.

Regulation of synaptic vesicle recycling by complex formation between intersectin 1 and the clathrin adaptor complex AP2

A. Pechstein, J. Bacetic, A. Vahedi-Faridi, K. Gromova, A. Sundborger, N. Tomlin, G. Krainer, O. Vorontsova, J.G. Schäfer, S.G. Owe, M.A. Cousin, W. Saenger, O. Shupliakov, V. Haucke

PNAS 2010, 107(9), 4206-4211

Clathrin-mediated synaptic vesicle (SV) recycling involves the spatiotemporally controlled assembly of clathrin coat components at phosphatidylinositiol (4, 5)-bisphosphate [PI(4,5)P2]-enriched membrane sites within the periactive zone. Such spatiotemporal control is needed to coordinate SV cargo sorting with clathrin/AP2 recruitment and to restrain membrane fission and synaptojanin-mediated uncoating until membrane deformation and clathrin coat assembly are completed. The molecular events underlying these control mechanisms are unknown. Here we show that the endocytic SH3 domain-containing accessory protein intersectin 1 scaffolds the endocytic process by directly associating with the clathrin adaptor AP2. Acute perturbation of the intersectin 1-AP2 interaction in lamprey synapses in situ inhibits the onset of SV recycling. Structurally, complex formation can be attributed to the direct association of hydrophobic peptides within the intersectin 1 SH3A-B linker region with the “side sites” of the AP2 α- and β-appendage domains. AP2 appendage association of the SH3A-B linker region inhibits binding of the inositol phosphatase synaptojanin 1 to intersectin 1. These data identify the intersectin-AP2 complex as an important regulator of clathrin-mediated SV recycling in synapses.

Native chemical ligation in the synthesis of internally modified oligonucleotide-peptide conjugates.

F. Diezmann, H. Eberhard, O. Seitz

Biopolymers 2010, 94, 397-404. ...

Peptide–oligonucleotide conjugates have frequently been synthesized to improve cellular delivery of antisense or antigene compounds, to allow the immobilization of peptide and protein conjugates on DNA arrays, or to decorate nucleic acid architectures with peptide functions. In such applications, the site of conjugation is of little importance, and peptides have predominantly been appended to one of the terminal ends of the oligonucleotide by using an oxime-, thioether-, or disulfide-linkage or native chemical ligation. We, herein, demonstrate the first coupling of peptides to sequence internal sites. This attachment mode provides better control of the spatial arrangement of peptides presented by self-assembled nucleic acid scaffolds. Internal modification requires special phosphoramidite building blocks that can be used in automated DNA synthesis. For this purpose, Fmoc/StBu-protected cysteine was attached via an aminopropargyl linker to the C5-position of uridine. The rigid triple bond conferred a high reactivity in native chemical ligation reactions of 5–6mer peptide thioesters with up to 15 nucleotides long oligonucleotides. The desired peptide–oligonucleotide conjugates were obtained in high yields after purification. UV melt experiments revealed that the peptide modification does not hamper nucleic acid hybridization. This finding marked an important step in our research program devoted to studies of multivalent presentation of peptides via modular assembly of nucleic acid complexes.

Acid-Mediated Transformations of Enantiopure 3,6-Dihydro-2H-1,2-oxazines into Functionalised Aminotetrahydrofuran Derivatives

V. Dekaris, B. Bressel, H.-U. Reissig

Synlett 2010, No. 1, 47-50 ...

Two new routes to substituted aminotetrahydrofuran derivatives have been investigated. Treatment of 3,6-dihydro-2H-1,2-oxazines with hydrochloric acid in the presence of zinc provided 4-benzylamino-5-hydroxy furanose derivatives which contain a quaternary anomeric centre with a vinyl unit. Upon mesylation and subsequent heating in aqueous media 5-hydroxy-3,4,5,6-tetrahydro-1,2-oxazines were converted into novel bicyclic 1,2-oxazines with complete regio- and stereoselectivity. Cleavage of the N-O bond and subsequent debenzylation furnished enantiopure polyhydroxylated aminotetrahydrofuran derivatives which are promising ligands for selectin inhibition studies.

Acidic and Basic Deprotection Strategies of Borane-protected Phosphinothioesters for the Traceless Staudinger Ligation

M. Mühlberg, D. Jaradat, R. Kleineweischede, I. Papp, D. Dechtrirat, S. Muth, M. Broncel, C.P.R. Hackenberger

Bioorg. Med. Chem. 2010, 18, 3679-3686 ...

The traceless Staudinger ligation has recently found various applications in the field of peptide synthesis and modification, including immobilization and cyclization strategies. In this report, we utilize the traceless Staudinger ligation in the formation of amide bonds, which allows the acquisition of acylated aminosugars and peptides as well as the cyclization of peptides. A key element in these synthetic procedures is the use of a borane-protected phosphinomethanethiol, which is demonstrated to be prone towards oxidation in its unprotected form, during the synthesis of phosphinothioesters. In combination with acidic and basic deprotection strategies for the borane-protected phosphinothioesters, amide bonds can be formed in the presence of azides in moderate to good overall yields.

A Novel Subtype of AP-1-binding Motif within the Palmitoylated trans-Golgi Network/Endosomal Accessory Protein Gadkin/γ-BAR

T. Maritzen, M.R. Schmidt, V. Kukhtina, V.A. Higman, H. Strauss, R. Volkmer, H. Oschkinat, C.G. Dotti, V. Haucke

J. Biol. Chem. 2010, 285(6), 4074-4086

Membrane traffic between the trans-Golgi network (TGN) and endosomes is mediated in part by the assembly of clathrin-AP-1 adaptor complex-coated vesicles. This process involves multiple accessory proteins that directly bind to the ear domain of AP-1γ via degenerate peptide motifs that conform to the consensus sequence ØG(P/D/E)(Ø/L/M) (with Ø being a large hydrophobic amino acid). Recently, γ-BAR (hereafter referred to as Gadkin for reasons explained below) has been identified as a novel AP-1 recruitment factor involved in AP-1-dependent endosomal trafficking of lysosomal enzymes. How precisely Gadkin interacts with membranes and with AP-1γ has remained unclear. Here we show that Gadkin is an S-palmitoylated peripheral membrane protein that lacks stable tertiary structure. S-Palmitoylation is required for the recruitment of Gadkin to TGN/endosomal membranes but not for binding to AP-1. Furthermore, we identify a novel subtype of AP-1-binding motif within Gadkin that specifically associates with the γ1-adaptin ear domain. Mutational inactivation of this novel type of motif, either alone or in combination with three more conventional AP-1γ binding peptides, causes Gadkin to mislocalize to the plasma membrane and interferes with its ability to render AP-1 brefeldin A-resistant, indicating its physiological importance. Our studies thus unravel the molecular basis for Gadkin-mediated AP-1 recruitment to TGN/endosomal membranes and identify a novel subtype of the AP-1-binding motif.

Bidirectional binding of invariant chain peptides to an MHC class II molecule

S. Günther, A. Schlundt, J. Sticht, Y. Roske, U. Heinemann, K.-H. Wiesmüller, G. Jung, K. Falk, O. Rötzschke, C. Freund

PNAS 2010, 107 (51), 22219-22224

T-cell recognition of peptides bound to MHC class II (MHCII) molecules is a central event in cell-mediated adaptive immunity. The current paradigm holds that prebound class II-associated invariant chain peptides (CLIP) and all subsequent antigens maintain a canonical orientation in the MHCII binding groove. Here we provide evidence for MHCII-bound CLIP inversion. NMR spectroscopy demonstrates that the interconversion from the canonical to the inverse alignment is a dynamic process, and X-ray crystallography shows that conserved MHC residues form a hydrogen bond network with the peptide backbone in both orientations. The natural catalyst HLA-DM accelerates peptide reorientation and the exchange of either canonically or inversely bound CLIP against antigenic peptide. Thus, noncanonical MHC-CLIP displays the hallmarks of a structurally and functionally intact antigen-presenting complex.

Amino sugars and their mimetics via 1,2-oxazines

F. Pfrengle, H.-U. Reissig

Chem.Soc.Rev. 2010, 39, 549-557 ...

This tutorial review covers recent research from our laboratory towards the synthesis of amino sugars and related carbohydrate mimetics employing 1,2-oxazines as crucial intermediates. The synthesis of new dideoxyamino carbohydrate derivatives, C2-branched 4-amino sugars and their mimetics, as well as sialic acid analogues, has been developed during the last few years. The results demonstrate that alkoxyallenes are ideal and versatile components for the chain elongation of carbohydrate derivatives. Our routes starting from 3,6-dihydro-2H-1,2-oxazines are presented in conjunction with two related examples from the recent literature. Finally, we discuss possible future applications of the described compounds as anti-inflammatory agents.

Addition of lithiated enol ethers to nitrones and subsequent Lewis acid induced cyclizations to enantiopure 3,6-dihydro-2H-pyrans – an approach to carbohydrate mimetics

F. Pfrengle, H.-U. Reissig

Beilstein J. Org. Chem. 2010, 6, No. 75 ...

A stereodivergent synthesis of enantiopure 3,6-dihydro-2H-pyrans is presented. The addition of lithiated enol ethers to carbohydrate-derived nitrones afforded syn- or anti-configured hydroxylamine derivatives 4a–d that were cyclized under Lewis acidic conditions to yield functionalized dihydropyrans cis- or trans-5a–d containing an enol ether moiety. This functional group was employed for a variety of subsequent reactions such as dihydroxylation or bromination. Bicyclic enol ether 19 was oxidatively cleaved to provide the highly functionalized ten-membered ring lactone 20. The synthesized enantiopure aminopyrans 24, 26, 28 and 30 can be regarded as carbohydrate mimetics. Trimeric versions of 24 and 28 were constructed via their attachment to a tricarboxylic acid core.

Polyglycerol nanogels: Highly functional scaffolds for biomedical applications.

A.L. Sisson, R. Haag

Softmatter, 2010, 6, 4968-4975.


Branched polyglycerols (PGs) are a versatile class of functionalisable, hydrophilic, hydroxylated polyethers with ideal properties for numerous applications. Historically, synthetic limitations have restricted the study of such materials to globular hyperbranched polymers (<10 nm) or macroscale biocompatible hydrogels (>1000 nm). In this Emerging Area minireview we describe how we use miniemulsion polymerisation to prepare polyglycerol nanogels on previously unobtainable length scales. Various cases are discussed with particle sizes that are highly tunable between 25 and 350 nm diameter; methods to surface functionalise such particles are also described. Biodegradable polyglycerol based nanogels have also been prepared by incorporating redox active disulfide branching points within the nanogel structure. Cell culture studies show that these nanogels are highly biocompatible. Additionally, dye labelled nanogels are shown by optical microscopy techniques to readily internalise into cells by endocytic mechanisms. We believe that these polyglycerol nanogels will emerge as excellent materials for use in a broad range of biomedical applications.

Novel Highly Monodisperse Water-Dispersable Iron Oxide Nanoparticles for Biomedical Applications.

A. Hofmann, S. Thierbach, A. Semisch, A. Hartwig, M. Taupitz, E. Rühl, C. Graf

J. Mater. Chem. 2010, 20, 7842-7853.


We demonstrate a unique approach for preparing high quality iron oxide (Fe3O4) nanoparticles functionalized by newly developed multifunctional dendron ligands for biomedical applications. These particles are suitable for magnetic resonance imaging (MRI), highly stable in aqueous solutions as well as physiological media and not cytotoxic. In particular, oleic acid capped Fe3O4 particles (d = 12 ± 0.8 nm) were modified in a ligand exchange process by investigating several dendron ligands of variable size and an adjustable number of polar end groups. The dendron based ligands lead only to a slight increase in hydrodynamic diameter of the nanoparticles after the ligand exchange process (∼6 nm). They also allow an adjustment of the particle polarity as well as a gradually variable surface functionalization. Light scattering, transmission electron microscopy, and visible spectroscopy studies show consistently that the dendron-capped iron oxide nanoparticles exhibit excellent stability in various physiological media as well as aqueous solutions in a broad pH range. It is also demonstrated by magnetic resonance studies that the magnetic relaxivity is almost not affected by the ligand exchange. Therefore, such small particles might be of specific interest for cardiovascular MRI and MRI of extravascular targets. In addition, the present approach opens new possibilities for the specific linking of biomolecules to the particle surface, which can be beneficial for various biological sensing and therapeutic applications. The cytotoxicity of the Fe3O4 nanoparticles was evaluated using the WST-8 assay. In the examined concentration range up to 100 μg Fe/mL no significant decrease in cell viability was detected.

New types of bifunctional hydroxamic acids as novel ligands for the hydrophilic stabilization of iron oxide nanoparticles.

A. Hofmann, C. Graf, S.-H. Kung, M. Kim, X. Peng, R. El-Aama, E. Rühl

Synthesis 2010, 42, 1150-1158


A general method for synthesizing bifunctional hydroxamic acids containing carboxylic acid or amino functionalities is reported. Various products from simple alkyl to complex dendrimer-like structures are described. Such molecules have recently been used in ligand-exchange reactions for the hydrophilic stabilization of originally oleic acid protected iron oxide nanoparticles.

Lysosomal Pathology and Osteopetrosis upon Loss of H+-Driven Lysosomal Cl- Accumulati

S. Weinert, S. Jabs, C. Supanchart, M. Schweizer, N. Gimber, M. Richter, J. Rademann, T. Stauber, U. Kornak, T. J. Jentsch

Science 2010, 1401-1403 ...

During lysosomal acidification, proton-pump currents are thought to be shunted by a chloride ion (Cl–) channel, tentatively identified as ClC-7. Surprisingly, recent data suggest that ClC-7 instead mediates Cl–/proton (H+) exchange. We generated mice carrying a point mutation converting ClC-7 into an uncoupled (unc) Cl– conductor. Despite maintaining lysosomal conductance and normal lysosomal pH, these Clcn7unc/unc mice showed lysosomal storage disease like mice lacking ClC-7. However, their osteopetrosis was milder, and they lacked a coat color phenotype. Thus, only some roles of ClC-7 Cl–/H+ exchange can be taken over by a Cl– conductance. This conductance was even deleterious in Clcn7+/unc mice. Clcn7–/– and Clcn7unc/unc mice accumulated less Cl– in lysosomes than did wild-type mice. Thus, lowered lysosomal chloride may underlie their common phenotypes.

Synthesis of Heterocycles via Alkoxyallenes.

T. Lechel, H.-U. Reissig

Pure Appl. Chem. 2010, 82, 1835-1844.


Lithiated alkoxyallenes are very versatile components for the synthesis of heterocycles such as furans, pyrroles, and 1,2-oxazines, easily allowing the preparation of natural products via these heterocyclic intermediates. A surprising three-component synthesis of N‑acylated enaminones allowed the synthesis of highly functionalized 4-hydroxypyridines, 5‑acetyloxazoles, and pyrimidines. All these heterocyclic products are ready for further functionalizations, in particular for palladium-catalyzed reactions, leading to libraries of new interesting heterocycles.

A simple method for the estimation of entropy differences

M. Weber, K. Andrae

MATCH Commun. Math. Comput. Chem., 2010, 63 (2), 319-332 ...

We present a simple method to calculate conformational entropy differences between different polymer structures from a thermostated molecular dynamics simulation. The mathematical concept is based on a special Monte Carlo quadrature approach combined with density estimation. First, this approach will be derived for a general integrant 𝑓. Then, we will apply the entropy estimator in order to investigate different protein resistant surfaces based on derivates of polyethylenglycol peg. For peg, the highest entropy value is found, which is an indicator for an optimal protein resistant function of peg. Such a preference is also seen in thermostated molecular dynamics simulations of larger chain lengths of peg.

Observation uncertainty in reversible Markov chains

P. Metzner, M. Weber, C. Schütte

Phys. Rev. e. Stat. Nonlin. Soft Matter Phys. 2010, 82 (3), 031114 ...

In many applications one is interested in finding a simplified model which captures the essential dynamical behavior of a real life process. If the essential dynamics can be assumed to be (approximately) memoryless then a reasonable choice for a model is a Markov model whose parameters are estimated by means of Bayesian inference from an observed time series. We propose an efficient Monte Carlo Markov chain framework to assess the uncertainty of the Markov model and related observables. The derived Gibbs sampler allows for sampling distributions of transition matrices subject to reversibility and/or sparsity constraints. The performance of the suggested sampling scheme is demonstrated and discussed for a variety of model examples. The uncertainty analysis of functions of the Markov model under investigation is discussed in application to the identification of conformations of the trialanine molecule via Robust Perron Cluster Analysis (PCCA+).

Synthesis, Reductive Cleavage, and Cellular Interaction Studies of Biodegradable, Polyglycerol Nanogels.

D. Steinhilber, A.L. Sisson, D. Mangoldt, P. Wilker, K. Licha, R. Haag

Adv. Funct. Mater. 2010, 20, 4133-4138.


In this paper we describe disulfide containing, polyglycerol nanogels as a new class of biodegradable materials. These nanoparticles are prepared in inverse miniemulsion via an acid catalyzed ring-opening polyaddition of disulfide containing polyols and polyepoxides. Varying conditions allow us to tune particle size and disulfide content within the polymer network; particles can be prepared with narrow polydispersities and diameters in the range from 25 to 350 nm. Particle degradation under reductive intracellular conditions is studied by various analytical techniques. Gel permeation chromatography indicates that final degradation products have relatively low molecular weights (≤ 5 kDa). In addition, studies in cell culture show these nanoscale materials to be highly biocompatible. Dye-labelled nanogels are shown by optical microscopy techniques to readily internalize into cells by endocytotic mechanisms. This study highlights the great potential of these particles to function as sophisticated nanotransporters that deliver cargo to a certain tissue or cell target and then biodegrade into smaller fragments which would be cleared from the body by the kidney. (with ≈ 30 kDa molecular weight cut off)

Kinetics Study of the Binding of Multivalent Ligands on Size-Selected Gold Nanoparticles

S. Perumal, A. Hofmann, N. Scholz, E. Rühl, C. Graf

Langmuir, 2011, 27 (8), 4456–4464 ...

The effect of ligand multivalency and nanoparticle size on the binding kinetics of thiol ligands on gold nanoparticles is investigated by exchanging monovalently bound pyrene on gold nanoparticles against flexible mono- and multivalent thiol ligands. Variable-sized gold nanoparticles of 2.2 ± 0.4, 3.2 ± 0.7, and 4.4 ± 0.9 nm diameter are used as substrates. The particles are coated by thiol functionalized pyrene ligands and the binding kinetics of the thiol ligands is studied by time-resolved fluorescence spectroscopy. The effect of multivalency on the binding kinetics is evaluated by comparing the rate constants of ligands of different valency. This comparison reveals that the multivalent ligands are exchanging substantially more rapidly than the monovalent ones. A particle size dependence of the rate constants is also observed, which is used to derive structural information on the binding of the mono- and multivalent ligands to the nanoparticle surface.

Chemically Tailored multivalent Virus Platforms: From Drug Delivery to Catalysis

A.K. Udit, C.P.R. Hackenberger, M.K. O'Reilly

Chem.Bio.Chem. 2010, 11, 481-484 ...

The new Jack of all trades: Phage! Virus particles have been used as vessels for drug delivery and for multivalent nanoscale display of light-harvesting complexes, thereby demonstrating their use as chemical platforms for applications spanning chemical biology to materials science.

Solid-phase synthesis of phosphoramidate-linked glycopeptide

D.M.M. Jaradat, H. Hamouda, C.P.R. Hackenberger

Eur. J. Org. Chem. 2010, 5004-5009 ...

The synthesis of stable glycopeptide mimetics is of particular interest in bioorganic chemistry to allow access of glycoconjugates for biological investigations. In this paper, a straightforward solid-phase synthesis of a novel glycopeptide mimetic by a Staudinger phosphite reaction is presented. Thereby, a dimethyl phosphite containing peptide, which is obtained by standard phosphitylation of a Ser residue, is treated on the solid support with glycosyl azides, delivering phosphoramidate-linked glycoconjugates. These artificial glycopeptides show excellent stability under acidic and physiological conditions.

Molecular Basis for Association of PIPKIγ-p90 with Clathrin Adaptor AP-2

N. Kahlfeldt, A. Vahedi-Faridi, S. Joo Koo, J.G. Schäfer, G. Krainer, S. Keller, W. Saenger, M. Krauss, v. Haucke

J. of Biological Chemistry 2010, 285 (4), 2734-2749

Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) is an essential determinant in clathrin-mediated endocytosis (CME). In mammals three type I phosphatidylinositol-4-phosphate 5-kinase (PIPK) enzymes are expressed, with the Iγ-p90 isoform being highly expressed in the brain where it regulates synaptic vesicle (SV) exo-/endocytosis at nerve terminals. How precisely PI(4,5)P2 metabolism is controlled spatially and temporally is still uncertain, but recent data indicate that direct interactions between type I PIPK and components of the endocytic machinery, in particular the AP-2 adaptor complex, are involved. Here we demonstrated that PIPKIγ-p90 associates with both the μ and β2 subunits of AP-2 via multiple sites. Crystallographic data show that a peptide derived from the splice insert of the human PIPKIγ-p90 tail binds to a cognate recognition site on the sandwich subdomain of the β2 appendage. Partly overlapping aromatic and hydrophobic residues within the same peptide also can engage the C-terminal sorting signal binding domain of AP-2μ, thereby potentially competing with the sorting of conventional YXXØ motif-containing cargo. Biochemical and structure-based mutagenesis analysis revealed that association of the tail domain of PIPKIγ-p90 with AP-2 involves both of these sites. Accordingly the ability of overexpressed PIPKIγ tail to impair endocytosis of SVs in primary neurons largely depends on its association with AP-2β and AP-2μ. Our data also suggest that interactions between AP-2 and the tail domain of PIPKIγ-p90 may serve to regulate complex formation and enzymatic activity. We postulate a model according to which multiple interactions between PIPKIγ-p90 and AP-2 lead to spatiotemporally controlled PI(4,5)P2 synthesis during clathrin-mediated SV endocytosis.

Ring Enlargement of Carbohydrate-Derived 1,2-Oxazines to Enantiopure 5-Bromo-1,2-oxazepines and Subsequent Palladium-Catalyzed Reactions

A. Al-Harrasi, S. Fischer, R. Zimmer, H.-U. Reissig

Synthesis 2010, 304-314 ...

Dibromocarbene addition to d-glyceraldehyde-derived 1,2-oxazines syn -1 and anti -1 provided dibromocyclopropane intermediates syn-3 and anti-3, which smoothly reacted with methanol under ring enlargement to furnish 5-bromo-1,2-oxazepine derivatives syn -4 and anti -4. Related 1,2-oxazines such as arabinose-derived­ compounds furnished the 1,2-oxazepine derivatives syn-4e and anti-4f with fair efficacy. The alkenyl bromide moiety of 1,2-oxazepine derivatives syn -4 and anti -4 was then exploited for the introduction of new substituents via palladium-catalyzed C-C bond forming processes (Sonogashira, Suzuki, Stille, and Heck reactions). These transformations led to a series of new highly substituted 1,2-oxazepine derivatives syn-5 or anti-5-11 being of considerable interest for further synthetic elaborations.

Stereocontrolled Syntheses of Enantiopure Polyhydroxylated Azetidines via 1,2-Oxazines

V. Dekaris, H.-U. Reissig

Synlett 2010, 1, 42 - 46


A set of new enantiopure polyhydroxylated azetidine derivatives has been prepared. The key starting materials, 3,6-dihydro-2H-1,2-oxazines, were subjected to a hydroboration-oxidation sequence to introduce the required 5-hydroxy group. Subsequent cleavage of the N-O bond with samarium diiodide, selective protection of the primary hydroxyl group, and ring closure after activation of the secondary hydroxyl group provided the protected azetidine derivatives. The efficacy of each individual step of this sequence depends on the configuration of the starting material. Three representative azetidine derivatives were converted into the deprotected polyhydroxylated azetidines which are interesting candidates as glycosidase inhibitors.

Templated Versus Non-Templated Synthesis of Benzo-21-Crown-7 and the Influence of Substituents on Its Complexing Properties.

W. Jiang, C.A. Schalley

Beilstein J. Org. Chem. 2010, 6, No. 14.


Two procedures for the synthesis of benzo-21-crown-7 have been explored. The [1+1] macrocyclization with KBF4 as the template was found to be more efficient than the intramolecular macrocyclization without template. Pseudorotaxanes form with secondary ammonium ions bearing at least one alkyl chain narrow enough to slip into the crown ether. Substitution on benzo-21-crown-7 or on the secondary ammonium axle alters the binding affinity and binding mode. Compared to dibenzo-24-crown-8, the complexing properties of benzo-21-crown-7 turn out to be more susceptible to modifications at the crown periphery.

Hemagglutinin of Influenza Virus Partitions into the Nonraft Domain of Model Membranes

J. Nikolaus, S. Scolari, E. Bayraktarov, N. Jungnick, S. Engel, A.P. Plazzo, M. Stöckl, R. Volkmer, M. Veit, A. Herrmann

Biophys. J. 2010, 99, 489-498 ...

The HA of influenza virus is a paradigm for a transmembrane protein thought to be associated with membrane-rafts, liquid-ordered like nanodomains of the plasma membrane enriched in cholesterol, glycosphingolipids, and saturated phospholipids. Due to their submicron size in cells, rafts can not be visualized directly and raft-association of HA was hitherto analyzed by indirect methods. In this study, we have used GUVs and GPMVs, showing liquid disordered and liquid ordered domains, to directly visualize partition of HA by fluorescence microscopy. We show that HA is exclusively (GUVs) or predominantly (GPMVs) present in the liquid disordered domain, regardless of whether authentic HA or domains containing its raft targeting signals were reconstituted into model membranes. The preferential partition of HA into ld domains and the difference between lo partition in GUV and GPMV are discussed with respect to differences in packaging of lipids in membranes of model systems and living cells suggesting that physical properties of lipid domains in biological membranes are tightly regulated by protein-lipid interactions.

Linear poly(methyl glycerol and linear polyglycerol as potent protein and cell resistant alternatives to poly(ethylene glycol).

M. Weinhart, I. Grunwald, M. Wyszogrodska, L. Gaetjen, A. Hartwig, R. Haag

Chem. Asian J., 2010, 5(9), 1992-2000.


The nonspecific interaction of proteins with surfaces in contact with biofluids leads to adverse problems and is prevented by a biocompatible surface coating. The current benchmark material among such coatings is poly(ethylene glycol) (PEG). Herein, we report on the synthesis of linear polyglycerol derivatives as promising alternatives to PEG. Therefore, gold surfaces as a model system are functionalized with a self-assembled monolayer (SAM) by a two-step anhydride coupling and a direct thiol immobilization of linear poly(methyl glycerol) and polyglycerol. Surface plasmon resonance (SPR) spectroscopy reveals both types of functionalized surfaces to be as resistant as PEG towards the adsorption of the test proteins fibrinogen, pepsin, albumin, and lysozyme. Moreover, linear polyglycerols adsorb even less proteins from human plasma than a PEG-modified surface. Additional cell adhesion experiments on linear poly(methyl glycerol) and polyglycerol-modified surfaces show comparable cell resistance as for a PEG-modified surface. Also, in the case of long-term stability, high cell resistance is observed for all samples in medium. Additional in vitro cell-toxicity tests add to the argument that linear poly(methyl glycerol) and polyglycerol are strong candidates for promising alternatives to PEG, which can easily be modified for biocompatible functionalization of other surfaces.

A New Family of Nonionic Dendritic Amphiphiles Displaying Unexpected Packing Parameters in Micellar Assemblies

B. Trappmann, K. Ludwig, M. R. Radowski, A. Shukla, A. Mohr, H. Rehage, C. Böttcher und R. Haag

J. Am. Chem. Soc. 2010, 132 (32), 11119–11124 ...

In this paper we report on the synthesis of a new family of nonionic dendritic amphiphiles that self-assemble into defined supramolecular aggregates. Our approach is based on a modular architecture consisting of different generations of hydrophilic polyglycerol dendrons [G1−G3] connected to hydrophobic C11 or C16 alkyl chains via mono- or biaromatic spacers, respectively. All amphiphiles complex hydrophobic compounds as demonstrated by solubilization of Nile Red or pyrene. The structure of the supramolecular assemblies as well as the aggregation numbers are strongly influenced by the type of the dendritic headgroup. While the [G1] amphiphiles form different structures such as ringlike and fiberlike micelles, the [G2] and [G3] derivatives aggregate toward spherical micelles of low polydispersity clearly proven by transmission electron microscopy (TEM) measurements. In the case of the biaromatic [G2] derivative, the structural persistence of the micelles allowed a three-dimensional structure determination from the TEM data and confirmed the aggregation number obtained by static light scattering (SLS) measurements. On the basis of these data, molecular packing geometries indicate a drastic mass deficit of alkyl chains in the hydrophobic core volume of spherical micelles. It is noteworthy that these highly defined micelles contain as little as 15 molecules and possess up to 74% empty space. This behavior is unexpected as it is very different from classical detergent micelles such as sodium dodecyl sulfate (SDS), where the hydrophobic core volume is completely filled by alkyl chains.

Complexes of Click-Derived Bistriazolylpyridines: Remarkable Electronic Influence of Remote Substituents on Thermodynamic Stability as well as Electronic and Magnetic Properties.

M. Ostermeier, M.-A. Berlin, R. Meudtner, S. Demeshko, F. Meyer, C. Limberg, S. Hecht

Chem. Eur. J. 2010, 16, 10202-10213.


2,6-Bis(1,2,3-triazol-4-yl)pyridine (btp) ligands with substitution patterns ranging from strongly electron-donating to strongly electron-accepting groups, readily prepared by means of Cu-catalyzed 1,3-dipolar cycloaddition (the “click” reaction), were investigated with regard to their complexation behavior, and the properties of the resulting transition-metal compounds were compared. Metal–btp complexes of 1:1 stoichiometry, that is, [Ru(btp)Cl2(dmso)] and [Zn(btp)Br2], could be isolated and were crystallographically characterized: they display octahedral and trigonal-bipyramidal coordination geometries, respectively, and exhibit high aggregation tendencies due to efficient π–π stacking leading to low solubilities. Metal–btp complexes of 1:2 stoichiometry, that is, [Fe(btp)2]2+ and [Ru(btp)2]2+, could also be synthesized and their metal centers show the expected octahedral coordination spheres. The iron compounds exhibit quite a complex magnetic behavior in the solid state including spin crossover near room temperature, and hysteresis and locking into high-spin states on tempering at 400 K, depending on the substituents on the btp ligands. Cyclic voltammetry studies of [Ru(btp)2]2+ reveal strong modulation of the oxidation potentials by more than 0.6 V and a clear linear correlation to the Hammett constant (σpara) of the substituent at the pyridine core. Isothermal titration calorimetry was used to measure the thermodynamics of the FeII–btp complexation process and enabled accurate determination of the complexation enthalpies, which display a linear relationship with the σpara values for the terminal phenyl substituents. Detailed NMR spectroscopic studies finally revealed that in the case of FeII complexation, dynamics are rapid for all investigated btp derivatives in acetonitrile, while replacing FeII by RuII or changing the solvent to dichloromethane effectively slows down ligand exchange. The results nicely demonstrate the utility of substituent parameters, originally developed for linear free-energy relationships to explain reactivity in organic reactions, in coordination chemistry, and to illustrate the potential to custom-design btp ligands and complexes thereof with predictable properties. The fast equilibration of the [Fe(btp)2]2+ complexes together with their tunable stability and interesting magnetic properties should enable the design of dynamic metallosupramolecular materials with advantageous properties.

From Peptides to their Alternating Ester-Urea Analogues: Synthesis and Influence of Hydrogen-Bonding Motif and Stereochemistry on Aggregation.

S. Hartwig, J. Schwarz, S. Hecht

J. Org. Chem. 2010, 75, 772-782.


Peptide-mimicking scaffolds with an incorporated ester-urea motif, replacing two adjacent amide residues, were synthesized and their aggregation behavior was studied in dependence of hydrogen bonding sites as well as backbone stereochemistry. Two oligomer series containing either 50% or 100% ester-urea units and either all-(l) or (d)-alt-(l) backbone configuration were prepared via ester and amide couplings, using a divergent/convergent exponential growth strategy. Their aggregation behavior in organic solution was investigated by means of concentration-dependent NMR spectroscopy and compared to the parent peptide series. Interestingly, the naturally occurring peptide scaffold exhibits the largest tendency to associate in combination with the strongest difference in aggregation behavior between all-(l) and (d)-alt-(l) backbone stereochemistry. With increasing incorporation of the ester-urea motif the aggregation strength decreases and become much less dependent on the backbone configuration. The obtained structure−aggregation relationships reveal the importance of the commensurability and multivalency of hydrogen bonding sites as well as conformational restriction for peptide association and should hence aid the design of peptide mimics, such as β-sheet breakers or gelators.

Polypseudopeptides with Variable Stereochemistry: Synthesis via Click-Chemistry, Postfuncionalization, and Conformational Behavior in Solution.

S. Hartwig, S. Hecht

Macromolecules 2010, 43, 242-248.


Polypseudopeptides with well-defined stereochemistries have been synthesized from readily available amino-acid-based building blocks by connecting (l,l)- or (l,d)-dipeptide AB-monomers carrying azide and alkyne termini via triazole amide-isosteres efficiently formed in the course of the “click” reaction. Deprotection of the thus-prepared lysine-based polypseudopeptides of both all-(l)- and (d)-alt-(l)-stereochemistries afforded water-soluble polymers with ionizable amino side chains, which could be fully labeled with pyrene chromophores via quantitative amide bond formation. The conformational behavior of the deprotected as well as the pyrene-labeled polymers was investigated using UV/vis, CD, and fluorescence spectroscopies. On one hand, the free polyamines display pH-dependent conformations in water. On the other hand, the pyrene-labeled polypseudopeptides change their conformation in response to varying organic solvent composition. Whereas the strictly alternating polypseudopeptides structurally resemble channel-forming peptides, such as the Gramicidin family, the incorporation of (d)-configured amino acids as well as triazole amide-isosteres should lead to interesting new materials for bioapplications.

Exponential growth of functional poly(glutamic acid) dendirmers with variable stereochemistry.

S. Hartwig, M.M. Nguyen, S. Hecht

Polym. Chem. 2010, 1, 69-71.


Polyglutamate dendrimers up to the fourth generation have been prepared via an accelerated iterative divergent/convergent binomial synthesis, which allows incorporation of either all-(L) or (D-alt-L) stereochemistry in the peptide backbone and enables versatile postfunctionalization of the dendritic core and periphery.

Dendritic polyglycerol sulfates as multivalent inhibitors of inflammation.

J. Dernedde, A. Rausch, M. Weinhart, S. Enders, R. Tauber, K. Licha, M. Schirner, U. Zügel, A. von Bonin, R. Haag

PNAS 2010, 107(46), 19679-19684.


Adhesive interactions of leukocytes and endothelial cells initiate leukocyte migration to inflamed tissue and are important for immune surveillance. Acute and chronic inflammatory diseases show a dysregulated immune response and result in a massive efflux of leukocytes that contributes to further tissue damage. Therefore, targeting leukocyte trafficking may provide a potent form of anti-inflammatory therapy. Leukocyte migration is initiated by interactions of the cell adhesion molecules E-, L-, and P-selectin and their corresponding carbohydrate ligands. Compounds that efficiently address these interactions are therefore of high therapeutic interest. Based on this rationale we investigated synthetic dendritic polyglycerol sulfates (dPGS) as macromolecular inhibitors that operate via a multivalent binding mechanism mimicking naturally occurring ligands. dPGS inhibited both leukocytic L-selectin and endothelial P-selectin with high efficacy. Size and degree of sulfation of the polymer core determined selectin binding affinity. Administration of dPGS in a contact dermatitis mouse model dampened leukocyte extravasation as effectively as glucocorticoids did and edema formation was significantly reduced. In addition, dPGS interacted with the complement factors C3 and C5 as was shown in vitro and reduced C5a levels in a mouse model of complement activation. Thus, dPGS represent an innovative class of a fully synthetic polymer therapeutics that may be used for the treatment of inflammatory diseases.

SnapShot: Endocytic Trafficking.

M. Wieffer, T. Maritzen, V. Haucke

Cell 2009, 137, 382 ...

Lysosomal localization of GLUT8 in the testis – the EXXXLL motif of GLUT8 is sufficient for its intracellular sorting via AP1- and AP2-mediated interaction

M.K. Diril, S. Schmidt, M. Krauß, V. Gawlik, H.-G. Joost, A. Schürmann, V. Haucke, R. Augustin

FEBS Journal, 2009, 276, 3729-3743 ...

The class III sugar transport facilitator GLUT8 co-localizes with the lysosomal protein LAMP1 in heterologous expression systems. GLUT8 carries a [D/E]XXXL[L/I]-type dileucine sorting signal that has been postulated to retain the protein in an endosomal/lysosomal compartment via interactions with clathrin adaptor protein (AP) complexes. However, contradictory findings have been described regarding the subcellular localization of the endogenous GLUT8 and the adaptor proteins that interact with its dileucine motif. Here we demonstrate that endogenous GLUT8 is localized in a late endosomal/lysosomal compartment of spermatocytes and spermatids, and that the adaptor complexes AP1 and AP2, but not AP3 or AP4, interact with its N-terminal intracellular domain (NICD). In addition, fusion of the GLUT8 NICD to the tailless lumenal domain of the IL-2 receptor alpha chain (TAC) protein (interleukin-2 receptor α chain) targeted the protein to intracellular membranes, indicating that its N-terminal dileucine signal is sufficient for endosomal/lysosomal targeting of the transporter. The localization and targeting of GLUT8 show striking similarities to sorting mechanisms reported for lysosomal proteins. Therefore, we suggest a potential role for GLUT8 in the so far unexplored substrate transport across intracellular membranes.

Dynamic, template-assisted strategies in fragment-based drug discovery

M. Schmidt, J. Rademann

Trends in Biotechnol. 2009, 27, 512-521 ...

Fragment-based methods for drug discovery are increasingly popular because they provide drug leads with greater ligand efficiency than conventional high-throughput screening. However, established methods for fragment detection do not address the central question in fragment-based ligand discovery: how can a primary ligand be optimally extended by a secondary fragment? Dynamic screening methods solve this issue by using a protein target as a template for ligand assembly, thus yielding high-affinity binders from low-affinity fragments. This review summarizes recent work on dynamic screening methodology, which resulted in the development of several high-affinity binders for various targets. Strengths and limitations of the published approaches are discussed and possible contributions of dynamic screening methodology to the drug discovery process are highlighted.

Energetics of the loop-to-helix transition leading to the coiled-coil structure of influenza virus hemagglutinin HA2 subunits

Q. Huang, T. Korte, S. Rachakonda, E. W. Knapp, A. Herrmann

Proteins 2009, 74, 291-303 ...

Fusion of influenza virus with the endosomal membrane of the host cell is mediated by the homotrimer-organized glycoprotein hemagglutinin (HA). Its fusion activity is triggered by a low pH-mediated conformational change affecting the structure of the HA1 and HA2 subunits. The HA2 subunits undergo a loop-to-helix transition leading to a coiled-coil structure, a highly conserved motif for many fusion mediating viral proteins. However, experimental studies showed that the HA2 coiled-coil structure is stable at neutral and low pH, implying that there is no direct relationship between low pH and the HA2 loop-to-helix transition. To interpret this observation, we used a computational approach based on the dielectric continuum solvent model to explore the influence of water and pH on the free energy change of the transition. The computations showed that the electrostatic interaction between HA2 fragments and water is the major driving force of the HA2 loop-to-helix transition leading to the coiled-coil structure, as long as the HA1 globular domain covering the HA2 subunits in the nonfusion competent conformation is reorganized and thereby allows water molecules to interact with the whole loop segments of the HA2 subunits. Moreover, we show that the energy released by the loop-to-helix transition may account for those energies required for driving the subsequent steps of membrane fusion. Such a water-driven process may resemble a general mechanism for the formation of the highly conserved coiled-coil motif of enveloped viruses.

Controlled Assembly of Vesicle-based Nanocontainers on Layer-by-Layer Particles via DNA Hybridization

M. Loew, J. Kang, L. Dähne, R. Hendus-Altenburger, O. Kaczmarek, J. Liebscher, D. Huster, K. Ludwig, C. Böttcher, A. Herrmann, A. Arbuzova

SMALL 2009, 5(3), 320-323 ...

Microscopic colloidal particles allow a precise regulation of chemical reactions in time and place. A controlled assembly of multiple layers of intact lipid vesicles on a solid support provided by layer-by-layer particles functionalized by a covalent attachment of DNA oligonucleotides is reported (see image). Lipophilic complementary oligonucleotides are incorporated into lipid vesicles. Fusion of liposomes and release can be triggered.

Molecular Rods with Oligospiroketal Backbone as Anchors in Biological Membranes

P. Müller, J. Nikolaus, S. Schiller, A. Herrmann, K. Möllnitz, S. Czapla, P. Wessig

Angew. Chem. Int.Ed. 2009, 48, 4433-4435 ...

Getting stuck in: A hydrophobic molecular rod with terminal fluorescent moieties has been synthesized. The insertion of the rod into membranes was investigated and shown to incorporate efficiently into model and biological membranes (see picture; gray C, blue N, red O). Those rods can be used as stable membrane-associated anchors for functionalization of membrane surfaces.

Lateral Distribution of the Transmembrane Domain of Influenza Virus Hemagglutinin Revealed by Time-resolved Fluorescence Imaging

S. Scolari, S. Engel, N. Krebs, A.P. Plazzo, R.F.M. De Almeida, M. Prieto, M. Veit, A. Herrmann

J. Biol. Chem. 2009, 284, 15708-15718

Influenza virus hemagglutinin (HA) has been suggested to be enriched in liquid-ordered lipid domains named rafts, which represent an important step in virus assembly. We employed Förster resonance energy transfer (FRET) via fluorescence lifetime imaging microscopy to study the interaction of the cytoplasmic and transmembrane domain (TMD) of HA with agly co sylphos pha tidyl ino si tol (GPI)-anchored peptide, an established marker for rafts in the exoplasmic leaflet of living mammalian plasma membranes. Cyan fluorescent protein (CFP) was fused to GPI, whereas the HA sequence was tagged with yellow fluorescent protein (YFP) on its exoplasmic site (TMD-HA-YFP), avoiding any interference of fluorescent proteins with the proposed role of the cytoplasmic domain in lateral organization of HA. Constructs were expressed in Chinese hamster ovary cells (CHO-K1) for which cholesterol-sensitive lipid nanodomains and their dimension in the plasma membrane have been described (Sharma, P., Varma, R., Sarasij, R. C., Ira, Gousset, K., Krishnamoorthy, G., Rao, M., and Mayor, S. (2004) Cell 116, 577–589). Upon transfection in CHO-K1 cells, TMD-HA-YFP is partially expressed as a dimer. Only dimers are targeted to the plasma membrane. Clustering of TMD-HA-YFP with GPI-CFP was observed and shown to be reduced upon cholesterol depletion, a treatment known to disrupt rafts. No indication for association of TMD-HA-YFP with GPI-CFP was found when palmitoylation, an important determinant of raft targeting, was suppressed. Clustering of TMD-HA-YFP and GPI-CFP was also observed in purified plasma membrane suspensions by homoFRET. We concluded that the pal mit oy lated TMD-HA alone is sufficient to recruit HA to cholesterol-sensitive nanodomains. The corresponding construct of the spike protein E2 of Semliki Forest virus did not partition preferentially in such domains.

Selective identification of cooperatively binding fragments in a high-throughput assay enables development of a picomolar caspase-3 inhibitor

M. Schmidt, A. El-Dahshan, S. Keller, J. Rademann

Angew. Chem. Int. Ed. 2009, 48, 6346-6349 ...

A potential anti-SARS drug has been developed by dynamic ligation screening (DLS), by which nucleophilic fragments are directed to the protein's active site by reversible reaction with an aldehyde inhibitor. Their inhibitory effect is detected by competition with a fluorogenic enzyme substrate. With this concept, low-affinity fragments binding specifically to the active site are quickly identified in a functional enzyme assay.

Selektive Detektion kooperativ bindender Fragmente in einem Hochdurchsatz-Ligationsassay zur Entwicklung eines pikomolaren Caspase-3-Inhibitors

M. Schmidt, A. El-Dahshan, S. Keller, J. Rademann

Angew. Chem. 2009, 121, 6464-6467 ...

Mit Fragmenten von Kooperativität zu Affinität: Eine chemisch reaktive Fluoreszenzpolarisations(FP)-Sonde ermöglicht die Detektion von kooperativen Fragmenten über die mehr als additive Bindung ihrer Ligationsprodukte. Zur Bestätigung wurde ein stabiles Derivat des Ligationsprodukts hergestellt, das aktiver war als alle bisher bekannten Inhibitoren von Caspase-3.

Metallfreie regioselektive Triazol-Ligationen liefern konformativ fixierte cis-Peptidmimetika

Ahsanullah, P. Schmieder, R. Kühne, J. Rademann

Angew. Chem. 2009, 121, 5143-5147 ...

Metallfreie Triazolschleifen: 1,5-Disubstituierte Peptidyltriazole werden regioselektiv durch eine 1,3-dipolare Cycloaddition von Peptidylphosphoranen und Aziden erhalten. So entstehen Peptidschleifen, die eine konformativ fixierte cis-Peptidbindung enthalten. Da die Reaktion regioselektiv und ohne Schwermetallzusatz verläuft, könnte sie Anwendung in der chemischen Biologie und der medizinischen Chemie finden.

Ein Selbstordnungsschema für Tetraharnstoffcalix[4]arene.

Y. Rudzevich, V. Rudzevich, F. Klautzsch, C. A. Schalley, V. Böhmer

Angew. Chem 2009, 121, 3925-3929.


Größe und Form sind wichtig: Eine äquimolare Mischung aus elf unterschiedlich substituierten Tetraharnstoff-Calix[4]arenen bildet in unpolaren Lösungsmitteln nur 6 von 35 möglichen Homo- und Heterodimeren. Da das Calixarengerüst und die vier Harnstoffreste in allen Fällen gleich sind, wird dieser Prozess der Selbstorganisation allein durch das Zusammenwirken von sterischen und stöchiometrischen Randbedingungen bestimmt.

Conformational Flexibility of Tetralactam Macrocycles and their Intermolecular Hydrogen-Bonding Patterns in the Solid State.

S.S. Zhu, M. Nieger, J. Daniels, T. Felder, I. Kossev, T. Schmidt, M. Sokolowski, F. Vögtle, C.A. Schalley

Chem. Eur. J. 2009, 15, 5040-5046.


Flexible rigidity: Tetralactam macrocycles of the Hunter type bear a rigid scaffold (see space-filling representation), but can still widely adapt to the properties of a guest molecule inside their cavities. X-ray crystal structures of a series of differently substituted macrocycles reveal a remarkably broad variety of intermolecular hydrogen-bonding patterns organizing the macrocycles in the crystals in intriguingly different ways. Despite their rigid scaffold, tetralactam macrocycles (TLMs) display a remarkable degree of conformational flexibility, as revealed by analysis of the corresponding X-ray crystal structures. This flexibility is not limited to the rotatability of the TLM amide groups but also applies to the m-xylene rings, and it thus has a great impact on the overall shape of the macrocycle cavity. The conformational properties of the TLMs give rise to a broad variety of intermolecular hydrogen-bonding patterns, including infinite ladders, an interesting catemer motif, and short C[BOND]H⋅⋅⋅O[DOUBLE BOND]C hydrogen bonds. These results are in accord with previous theoretical calculations, support a structural model proposed earlier for an interpretation of scanning tunneling microscopy images, and substantially contribute to the understanding of the adaptability of macrocyclic scaffolds, which is crucial for guest binding or templated syntheses with TLMs.

Unusual Enantiopure Heterocyclic Skeletons via Lewis Acid Promoted Rearrangements of 1,3-Dioxolanyl-Substituted 1,2-Oxazines

F. Pfrengle, A. Al-Harrasi, I. Brüdgam, H.-U. Reissig

Eur. J. Org. Chem. 2009, 2, 282 - 291


Lewis acid promoted rearrangements of different 4-alkoxy-substituted 1,2-oxazines syn-1 are reported. Depending on the nature of this alkoxy group different reaction pathways are possible either providing bicyclic 1,2-oxazinones 2 or the novel tricyclic products 3–5. A mechanistic rational describing the role of the 4-alkoxy group is presented. The key step for formation of tricyclic skeletons 3–5 is a 1,2-alkyl shift. Hydrogenation reactions of these tricyclic compounds gave unsaturated 1,2-oxazines 12 and 13 or tetrahydrofurans 15a–c/16a–c depending on the time of hydrogenolysis. Tetrahydrofuryl-annulated 1,2-oxazine 12 was used for further transformations into complex substituted tetrahydrofurans. Reduction with sodium cyanoborohydride and subsequent cleavage of the N,O-bond by hydrogenation furnished aminofuran derivative 19. Alternatively treatment with a strong base such as n-butyllithium afforded imidoester 21 via a Beckmann-type fragmentation.

Metal-free, regioselective triazole ligations deliver locked cis-peptide mimetics

Ahsanullah, P. Schmieder, R. Kühne, J. Rademann

Angew. Chem. Int. Ed. 2009, 48, 5042-5045 ...

Metal-free triazole turns: 1,5-Disubstituted peptidyl triazoles are obtained regioselectively from the 1,3-dipolar cycloaddition of peptidyl phosphoranes and azides. Peptide turns are thus formed that contain a conformationally locked cis peptide bond. Being regioselective and free of heavy metals, this reaction may find broad application in chemical biology and medicinal chemistry.

Integrative self-sorting is a programming language for high level self-assembly.

W. Jiang, C.A. Schalley

Proc. Natl. Acad. Sci 2009, 106, 10425-10429.


Starting from the basis of a simple 4-component self-sorting system of crown ethers and ammonium ions, we design 6 building blocks in which 2 identical or different binding sites are incorporated. These building blocks can be mixed in many different ways to yield quite distinctly different pseudorotaxane assemblies. The self-sorting process integrates all building blocks in specific places so that this approach permits us to exert positional control and can widely influence the resulting assemblies with respect to the details of their structures. At maximum, we report quadruply interlocked species with up to 5 subunits that form specific assemblies. Although NMR methods are limited to the analysis of simpler complexes, ESI-MS and, in particular, tandem mass spectrometry is highly useful to analyze the assemblies' connectivities.

Chemoselektive Staudinger-Phosphit-Reaktion von Aziden für die Phosphorylierung von Proteinen

R. Serwa, I. Wilkening, G. del Signore, M. Mühlberg, I. Claußnitzer, C. Weise, M. Gerrits, C.P.R. Hackenberger

Angew. Chem. 2009, 121, 8382-8387 ...

Hohe Ausbeuten bei Raumtemperatur werden mit der Titelreaktion zur Modifizierung von Peptiden und Proteinen in einer Reihe von Lösungsmitteln, darunter Puffer bei physiologischem pH-Wert, erzielt. In Kombination mit nichtnatürlicher Proteintranslation ermöglicht die Reaktion die ortsspezifische Einführung phosphorylierter Tyr-Analoga in Proteine

Chemoselective Staudinger-Phosphite Reaction of Azides for the Phosphorylation of Proteins

R. Serwa, I. Wilkening, G. del Signore, M. Mühlberg, I. Claußnitzer, C. Weise, M. Gerrits, C.P.R. Hackenberger

Angew. Chem. Int. Ed. 2009, 47, 8234-8239 ...

Extending the toolbox: The title reaction was identified as a chemoselective means to modify azides in peptides and proteins in high yields at room temperature in various solvents including aqueous buffers at physiological pH. In combination with nonnatural protein translation the Staudinger-phosphite reaction allows the site-specific incorporation of phosphorylated Tyr analogues in proteins.

Uncovering Individual Hydrogen Bonds in Rotaxanes by Frequency Shifts

B. Kirchner, C. Spickermann, W. Reckien, C.A. Schalley

J. Am. Chem. Soc. 2010, 132, 484-494


We present a theoretical investigation of amide pseudorotaxane IR spectra in the harmonic approximation. In particular, we focus on the effect of axle substitution on the hydrogen bonds that are formed between axle and wheel. Two types of pseudorotaxanes are studied: one with the substituent affecting mostly the axle’s carbonyl group and one with the effect influencing primarily the amide NH group. Sizeable red shifts are predicted for the carbonyl stretching frequencies, and large red shifts for the NH stretching frequencies. For the wheel amide groups involved in hydrogen bonding merely with their NH hydrogens, a small shift is observed for the carbonyl stretch mode. A clear relation is observed between the NH stretch shifts and individual hydrogen bond energies. This is confirmed by correlations of the shared electron number with the NH stretch shift showing that this quantity can be taken as an indicator for individual hydrogen bond energies. Axle substitution influences the strengths of the individual hydrogen bonds which is again reflected in the NH stretch frequency shifts. A linear relationship of Hammett’s substituent parameters with the NH frequency shifts can be established.

Inhibition of selectin binding by colloidal gold with functionalized shells.

J. Dernedde, S. Enders, H.-U. Reissig, M. Roskamp, S. Schlecht, S. Yekta

Chem. Comm. 2009, 8, 932-934


Gold colloids with terminally functionalized sulfated thiol shells were found to bind to P- and L-selectins with IC50 values in the picomolar range; branched acyclic epitopes show the highest affinity, whereas a sulfated carbohydrate mimetic provides the best selectivity.

Qualitative detection of single submicron and nanoparticles in human skin by scanning transmission x-ray microscopy.

C. Graf, M. Meinke, Q. Gao, S. Hadam, J. Raabe, W. Sterry, U. Blume-Peytavi, J. Lademann, E. Rühl, A. Vogt

J. Biomed. Opt. 2009, 14(2), 021015/9.


First results on single particle detection in human skin samples by x-ray microscopy are reported. 94±6 and 161±13nm gold core particles with silica shells and 298±11nm silica particles coated with a gold shell on ultramicrotome sections of human skin were determined. The particles were applied on fresh intact skin samples, which were sectioned prior to imaging. After screening the sections by conventional microscopy techniques, defined areas of interest were qualitatively investigated by scanning transmission x-ray microscopy at the Swiss Light Source. In studies on the percutaneous penetration of 161±13nm particles on human skin samples, x-ray microscopy yielded high-resolution images of single particles spreading on the superficial layer of the stratum corneum and on the epithelium in superficial parts of hair follicles. No deeper penetration was observed. The present work using x-ray microscopy provides the unique opportunity to study qualitative penetration processes and membrane-particle interactions on the level of single particles. This goes beyond present approaches using optical microscopy. Further improvement of this approach will allow one to study particles with different physicochemical properties and surface modifications, including responses of the exposed tissue.

Highly Functionalised Enantiopure 4-Hydroxypyridine Derivatives by a Versatile Three-Component Synthesis.

C. Eidamshaus, H.-U. Reissig

Adv. Synth. Catal. 2009, 351, 1162-1166.


The scope of a novel alkoxyallene-based pyridine synthesis was expanded to enantiopure carboxylic acids and nitriles as starting materials. The enantiomeric purity of the chiral α-secondary carboxylic acids and nitriles was completely preserved during this reaction sequence thus allowing the one-pot preparation of a whole range of 4-hydroxypyridines or their 4-pyridinone tautomers in good yields.

Three-Component Synthesis of Highly Functionalized 5-Acetyloxazoles.

T. Lechel, D. Lentz, H.-U. Reissig

Chemistry-A European Journal 2009, 15, 5432-5435.


By a flexible three-component synthesis, alkoxy-substituted enamides are easily available from lithiated alkoxyallenes, nitriles and carboxylic acids (see scheme). The treatment of these versatile intermediates with trifluoroacetic acid provided 5-acetyloxazoles in moderate to good yields. Different substituents are possible at C-2 and C-5 and the 5-acetyl group is a suitable handle for further synthetic transformations.

Magnetic and Structural Investigation of ZnSe Semiconductor Nanoparticles Doped With Isolated and Core-Concentrated Mn2R Ions

C. Graf, A. Hofmann, T. Ackermann, C. Boeglin, R. Viswanatha, X. Peng, A. F. Rodríguez, F. Nolting, E. Rühl

Adv. Funct. Mater. 2009, 19, 2501-2510.


X-Ray magnetic circular dichroism (XMCD) experiments on diluted magnetic semiconductor nanocrystals (2–7 nm) are reported in order to study their local electronic structure and magnetic properties. ZnSe nanoparticles containing either single manganese ions (Mn2+) distributed in the lattice of the entire particle or a MnSe core in the center are prepared using high temperature approaches. The Mn2+ concentration is varied between less than one to several tens of manganese ions per nanocrystal. For all samples it is shown that the Mn2+ is exclusively present in the bulk of ZnSe nanoparticles with no evidence for oxidation to higher Mn-oxidation states. The magnetic ions are highly polarized inside the nanocrystals reaching about 80% of the theoretical value of a pure d5 state under identical conditions for the case of isolated manganese ions. Nanocrystals with a MnSe core ZnSe shell structure reach <50% of this value. Thus, their polarization is significantly more hindered, which is due to the significantly enhanced Mn–Mn interactions and a more distorted crystalline lattice. In contrast, no coupling between the manganese centers is observed in the nanoparticles doped samples with low concentrations of Mn2+, indicating that these ions are isolated in the bulk of the nanoparticles.

Proline-rich sequence recognition II: proteomic analysis of TSG101-UEV interactions

A. Schlundt, J. Sticht, K. Piotukh, D. Kosslick, N. Jahnke, S. Keller, M. Schuemann, E. Krause, C. Freund

Mol. Cell. Proteom. 2009, 8, 2474-2486 ...

The tumor maintenance protein Tsg101 has recently gained much attention because of its involvement in endosomal sorting, virus release, cytokinesis, and cancerogenesis. The ubiquitin-E2-like variant (UEV) domain of the protein interacts with proline-rich sequences of target proteins that contain P(S/T)AP amino acid motifs and weakly binds to the ubiquitin moiety of proteins committed to sorting or degradation. Here we performed peptide spot analysis and phage display to refine the peptide binding specificity of the Tsg101 UEV domain. A mass spectrometric proteomics approach that combines domain-based pulldown experiments, binding site inactivation, and stable isotope labeling by amino acids in cell culture (SILAC) was then used to delineate the relative importance of the peptide and ubiquitin binding sites. Clearly “PTAP” interactions dominate target recognition, and we identified several novel binders as for example the poly(A)-binding protein 1 (PABP1), Sec24b, NFκB2, and eIF4b. For PABP1 and eIF4b the interactions were confirmed in the context of the corresponding full-length proteins in cellular lysates. Therefore, our results strongly suggest additional roles of Tsg101 in cellular regulation of mRNA translation. Regulation of Tsg101 itself by the ubiquitin ligase TAL (Tsg101-associated ligase) is most likely conferred by a single PSAP binding motif that enables the interaction with Tsg101 UEV. Together with the results from the accompanying article (Kofler, M., Schuemann, M., Merz, C., Kosslick, D., Schlundt, A., Tannert, A., Schaefer, M., Lührmann, R., Krause, E., and Freund, C. (2009) Proline-rich sequence recognition: I. Marking GYF and WW domain assembly sites in early spliceosomal complexes. Mol. Cell. Proteomics 8, 2461–2473) on GYF and WW domain pathways our work defines major proline-rich sequence-mediated interaction networks that contribute to the modular assembly of physiologically relevant protein complexes.

Efficient Simulation of Ligand-Receptor Binding Processes Using the Conformation Dynamics Approach

A. Bujotzek, M. Weber

J. Bioinform. Comput. Biol., 2009, 7 (5), 811-831 ...

The understanding of biological ligand–receptor binding processes is relevant for a variety of research topics and assists the rational design of novel drug molecules. Computer simulation can help to advance this understanding, but, due to the high dimensionality of according systems, suffers from the severe computational cost. Based on the framework provided by conformation dynamics and transition state theory, a novel heuristic approach of simulating ligand–receptor binding processes is introduced, which is not dependent on calculating lengthy molecular dynamics trajectories. First, the relevant portion of conformational space is partitioned with meshless methods. Then, each region is sampled separately, using hybrid Monte Carlo. Finally, the dynamical binding process is reconstructed from the static overlaps between the partial densities obtained in the sampling step. The method characterizes the metastable steps of the binding process and can yield the corresponding transition probabilities.

Proline-rich sequence recognition I: Marking GYF and WW domain assembly sites in early spliceosomal complexes

M. Kofler, M. Schümann, C. Merz, D. Kosslick, A. Schlundt, A. Tannert,. M. Schaefer, R. Lührmann, E. Krause, C. Freund

Mol. Cell. Proteom. 2009, 8, 2461-2473 ...

Proline-rich sequences (PRS) and their recognition domains have emerged as transposable protein interaction modules during eukaryotic evolution. They are especially abundant in proteins associated with pre-mRNA splicing and likely assist in the formation of the spliceosome by binding to GYF and WW domains. Here we profile PRS-mediated interactions of the CD2BP2/52K GYF domain by a site-specific peptide inhibitor and stable isotope labeling/mass spectrometry analysis. Several PRS hubs with multiple proline-rich motifs exist that can recruit GYF and/or WW domains. Saturating the PRS sites by an isolated GYF domain inhibited splicing at the level of A complex formation. The interactions mediated by PRS are therefore important to the early phases of spliceosomal assembly.

A New Ring Closure Approach to Enantiopure 3,6-Dihydro-2H-pyrans: Stereodivergent Access to Carbohydrate Mimetics

F. Pfrengle, D. Lentz, H.-U. Reissig

Org. Lett. 2009, Vol. 11, No. 23, 5534-5537 ...

A set of enantiopure carbohydrate mimetics has been synthesized via Lewis acid promoted cyclization of 1,3-dioxolanyl-substituted enol ethers as a crucial new step providing highly functionalized 3,6-dihydro-2H-pyran derivatives. The flexible approach starting from glyceraldehyde-derived nitrone is comprised of only six simple steps smoothly allowing synthetic modifications at the different stages of the sequence. All reactions proceeded with good to excellent stereocontrol and can be performed with either of the two enantiomers.

Switchable electrostatic interactions between gold nanoparticles and coiled coil peptides direct colloid assembly.

S.C. Wagner, M. Roskamp, C. Böttcher, S. Schlecht, B. Koksch

Org. Biomol. Chem 2009, 7, 46-51.


The nanoparticle–peptide interaction described here is based on electrostatic forces and the pH value can act as a trigger to direct the organization of functionalized nanoparticles in a reversible and repeatable manner. The ability of the peptide to interact with the charged gold nanoparticles is directly related to its helical structure and was not found for a random coil peptide with the same net charge. Interestingly, the interaction with nanoparticles seems to induce a fibrillation of the coiled coil peptide.

Highly dynamic motion of crown ethers along oligolysine peptide chains.

D.P. Weimann, H.D.F. Winkler, J.A. Falenski, B. Koksch, C.A. Schalley

Nat. Chem., 2009, 1(7), 573-577.


Molecular mobility has attracted considerable attention in supramolecular chemistry and biochemistry, but the simple question of whether a small molecule can move directly between different binding sites of a multitopic host without intermediate dissociation has not been addressed so far. To study such processes, we consider hydrogen/deuterium exchange experiments on a model system comprising complexes formed between 18-crown-6 and oligolysine peptides. Because direct binding-site hopping is indistinguishable in solution from a dissociation/reassociation mechanism, here we show that the high vacuum of a mass spectrometer offers a unique environment for probing such processes. The highly dynamic motion of crown ethers along oligolysine peptide chains proceeds mechanistically by a simultaneous transfer of the crown ether from its ammonium ion binding site to a nearby amino group together with a proton. Furthermore, the exchange experiments unambiguously reveal the zwitterionic structure of the 18-crown-6/oligolysine complexes, highlighting the versatility and potential of gas-phase experiments for investigating non-covalent interactions.

Enantiopure Aminopyrans by a Lewis Acid Promoted Rearrangement of 1,2-Oxazines: Versatile Building Blocks for Oligosaccharide and Sugar Amino Acid Mimetics

A. Al-Harrasi, F. Pfrengle, V. Prisyazhnyuk, S. Yekta, P. Koós, H.-U. Reissig

Chem. Eur. J. 2009, 15, 11632-11641 ...

1,3-Dioxolanyl-substituted 1,2-oxazines, such as syn-1 and anti-1, rearrange under Lewis acidic conditions to provide bicyclic products 2–5. Subsequent reductive transformations afforded enantiopure 3-aminopyran derivatives such as 7 and 9 or their protected diastereomers 16 and 18, which can be regarded as carbohydrate mimetics. An alternative sequence of transformations including selective oxidation of the primary hydroxyl groups in 21 and 24 led to two protected β-amino acid derivatives with carbohydrate-like backbone (sugar amino acids). Treatment of bicyclic ester 23 with samarium diiodide cleaved the N[BOND]O bond and furnished the unusual β-lactam 27 in excellent yield. Alternatively, γ-amino acid derivative 29 was efficiently prepared in a few steps. Fairly simple transformations gave azides 32 and 35 or alkyne 30 which are suitable substrates for the construction of oligosaccharide mimetics such as 34 by copper iodide catalyzed cycloadditions. With this report we demonstrate that enantiopure rearrangement products 2–5 are protected precursors of a variety of polyfunctionalized pyran derivatives with great potential for chemical biology.

Application of the Method of Increments to the Adsorption of H2S on Graphene

B. Paulus, K. Rosciszewski

Int. J. Quantum Chem. 2009, 109, 3055-3062 ...

The method of increments is applied to the adsorption energy of H2S on a graphene layer using a CCSD(T) correlation treatment. We determine the incremental expansion for the correlation contribution to the adsorption energy in terms of localized orbitals of the molecule and the surface. The changes in the correlation energy of the molecule and the individual localized surface orbitals are repulsive and small, the major contributions arise from the joint correlation of molecule and surface orbitals.

Stereodivergent De Novo Synthesis of Branched Amino Sugars by Lewis Acid Promoted Rearrangement of 1,2-Oxazines

F. Pfrengle, D. Lentz, H.-U. Reissig

Angew. Chem Int. Ed., 2009, 48, 3165-3169 ...

Well concealed: 1,2-oxazines such as 1 rearrange under Lewis acidic conditions to bicyclic products of type 2, which can be incorporated into oligosaccharides as protected amino sugar equivalents. Subsequent reductive steps provide unusual oligosaccharides 3 having C2-branched 4-amino sugar units. Most of the reactions proceed with excellent stereocontrol and allow the synthesis of a collection of stereoisomers.

Stereodivergente De-novo-Synthese verzweigter Aminozucker durch Lewis-Säure-induzierte Umlagerung von 1,2-Oxazinen

F. Pfrengle, D. Lentz, H.-U. Reissig

Angew. Chem., 2009, 121, 3211 - 3215 ...

Gut getarnt: 1,2-Oxazine wie 1 lagern unter Einwirkung von Lewis-Säuren in bicyclische Produkte vom Typ 2 um, die als geschützte Äquivalente von Aminozuckern in Oligosaccharide eingebaut werden können. Nachfolgende reduktive Schritte erzeugen ungewöhnliche Oligosaccharide 3 mit C-2-verzweigten 4-Aminozuckereinheiten. Die meisten Reaktionen erfolgen unter exzellenter Stereokontrolle und ermöglichen die Synthese einer Kollektion von Stereoisomeren.

DNA and RNA-Controlled Switching of Protein Kinase Activity.

L. Röglin, F. Altenbrunn, O. Seitz

ChemBioChem. 2009, 10, 758-765. ...

Constrained: The readily programmable nucleic acid mediated recognition is used to constrain a phosphopeptide that was flanked by PNA segments. RNA-based switching allows control over the activity of target enzymes such as the protein kinase Src. It might thus be feasible to transduce changes of the concentration of selected RNA molecules to changes of the activity of signal transduction proteins. Protein switches use the binding energy gained upon recognition of ligands to modulate the conformation and binding properties of protein segments. We explored whether the programmable nucleic acid mediated recognition might be used to design or mimic constraints that limit the conformational freedom of peptide segments. The aim was to design nucleic acid–peptide conjugates in which the peptide portion of the conjugate would change the affinity for a protein target upon hybridization. This approach was used to control the affinity of a PNA–phosphopeptide conjugate for the signal transduction protein Src kinase, which binds the cognate phosphopeptides in a linear conformation. Peptide–nucleic acid arms were attached to known peptide binders. The chimeric molecules were studied in three modes: 1) as single strands, 2) constrained by intermolecular hybridization (duplex formation) and 3) constrained by intramolecular hybridization (hairpin formation). Of note, duplexes that were designed to accommodate bulged peptide structures (for example, in hairpins or bulges) had lower binding affinities than duplexes in which the peptide was allowed to adopt a more relaxed conformation. Greater than 90-fold differences in binding affinities were observed. It was, thus, feasible to make use of DNA hybridization to reversibly switch from no to almost complete inhibition of Src-SH2–peptide binding, and vice versa. A series of DNA and PNA-based hybridization experiments revealed the importance of charges and conformational effects. Nucleic acid mediated switching was extended to the use of RNA; this enabled a regulation of the enzymatic activity of the Src kinase. The proof-of-principle results demonstrate for the first time that PNA–peptide chimeras can transduce changes of the concentration of a given RNA molecule to changes of the activity of a signal transduction enzyme.

Sugars, Alkaloids, and Heteroaromatics: Exploring Heterocyclic Chemistry with Alkoxyallenes.

M. Brasholz, H.-U. Reissig, R. Zimmer

Acc. Chem. Res 2009, 42 (1), 45-56 ...

As master craftsmen, modern synthetic chemists are challenged to achieve remarkable feats of efficiency and elegance toward molecular targets. The nature of this pursuit necessitates the collection of synthetic repertoires that are tried and true. With methodologies and pathways increasingly scrutinized, the adept chemist must seek out propitious tools to incorporate into the arsenal. With this in mind, this Account highlights the versatility of alkoxyallenes as precursors to valuable heterocyclic building blocks for such efforts as natural product synthesis. Accessed by the etherification of either propargyl alcohols or propargylic halides, alkoxyallenes are obtained after base-catalyzed isomerizations of the propargylic ethers. A host of umpolung synthons are available through this scheme after metalation, generating C3 nucleophiles synthetically equivalent to vital anionic and zwitterionic synthons. Reactions with a diverse set of heteroatomic electrophiles yield carbohydrates, spiroketals, alkaloids, and heteroaromatics via [3 + 2] or [3 + 3] cyclizations. By employing lithiated alkoxyallenes into transformation routes, the natural product chemist can utilize this methodology as a viable resource in stereoselective synthesis. A survey of our own utilization of alkoxyallenes along synthetic pathways toward natural product targets reveals their suitability for generating advantageous precursors. A set of four stereoisomeric 2,6-dideoxyhexoses were stereoselectively obtained after an initial lithiated alkoxyallene and lactaldehyde cyclization, followed by the oxidative ring opening of the dihydrofurans. Through the addition of a lithiated alkoxyallene to a functionalized benzaldehyde, an essential spiroketal diastereomer was rapidly achieved in a few steps. We greatly benefitted from alkoxyallenes in the construction of complex nitrogen-containing synthetic targets, whether pyrrolidine alkaloids, substituted imidazole derivatives, or functionalized pyridines. A pinnacle example of their utility came from the coupling of alkoxyallenes to nitrones affording 1,2-oxazines, which served as a gateway to an array of novel polyfunctionalized compounds such as aminopolyols, hydroxylated pyrrolidines, or carbohydrate mimetics. Alkoxyallenes have proven themselves to be powerful C3 building blocks toward complex molecular targets, revealing novel pathways to a variety of desirable highly functionalized heterocycles. In our view, the full extent of their synthetic utility has yet to be truly realized.

A Self-Sorting Scheme based on Tetra-Urea Calix[4]arenes.

Y. Rudzevich, V. Rudzevich, F. Klautzsch, C. A. Schalley, V. Böhmer

Angew. Chem. Int. Ed. 2009, 48, 3867-3871


Size and shape do matter: When dimerized in nonpolar solvents, an equimolar mixture of eleven tetra-urea calix[4]arenes with different wide-rim substituents self-sorts into only six out of 35 different homo- and heterodimers (see picture). Since the calixarene scaffold and the four urea units are the same in all cases, the self-sorting process is driven only by the cooperative action of steric requirements and stoichiometry.

Hyperbranched PEI with various oligosaccharide architectures: Synthesis, characterization, ATP complexation and cellular uptake properties.

D. Appelhans, H. Komber, M.A. Quadir, S. Richter, S. Schwarz, J. van der Vlist, A. Aigner, M. Müller, K. Loos, J. Seidel, K.-F. Arndt, R. Haag, B. Voit

Biomacromolecules 2009, 10(5), 1114-1124.


We present a rapid synthetic method for the development of hyperbranched PEIs decorated with different oligosaccharide architectures as carrier systems (CS) for drugs and bioactive molecules for in vitro and in vivo experiments. Reductive amination of hyperbranched PEI with readily available oligosaccharides results in sugar functionalized PEI cores with oligosaccharide shells of different densities. These core−shell architectures were characterized by NMR spectroscopy, elemental analysis, SLS, DLS, IR, and polyelectrolyte titration experiments. ATP complexation of theses polycations was examined by isothermal titration calorimetry to evaluate the binding energy and ATP/CS complexation ratios under physiological conditions. In vitro experiments showed an enhanced cellular uptake of ATP/CS complexes compared to those of the free ATP molecules. The results arise to initiate further noncovalent complexation studies of pharmacologically relevant molecules that may lead to the development of therapeutics based on this polymeric delivery platform.

Functional Nanoparticles from Dendritic Precursors: Hierarchical Assembly in Miniemulsion.

A. Sisson, I. Papp, K. Landfester, R. Haag

Macromolecules 2009, 42, 556-559.


Biocompatible Functionalized Polyglycerol Microgels with Cell Penetrating Properties

A. Sisson, D. Steinhilber, T. Rossow, P. Welker, K. Licha, R. Haag

Angew. Chem. Int. Ed. 2009, 48, 7540-7545.


Gels for cells: A new method to produce biocompatible polyglycerol microgel nanoparticles with controllable size (25–100 nm, see picture, top) and a readily functionalizable surface is developed. Fluorescence imaging shows clear evidence for particle uptake by cells through endocytotic pathways and localization of the particles in the perinuclear region.

Formation of chiral aggregates of tetralactam macrocycles on the Au(111) surface.

I. Kossev, T. Felder, C.A. Schalley, F. Vögtle, M. Sokolowski

Springer Proc. Phys. 2009, 127, 235-245.


Monolayers of a large tetralactam macrocycle were prepared by vacuum sublimation on the Au(111) surface and investigated by scanning tunnelling microscopy. The macrocycles form three different highly ordered monolayer structures α, β, and η. The α and β structure are stable at room temperature and can be understood as two dimensional networks which are held together by hydrogen bonds between the next neighbour molecules. These structures were described in detail before [Kossev et al., Adv. Mat. 17, 513 (2007)]. The third structure, which is described here, is only observed after heating at 400 K and rapid cooling to low temperatures. It consists of chiral aggregates, composed of three molecules. These aggregates are either left or right handed. The surface is covered by a racemic mixture of long range ordered domains with either left or right handed aggregates.

Chemoselektive Peptidcyclisierung über spurlose Staudinger-Ligation

R. Kleineweischede, C.P.R. Hackenberger

Angew. Chem. Int. Ed. 2008, 47, 5984-5988 ...

Zwei Fliegen mit einer Klappe! Eine intramolekulare spurlose Staudinger-Ligation wurde für die Synthese cyclischer Peptide entwickelt. Die abschließende Amidbildung wird durch Entschützung des Azido-Peptid-Phosphanylthioesters ermöglicht. Unter Trifluoressigsäure(TFA)-Bedingungen werden alle Schutzgruppen gleichzeitig entfernt, sodass eine chemoselektive Amidbildung eingeleitet werden kann.

Novel Furo-pyridine Derivatives via Sonogashira Reactions of Functionalized Pyridines.

T. Lechel, J. Dash, I. Brüdgam, H.-U. Reissig

Eur. J. Org. Chem. 2008, 3647-3655.


A series of 4-pyridyl nonaflates was coupled with several terminal alkynes to efficiently provide new 4-alkynyl-substituted 3-alkoxypyridine derivatives. Apt conditions were developed for their conversion into furo[2,3-c]pyridines. Sonogashira reactions of 4-alkoxy-substituted 3-pyridyl nonaflates allowed an access to regioisomeric furo[3,2-c]pyridines. For both types of alkynyl-substituted alkoxypyridinesan alternative method for cyclization employing iodine monochloride furnished iodinated furo[2,3-c]- or furo[3,2-c]pyridines, which can undergo a second palladium-catalyzed step. Iodination of 4-hydroxypyridine derivative 24 with iodine afforded a pentasubstituted pyridine which after Sonogashira reaction immediately undergoes a cyclization to furo-pyridine 25. Thus, three different types of furo-pyridines can be prepared starting from one precursor. Several compounds prepared are fluorescent and show strong Stokes shifts.

A Modular "Toolbox" Approach to Flexible Branched Multi-Macrocyclic Hosts as Precursors for Multiply Interlocked Architectures."

B. Baytekin, S.S. Zhu, B. Brusilowskij, J. Illigen, J. Ranta, J. Huuskonen, K. Rissanen, L. Kaufmann, C.A. Schalley

Chem. Eur. J. 2008, 14, 10012-10028.


Tetralactam macrocycles can be functionalized by a variety of cross-coupling reactions. A modular “toolbox” strategy is presented that allows 1) several tetralactam macrocycles to be covalently connected with each other or with a central spacer, 2) the macrocycles to be substituted with or connected to different chromophores, and 3) metal-coordination sites to be attached to the macrocycles. With this approach a series of different oligo-macrocyclic hosts was obtained with great structural diversity and enormous potential for further functionalization. Rotaxanes made on the basis of these macrocycles have been synthesized to demonstrate their utility in building more complex supramolecular architectures.

Helixinversion in responsiven Foldameren durch achirale Gastmoleküle (Halogenidionen).

R.M. Meudtner, S. Hecht

Angew. Chem. 2008, 120, 5004-5008.


Überraschendes Spiegelbild: Die Anwendung von Clickchemie führt zur effizienten Synthese einer neuen, als Clickamere bezeichneten Klasse von Foldameren mit außergewöhnlichen Faltungs- und Erkennungseigenschaften: Sie zeigen in Gegenwart von Halogenidionen – achiralen Spezies also – eine unerwartete Helixinversion

Helicity Inversion in Novel Responsive Foldamers Induced by Achiral Halide Anions.

R.M. Meudtner, S. Hecht

Angew. Chem. Int. Ed. 2008, 47, 4926-4930


A surprising about-face: Click chemistry was used for the efficient synthesis of a new class of foldamers, referred to as clickamers, that display unusual folding and recognition properties. In the presence of halide ions—achiral species—they undergo an unprecedented helix inversion.

Responsive Backbones Based on Alternating Triazole-Pyridine/Benzene Copolymers: From Helically Folding Polymers to Metallosupramolecularly Crosslinked Gels.

R.M. Meudtner, S. Hecht

Macromol. Rapid. Commun. 2008, 29, 347-351.


Step-growth polymerization using Cu-catalyzed 1,3-dipolar cycloaddition reactions, commonly referred to as “click chemistry,” has been used to prepare poly[(1,2,3-triazol-4-yl-1,3-pyridine)-alt-(1,2,3-triazol-1-yl-1,3-phenylene)]s. The recently discovered strong preference of the 2,6-bis(1,2,3-triazol-4-yl)pyridine (BTP) subunits to adopt an anti–anti conformation enables the extended heteroaromatic polymer strands to adopt a helical conformation, as shown by circular dichroism (CD) spectroscopy. Addition of various transition metal ions leads to coordinative crosslinking and therefore efficient gelation of the polymer solutions. The integration of the BTP scaffold in the main chain of the described polymers illustrates a synthetically inspired approach to readily access new functional macromolecules with potential applications as sensing and magnetic/emissive materials.

Photoactivation of CdSe/ZnS Quantum Dots Embedded in Silica Colloids.

S. Dembski, C. Graf, T. Krüger, U. Gbureck, A. Ewald, A. Bock, E. Rühl

Small 2008, 4, No. 9, 1516-1526.


A study of the influence of the local environment on the light-induced luminescence enhancement of CdSe/ZnS quantum dots (QD) embedded in silica colloids that are dispersed in various solvents is presented. The photoluminescence of the embedded QD is enhanced up to a factor of ten upon photoactivation by ultraviolet or visible light. This enhancement is strongly dependent on the local environment. The thickness-dependent permeability of the silica shell covering the QD controls the influence of the solvent on the QD. If foreign ions are present the activation state is stabilized after termination of the activation, whereas in their absence the process is partially reversible. A new qualitative model for the photoactivation of QD in various environments is developed. It comprises light-induced passivation and subsequent oxidation processes. The embedded QD also retain their fluorescence quantum yield inside living cells. Moreover, they can be activated for many hours in living cells by laser radiation in the visible regime.

A Sequential Assignment Procedure for Proteins that have Intermediate Line Widths in MAS NMR Spectra: Amyloid Fibrils of Human CA150.WW2

J. Becker, N. Ferguson, J. Flinders, B.-J. van Rossum, A.R. Fersht, H. Oschkinat

ChemBioChem 2008, 9, 1946-1952 ...

The second WW domain (WW2) of CA150, a human transcriptional activator, forms amyloid fibrils in vitro under physiological conditions. Based on experimental constraints from MAS NMR spectroscopy experiments, alanine scanning and electron microscopy, a structural model of CA150.WW2 amyloid fibrils was calculated earlier. Here, the assignment strategy is presented and suggested as a general approach for proteins that show intermediate line width. The 13C,13C correlation experiments were recorded on fully or partially 13C-labelled fibrils. The earlier 13C assignment (26 residues) was extended to 34 of the 40 residues by direct 13C-excitation experiments by using a deuterated sample that showed strongly improved line width. A 3D HNC-TEDOR (transferred-echo double-resonance) experiment with deuterated CA150.WW2 fibrils yielded 14 amide nitrogen and proton resonance assignments. The obtained chemical shifts were compared with the chemical shifts determined with the natively folded WW domain. TALOS (Torsion angle likelihood obtained from shift and sequence similarity) predictions confirmed that, under physiological conditions, the fibrillar form of CA150.WW2 adopts a significantly different β structure than the native WW-domain fold.

Synthesis of N,N-disubstituted phosphoramidates via a Lewis acid-catalyzed phosphorimidate rearrangement

I. Wilkening, G. del Signore, C.P.R. Hackenberger

Chem.Comm 2008, 2932-2934 ...

A Lewis acid-catalyzed rearrangement of phosphorimidates allows a direct, high-yielding transformation of azides with commercially available phosphites into secondary phosphoramidates.

MolecularWorkbench for Imaging andManipulation of Single Macromolecules and Their Complexes with the Scanning Force Microscope

J.P. Rabe

Top Curr Chem 2008, 285, 77-102 ...

The structure and properties of single macromolecules are key to understanding function in biologicalmolecular systems, as well as to developing artificial functional systems. In order to systematicallyinvestigate and control the conformations of single macromolecules and their complexes a “molecularworkbench” has been developed. It consists of an atomically flat, inert solid substrate suchas the basal plane of highly oriented pyrolytic graphite (HOPG), coated with a layer of moleculessuch as alkanes or alkyl chains containing amphiphiles that control the interaction between the substrateand adsorbed macromolecules. A scanning force microscope (SFM) operated in tapping or contactmode is used to both image and manipulate the macromolecules to correlate their structure with mechanicalproperties, and to assemble macromolecular systems that would not form spontaneously.

A Convergent Approach to Biocompatible Polyglycerol Click Dendrons for the Synthesis of Modular Core-Shell Architectures and Their Transport Behavior.

M. Wyszogrodzka, R. Haag

Chem. Eur. J. 2008, 14(30), 9202-9214.


Dendrimers are an important class of polymeric materials for a broad range of applications in which monodispersity and multivalency are of interest. Here we report on a highly efficient synthetic route towards bifunctional polyglycerol dendrons on a multigram scale. Commercially available triglycerol (1), which is highly biocompatible, was used as starting material. By applying Williamson ether synthesis followed by an ozonolysis/reduction procedure, glycerol-based dendrons up to the fourth generation were prepared. The obtained products have a reactive core, which was further functionalized to the corresponding monoazido derivatives. By applying copper(I)-catalyzed 1,3-dipolar cycloaddition, so-called “click” coupling, a library of core–shell architectures was prepared. After removal of the 1,2-diol protecting groups, water-soluble core–shell architectures 24–27 of different generations were obtained in high yields. In the structure–transport relationship with Nile red we observe a clear dependence on core size and generation of the polyglycerol dendrons.

Modular synthesis of multivalent glycoarchitectures and their unique selectin binding behavior.

I. Papp, J. Dernedde, S. Enders, R. Haag

Chem. Commun. 2008, 5851-5853.


Hyperbranched polyglycerols (HPGs) are ideal scaffolds for the multivalent presentation of saccharides, due to their biocompatible, carbohydrate-like properties; here, we report the conjugation of galactose sugar moieties to HPG, and the multivalent effect of these constructs on selectin binding.

Integrative Self-Sorting: Construction of a Cascade-Stoppered Hetero[3]Rotaxane.

W. Jiang, H.D.F. Winkler, C.A. Schalley

J. Am. Chem. Soc. 2008,130,13852-13853.


In this Communication, a competing self-sorting system containing benzo-21-crown-7, dibenzo-24-crown-8 and two secondary ammonium salts is constructed, which is then modified to achieve a hetero[3]pseudorotaxane with a specific sequence of wheels. With these two systems, we successfully demonstrate the concept of integrative self-sorting, and their relation. Furthermore, based on this self-sorting scheme, a hetero[3]rotaxane with an efficient stopper cascade has been synthesized.

Preserving the Markov Property of Reduced Reversible Markov Chains

M. Weber, S. Kube

AIP Conf. Proc., 2008, 1048, 593-596 ...

The computation of essential dynamics of molecular systems by conformation dynamics turned out to be very successful. This approach is based on Markov chain Monte Carlo simulations. Conformation dynamics aims at decomposing the state space of the system into metastable subsets. The set‐based reduction of a Markov chain, however, destroys the Markov property. We will present an alternative reduction method that is not based on sets but on membership vectors, which are computed by the Robust Perron Cluster Analysis (PCCA+). This approach preserves the Markov property.

Chemoselektive Ligations- und Modifikationsstrategien für Peptide und Proteine

C.P.R. Hackenberger, D. Schwarzer

Angew. Chem 2008, 120, 10182-10228 ...

Die Untersuchung biologischer Fragestellungen mit chemischen Methoden wird meist als “Chemische Biologie” bezeichnet und setzt voraus, dass biologisch relevante Makromoleküle, wie Peptide und Proteine, chemisch zugänglich sind. Auf der Festphasensynthese von Peptiden aufbauend wurden viele chemoselektive Ligations- und Modifikationstechniken zur Verknüpfung synthetischer Peptide oder funktionaler Einheiten zu größeren synthetischen, biologisch relevanten Makromolekülen entwickelt. Dieser Aufsatz fasst die aktuellen Entwicklungen auf dem Gebiet der chemoselektiven Ligations- und Modifikationsstrategien zusammen und illustriert ihre Anwendbarkeit an Beispielen aus der chemischen Totalsynthese von Proteinen bis hin zur Semisynthese natürlicher modifizierter Proteine.

Chemoselective Peptide Cyclization by Traceless Staudinger Ligation

R. Kleineweischede, C.P.R. Hackenberger

Angew. Chem. Int. Ed. 2008, 120, 6073-6077 ...

Two birds with one stone! An intramolecular traceless Staudinger ligation was employed to synthesize cyclic peptides. The final amide bond formation was induced by deprotection of the azidopeptide phosphinothioesters. Treatment with trifluoroacetic acid (TFA) results in the simultaneous removal of the borane along with the protecting groups on the peptide side chains, and chemoselective amide bond cyclization ensues.

N?O-Acyl shift in Fmoc-based syntheses of phosphopeptides.

H. Eberhard, O. Seitz

Org. Biomol. Chem. 2008, 6, 1349-1355. ...

Synthetic phosphopeptides are frequently used as chemical probes to explore protein–protein interactions involved in cellular signal transduction. Most commonly, the solid-phase synthesis of phosphotyrosine-containing peptides is performed by applying the Fmoc-strategy and N-Fmoc-protected tyrosine derivatives bearing acid-labile phospho protecting groups. We observed a side-reaction, the isomerisation at threonine, which furnishes depsipeptides. It is shown that the rate of N→O-acyl migration depends on the sequence context. Depsipeptides were formed most rapidly when the phosphotyrosine was located in the +2 position. Furthermore, different phosphotyrosine building blocks were compared and a suitable method that provides phosphopeptides in enhanced purity and yield is suggested.

O-Allyl protection in the Fmoc-based synthesis of difficult PNA.

F. Altenbrunn, O. Seitz

Org. Biomol. Chem. 2008, 6, 2493-2498. ...

The synthesis of homothymine PNA-oligomers can be plagued by the occurrence of a significant amount of truncation products, probably because on-resin aggregation hinders access during the coupling reactions. The use of low resin loading and the addition of the chaotropic salt KSCN in DMF allowed a partial remedy by conferring enhancements to the coupling yields. However, protection of the imide group by using O-allyl-protected thymine Fmoc-tAll provided the most significant improvements to the yields, even in cases where the use of non-protected thymine building blocks resulted in 70% truncation products. Deallylation occurs during the TFA cleavage step. Thus, O-allyl-protection can be applied in combination with standard protocols used in automated PNA synthesis.

Controlling the activity of peptides and proteins with smart nucleic acid–protein hybrids.

L. Röglin, O. Seitz

Org. Biomol. Chem. 2008, 6, 3881-3887. ...

Oligonucleotide–peptide conjugates have frequently been used to control the localisation of the conjugate molecule. For example, the oligonucleotide segment has allowed spatially addressed immobilization of peptides and proteins on DNA-arrays via hybridisation while the peptide part has most frequently been used to confer transfer of oligonucleotide cargo into live cells. The regulation of functional properties such as the affinity of these bioconjugates for protein targets has rarely been addressed. This review article describes the current developments in the application of smart oligonucleotide–peptide hybrids. The mutual recognition between nucleic acid segments is used to constrain the structure or control the distance between peptide and protein segments. Application of these new type of oligonucleotide–peptide hybrids allowed not only the regulation of binding affinity of peptide ligands but also control of enzymatic and optical activity of proteins.

Indium Trichloride Mediated Cleavage of Acetonides in the Presence of Acid-Labile Functional Groups – Enhancing the Synthetic Utility of 1,3-Dioxolanyl-Substituted 1,2-Oxazines.

F. Pfrengle, V. Dekaris, L. Schefzig, R. Zimmer, H.-U. Reissig,

Synlett 2008, 2965-2968 ...

Indium trichloride in an acetonitrile-water mixture chemoselectively cleaved the isopropylidene acetals of various 1,3-dioxolanyl-substituted 1,2-oxazines as well as carbohydrate derivatives. Deprotection of acetonides can be achieved in substrates susceptible for acid-induced cyclizations. Most importantly, enol ether moieties are not attacked and the presence of glycosidic linkages or acid-sensitive protecting groups such as tert-butyldimethylsilyl, 2-(trimethylsilyl)ethyl, or tert-butoxycarbonyl is also tolerated.

Practical Routes to 2,6-Disubstituted Pyridine Derivatives.

L. Vandromme, H.-U. Reissig, S. Gröper, J. P. Rabe

Eur. J. Org. Chem. 2008, 12, 2049.


We report the synthesis of a series of 2,6-disubstitutedpyridines in a straightforward manner starting from readily available 2-substituted pyridines. The main sequence involves a selective α-lithiation reaction with halogen functionalization followed by a Grignard reaction catalyzed by Fe(acac)3. After demonstration of the easy feasibility of this strategy by synthesizing 2,6-disubstituted pyridines 1, the route was applied to obtain pyridine derivatives bearing electron-donating or electron-withdrawing groups. Thus, a series of pyridines bearing an aryl moiety in the 6-position and an alkyl chain in the 2-position was obtained. The pyridines were studied for their self-assembly abilities at the interface between an organic solution and the basal plane of graphite. Preliminary STM results for one compound are reported.

Chemoselective Ligation and Modification Strategies for Peptides and Proteins

C.P.R. Hackenberger, D. Schwarzer

Angew. Chem. Int. Ed. 2008, 47/52, 10030-10074 ...

The investigation of biological processes by chemical methods, commonly referred to as chemical biology, often requires chemical access to biologically relevant macromolecules such as peptides and proteins. Building upon solid-phase peptide synthesis, investigations have focused on the development of chemoselective ligation and modification strategies to link synthetic peptides or other functional units to larger synthetic and biologically relevant macromolecules. This Review summarizes recent developments in the field of chemoselective ligation and modification strategies and illustrates their application, with examples ranging from the total synthesis of proteins to the semisynthesis of naturally modified proteins.

HPMA as a scaffold for the modular assembly of functional peptide polymers by native chemical ligation

I. R. Ruttekolk, F. Duchardt, R. Fischer, K.-H. Wiesmüller, J. Rademann, R. Brock

Bioconj. Chem. 2008, 19, 2081-2087 ...

Synthetic peptides are valuable tools in fundamental and applied biomedical research. On one hand, these molecules provide highly efficient access to competitive inhibitors of molecular interactions and enzyme substrates by rational design. On the other hand, peptides may serve as powerful vectors to mediate cellular uptake of molecules that otherwise enter cells only poorly. The coupling of both such functionalities provides access to molecules interfering with molecular processes inside the cell. However, the combination of several functionalities on one synthetic peptide may be compromised by problems associated with the synthesis of long peptides. Native chemical ligation enables the chemoselective coupling of fully deprotected functional building blocks. However, peptide thioesters are still not accessible by standard solid-phase peptide synthesis. Here, we demonstrate the cofunctionalization of a thioester-activated N-hydroxypropyl methacrylamide (HPMA) copolymer (28 500 Da) with the cell-penetrating peptide (CPP) nonaarginine and a bioactive peptide as independent building blocks by native chemical ligation. Nonaarginine was employed as a cell-penetrating peptide (CPP), a fluorescein-labeled analogue of a pro-apoptotic peptide as a biofunctional cargo. Incorporation of the fluorescein label enabled the highly sensitive quantification of the coupling stoichiometry by fluorescence correlation spectroscopy (FCS) using 0.4 pmol/12 ng of labeled construct. A construct only bearing the functional cargo peptide required cellular import by electroporation in order to show activity. In contrast, a construct combining all functionalities was active upon incubation of cells, validating the modular nature of the approach.

Sensitized Detection of Inhibitory Fragments and Iterative Development of Non-Peptidic Protease Inhibitors by Dynamic Ligation Screening

M. Schmidt, A. Isidro-Llobet, M. Lisurek, A. El-Dahshan, J. Tan, R. Hilgenfeld, J. Rademann

Angew. Chem. Int. Ed. 2008, 47, 3275-3278 ...

Synthetic peptides are valuable tools in fundamental and applied biomedical research. On one hand, these molecules provide highly efficient access to competitive inhibitors of molecular interactions and enzyme substrates by rational design. On the other hand, peptides may serve as powerful vectors to mediate cellular uptake of molecules that otherwise enter cells only poorly. The coupling of both such functionalities provides access to molecules interfering with molecular processes inside the cell. However, the combination of several functionalities on one synthetic peptide may be compromised by problems associated with the synthesis of long peptides. Native chemical ligation enables the chemoselective coupling of fully deprotected functional building blocks. However, peptide thioesters are still not accessible by standard solid-phase peptide synthesis. Here, we demonstrate the cofunctionalization of a thioester-activated N-hydroxypropyl methacrylamide (HPMA) copolymer (28 500 Da) with the cell-penetrating peptide (CPP) nonaarginine and a bioactive peptide as independent building blocks by native chemical ligation. Nonaarginine was employed as a cell-penetrating peptide (CPP), a fluorescein-labeled analogue of a pro-apoptotic peptide as a biofunctional cargo. Incorporation of the fluorescein label enabled the highly sensitive quantification of the coupling stoichiometry by fluorescence correlation spectroscopy (FCS) using 0.4 pmol/12 ng of labeled construct. A construct only bearing the functional cargo peptide required cellular import by electroporation in order to show activity. In contrast, a construct combining all functionalities was active upon incubation of cells, validating the modular nature of the approach.

Computation of equilibrium densities in metastable dynamical systems by domain decomposition

S. Kube, M. Weber

AIP Conf. Proc., 2008, 1048, 339-342 ...

Whenever the stationary density of molecular dynamical systems decomposes into almost invariant partial densities, its computation from long‐time dynamics simulations is infeasible within the available computer time due to the well‐known “trapping problem.” In order to avoid this computational difficulty, we suggest a domain decomposition approach that is similar to umbrella sampling methods. In contrast to standard umbrella sampling techniques, our decomposition forms a partition of unity such that the corresponding stationary density can be computed as eigenvector of some mass matrix. This approach has many advantages over traditional approaches used to unbias and recombine the umbrella sampling calculations. The theoretical analysis is illustrated by a two‐dimensional example.

Mechanical stability of bivalent transition metal complexes analyzed by single-molecule force spectroscopy

M. Gensler, C. Eidamshaus, M. Taszarek, H.-U. Reissig, J. P. Rabe

Beilstein J. Org. Chem. 2015, 11, 817–827 ...

Multivalent biomolecular interactions allow for a balanced interplay of mechanical stability and malleability, and nature makes widely use of it. For instance, systems of similar thermal stability may have very different rupture forces. Thus it is of paramount interest to study and understand the mechanical properties of multivalent systems through well-characterized model systems. We analyzed the rupture behavior of three different bivalent pyridine coordination complexes with Cu2+ in aqueous environment by single-molecule force spectroscopy. Those complexes share the same supramolecular interaction leading to similar thermal off-rates in the range of 0.09 and 0.36 s−1, compared to 1.7 s−1 for the monovalent complex. On the other hand, the backbones exhibit different flexibility, and we determined a broad range of rupture lengths between 0.3 and 1.1 nm, with higher most-probable rupture forces for the stiffer backbones. Interestingly, the medium-flexible connection has the highest rupture forces, whereas the ligands with highest and lowest rigidity seem to be prone to consecutive bond rupture. The presented approach allows separating bond and backbone effects in multivalent model systems.

Synthesis of Divalent Carbohydrate Mimetics by Reductive Amination with Enantiopure 1,2-Oxazines as Precursors

J. Salta, H.-U. Reissig

Synthesis 2015, 47(13), 1893-1898 ...

A direct approach to mono- and divalent carbohydrate mimetics starting from an enantiopure 1,2-oxazine derivative is described. After the Lewis acid induced rearrangement and subsequent reduction to provide the expected bicyclic 1,2-oxazine derivative as major component, a new tricyclic compound resulting from a different rearrangement pathway was isolated in small amounts. A smooth and optimized method for the hydrogenation of the bicyclic 1,2-oxazine derivative is presented, affording the desired aminopyran with d-idopyranose configuration. By reductive amination this aminopyran was connected with different aldehydes to furnish N-alkylated compounds. Reductive amination using 1,5-pentanedial resulted in the formation of a piperidine ring. With rigid aromatic dialdehydes the desired divalent compounds were obtained in good to excellent yields. Similar divalent carbohydrate mimetics were prepared from serinol.

Adsorption mechanism and valency of catechol-functionalized hyperbranched polyglycerols

S. Krysiak, Q. Wei, K. Rischka, A. Hartwig, R. Haag, T. Hugel

Beilstein J. Org. Chem. 2015, 11, 828–836 ...

Nature often serves as a model system for developing new adhesives. In aqueous environments, mussel-inspired adhesives are promising candidates. Understanding the mechanism of the extraordinarily strong adhesive bonds of the catechol group will likely aid in the development of adhesives. With this aim, we study the adhesion of catechol-based adhesives to metal oxides on the molecular level using atomic force microscopy (AFM). The comparison of single catechols (dopamine) with multiple catechols on hyperbranched polyglycerols (hPG) at various pH and dwell times allowed us to further increase our understanding. In particular, we were able to elucidate how to achieve strong bonds of different valency. It was concluded that hyperbranched polyglycerols with added catechol end groups are promising candidates for durable surface coatings.