Research Overview: Chemical Biology of Carbohydrates

Carbohydrates are involved in numerous biologically important recognition and signaling processes. However, our understanding of the underlying mechanisms is just at the beginning and methods for their elucidation need further development. The general aim of our group is to elucidate the biological functions of carbohydrates employing the methods of organic synthesis in combination with biological assays and to use carbohydrates as starting materials for the synthesis of biologically active compounds.

Employing combinatorial approaches we identified high-affinity ligands for proteins, especially plant and human lectins, and RNA. Multivalent carbohydrate-protein interactions are mechanistically investigated and structurally characterized by X-ray crystallography and (in solution) by EPR spectroscopy. In addition, the group is involved in the development of new synthetic methods for glycoside bond formation, preparation of glycopeptides, preparation of carbohydrate microarrays, and bioorthogonal ligation reactions. The latter are employed for metabolic oligosaccharide engineering in living cells. Selected examples of our research are depicted in the following paragraphs.

 

1. Multivalent Carbohydrate-Protein Interactions

1.1 Combinatorial Synthesis of Neoglycopeptides

We developed a new linker for the quantitative and reversible attachment of carbohydrates to amino groups (Figure 1A). Employing this linker, conformationally restricted, multivalent cyclic neoglycopeptides, such as 1, were obtained by solid-phase synthesis in a highly convergent fashion (Figure 1B). Libraries of glycoclusters of this type are suited for screening for lectin-binding properties.

Figure 1. (A) Aloc-based linker for reversible glycoconjugation. (B) Cyclic neoglycopeptide 1 obtained by solid-phase peptide synthesis.
 

V. Wittmann, S. Seeberger. Combinatorial Solid-Phase Synthesis of Multivalent Cyclic Neoglycopeptides. Angew. Chem. 2000, 112, 4508-4512; Angew. Chem. Int. Ed. 2000, 39, 4348-4352.

V. Wittmann, S. Seeberger, H. Schägger. Temporary Attachment of Carbohydrates to Cyclopeptide Templates: A New Strategy for Single-Bead Analysis of Multivalent Glycopeptides. Tetrahedron Lett. 2003, 44, 9243-9246.

 

1.2 Spatial Screening of Multivalent Lectin Ligands

High-affinity lectin ligands are of considerable medicinal interest in the diagnosis and manipulation of biological recognition processes that occur during inflammation, adhesion of viruses and bacteria to host cells, fertilization, and others. A promising approach to arrive at such ligands is the generation of multivalent carbohydrate derivatives which can simultaneously bind to several binding sites of a multivalent lectin. However, in many cases the required orientation of the carbohydrate epitopes for a multivalent interaction to take place is not known. We developed a screening procedure for the identification of the required orientation of the carbohydrate epitopes (spatial screening) comprising four steps (Figure 2).Potent ligands are resynthesized as pure compounds and investigated by an enzyme-linked lectin assay (ELLA).

Figure 2. Process for the spatial screening of multivalent lectin ligands comprising three steps: 1.) Preparation of a one-bead one-compound library of spatially diverse cyclic neoglycopeptides. 2.) Screening of the bead-bound glycoclusters for binding affinity to the biotinylated lectin (here: wheat germ agglutinin). 3.) Identification of potent ligands by automated Edman sequencing of neoglycopeptides bound to single resin beads.
 

V. Wittmann, S. Seeberger. Spatial Screening of Cyclic Neoglycopeptides: Identification of Polyvalent Wheat-Germ Agglutinin Ligands. Angew. Chem. 2004, 116, 918-921; Angew. Chem. Int. Ed. 2004, 43, 900-903.

V. Wittmann. Synthetic Approaches to Study Multivalent Carbohydrate-Lectin Interactions. In Highlights in Bioorganic Chemistry: Methods and Applications (Eds.: C. Schmuck, H. Wennemers), Wiley-VCH, Weinheim, 2004, 203-213.

C. Maierhofer, K. Rohmer, V. Wittmann. Probing Multivalent Carbohydrate-Lectin Interactions by an Enzyme-Linked Lectin Assay Employing Covalently Immobilized Carbohydrates. Bioorg. Med. Chem. 2007, 15, 7661-7676.

S. André, D. Specker, N. V. Bovin, M. Lensch, H. Kaltner, H.-J. Gabius, V. Wittmann. Carbamate-Linked Lactose: Design of Clusters and Evidence for Selectivity to Block Binding of Human Lectins to (Neo)Glycoproteins with Increasing Degree of Branching and to Tumor Cells. Bioconjugate Chem. 2009, 20, 1716-1728.

A. Semmler, R. Weber, M. Przybylski, V. Wittmann. De novo Sequencing of Peptides on Single Resin Beads by MALDI-FTICR Tandem Mass Spectrometry. J. Am. Soc. Mass Spectrom. 2010, 21, 215-219.

H. S. G. Beckmann, H. M. Möller, V. Wittmann. High-Affinity Multivalent Wheat Germ Agglutinin Ligands by One-Pot Click Reaction. Beilstein J. Org. Chem. 2012, 8, 819-826.

 

1.3 X-ray Crystallography Studies of Multivalent Carbohydrate-Protein Interactions

To understand the structural basis of multivalent binding of several high-affinity ligands identified from combinatorial screening, we employed X-ray crystallography. We could solve the crystal structure of a complex of the divalent ligand 2 binding to wheat germ agglutinin (WGA) (Figure 3). Four molecules of 2 simultaneously bind to WGA with each ligand bridging adjacent binding sites. This showed for the first time that all eight sugar binding sites of the WGA dimer are simultaneously functional. We also investigated a tetravalent neoglycopeptide with a binding potency 25500 times higher than that of GlcNAc (6400 times per contained sugar). X-Ray structure analysis of its complex with glutaraldehyde-crosslinked WGA and comparison of the crystal structure with the solution NMR structure of the neoglycopeptide (Figure 4) suggest that the conformation of the glycopeptide in solution is already pre-organized in a way supporting multivalent binding to the protein.

Figure 3. 1.7 structure of divalent ligand 2 in complex with WGA isolectin 3 (PDB ID: 2X52).

Figure 4. Superposition of the dominant conformational family of neoglycopeptide 3 determined by solution NMR spectroscopy and the red colored substructure of 3 visible in the X-ray Structure.
 

D. Schwefel, C. Maierhofer, J. G. Beck, S. Seeberger, K. Diederichs, H. M. Möller, W. Welte, V. Wittmann. Structural Basis of Multivalent Binding to Wheat Germ Agglutinin. J. Am. Chem. Soc. 2010, 132, 8704-8719.

 

1.4 EPR Studies of Multivalent Carbohydrate-Protein Interactions

It is well established that the structure of biomolecules determined by X-ray crystallography is not necessarily identical to their solution structure. Furthermore, binding mechanisms in a densely packed crystal and in solution may differ. Therefore, we applied electron paramagnetic resonance (EPR) spectroscopy of spin-labeled ligands to provide, for the first time, structural evidence for chelating binding of divalent ligands to the same protein in (frozen glassy) solution. Double electron-electron resonance techniques (DEER or PELDOR) deliver distance distributions between two nitroxide labels at opposite ends of divalent ligands. Analyses of the distance distributions show a detailed picture of the binding mechanisms of the divalent ligands (Figure 5). Chelating binding is directly detected and can be differentiated from monovalent binding of multiple molecules.

Figure 5. (A) Divalent WGA ligand labeled with two nitroxide spin markers. (B) Chelating binding of the divalent ligand to wheat germ agglutinin. (C) Distance distributions between the two spin labels of the divalent ligand. Top: In absence of protein a broad distance distribution due to the flexibility of the linker between the sugar moieties is observed. Bottom: In presence of an excess of protein, the stretched divalent ligand simultaneously binds to two adjacent binding sites (chelating binding). This results in a narrower distance distribution. The increased peak maximum of 2.3 nm is indicative of chelating binding.
 

P. Braun, B. Nägele, V. Wittmann, M. Drescher. Mechanism of Multivalent Carbohydrate-Protein Interactions Studied by EPR Spectroscopy. Angew. Chem. 2011, 123, 8579-8582; Angew. Chem. Int. Ed. 2011, 50, 8428-8431.

 

2.Carbohydrate Microarrays

Carbohydrate microarrays are an emerging tool for the high-throughput screening of carbohydrate-protein interactions. The crucial step in the preparation of carbohydrate arrays is the attachment of carbohydrate probes to the surface. We examined the Diels-Alder reaction with inverse electron demand (DARinv) as an irreversible, chemoselective ligation reaction for that purpose (Figure 6). For the immobilization of non-functionalized reducing oligosaccharides we developed a bifunctional chemoselective linker that enables the attachment of a dienophile tag to the oligosaccharides via an oxime ligation. Both immobilization strategies facilitate the preparation of high-quality carbohydrate microarrays.

Figure 6. Preparation of carbohydrate microarrays employing DARinv. Carbohydrates with a dienophile (norbornene derivative) are printed on tetrazine-modified glass slides. Binding of fluorescently labeled lectins can be detected by an array scanner. Different printing densities and different carbohydrate/lectin pairs (A-F) lead to varying fluorescence intensities.
 

H. S. G. Beckmann, A. Niederwieser, M. Wiessler, V. Wittmann. Preparation of Carbohydrate Arrays Using Diels-Alder Reactions with Inverse Electron Demand. Chem. Eur. J. 2012, 18, 6548-6554.

 

3. Development and Application of Ligation Reactions

3.1 One-Pot Procedure for Diazo Transfer and Azide-Alkyne Cycloaddition

In the field of ligation chemistry we introduced a new one-pot reaction for Cu(II)-catalyzed diazo transfer and Cu(I)-catalyzed azide-alkyne [3+2] dipolar cycloaddition (CuAAC, also known as click reaction). By this procedure, 1,4-disubstituted 1,2,3-triazoles are obtained in excellent yields from a variety of readily available amines without the need for isolation of the azide intermediates. The reaction has a broad scope and is especially practical for the synthesis of multivalent structures because compounds substituted with multiple azides are potentially unstable. The reaction was applied for the synthesis of a number of compounds including multivalent glycoconjugates (Figure 7) that were investigated as lectin ligands.

Figure 7. Synthesis of a divalent glycoconjugate using the one-pot procedure.
 

H. S. G. Beckmann, V. Wittmann.One-Pot Procedure for Diazo Transfer and Azide-Alkyne Cycloaddition: Triazole Linkages from Amines. Org. Lett. 2007, 9, 1-14.

 

3.2 Efficient N-Terminal Glycoconjugation of Proteins by the N-End Rule

The CuAAC reaction was also employed for the N-terminal glycoconjugation of the RNase inhibitor barstar. This protein contains only one methionine residue at the N terminus (Met1). Making use of the N-end rules, Met1 was replaced with azidohomoalanine by an auxotrophy-based residue-specific method. Subsequently, azide-containing barstar was ligated to several propargyl glycosides through CuAAC reactions in almost quantitative yields, giving access to stable and active homogeneous triazole-linked glycoproteins (Figure 8). Carbohydrate epitopes incorporated by this strategy can serve as recognition motifs for lectins, as was shown by surface plasmon resonance experiments.

Figure 8. Preparation of a triazole-linked glycoprotein by Cu(I)-catalyzed azide-alkyne cycloaddition between azide-containing barstar the propargyl glycoside of N,N'-diacetylchitobiose.
 

L. Merkel, H. S. G. Beckmann, V. Wittmann, N. Budisa. Efficient N-Terminal Glycoconjugation of Proteins by the N-End Rule. ChemBioChem 2008, 9, 1220-1224.

 

3.3 Metabolic Oligosaccharide Engineering

Currently, we develop chemoselective ligation reactions for metabolic oligosaccharide engineering (MOE). MOE offers a possibility to introduce carbohydrate residues with non-natural functional groups into the glycan chains of glycoproteins within living cells. The method relies on the promiscuity of the enzymes involved in glycan biosynthesis leading to processing of the modified monosaccharides analogously to the natural sugar. The functional groups incorporated into the glycan chains can subsequently be ligated to an exogenously delivered detectable probe (fluorescence dye, biotin moiety) by a bioorthogonal ligation reaction. In this way, glycosylation can be visualized in living cells.

As a bioorthogonal ligation reaction we employ inverse-electron-demand Diels-Alder reactions (DARinv) between tetrazines and terminal or strained alkenes. As the DARinv can be carried out in the presence of azides, the combination of click and Diels-Alder chemistry becomes feasible allowing simultaneous detection of two different sugars (Figures 9 and 10).

Figure 9. Dual-labeling strategy for MOE employing alkene- and azide-labeled monosaccharides followed by a Diels-Alder reaction with inverse electron demand (DARinv) and a strain-promoted azide-alkyne cycloaddition (SPAAC) with two different labels.

Figure 10. HeLa S3 cells were grown for 3 days with 100 µM Ac4ManNPtl and 25 µM Ac4GalNAz (a), without additional sugar (b), with 100 µM Ac4ManNPtl only (c), and with 25 µM Ac4GalNAz only (d). Living cells were labeled by DARinv with a tetrazine-biotin conjugate followed by incubation with fluorescently labeled streptavidin (red channel) and by SPAAC with a fluorescently labeled dibenzocyclooctyne (green channel).
 

A. Niederwieser, A.-K. Späte, L. D. Nguyen, C. Jüngst, W. Reutter, V. Wittmann. Two-Color Glycan Labeling of Live Cells by a Combination of Diels-Alder and Click Chemistry. Angew. Chem. 2013, 125, 4359-4363; Angew. Chem. Int. Ed. 2013, 52, 4265-4268.

 

4. Synthesis of 2-Acetamido-2-deoxy-glucopyranosides

2-Acetamido-2-deoxy-D-glucose (N-acetylglucosamine, GlcNAc) is an ubiquitous constituent of biologically important oligosaccharides and glycoconjugates including glycoproteins, glycolipids, glycosaminoglycans, and peptidoglycan. Glycosylation protocols for the preparation of 2-acetamido-2-deoxyglycosides differ significantly from those applied to the synthesis of other glycosides due to the presence of the 2-acetamido group. The activation of sugar oxazolines for example, which directly leads to the desired 2-acetamido-2-deoxyglycosides, requires rather harsh reaction conditions resulting in low yields. We have discovered that sugar oxazolines can also be efficiently activated under significantly milder conditions employing copper(II) salts such as CuBr2 and CuCl2 at elevated temperatures (Figure 11). Under these conditions, even reaction times of several days do not lead to decomposition of oxazoline or reaction product.

Figure 11. Synthesis of 2-acetamido-2-deoxy-glucopyranosides by copper(II)-mediated activation of sugar oxazolines.
 

V. Wittmann, D. Lennartz. Copper(II)-mediated Activation of Sugar Oxazolines: Mild and Efficient Synthesis of ß-Glycosides of N-Acetylglucosamine. Eur. J. Org. Chem. 2002 , 1363-1367.

 

5. RNA Targeting with Aminoglycoside Mimetics

Naturally occurring aminoglycosides, such as neomycin B, are an important class of antibiotics. This property is based on binding to specific regions within the 16S rRNA of bacterial ribosomes, which leads to reduced accuracy during protein biosynthesis. The preparation of aminoglycoside mimetics is a promising approach to overcome emerging antibiotic resistances and to obtain ligands for new RNA targets.

We developed a combinatorial approach to a new class of RNA ligands based on suitably protected sugar diamino acids (SDAs). Employing well-established peptide coupling reactions, complex libraries of linear and branched amide-linked oligomers are obtained with only a small set of SDA building blocks (Figure 12). A small library of 26 oligomers was screened for RNA binding properties and several inhibitors of the RNA editing reaction of Trypanosoma brucei could be identified.

Figure 12. Synthesis of linear and branched amide-linked oligomers from orthogonally protected sugar diamino acid (SDA) building blocks. PG = protecting group
 

Riboswitches are RNA elements found in bacterial mRNA molecules. Upon binding to metabolites they undergo conformational changes that, in turn, result in the modulation of gene expression. The glmS-riboswitch is unique among riboswitch families as it represents a metabolite-dependent ribozyme that undergoes self-cleavage upon recognition of glucosamine-6-phosphate. The glmS-riboswitch is located in the 5'-untranslated region of bacterial genes involved in cell wall biosynthesis. Therefore, this riboswitch represents a promising target for developing new antibiotics. We discovered and synthesized a carba-sugar with potency similar to that of the native metabolite glucosamine-6-phosphate in modulating the activity of the glmS-riboswitch of pathologically relevant and vancomycin-resistant Staphylococcus aureus. This compound represents a valuable lead structure for the development of antibiotics with a novel mode of action.
 

V. Wittmann. RNA-Targeting mit Aminoglycosid-Analoga. Nachr. Chem. 2002, 50, 1364-1368.

F. Sicherl, V. Wittmann. Orthogonally Protected Sugar Diamino Acids as Building Blocks for Oligosaccharide Mimetics. In Peptide Revolution: Genomics, Proteomics & Therapeutics (Proc. 18th American Peptide Symposium) (Eds.: M. Chorev, T. K. Sawyer), American Peptide Society, San Diego, 2004, 161-162.

F. Sicherl, V. Wittmann. Orthogonally Protected Sugar Diamino Acids as Building Blocks for Linear and Branched Oligosaccharide Mimetics. Angew. Chem. 2005, 117, 2133-2136; Angew. Chem. Int. Ed. 2005, 44, 2096-2099.

C. E. Lünse, M. S. Schmidt, V. Wittmann, G. Mayer. Carba-sugars Activate the glmS-Riboswitch of Staphylococcus aureus. ACS Chem. Biol. 2011, 6, 675-678.

 

Last update: October 30, 2013 by