Peptide and glycopeptide dendrimers. Part I

Recent progress in peptide and glycopeptide chemistry make the preparation of peptide and glycopeptide dendrimers of acceptable purity, with designed structural and immunochemical properties reliable. New methodologies using unprotected peptide building blocks have been developed to further increase possibilities of their design and improve their preparation and separation. Sophisticated design of peptide and glycopeptide dendrimers has led to their use as antigens and immunogens, for serodiagnosis and other biochemical uses including drug delivery. Dendrimers bearing peptide with predetermined secondary structures are useful tools in protein de novo design. This article covers synthesis and applications of multiple antigen peptides (MAPs), multiple antigen glycopeptides (MAGs), multiple antigen peptides based on sequential oligopeptide carriers (MAP‐SOCs), glycodendrimers and template‐assembled synthetic proteins (TASPs). Part I deals with the development of various structural forms of MAPs as well as their application as antigens, immunogens, and for immunodiagnostic and biochemical purposes. Copyright © 1999 European Peptide Society and John Wiley & Sons, Ltd.     

Peptide and glycopeptide dendrimers. Part II

Recent progress in peptide and glycopeptide chemistry make the preparation of peptide and glycopeptide dendrimers of acceptable purity, with designed structural and immunochemical properties reliable. New methodologies using unprotected peptide building blocks have been developed to further increase the possibilities of their design and improve their preparation and separation. The sophisticated design of peptide and glycopeptide dendrimers has led to their use as antigens and immunogens, for serodiagnosis and other biochemical uses including drug delivery. Dendrimers bearing peptide with predetermined secondary structures are useful tools in protein de novo design. This article covers synthesis and applications of multiple antigen peptides (MAPs), multiple antigen glycopeptides (MAGs), multiple antigen peptides based on sequential oligopeptide carriers (MAP‐SOCs), glycodendrimers and template‐assembled synthetic proteins (TASPs). In part II the preparation of MAPs, and the utility of glycodendrimers and TASPs are discussed. Copyright © 1999 European Peptide Society and John Wiley & Sons, Ltd.     

Efficient synthesis of O-linked glycopeptide by a transglycosylation using endo alpha-N-acetylgalactosaminidase from Streptomyces sp.

Gal beta-(1-->3)-GalNAc-linked hexapeptide was synthesized by a transglycosylation using Gal beta-(1-->3)-GalNAc beta-pNP as a donor and a serine-containing hexapeptide as an acceptor using endo GalNAc-ase from Streptomyces sp.. The Gal beta-(1-->3)-GalNAc residue was transferred to the hydroxyl group of the serine residue of the peptide. The total yield of the glycopeptide via this process was better than that of the chemoenzymatic method. This process was confirmed to be a versatile method for the synthesis of O-linked glycopeptides.     

Cysteine S-glycosylation, a new post-translational modification found in glycopeptide bacteriocins

O-glycosylation is a ubiquitous eukaryotic post-translational modification, whereas early reports of S-linked glycopeptides have never been verified. Prokaryotes also glycosylate proteins, but there are no confirmed examples of sidechain glycosylation in ribosomal antimicrobial polypeptides collectively known as bacteriocins. Here we show that glycocin F, a bacteriocin secreted by Lactobacillus plantarum KW30, is modified by an N-acetylglucosamine β-O-linked to Ser18, and an N-acetylhexosamine S-linked to C-terminal Cys43. The O-linked N-acetylglucosamine is essential for bacteriostatic activity, and the C-terminus is required for full potency (IC₅₀ 2 nM). Genomic context analysis identified diverse putative glycopeptide bacteriocins in Firmicutes. One of these, the reputed lantibiotic sublancin, was shown to contain a hexose S-linked to Cys22.     

Random glycopeptide bead libraries for seromic biomarker discovery

Identification of disease-specific biomarkers is important to address early diagnosis and management of disease. Aberrant post-translational modifications (PTM) of proteins such as O-glycosylations (O-PTMs) are emerging as triggers of autoantibodies that can serve as sensitive biomarkers. Here we have developed a random glycopeptide bead library screening platform for detection of autoantibodies and other binding proteins. Libraries were build on biocompatible PEGA beads including a safety-catch C-terminal amide linker (SCAL) that allowed mild cleavage conditions (I(2)/NaBH(4) and TFA) for release of glycopeptides and sequence determination by ESI-Orbitrap-MS(n). As proof-of-principle, tumor -specific glycopeptide reporter epitopes were built-in into the libraries and were detected by tumor-specific monoclonal antibodies and autoantibodies from cancer patients. Sequenced and identified glycopeptides were resynthesized at the preparative scale by automated parallel peptide synthesis and printed on microarrays for validation and broader analysis with larger sets of sera. We further showed that chemical synthesis of the monosaccharide O-glycopeptide library (Tn-glycoform) could be diversified to other tumor glycoforms by on-bead enzymatic glycosylation reactions with recombinant glycosyltransferases. Hence, we have developed a high-throughput flexible platform for rapid discovery of O-glycopeptide biomarkers and the method has applicability in other types of assays such as lectin/antibody/enzyme specificity studies as well as investigation of other PTMs.     

Bi- and trivalent glycopeptide mannopyranosides as inhibitors of type 1 fimbriae-mediated bacterial adhesion: variation of valency, aglycon and scaffolding

In order to test relevant structural parameters for effective inhibition of mannose-specific bacterial adhesion, bi- and trivalent glycopeptide α-d-mannopyranosides were synthesized that differ in their conformational properties as well as in the spatial arrangement of attached mannosyl residues. They were tested in an inhibition adhesion assay with fluorescent Escherichia coli bacteria and testing results were referenced to the inhibitory potency of methyl α-d-mannopyranoside. It was shown, that besides the nature of the mannoside aglycon moiety, scaffolding of α-d-mannopyranosides on a peptide backbone was important for the performance of the synthesized glycopeptides as inhibitors of bacterial adhesion.     

Biosynthetic systems for nonribosomal peptide antibiotic assembly

Modular peptide synthetases, which act as the protein templates for the synthesis of a large number of peptide antibiotics and siderophores, hold great potential for the development of novel compounds. Recently, significant progress has been made towards understanding their molecular architecture and substrate specificity. The first crystal structure of a peptide synthetase has been solved, and the enzymes responsible for post-translational modification of peptide synthetases have recently been discovered. These will allow addressing important yet poorly understood mechanistic aspects.     

Comparative analysis and insights into the evolution of gene clusters for glycopeptide antibiotic biosynthesis

The bal, cep, dbv, sta and tcp gene clusters specify the biosynthesis of the glycopeptide antibiotics balhimycin, chloroeremomycin, A40926, A47934 and teicoplanin, respectively. These structurally related compounds share a similar mechanism of action in their inhibition of bacterial cell wall formation. Comparative sequence analysis was performed on the five gene clusters. Extensive conserved synteny was observed between the bal and cep clusters, which direct the synthesis of very similar compounds but originate from two different species of the genus Amycolatopsis. All other cluster pairs show a limited degree of conserved synteny, involving biosynthetically functional gene cassettes: these include those involved in the synthesis of the carbon backbone of two non-proteinogenic amino acids; in the linkage of amino acids 1–3 and 4–7 in the heptapeptide; and in the formation of the aromatic cross-links. Furthermore, these segments of conserved synteny are often preceded by conserved intergenic regions. Phylogenetic analysis of protein families shows several instances in which relatedness in the chemical structure of the glycopeptides is not reflected in the extent of the relationship of the corresponding polypeptides. Coherent branchings are observed for all polypeptides encoded by the syntenous gene cassettes. These results suggest that the acquisition of distinct, functional genetic elements has played a significant role in the evolution of glycopeptide gene clusters, giving them a mosaic structure. In addition, the synthesis of the structurally similar compounds A40926 and teicoplanin appears as the result of convergent evolution.     

A designed glycopeptide array for characterization of sugar-binding proteins toward a glycopeptide chip technology

For the realization of a practical high-throughput protein detection and analysis system, a novel peptide array has been constructed using a designed glycopeptide model library with an α-helical secondary structure. This study will contribute the increment of the diversity of such an array system and the application to focused proteomics and ligand screening by effective detection of sugar-binding proteins. Fluorescent glycopeptides with an α-helix, a β-strand, or a loop structure were designed initially to select a suitable scaffold for the detection of a model protein. After selection of the α-helical structure as the best scaffold, a small model library with various saccharides was constructed to have charge and hydrophobicity variations in the peptide sequences. When various sugar-binding proteins were added to the peptide library array, the fluorescent peptides showed different responses in fluorescence intensities depending on their sequences as well as saccharides. The patterns of these responses could be regarded as “protein fingerprints” (PFPs), which are able to establish the identities of the target proteins. The resulting PFPs reflected the recognition properties of the proteins. Furthermore, statistical data analysis from obtained PFPs was performed using a cluster analysis. The PFPs of sugar-binding proteins were clustered successfully depending on their families and binding properties. These studies demonstrate that arrays with glycopeptide libraries based on designed structures can be promising tools to detect and analyze the target proteins. Designed peptides with functional groups such as sugars will play roles as the capturing agents of high-throughput protein nano/micro arrays for focused proteomics and ligand screening studies.     

Selection of biologically active peptides by phage display of random peptide libraries

Random peptide libraries displayed on phage are used as a source of peptides for epitope mapping, for the identification of critical amino acids responsible for protein—protein interactions and as leads for the discovery of new therapeutics. Efficient and simple procedures have been devised to select peptides binding to purified proteins, to monoclonal and polyclonal antibodies and to cell surfaces in vivo and in vitro.     

Isolation and structural studies on a galactofuranosyl-containing glycopeptide fromAscobolus furfuraceus

A galactofuranosyl-containing glycopeptide has been isolated from mycelium ofAscobolus furfuraceus by extraction with water. The glycoconjugate was purified by DEAE-cellulose chromatography followed by gel filtration. A molecular weight of about 20 000 was determined by the latter method using standard dextrans. Neutral sugars accounted for 94.5% of the glycopeptide and were characterized as mannose, galactose, and glucose. Glucosamine was estimated colorimetrically (1.8%). The molar ratio of Man:Gal:Glc:GlcNH₂ was 68:32:16:2. A trace amount of total phosphorus (0.2%) was found. The predominant amino acids were threonine and serine. The peptide moiety was labeled with [¹⁴C]formaldehyde and the elution of radioactivity was coincident with sugar on gel filtration in the presence of sodium dodecyl sulfate. The peak of radioactivity was retarded on release of galactose by mild acid hydrolysis. These results confirm the sugar-peptide linkage.     

Capillary zone electrophoresis and MALDI–mass spectrometry for the monitoring of in vitro O-glycosylation of a threonine/serine-rich MUC5AC hexadecapeptide

The in vitro N-acetylgalactosaminylation by human gastric UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases was assessed using the peptide motif GTTPSPVPTTSTTSAP, which is found naturally in the tandem repeat domains of the apomucin encoded by the gene MUC5AC. This peptide appeared to be an excellent tool for obtaining an insight into the extensive O-glycosylation processes of apomucins. Up to six N-acetylgalactosamines were added and the given glycopeptide species were well separated by capillary zone electrophoresis. Moreover, the degree of glycosylation (number of monosaccharide O-linked attachments) could be determined by MALDI–mass spectrometry without prior separation. Using different incubation times, we evidenced the accumulation of various glycopeptides, suggesting that the total glycosylation of an apomucin-peptide requires orderly N-acetylgalactosaminylation processing. This information was completed by experimental data showing that N-acetylgalactosaminylated octapeptides (the peptide backbones of which are part of GTTPSPVPTTSTTSAP) were able to selectively inhibit some N-acetylgalactosaminyltransferases. Our results suggest that this inhibition may influence the quality of the intermediate products appearing during the in vitro O-glycosylation process.     

Chemical synthesis of analogs of the glycopeptide contulakin-G, an analgetically active conopeptide from Conus geographus

Cone snails are marine predators that use immobilizing venoms for catching prey. Chemical analysis of the venoms has revealed a variety of biologically active small and intermediate size peptides rich in post-translational modifications (modified amino acids, glycosylation). The glycopeptide contulakin-G (pGlu-Ser-Glu-Glu-Gly-Gly-Ser-Asn-Ala-[beta-D-Galp-(1-->3)-alpha-D-GalpNAc-(1-->]Thr-Lys-Lys-Pro-Tyr-Ile-Leu-OH) is a potent analgesic from Conus geographus venom. The in vivo activity of synthetic contulakin-G was previously found to be significantly higher compared to that of a peptide lacking the glycan. In order to further investigate the importance of the glycan, we have now synthesized analogs of contulakin-G where the glycan chain O-linked to threonine has been altered either to beta-D-Galp-(1-->3)-beta-D-GalpNAc-, alpha-D-Galp-(1-->3)-alpha-D-GalpNAc-, or beta-D-Galp-(1-->6)-alpha-D-GalpNAc-. The glycopeptides were assembled on a Wang resin using commercially available Fmoc amino acids and synthetically prepared Fmoc-protected threonine derivatives carrying O-acetyl protected sugar chains. The final products were thoroughly characterized by NMR and mass spectroscopy.     

Genetically-encoded fragment-based discovery of glycopeptide ligands for DC-SIGN

We have employed genetically-encoded fragment-based discovery to identify novel glycopeptides with affinity for the dendritic cell receptor DC-SIGN. Starting from libraries of 10⁸ mannose-conjugated peptides, we identified glycopeptides that exhibited up to a 650-fold increase in multivalent binding affinity for DC-SIGN, which is also preserved in cells. Monovalently, our most potent glycopeptides have a similar potency to a Man₃ oligosaccharide, representing a 15-fold increase in activity compared to mannose. These compounds represent the first examples of glycopeptide ligands that target the CRD of DC-SIGN. The natural framework of glycopeptide conjugates and the simplicity of orthogonal conjugation to make these glycopeptides anticipates a promising future for development of DC-SIGN-targeting moieties.     

Convenient synthesis of a sialylglycopeptide-thioester having an intact and homogeneous complex-type disialyl-oligosaccharide

Access to glycopeptides with C-terminal thioester functionality is essential for the synthesis of large glycopeptides and glycoproteins through the use of native chemical ligation. Toward that end, we have developed a concise method for the synthesis of a glycopeptide thioester having an intact complex-type dibranched disialyl-oligosaccharide. The synthesis employed a coupling reaction between benzylthiol and a free carboxylic acid at the C-terminus of a glycopeptide in which the peptide side chains are protected. After construction of glycopeptide on the HMPB-PEGA resin through the Fmoc-strategy, the protected glycopeptide was released upon treatment with acetic acid/trifluoroethanol and then the C-terminal carboxylic acid was coupled with benzylthiol at -20 degrees C in DMF. For this coupling, PyBOP/DIPEA was found to be the best for the formation of the thioester, while avoiding racemization. Finally, the protecting groups were removed in good yield with 95% TFA, thus affording a glycopeptide-thioester having an intact and homogeneous complex-type disialyl-oligosaccharide.     

Intact glycopeptide characterization using mass spectrometry

Glycosylation is one of the most prominent and extensively studied protein post-translational modifications. However, traditional proteomic studies at the peptide level (bottom-up) rarely characterize intact glycopeptides (glycosylated peptides without removing glycans), so no glycoprotein heterogeneity information is retained. Intact glycopeptide characterization, on the other hand, provides opportunities to simultaneously elucidate the glycan structure and the glycosylation site needed to reveal the actual biological function of protein glycosylation. Recently, significant improvements have been made in the characterization of intact glycopeptides, ranging from enrichment and separation, mass spectroscopy (MS) detection, to bioinformatics analysis. In this review, we recapitulated currently available intact glycopeptide characterization methods with respect to their advantages and limitations as well as their potential applications.     

Synthesis of mimetic peptides containing glucosamine

A convenient synthesis of 2-amino-3,4,6-tri-O-benzyl-2-deoxy-β-d-glucopyranoside was described from the readily available starting material 2-acetamido-2-deoxy-d-glucose (N-acetyl-d-glucosamine). Herein, the coupling of different lipophilic amino acids with 2-amino-3,4,6-tri-O-benzyl-2-deoxy-β-d-glucose was reported via an amide linkage as useful building blocks for the synthesis of glycopeptides. Of particular interest, bioactive peptide Arg-Gly-Asp (RGD) was incorporated into the building block containing valine was also reported. The 15 examples of corresponding di-, tri- and tetra-peptides were obtained as single αanomers.     

Design, synthesis, and immunochemical characterization of a chimeric glycopeptide corresponding to the Shigella flexneri Y O-polysaccharide and its peptide mimic MDWNMHAA

Two glycopeptide chimeras corresponding to the Shigella flexneri Y O-polysaccharide and its peptide mimic were designed in an attempt to improve the binding affinity by increasing the entropy of binding relative to the original octapeptide mimic of the O-polysaccharide. The design was based on the X-ray crystal structures of a monoclonal antibody SYA/J6 in complex with its cognate ligands, a pentasaccharide corresponding to the S. flexneri Y O-polysaccharide and the octapeptide mimic, MDWNMHAA. Both chimeric molecules consist of a rhamnose trisaccharide linked through an α- or β-thioglycosidic linkage to a MDW moiety in which the W unit has been modified. We predicted that omission of the NMHAA moiety would obviate the bound water molecules that provided complementarity with the antibody-combining site, and the conformational restriction resulting from imposition of an α-turn at the C-terminus of the peptide. The glycopeptides were then docked into the active site of SYA/J6 using the program autodock 3.0, and the structures were optimized. The best models obtained in each case showed that the chimeric molecules, with either an α- or β-thioglycosidic linkage, might be reasonable surrogate ligands for the antibody. We report here the synthesis of the α-glycopeptide employing solution and solid-phase strategies. Immunochemical characterization indicated that the α-glycopeptide unfortunately did not inhibit binding of SYA/J6 to the S. flexneri Y lipopolysaccharide.     

Localization and overall structure of a mannose-rich glycopeptide from a pathologic immunoglobulin

The structure of a mannose-rich glycopeptide from a human pathological IgM has been investigated. It belongs to the group I (simple) glycopeptides and contains only mannose and N-acetylglucosamine residues in a molar ratio of 10:2. The structures of its oligosaccharide moiety and peptide chain have been determined: its molecular localization is specified and the relation between its biosynthesis and the oligosaccharide structure determine is discussed. Based on the alpha- and beta-mannosidase digestions and permethylation studies for the oligosaccharide moiety, and on the results obtained after sequential analysis of the peptide chain, the following structure is proposed for the mannose-rich IgM Du glycopeptide: (Formula: see text). The recovery of one molecule of this glycopeptide per molecule of heavy chain and the determination of the amino acid sequence have led us to locate this glycopeptide on asparagine 402 of the Fc portion of the heavy chain mu of IgM Du.     

Understanding and manipulating glycopeptide pathways: the example of the dalbavancin precursor A40926

Glycopeptide antibiotics represent an important class of microbial compounds produced by several genera of actinomycetes. The emergence of resistance to glycopeptides among enterococci and staphylococci has prompted the search for second-generation drugs of this class and semi-synthetic derivatives are currently under clinical trials. Dalbavancin is obtained by chemical modification of the natural glycopeptide A40926, produced by a Nonomuraea sp. Recently, there has been considerable progress in the elucidation of biosynthesis of glycopeptide antibiotics; several gene clusters have been characterized, thus providing an understanding of the biosynthesis of these chemically complex molecules. Furthermore, such investigations have yielded the first glycopeptide derivatives produced by genetic or enzymatic intervention. We have isolated and characterized the dbv clusters, involved in the formation of the glycopeptides A40926. The development of a gene-transfer system for Nonomuraea sp. has allowed the manipulation of the A40926 pathway. New derivatives were obtained by inactivating selected dbv genes. In addition, our data suggest differences in the biosynthetic routes for heptapeptide formation between the vancomycin and the teicoplanin families of glycopeptides.