Synthesis Of Glycopeptides Research Articles

Synthesis of Glycopeptide and Glycoprotein Remodelling for Immunological Studies

Over the past few decades, immunologists have begun to try and develop vaccines against tumour cells. One of approach is targeting an endocytic receptor on antigen-presenting cells, which results in enhanced antigen cross-presentation. To date, there is no clear picture of the factors that are important in controlling antigen cross-presentation. The main aim of this project was to determine which structural parameters of the glycoprotein-antigen conjugate resulted in enhanced cross-presentation upon MR-ligation. Therefore, concerns the chemoenzymatic synthesis of defined glycopeptides and glycoproteins as chemical biology tools to help unravel the role(s) of the MR in antigen cross presentation. Enzymatic degradation of locust bean gum provided a Manβ(1→4)Man disaccharide building block which in turn allowed the synthesis of N-glycan disaccharide and tetrasaccharide oxazolines. In addition, large N-glycan oligosaccharide oxazoline, Man9GlcNAc-high mannose (from soybean) was accessed by semi-synthetic approaches. After this successful synthesis of N-glycans, Native glycoforms of immunological probe peptides were made by the use of ENGase enzymes (EndoA-N171A), which attached sugar oxazolines to a peptide (OVA247-264A5K) containing a GlcNAc handle. Finally, glycoprotein remodelling of ovalbumin (OVA) was achieved with the N-glycan tetrasaccharide oxazoline donor using WT Endo A as catalyst. The synthesis of these glycopeptides and glycoproteins in homogenous form should facilitate future analysis to help define the pathway taken by an antigen after uptake by the MR.

Chemoenzymatic Synthesis of Glycopeptides Bearing Galactose-Xylose Disaccharide from the Proteoglycan Linkage Region.

Proteoglycans have important biological activities. To improve the overall synthetic efficiency, a new chemoenzymatic route has been established for the proteoglycan linkage region bearing a galactose-xylose disaccharide. The xylosylated glycopeptides were synthesized via solid phase synthesis, which was followed by the addition of the galactose unit by the galactosyl transferase β4GalT7. This work leads to a better understanding of the acceptor preference of β4GalT7 and opens the door for expeditious synthesis of the proteoglycan linkage region.

Exploration of human xylosyltransferase for chemoenzymatic synthesis of proteoglycan linkage region.

Proteoglycans (PGs) play important roles in many biological processes including tumor progression, cell adhesion, and regulation of growth factor activities. With glycosaminoglycan chains attached to the core proteins in nature, PGs are highly challenging synthetic targets due to the difficulties in integrating the sulfated glycans with the peptide backbone. To expedite the synthesis, herein, the utility of human xylosyltransferase I (XT-I), the enzyme responsible for initiating PG synthesis, has been explored. XT-I was found to be capable of efficiently installing the xylose unit onto a variety of peptide structures on mg scales. Furthermore, an unnatural sugar, i.e., 6-azidoglucose can be transferred by XT-I introducing a reactive handle onto the glycopeptide for selective functionalization. XT-I can be coupled with β-4-galactosyl transferase-7 for one pot synthesis of glycopeptides bearing galactose-xylose disaccharide, paving the way toward efficient chemoenzymatic synthesis of PG glycopeptides and glycoproteins.

Studies in glycopeptide synthesis

Studies in glycopeptide synthesis

Development of Strategies for Glycopeptide Synthesis: An Overview on the Glycosidic Linkage

Glycoproteins and glycopeptides are an interesting focus of research, because of their potential use as therapeutic agents, since they are related to carbohydrate-carbohydrate, carbohydrate-protein, and carbohydrate-lipid interactions, which are commonly involved in biological processes. It has been established that natural glycoconjugates could be an important source of templates for the design and development of molecules with therapeutic applications. However, isolating large quantities of glycoconjugates from biological sources with the required purity is extremely complex, because these molecules are found in heterogeneous environments and in very low concentrations. As an alternative to solving this problem, the chemical synthesis of glycoconjugates has been developed. In this context, several methods for the synthesis of glycopeptides in solution and/or solid-phase have been reported. In most of these methods, glycosylated amino acid derivatives are used as building blocks for both solution and solid-phase synthesis. The synthetic viability of glycoconjugates is a critical parameter for allowing their use as drugs to mitigate the impact of microbial resistance and/or cancer. However, the chemical synthesis of glycoconjugates is a challenge, because these molecules possess multiple reaction sites and have a very specific stereochemistry. Therefore, it is necessary to design and implement synthetic routes, which may involve various protection schemes but can be stereoselective, environmentally friendly, and high-yielding. This review focuses on glycopeptide synthesis by recapitulating the progress made over the last 15 years.

Chemical Synthesis of Homogeneous Human E-Cadherin N-Linked Glycopeptides: Stereoselective Convergent Glycosylation and Chemoselective Solid-Phase Aspartylation.

We report herein an efficient chemical synthesis of homogeneous human E-cadherin N-linked glycopeptides consisting of a heptapeptide sequence adjacent to the Asn-633 N-glycosylation site with representative N-glycan structures, including a conserved trisaccharide, a core-fucosylated tetrasaccharide, and a complex-type biantennary octasaccharide. The key steps are a chemoselective on-resin aspartylation using a pseudoproline-containing peptide and stereoselective glycosylation using glycosyl fluororide as a donor. This synthetic strategy demonstrates potential utility in accessing a wide range of homogeneous N-linked glycopeptides for the examination of their biological function.

Synthesis of MUC1-derived glycopeptide bearing a novel triazole STn analog

The synthesis of MUC1 glycopeptides bearing modified tumor-associated carbohydrate antigens (TACAs) represents an effective strategy to develop potential antitumor vaccines that trigger strong immune response. In this context, we present herein the multistep synthesis of the triazole glycosyl amino acid Neu5Ac-α/β2-triazole-6-βGalNAc-ThrOH 1 as STn antigen analog, along with its assembly on the corresponding MUC1 peptide to give NAcProAsp [Neu5Acα/β2-triazole-6-βGalNAc]ThrArgProGlyOH 2. Despite interacting differently with SM3 monoclonal antibody, as shown by molecular dynamic simulations, this unnatural triazole glycopeptide may represent a promising candidate for cancer immunotherapy.

Machine-Driven Chemoenzymatic Synthesis of Glycopeptide.

Historically, researchers have put considerable effort into developing automation systems to prepare natural biopolymers such as peptides and oligonucleotides. The availability of such mature systems has significantly advanced the development of natural science. Over the past twenty years, breakthroughs in automated synthesis of oligosaccharides have also been achieved. A machine-driven platform for glycopeptide synthesis by a reconstructed peptide synthesizer is described. The designed platform is based on the use of an amine-functionalized silica resin to facilitate the chemical synthesis of peptides in organic solvent as well as the enzymatic synthesis of glycan epitopes in the aqueous phase in a single reaction vessel. Both syntheses were performed by a peptide synthesizer in a semiautomated manner.

Machine‐Driven Chemoenzymatic Synthesis of Glycopeptide

AbstractHistorically, researchers have put considerable effort into developing automation systems to prepare natural biopolymers such as peptides and oligonucleotides. The availability of such mature systems has significantly advanced the development of natural science. Over the past twenty years, breakthroughs in automated synthesis of oligosaccharides have also been achieved. A machine‐driven platform for glycopeptide synthesis by a reconstructed peptide synthesizer is described. The designed platform is based on the use of an amine‐functionalized silica resin to facilitate the chemical synthesis of peptides in organic solvent as well as the enzymatic synthesis of glycan epitopes in the aqueous phase in a single reaction vessel. Both syntheses were performed by a peptide synthesizer in a semiautomated manner.

Palladium-Catalyzed Synthesis of Glycopeptides through C–H Activation

Key words palladium catalysis - C–H activation - glycosylation - glycopeptides

Synthesis of C‐Glycosyl Amino Acid Building Blocks Suitable for the Solid‐Phase Synthesis of Multivalent Glycopeptide Mimics

Five C‐glycosyl functionalized lysine building blocks, featuring C‐glycosidic derivatives of α‐rhamnose, α‐mannose, α‐galactose, β‐galactose, and β‐N‐acetyl glucosamine have been designed and synthesized. These derivatives, equipped with acid‐labile protecting groups, are eminently suitable for solid‐phase synthesis of multivalent glycopeptides. The lysine building blocks were prepared from C‐allyl glycosides that underwent a Grubbs cross‐metathesis with an acrylate, followed by a reduction of the C=C double bond in the resulting α,β‐unsaturated esters, and liberation of the carboxylate to allow condensation with a lysine side chain. The thus obtained C‐glycosides, five in total, were applied in the solid‐phase peptide synthesis (SPPS) of three glycopeptides, showing the potential of the described building blocks in the assembly of well‐defined mimics of homo‐ and heteromultivalent glycopeptides and glycoclusters.

Generation of a glycosylated asparagine residue through chemoselective acylation of a glycosylhydrazide

Herein, we report the first selective anomeric N-acylation of a glycosylhydrazide. We show that this transformation can be harnessed to generate amino acid building blocks including FmocAsn(GlcNAc)OH (1), a residue that has been previously shown to be a competent reagent in the solid-phase peptide synthesis of N-linked glycopeptides.

Synthesis of new asparagine-based glycopeptides for future scanning tunneling microscopy investigations.

For investigations on the biological functions of oligosaccharides and peptidomimetics, new asparagine-based mono- and disaccharides containing glycopeptides were prepared in solution. The applicability of two common peptide coupling reagents, using an orthogonal Fmoc/t-Bu strategy along with acetyl protecting groups for the carbohydrate moiety, was studied. Thus, the prepared libraries of glycopeptides were designed as model systems of cell surfaces for future investigations by combined preparative mass spectroscopy and scanning tunneling microscopy (STM) using soft-landing electrospray beam deposition (ES-IBD), on metal surfaces.

A new strategy for the chemoenzymatic synthesis of glycopeptides by De-O-acetylation with an esterase and glycosylations with glycosyltransferases

Glycopeptides are fragments of glycoproteins and are important in evaluating the biological roles of carbohydrates in glycoproteins. Fmoc solid-phase peptide synthesis using acetyl-protected glycosylated amino acids is a common strategy for the preparation of glycopeptides, but this approach normally requires chemical de-O-acetylation with a base that β-eliminates sugar residues and epimerizes the peptide backbone. Here we demonstrate a facile new chemoenzymatic synthetic strategy for glycopeptides, using an esterase for the de-O-acetylation of sugar residues and glycosyltransferases for successive sugar elongations at neutral pH.

Synthesis of C-Terminal Glycopeptides

Synthesis of C-Terminal Glycopeptides

Palladium‐Catalysed C(sp3)−H Glycosylation for the Synthesis of C‐Alkyl Glycoamino Acids

AbstractWe have developed a highly efficient and practical approach for palladium‐catalyzed trifluoroacetate‐promoted N‐quinolylcarboxamide‐directed glycosylation of inert β‐C(sp3)−H bonds of N‐phthaloyl α‐amino acids with glycals under mild conditions. For the first time, C(sp3)−H activation for glycosylation was achieved to build C‐alkyl glycosides. This method facilitates the synthesis of various β‐substituted C‐alkyl glycoamino acids and offers a tool for glycopeptide synthesis.

Palladium-Catalysed C(sp3 )-H Glycosylation for the Synthesis of C-Alkyl Glycoamino Acids.

We have developed a highly efficient and practical approach for palladium-catalyzed trifluoroacetate-promoted N-quinolylcarboxamide-directed glycosylation of inert β-C(sp3 )-H bonds of N-phthaloyl α-amino acids with glycals under mild conditions. For the first time, C(sp3 )-H activation for glycosylation was achieved to build C-alkyl glycosides. This method facilitates the synthesis of various β-substituted C-alkyl glycoamino acids and offers a tool for glycopeptide synthesis.

Design and synthesis of glyco-peptides as anti-cancer agents targeting thrombin-protease activated receptor-1 interaction.

Thrombin activates protease-activated receptor-1 (PAR-1) through binding to exosite I and the active site to promote tumor growth. We have developed a new class of anti-cancer glyco-peptides to target exosite I selectively without affecting the active-site-mediated coagulation activity and showed the importance of glycans for the stability and anti-cancer activity of the glyco-peptides.

Chemical synthesis of human syndecan-4 glycopeptide bearing O-, N-sulfation and multiple aspartic acids for probing impacts of the glycan chain and the core peptide on biological functions.

Proteoglycans are a family of complex glycoproteins with glycosaminoglycan chains such as heparan sulfate (HS) attached to the core protein backbone. Due to the high structural heterogeneity of HS in nature, it is challenging to decipher the respective roles of the HS chain and the core protein on proteoglycan functions. While the sulfation patterns of HS dictate many activities, the core protein can potentially impact HS functions. In order to decipher this, homogeneous proteoglycan glycopeptides are needed. Herein, we report the first successful synthesis of proteoglycan glycopeptides bearing multiple aspartic acids in the core peptide and O- and N-sulfations in the glycan chain, as exemplified by the syndecan-4 glycopeptides. To overcome the high acid sensitivities of sulfates and base sensitivities of the glycopeptide during synthesis, a new synthetic approach has been developed to produce a sulfated glycan chain on a peptide sequence prone to the formation of aspartimide side products. The availability of the structurally well-defined synthetic glycopeptide enabled the investigation of their biological functions including cytokine, growth factor binding and heparanase inhibition. Interestingly, the glycopeptide exhibited context dependent enhancement or decrease of biological activities compared to the peptide or the glycan alone. The results presented herein suggest that besides varying the sulfation patterns of HS, linking the HS chain to core proteins as in proteoglycans may be an additional approach to modulate biological functions of HS in nature.

Products of Chemoenzymatic Synthesis Representing MUC1 Tandem Repeat Unit with T-, ST- or STn-antigen Revealed Distinct Specificities of Anti-MUC1 Antibodies

Anti-mucin1 (MUC1) antibodies have long been used clinically in cancer diagnosis and therapy and specific bindings of some of them are known to be dependent on the differential glycosylation of MUC1. However, a systematic comparison of the binding specificities of anti-MUC1 antibodies was not previously conducted. Here, a total of 20 glycopeptides including the tandem repeat unit of MUC1, APPAHGVTSAPDTRPAPGSTAPPAHGV with GalNAc (Tn-antigen), Galβ1-3GalNAc (T-antigen), NeuAcα2-3Galβ1-3GalNAc (sialyl-T-antigen), or NeuAcα2-6GalNAc (sialyl-Tn-antigen) at each threonine or serine residue were prepared by a combination of chemical glycopeptide synthesis and enzymatic extension of carbohydrate chains. These glycopeptides were tested by the enzyme-linked immunosorbent assay (ELISA) for their capacity to bind 13 monoclonal antibodies (mAbs) known to be specific for MUC1. The results indicated that anti-MUC1 mAbs have diverse specificities but can be classified into a few characteristic groups based on their binding pattern toward glycopeptides in some cases having a specific glycan at unique glycosylation sites. Because the clinical significance of some of these antibodies was already established, the structural features identified by these antibodies as revealed in the present study should provide useful information relevant to their further clinical use and the biological understanding of MUC1.