Solid-phase Glycopeptide Synthesis Research Articles

Divergent synthesis of amino acid-linked O-GalNAc glycan core structures.

O-GalNAc glycans, also known as mucin-type O-glycans, are primary constituents of mucins on various mucosal sites of the body and also ubiquitously expressed on cell surface and secreted proteins. They have crucial roles in a wide range of physiological and pathological processes, including tumor growth and progression. In addition, altered expression of O-GalNAc glycans is frequently observed during different disease states. Research dedicated to unraveling the structure-function relationships of O-GalNAc glycans has led to the discovery of disease biomarkers and diagnostic tools and the development of O-glycopeptide-based cancer vaccines. Many of these efforts require amino acid-linked O-GalNAc core structures as building blocks to assemble complex O-glycans and glycopeptides. There are eight core structures (cores one to eight), from which all mucin-type O-glycans are derived. In this protocol, we describe the first divergent synthesis of all eight cores from a versatile precursor in practical scales. The protocol involves (i) chemical synthesis of the orthogonally protected precursor (3 days) from commercially available materials, (ii) chemical synthesis of five unique glycosyl donors (1-2 days for each donor) and (iii) selective deprotection of the precursor and assembly of the eight cores (2-4 days for each core). The procedure can be adopted to prepare O-GalNAc cores linked to serine, threonine and tyrosine, which can then be utilized directly for solid-phase glycopeptide synthesis or chemoenzymatic synthesis of complex O-glycans. The procedure empowers researchers with fundamental organic chemistry skills to prepare gram scales of any desired O-GalNAc core(s) or all eight cores concurrently.

Facile preparation of the N-acetyl-glucosaminylated asparagine derivative with TFA-sensitive protecting groups useful for solid-phase glycopeptide synthesis.

In this study, a novel N-acetyl-glucosaminylated asparagine derivative was developed. This derivative carried TFA-sensitive protecting groups and was derived from commercially available compounds only in three steps. It was applicable to the ordinary 9-fluorenylmethoxycarbonyl (Fmoc)-based solid-phase peptide synthesis (SPPS) method, and the protecting groups on the carbohydrate moiety could be removed by a single step of TFA cocktail treatment generally used for the final deprotection step in Fmoc-SPPS.

Straightforward entry to S-glycosylated fmoc-amino acids and their application to solid phase synthesis of glycopeptides and glycopeptidomimetics.

Streamlined access to S-glycosylated Fmoc-amino acids was developed. The process provides diverse glycosylated modified amino acids in high yield and stereoselectivity taking advantage of the in situ generation of a glycosylthiolate obtained from carbohydrate acetates in a few steps. Mild basic conditions make the conjugation reaction compatible with Fmoc-iodo-amino acids. To validate the strategy the glycosylated building blocks were used for SPPS and the unprecedented incorporation of a long thio-oligosaccharide to the peptide chain was demonstrated.

Stereoselective Synthesis of Sialylated Tumor-Associated Glycosylamino Acids

Suitably protected sialyl T(N) and 2,6-sialyl T tumor-associated carbohydrate antigen-derived amino acids have been prepared stereoselectively using an oxazolidinone-derived sialoside donor. These glycosylamino acids can be employed directly in the solid-phase synthesis of glycopeptides, as demonstrated by the efficient preparation of tumor-associated MUC1 glycopeptide fragments.

Solid phase synthesis of glycopeptides using Shoda's activation of unprotected carbohydrates

An expedient and simple protocol to access S-linked glycopeptides by Fmoc SPPS using unprotected carbohydrates is reported. The utility of the method was demonstrated with the solid phase synthesis of a MUC1 fragment (20 mer) containing two glycosylation sites that were substituted with S-linked glycans.

Design and Synthesis of Multifunctional Gold Nanoparticles Bearing Tumor-Associated Glycopeptide Antigens as Potential Cancer Vaccines

The development of vaccines against specific types of cancers will offer new modalities for therapeutic intervention. Here, we describe the synthesis of a novel vaccine construction prepared from spherical gold nanoparticles of 3-5 nm core diameters. The particles were coated with both the tumor-associated glycopeptides antigens containing the cell-surface mucin MUC4 with Thomsen Friedenreich (TF) antigen attached at different sites and a 28-residue peptide from the complement derived protein C3d to act as a B-cell activating "molecular adjuvant". The synthesis entailed solid-phase glycopeptide synthesis, design of appropriate linkers, and attachment chemistry of the various molecules to the particles. Attachment to the gold surface was mediated by a novel thiol-containing 33 atom linker which was further modified to be included as a third "spacer" component in the synthesis of several three-component vaccine platforms. Groups of mice were vaccinated either with one of the nanoplatform constructs or with control particles without antigen coating. Evaluation of sera from the immunized animals in enzyme immunoassays (EIA) against each glycopeptide antigen showed a small but statistically significant immune response with production of both IgM and IgG isotypes. Vaccines with one carbohydrate antigen (B, C, and E) gave more robust responses than the one with two contiguous disaccharides (D), and vaccine E with a TF antigen attached to threonine at the 10th position of the peptide was selected for IgG over IgM suggesting isotype switching. The data suggested that this platform may be a viable delivery system for tumor-associated glycopeptide antigens.

Synthetic, Structural, and Biosynthetic Studies of an Unusual Phospho-Glycopeptide Derived from α-Dystroglycan

Aberrant glycosylation of α-dystroglycan (α-DG) results in loss of interactions with the extracellular matrix and is central to the pathogenesis of several disorders. To examine protein glycosylation of α-DG, a facile synthetic approach has been developed for the preparation of unusual phosphorylated O-mannosyl glycopeptides derived from α-DG by a strategy in which properly protected phospho-mannosides are coupled with a Fmoc protected threonine derivative, followed by the use of the resulting derivatives in automated solid-phase glycopeptide synthesis using hyper-acid-sensitive Sieber amide resin. Synthetic efforts also provided a reduced phospho-trisaccharide, and the NMR data of this derivative confirmed the proper structural assignment of the unusual phospho-glycan structure. The glycopeptides made it possible to explore factors that regulate the elaboration of critical glycans. It was established that a glycopeptide having a 6-phospho-O-mannosyl residue is not an acceptor for action by the enzyme POMGnT1, which attaches β(1,2)-GlcNAc to O-mannosyl moietes, whereas the unphosphorylated derivate was readily extended by the enzyme. This finding implies a specific sequence of events in determining the structural fate of the O-glycan. It has also been found that the activity of POMGnT1 is dependent on the location of the acceptor site in the context of the underlying polypeptide/glycopeptide sequence. Conformational analysis by NMR has shown that the O-mannosyl modification does not exert major conformational effect on the peptide backbone. It is, however, proposed that these residues, introduced at the early stages of glycoprotein glycosylation, have an ability to regulate the loci of subsequent O-GalNAc additions, which do exert conformational effects. The studies show that through access to discrete glycopeptide structures, it is possible to reveal complex regulation of O-glycan processing on α-DG that has significant implications both for its normal post-translational maturation, and the mechanisms of the pathologies associated with hypoglycosylated α-DG.

Synthesis of Biantennary Complex-Type Nonasaccharyl Asn Building Blocks for Solid-Phase Glycopeptide Synthesis

The biantennary complex-type N-glycans bearing LacNAc and LacdiNAc as the nonreducing end motif were synthesized in a protected form suitable to use in the Fmoc solid-phase peptide synthesis studies. Two approaches for the nonasaccharide synthesis were examined by taking advantage of the highly β-selective glycosylation with GlcNTCA (N-phenyl)trifluoroacetimidate. An earlier approach, which involved the reaction of the trisaccharide donor (Gal-GlcNTCA-Man) and trisaccharide acceptor (Man-GlcNPhth(2)-N(3)), produced a mixture of nonasaccharide isomers. On the other hand, mannosylation of the trisaccharide acceptor (Man-GlcNPhth(2)-N(3)) stereoselectively afforded the known pentasaccharide (Man(3)-GlcNPhth(2)-N(3)), which was reacted with the disaccharyl glycosyl donor (Gal-GlcNTCA or GalNTCA-GlcNTCA) to produce the desired nonasaccharide as a single stereoisomer. Selective dephthaloylation followed by N-acetylation furnished the GlcNAc(2) functionality. The resulting nonasaccharyl azides were condensed with Fmoc-Asp(OPfp)-OBu(t) or Fmoc-Asp(OPfp)-OPac in the presence of Ph(CH(3))(2)P and HOOBt. Finally, the Zn reduction and cleavage of the tert-butyl ester or Zn reduction alone produced the targeted nonasaccharyl Asn building blocks.

A High-Throughput O-Glycopeptide Discovery Platform for Seromic Profiling

Biomarker microarrays are becoming valuable tools for serological screening of disease-associated autoantibodies. Post-translational modifications (PTMs) such as glycosylation extend the range of protein function, and a variety of glycosylated proteins are known to be altered in disease progression. Here, we have developed a synthetic screening microarray platform for facile display of O-glycosylated peptides (O-PTMs). By introduction of a capping step during chemical solid-phase glycopeptide synthesis, selective enrichment of N-terminal glycopeptide end products was achieved on an amine-reactive hydrogel-coated microarray glass surface, allowing high-throughput display of large numbers of glycopeptides. Utilizing a repertoire of recombinant glycosyltransferases enabled further diversification of the array libraries in situ and display of a new level of potential biomarker candidates for serological screening. As proof-of-concept, we have demonstrated that MUC1 glycopeptides could be assembled and used to detect autoantibodies in vaccine-induced disease-free breast cancer patients and in patients with confirmed disease at time of diagnosis.

ChemInform Abstract: Glycosylation of Phenols: Preparation of 1,2-cis and 1,2-trans Glycosylated Tyrosine Derivatives to be used in Solid-Phase Glycopeptide Synthesis.

The synthesis of four building blocks, Nα-Fmoc-Tyr(Ac4-β-D-Glc)-OPfp 6, Nα- Fmoc-Tyr(Bz4-α-D-Glc)-OPfp 16, Nα-Fmoc-Tyr[Ac3-α-D-Glc]-(1→4)-Ac3-[β-D-Glc]-OPfp 9 and Nα-Fmoc-Tyr[Bz4-α-D-Glc-(1 →4)-Bz3-α-D-Glc]-OPfp 19, suitable for solid-phase glycopeptide synthesis is described. Several different glycosylation procedures were evaluated for this purpose. A remarkable solvent effect on the α:β ratio was observed on going from dichloromethane to the nucleophilic solvent acetonitrile for the glycosylation reactions promoted by silver triflate with participating protecting groups in the 2-position of the glycosyl donor.

ChemInform Abstract: An Improved Synthesis of a Galactosylated Hydroxylysine Building Block and Its Use in Solid-Phase Glycopeptide Synthesis.

ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

Synthesis of glycosylated β3-homo-threonine conjugates for mucin-like glycopeptide antigen analogues

Glycopeptides from the mucin family decorated with tumour-associated carbohydrate antigens (TACA) have proven to be important target structures for the development of molecularly defined anti-cancer vaccines. The strategic incorporation of β-amino acid building blocks into such mucin-type sequences offers the potential to create pseudo-glycopeptide antigens with improved bioavailability for tumour immunotherapy. Towards this end, TN and TF antigen conjugates O-glycosidically linked to Fmoc-β3-homo-threonine were prepared in good yield via Arndt–Eistert homologation of the corresponding glycosyl α-amino acid derivative. By incorporation of TN-Fmoc-β3hThr conjugate into the 20 amino acid tandem repeat sequence of MUC1 using sequential solid-phase glycopeptide synthesis, a first example of a mixed α/β-hybrid glycopeptide building block was obtained. The latter is of interest for the development of novel glycoconjugate mimics and model structures for anti-cancer vaccines with increased biological half-life.

Role of a Cytoplasmic Dual-function Glycosyltransferase in O2 Regulation of Development in Dictyostelium

In the social amoeba Dictyostelium, a terminal step in development is regulated by environmental O(2). Prolyl 4-hydroxylase-1 (P4H1) was previously implicated in mediating the O(2) signal, and P4H1-null cells require elevated O(2) to culminate. The E3-ubiquitin ligase adaptor Skp1 is a P4H1 substrate, and here we investigate the function of PgtA, a dual function beta3-galactosyltransferase/alpha2-fucosyltransferase that contributes the 2nd and 3rd sugars of the pentasaccharide cap formed on Skp1 hydroxyproline. Although pgtA-null cells, whose Skp1 contains only a single sugar (N-acetylglucosamine or GlcNAc), show wild-type O(2) dependence of culmination, cells lacking AgtA, an alpha3-galactosyltransferase required to extend the trisaccharide, require elevated O(2) as for P4H1-null cells. Skp1 is the only detectable protein modified by purified PgtA added to pgtA-null extracts. The basis for specificity of PgtA was investigated using native Skp1 acceptor glycoforms and a novel synthetic peptide containing GlcNAcalpha1,4-hydroxy(trans)proline. Cysteine-alkylation of Skp1 strongly inhibited modification by the PgtA galactosyltransferase but not the fucosyltransferase. Furthermore, native and synthetic Skp1 glycopeptides were poorly galactosylated, not processively fucosylated, and negligibly inhibitory, whereas the fucosyltransferase was active toward small substrates. In addition, the galactosyltransferase exhibited an atypical concentration dependence on UDP-galactose. The results provide the first evidence that Skp1 is the functional target of P4H1 in O(2) regulation, indicate a gatekeeper function for the beta3-galactosyltransferase in the PgtA dual reaction, and identify an unexpected P4H1-dependent yet antagonistic function for PgtA that is reversed by AgtA.

Solid-phase synthesis of O-sulfated glycopeptide by the benzyl-protected glycan strategy

To expand the repertoire of our benzyl-protection strategy for solid-phase glycopeptide synthesis, an O-sulfated glycopeptide was chosen as the synthetic target. Trisaccharyl serine derivatives (Galβ1–4-GlcNAcβ1–2-Manα1–3-Ser) carrying (4-methoxyphenyl)methyl (MPM) groups at either 3-O or 6-O of the Gal residue were prepared through three stereoselective glycosylations. Cleavage of MPM followed by reaction with Me3N·SO3 efficiently afforded 3-O- and 6-O-sulfo-glycoserines, respectively. A preliminary debenzylation study using the sulfated glycoserines revealed that the sulfate groups persisted under ‘low-acidity TfOH’ conditions, when using a limited amount of TfOH and extending the reaction period. The 3-O-sulfo-glycoserine was then introduced into an icosapeptide modeled after an α-dystroglycan fragment by a combination of automated and manual solid-phase peptide synthesis procedures. The synthesized glycopeptide was successfully debenzylated by the low-acidity TfOH cocktail with slight damage to the sulfate functionality.

Efficient Substitution Reaction from Cysteine to the Serine Residue of Glycosylated Polypeptide: Repetitive Peptide Segment Ligation Strategy and the Synthesis of Glycosylated Tetracontapeptide Having Acid Labile Sialyl-TN Antigens

This paper reports the synthesis of a 40-residue glycopeptide having two antigenic sialyl-T(N) (NeuAc-alpha-(2,6)-GalNAc-Thr) residues in the MUC1 sequence. This target glycopeptide is a tandem repeat form of 20-residue glycopeptides. For the synthesis of this large molecule, native chemical ligation (NCL) at the serine site was used ((Cys)NCL(Ser)). The concept of (Cys)NCL(Ser) relies on the following: (1) conventional NCL between peptide-alpha-thioester and the cysteine residue of another peptide segment; (2) methylation of the thiol that was used for NCL; (3) acidic CNBr conversion of the cysteine residue to the serine residue forming an O-ester linkage; and (4) an O- to N-acyl shift to couple the two glycopeptides through a native amide bond. To synthesize glycopeptide having an acid-labile sugar moiety, a 20-residue glycopeptide-alpha-thioester and 20-residue glycopeptide having a cysteine residue at the N-terminal were synthesized by solid phase glycopeptide synthesis, and then coupled by (Cys)NCL(Ser). As the result of extensive investigation, CNBr activation with an additional acid (trifluoroacetic acid) was found to be essential to obtain good reactivity and yield, and this condition afforded a tandem repeat form of 40-residue sialylglycopeptide having two sialyl-T(N) residues. In addition to this, it was demonstrated that the cysteine thiol protected by the acetoamidomethyl (Acm) group did not react with the CNBr reagent, and therefore (Cys)NCL(Ser) can be used for repetitive native chemical ligation in the presence of a protecting N-terminal cysteine residue with an Acm group.

Efficient Synthesis of MUC4 Sialylglycopeptide through the New Sialylation Using 5-Acetamido-Neuraminamide Donors

Sialylation reactions using a new sialyl donor, diethyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-2-O-beta-D-glycero-D-galacto-2-nonulopyranosylonamide phosphite (Neu5Ac-1-amide-2-phosphite) derivatives, and the synthesis of the sialyl-T N-MUC4 glycopeptide are described. The sialylation was performed in CH2Cl2 solvent toward the 6-hydroxyl group of several monosugar acceptors and generated alpha-sialoside in good yield under low temperature and TMSOTf activation system. Amide derivatives of sialoside were easily converted into naturally occurring sialoside after hydrolysis of the amide group. Sialyl-alpha(2,6)-GalN3 was also prepared by this new sialylation protocol, and then this sialoside was further converted into a Fmoc-protected sialyl-TN serine derivative for solid-phase glycopeptides synthesis. The solid-phase glycopeptide synthesis using this sialyl-TN serine derivative in which the sugar hydroxyl group was free afforded the target sialyl-TN-MUC4 glycopeptide.

A comparative study of different glycosylation methods for the synthesis of d-mannopyranosides of Nα-fluorenylmethoxycarbonyl- trans-4-hydroxy- l-proline allyl ester

The synthesis of Nα-fluorenylmethoxycarbonyl- trans-4-hydroxy-4- O-[(2,3,4,6-tetra- O-acetyl)-α- d-mannopyranosyl]- l-proline allyl ester and Nα-fluorenylmethoxycarbonyl- trans-4-hydroxy-4- O-[(2,3,4,6-tetra- O-benzoyl)-α- d-mannopyranosyl]- l-proline allyl ester is described. Glycosylation using Königs–Knorr conditions with a benzoyl protected glycosyl donor provided the optimum method. Removal of the allyl ester gave two mannosylated building blocks suitable for solid phase glycopeptide synthesis.

Ritter-based glycoconjugation of amino acids and peptides—access to novel glycoconjugates displaying a β-amide linkage between amino acid and sugar moiety

β-Peptidic- d- gluco-, d- galacto-, and l- fuco-configured glycosyl amino acids can be prepared from the corresponding 2-deoxy-oct-3-ulopyranosonic acids via a one-pot intramolecular Ritter reaction. Initially, a ketopyranoside-based acid condenses under Lewis acid promoted conditions with nitriles (PhCN, MeCN) and a partially protected diamino ester (Boc-DAB- O- t-Bu, Boc-Orn- O- t-Bu) to form a β-peptidic glycosyl amino t-butylesters. The glycosyl amino t-butylesters can be converted into Fmoc-protected glycosyl amino acids that are suitably protected for solid-phase glycopeptide synthesis. Furthermore, replacement of the protected diamino ester by immobilized peptide amines permits post-synthetic N-terminal- and N(ε)-glycoconjugation of peptides on the solid phase.

Highly regioselective synthesis of a 3-O-sulfonated arabino Lewis a asparagine building block suitable for glycopeptide synthesis

Using the stannylene method, the trisaccharide 2-acetamido-3- O-[6- O-benzyl-β- d-galactopyranosyl]-4- O-[2,3,4-tri- O-benzyl-β- d-arabinopyranosyl]-6- O-benzyl-2-deoxy-β- d-glucopyranosyl azide was regioselectively sulfonated and, after reduction of the anomeric azide, coupled to Fmoc α-allyl aspartate. After Pd(0)-catalyzed deallylation, the sulfatyl Lewis a asparagine building block was obtained, suitable for solid-phase glycopeptide synthesis applying the fluoride labile PTMSEL linker system.

Combinatorial Synthesis of MUC1 Glycopeptides: Polymer Blotting Facilitates Chemical and Enzymatic Synthesis of Highly Complicated Mucin Glycopeptides

The chemoselective polymer blotting method allows for rapid and efficient synthesis of glycopeptides based on a "catch and release" strategy between solid-phase and water-soluble polymer supports. We have developed a heterobifunctional linker sensitive to glutamic acid specific protease (BLase). The general procedure consists of five steps, namely (i) the solid-phase synthesis of glycopeptide containing BLase sensitive linker, (ii) subsequent deprotections and the release of the glycopeptide from the resin, (iii) chemoselective blotting of the glycopeptide intermediates in the presence of water-soluble polymers with oxylamino functional groups, (iv) sugar elongations using glycosyltransferases, and (v) the release of target glycopeptides from the polymer platform by selective BLase promoted hydrolysis. The combined use of the solid-phase chemical syntheses of peptides and the enzymatic syntheses of carbohydrates on water-soluble polymers would greatly contribute to the production of complicated glycopeptide libraries, thereby enhancing applicative research. We report here a high-throughput synthetic system for the various types of MUC1 glycopeptides exhibiting a variety of sugar moieties. It is our belief that this concept will become part of the entrenched repertoire for the synthesis of biologically important glycopeptides on the basis of glycosyltransferase reactions in automated and combinatorial syntheses.