Recent progress in the solid-phase synthesis of glycopeptide

The recent understanding of the biological role of glycoproteins has brought about a demand for the highly homogeneous glycopeptides as the functional model for glycoproteins. Thus, much efforts have been made to establish easy and efficient method for glycopeptide synthesis. In this paper, we briefly review the recent advances in the synthesis of O- and N-linked glycopeptide based on the solid-phase method. In O-glycopeptide section, the preparation of glycosylated amino acid units with mucin type and other O-linked carbohydrate chains and their use for solid-phase synthesis are summarized. Other approaches, such as the glycosylation of resin bound peptide are also overviewed. In N-glycopeptide section, the synthesis using glycosylated amino acid units as well as other methods are described.     

Enzymatic Synthesis of Neoglycopeptide Building Blocks

A transglutaminase-catalyzed transacylation is the major step in a practical, regioselective synthesis of N-linked spacer-modified glycopeptide analogues. The process requires minimal modification of the saccharide during the N-glycosylation, photoinduced coupling of cysteamine, and final enzymatic reaction (see scheme).     

Solid-phase synthesis of thioether-linked glycopeptide mimics for application to glycoprotein semisynthesis

[reaction: see text]. Glycoproteins are particularly suited to protein semisynthesis since homogeneous samples for biological analyses are not readily available using traditional recombinant techniques. Here we apply glycosyl iodoacetamides, normally used for the modification of bacterially derived proteins, to solid-phase glycopeptide synthesis. This provides access to glycopeptide alpha-thioesters, which may lend themselves to the semisynthesis of homogeneous N-linked glycoprotein mimics and novel glycopeptide libraries.     

Mature homogeneous erythropoietin building blocks by chemical synthesis: the EPO 22-37 glycopeptide domain presenting the full N-linked dodecasaccharide

A synthesis of EPO 22-37 glycopeptide (1), presenting the N-linked dodecasaccharide of erythropoietin, is described.     

A versatile strategy for the synthesis of N-linked glycoamino acids from glycals

A versatile route for the synthesis of N-linked glycoamino acids from readily available glycals is reported. A variety of glycals possessing different carbohydrate templates (mono-, di- and trisaccharide glycals) were shown to undergo a novel iodine catalyzed stereoselective diamination reaction with chloramine-T. Taking advantage of the difference in the reactivity between the anomeric and C2 sulfonamido groups of these diamines 7, 13, 15, 17 and 19, they could be protected differentially at the C2 and anomeric nitrogen atoms. Thus, chemoselective acetylation of these diamines installed the C2 acetamido group, an essential functionality that plays a crucial role in inducing a beta-turn in N-linked glycoproteins. Subsequent protection of the anomeric nitrogens of 20a,b,e as their Alloc (allyloxycarbonyl) derivatives followed by SmI(2) mediated facile didetosylation afforded 24a-c. Deprotection of the Alloc group of 24a and 24c and coupling of the liberated free amine with a variety of protected amino acids provided N-linked glycoamino acids 25 and 27 in high yields. An illustrative synthesis of an N-linked glycopeptide 29 is also reported.     

Toward a prostate specific antigen-based prostate cancer diagnostic assay: preparation of keyhole limpet hemocyanin-conjugated normal and transformed prostate specific antigen fragments

Prostate specific antigen (PSA) molecules secreted by cancerous and normal prostate cells differ in their N-linked glycan composition, while the peptide backbone appears to be conserved. Antibodies selectively recognizing such differentially glycosylated PSA structures could form a basis for a new diagnostic assay for prostate cancer. Twenty-amino acid PSA fragments carrying di-, tri-, and tetrabranched complex-type glycans were prepared by total synthesis and conjugated to maleimide-modified keyhole limpet hemocyanin (KLH) carrier protein through backbone Cys residues. These glycopeptide/KLH conjugates were then used for antibody generation.     

Simple modification of a protein database for mass spectral identification of N-linked glycopeptides

We describe an algorithm which modifies a protein database such that during a database search deamidation is limited to asparagines strictly contained within the N-glycosylation consensus sequence. The modified database was evaluated using a dataset created from the shotgun proteomic analysis of N-linked glycopeptides from human blood serum. We demonstrate that the application of the modified database eliminates incorrect glycopeptide assignments, reduces the peptide false-discovery rate, and eliminates the need for manual validation of glycopeptide identifications.     

Synthesis and evaluation of carboxymethyl chitosan for glycopeptide enrichment

Glycans are known to be involved in a variety of biological processes throughout human physiology. Mass spectrometry has demonstrated itself as powerful analytical tool for quantitative and structural characterization of glycans. Studying these molecules at the glycopeptide level however, offers distinct advantages, namely the ability to characterize both the glycan and peptide fragments simultaneously, and moreover the ability to assign site specific heterogeneity. In light of this, peptides often dominate the spectrum and hinder the ionization efficiency of glycopeptides. For this reason, enrichment protocols prior to downstream MS analysis need to be developed. Here, we discuss the synthesis and use of carboxymethyl chitosan (CMCH) to enrich glycopeptides from a 12 protein mixture for MS analysis. This protocol was compared to a commercially available glycopeptide enrichment kit offered by EMD Millipore through the use of tandem mass tags (TMT) for relative quantification. Using this approach, we identified 98 unique N-linked glycopeptides and observed, that CMCH was able to enrich more sialylation than the commercial kit. In addition, we observed a trend based on TMT reporter ratios with respect to increasing sialylation. This corroborated that this stationary phase was exhibiting a mixed-mode enrichment through both hydrophilic interaction liquid chromatography (HILIC) and weak anion exchange (WAX) principles.     

Synthesis of glycopeptide dendrimer by a convergent method

Glycopeptide thioester comprising the sequence of extracellular matrix metalloproteinase inducer (emmprin) (34-58) was prepared and condensed with a dendrimer core having eight amino groups by the thioester method. The desired product, a glycopeptide dendrimer carrying an N-linked core pentasaccharide of about 30 kDa, was successfully isolated by preparative electrophoresis and characterized by mass analysis.     

Structure determination of glycopeptide of p-momorcharin

The structure of the N-linked oligosaccharide chain of β-momorcharin, a ribosome-inactivating protein from the seeds of Momordica charantia Linn (Cucurbitaceae) was determined. A glycopeptide liberated by pronase digestion of the glycoprotein was subjected to amino acid and neutral carbohydrate analysis to establish the composition of amino acid and sugar residues. The sequences and glycosylation linkages of the sugar and amino acid residues in the glycopeptide were determined as Manal-6(Xylβ1-2)-Manβ1-4GlcNAcβ1-4(Fucal-3)-GlcNAc-Asn-Leu by 2D-NMR spectroscopy and FAB-MS data.     

Characterization of glycosylation sites for a recombinant IgG1 monoclonal antibody and a CTLA4-Ig fusion protein by liquid chromatography-mass spectrometry peptide mapping

Liquid chromatography mass spectrometry (LC-MS) peptide mapping can be a versatile technique for characterizing protein glycosylation sites without the need to remove the attached glycans as in conventional oligosaccharide mapping methods. In this way, both N-linked and O-linked sites of glycosylation can each be directly identified, characterized, and quantified by LC-MS as intact glycopeptides in a single experiment. LC-MS peptide mapping of the individual glycosylation sites avoids many of the limitations of preparing and analyzing an entire pool of released N-linked oligosaccharides from all sites mixed together. In this study, LC interfaced to a linear ion trap mass spectrometer (ESI-LIT-MS) were used to characterize the glycosylation of a recombinant IgG1 monoclonal antibody and a CTLA4-Ig fusion protein with multiple sites of N-and O-glycosylation. Samples were reduced, S-carboxyamidomethylated, and cleaved with either trypsin or endoproteinase Asp-N. Enhanced detection for minor IgG1 glycoforms (～0.1 to 1.0 mol% level) was obtained by LC-MS of the longer 32-residue Asp-N glycopeptide (4+ protonated ion) compared to the 9-residue tryptic glycopeptide (2+ ion). LC-MS peptide mapping was run according to a general procedure: (1) Locate N-linked and/or O-linked sites of glycosylation by selected-ion-monitoring of carbohydrate oxonium fragment ions generated by ESI in-source collision-induced dissociation (CID), i.e. 204, 366, and 292 Da marker ions for HexNAc, HexNAc-Hex, and NeuAc, respectively; (2) Characterize oligosaccharides at each site via MS and MSMS. Use selected ion currents (SIC) to estimate relative amounts of each glycoform; and (3) Measure the percentage of site-occupancy by searching for any corresponding nonglycosylated peptide.     

Rapid characterization of N-linked glycosylation site using non-specific protease digestion of gel-separated glycoproteins and matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry

Rapid identification of glycosylation sites of glycoproteins is urgently needed in glycoproteomics study. In the present work, a rapid and simple method based on non-specific digestion of gel-separated glycoproteins and matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry was described, which can efficiently identify the N-linked glycosylation sites. One-step in-gel digestion of Ribonuclease B (RNase B) by proteinase K was employed to generate glycopeptides with short and discrepant peptide composition. When compared with glycopeptides prepared by two-step in gel-digestion using trypsin-proteinase K or trypsin-pronase, the direct proteinase K treatment showed obvious superiority in both glycopeptide recovery and preparation simplicity. Most importantly, it helps to generate greater variety of glycopeptide series with rich information for glycosylation site identification. In addition, binary matrices 5-chloro-2-mercaptobenzothiazole (CMBT) /2,5-dihydroxybenzoic acid (DHB) were found to form homogeneous microcrystal on the target with the purified glycopeptides, leading to improved detection sensitivity. Thus, the present work provides an optimized solution to speed up the characterization of N-linked glycosylation sites in glycoproteins.     

Structural analysis of a glycoprotein by liquid chromatography-mass spectrometry and liquid chromatography with tandem mass spectrometry. Application to recombinant human thrombomodulin

Using recombinant human thrombomodulin (rhTM) expressed in Chinese hamster ovary (CHO) cells, we studied the structural analysis of a glycoprotein by liquid chromatography-mass spectrometry (LC-MS) and liquid chromatography with tandem mass spectrometry (LC-MS-MS). First, we analyzed the structure of both the O- and N-linked glycans in rhTM by oligosaccharide mapping using LC-MS equipped with a graphitized carbon column (GCC-LC-MS). Major O- and N-linked glycans were determined to be core 1 structure and fucosyl biantennary containing NeuAc(0-2) respectively. Next, the post-translational modifications and their heterogeneities, including the site-specific glycosylation, were analyzed by mass spectrometric peptide/glycopeptide mapping of trypsin-digested rhTM and precursor-ion scanning. Precursor-ion scanning was successful in the detection of five glycopeptides. Four N-glycosylation sites and their site-specific carbohydrate heterogeneity were determined by their mass spectra. O-Glycosylation could be estimated on the basis of its mass spectrum. We were able to identify partial beta-hydroxylation on Asn324 and Asn439, and O-linked glucose on Ser287 from the peptide/glycopeptide map and their mass spectra. We demonstrated that a sequential analysis of LC-MS and LC-MS-MS are very useful for the structural analysis of O- and N-linked glycans, polypeptides, and post-translational modifications and their heterogeneities, including site-specific glycosylation in a glycoprotein. Our method can be applied to a glycoprotein in biological samples.     

Combined use of lectin affinity chromatography and endo-beta-galactosidase to study polylactosamine sequences isolated from haemopoietic cell surfaces

The trypsin-sensitive glycopeptides from cell surfaces of a multipotential murine haemopoietic cell line (DE) have been studied using serial lectin affinity chromatography on columns of immobilized lentil lectin (LCA), concanavalin A (Con A), and wheat-germ agglutinin (WGA). WGA-binding material consisted of glycopeptides that failed to bind to LCA and Con A. Step elution from the WGA-column with 0.01-, 0.1-, 0.5- and 1.0 M N-acetyl-D-glucosamine yielded four affinity classes of glycopeptide (WGA-W, WGA-I, WGA-S and WGA-SS respectively). WGA-W, WGA-I and WGA-S contained both alkali-stable (N-linked) and alkali-labile (O-linked) carbohydrate on high molecular weight glycopeptides. The WGA-SS fraction contained only N-linked carbohydrate. N-linked glycopeptides isolated from each WGA-binding class differed in molecular size, relative N-acetylneuraminic acid content and affinity for Ricinus communis 120 agglutinin. endo-beta-Galactosidase digestion showed that these glycopeptides contained polylactosamine-type glycans. Gel filtration profiles of the enzyme treated materials were different for each WGA-binding population suggesting variation in branching patterns and/or substitution with fucose residues. Affinity chromatography has shown that the WGA binding molecules are the major glycopeptide group at DE cell surfaces.     

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.     

Identification of alpha-galactosyl epitope mimetics through rapid generation and screening of C-linked glycopeptide library

A general methodology has been established for rapid generation and screening of combinatorial glycopeptide library and subsequent mass spectrometric sequencing to identify the mimetics of Galalpha(1,3)Gal epitopes. Using this approach, several active glycopeptide sequences were recognized and found to inhibit the binding of human natural anti-Gal antibodies with comparable IC(50)s to synthetic Galalpha(1,3)Gal oligosaccharides. The most active glycopeptides detected from the library included Gal-Tyr-Trp-Arg-Tyr, Gal-Thr-Trp-Arg-Tyr, and Gal-Arg-Trp-Arg-Tyr. These glycopeptides showed higher affinities to anti-Gal antibodies than known Galalpha(1,3)Gal peptide mimetics, such as DAHWESWL and SSLRGF. Our results suggest that, by combining a peptide sequence (the "functional" mimic part) with a terminal alpha-linked galactose moiety (the "structural" mimic part), the resulting glycopeptide could be a very good Galalpha(1,3)Gal mimetic. Analysis of these active glycopeptides provided first-hand information regarding the binding site of anti-Gal antibodies to facilitate the structurally based design of more potent and stable inhibitors.     

Chemoselective elaboration of O-linked glycopeptide mimetics by alkylation of 3-thioGalNAc.

A critical branch point in mucin-type oligosaccharides is the beta 1-->3 glycosidic linkage to the core alpha-N-acetylgalactosamine (GalNAc) residue. We report here a strategy for the synthesis of O-linked glycopeptide analogues that replaces this linkage with a thioether amenable to construction by chemoselective ligation. The key building block was a 2-azido-3-thiogalactose-Thr analogue that was incorporated into a peptide by fluorenylmethoxycarbonyl (Fmoc)-based solid-phase peptide synthesis. Higher order oligosaccharides were readily generated by alkylation of the corresponding 3-thioGalNAc with N-bromoacetamido sugars. The rapid assembly of "core 1"and "core 3" O-linked glycopeptide mimetics was accomplished in this fashion.     

Reversed-phase depletion coupled with hydrophilic affinity enrichment for the selective isolation of N-linked glycopeptides by using Click OEG-CD matrix

Selective enrichment of glycopeptides is of great importance for protein glycosylation analysis using mass spectrometry since the signals of glycopeptides could be severely suppressed by the coexisting non-glycosylated peptides in the protein digest. In the present work, a strategy for N-linked glycopeptide enrichment through reversed-phase depletion coupled with hydrophilic affinity enrichment by applying the customized matrix named Click OEG-CD is developed. Compared with single hydrophilic interaction liquid chromatography (HILIC) mode, the strategy exhibited remarkably higher selectivity for N-linked glycopeptides. As many as 22, 18, and eight glycopeptides were detected in the glycopeptide fraction enriched with the strategy from the digests of human immunoglobulin G, horseradish peroxidase and bovine ribonuclease B, respectively. In addition, the strategy also showed high glycosylation microheterogeneity coverage for the enrichment of human α₁-acid glycoprotein glycopeptides. More than 170 glycopeptides covering all the glycosylation sites were detected in the enriched fraction. The revered-phase liquid chromatography depletion coupled with HILIC enrichment strategy by using Click OEG-CD matrix is expected to show more potential in further applications in glycosylation analysis.     

α‐O‐Linked Glycopeptide Mimetics: Synthesis,Conformation Analysis,and Interactions with Viscumin,a Galactoside‐Binding Model Lectin

Efficient cycloaddition of a silylidene‐protected galactal with a suitable heterodiene yielded the basis for a facile diastereoselective route to a glycopeptide‐mimetic scaffold. Its carbohydrate part was further extended by β1–3‐linked galactosylation. The pyranose rings retain their ⁴C₁ chair conformation, as shown by molecular modeling and NMR spectroscopy, and the typical exo‐anomeric geometry was observed for the disaccharide. The expected bioactivity was ascertained by saturation‐transfer‐difference NMR spectroscopy by using the galactoside‐specific plant toxin viscumin as a model lectin. The experimental part was complemented by molecular docking. The described synthetic route and the strategic combination of computational and experimental techniques to reveal conformational properties and bioactivity establish the prepared α‐O‐linked glycopeptide mimetics as promising candidates for further exploitation of this scaffold to give O‐glycans for lectin blocking and vaccination.     

Characterization of the N-linked glycosylation site of recombinant pectate lyase

Recombinant pectate lyase from Aspergillus niger was overexpressed in Aspergillus nidulans. The two recombinant proteins produced differed in molecular mass by 1200 Da, which suggested that the larger molecular weight protein was glycosylated. The deduced amino acid sequence was searched for potential N-linked glycosylation sites, and one potential site was identified at residue 64. The proteins were analyzed for their ability to bind various lectins as an assay for the presence of carbohydrates. The proteins were then digested with trypsin to facilitate the isolation of the potential glycosylation site. The resulting digestion products were subsequently analyzed by liquid chromatography/mass spectrometry using in-source collision induced dissociation to detect glycopeptides. Once the glycopeptide had been identified, treatment with an endoglycosidase both verified the location of glycosylation and identified the mass of the glycan. The Complex Carbohydrate Structural Database was searched for possible N-linked structures with the same mass, and the suggested primary sequence was confirmed by an exoglycosidase digestion. The data demonstrated that the larger recombinant protein contained a high mannose N-linked structure (Man(5)GlcNAc(2)) attached to N-64, while this site was not occupied in the smaller protein.