Selective electrochemical glycosylation by reactivity tuning

Electrochemical glycosylation of a selenoglycoside donor proceeds efficiently in an undivided cell in acetonitrile to yield beta-glycosides. Measurement of cyclic voltammograms for a selection of seleno-, thio-, and O-glycosides indicates the dependence of oxidation potential on the anomeric substituent allowing the possibility for the rapid construction of oligosaccharides by selective electrochemical activation utilising variable cell potentials in combination with reactivity tuning of the glycosyl donor. A variety of disaccharides are readily synthesised in high yield, but limitations of the use of selenoglycosides as glycosyl donors for selective glycosylation of thioglycoside acceptors are exposed. The first electrochemical trisaccharide synthesis is described.     

In Situ Preactivation Strategies for the Expeditious Synthesis of Oligosaccharides: A Review

Carbohydrates have gained increasing appreciation over the last few decades for their fundamental roles in all essential areas of life. As a result, there has been a surge of activity in synthetic glycosylation strategies to construct useful oligosaccharides. This review evaluates the advances in synthetic carbohydrate chemistry, specifically preactivation methodologies, stereoselective β-mannosylations, and an automated, electrochemical preactivation method. Also discussed are the use of preactivation as a tool to study reactive intermediates and applications of preactivation protocols in the one-pot synthesis of a hyaluronic acid decasaccharide and one-pot synthesis of a tristearoyl lipomannan containing a pseudotrisaccharide.     

寡糖合成中的“预活化”策略

寡糖及其缀合物因其重要的生物学功能而日益受到人们的关注,由于糖链结构的复杂性与多样性,寡糖的化学合成具有很大的挑战性。为了减少合成及分离步骤,提高寡糖合成的效率,糖基化策略十分重要。"一釜合成法"由于进行多个连续的糖基化反应但不需分离中间体而具有很大优势,但传统"一釜法"在设计单糖模块时需要进行精细复杂的保护基操作和离去基调整而影响其合成效率。"预活化"寡糖合成策略不依赖于糖基供体与糖基受体的活性差异,无需复杂的保护基操作,所有偶联反应在同一条件下一釜完成,实现了寡糖的高效、快速合成。本文在简要介绍传统"一釜合成法"的基础上,对"预活化"策略的研究进展进行综述,重点介绍"预活化"策略的基本原理,发展过程及其在生物活性寡糖合成上的应用。     

Oligosaccharide synthesis with glycosyl phosphate and dithiophosphate triesters as glycosylating agents

Described is an efficient one-pot synthesis of alpha- and beta-glycosyl phosphate and dithiophosphate triesters from glycals via 1,2-anhydrosugars. Glycosyl phosphates function as versatile glycosylating agents for the synthesis of beta-glucosidic, beta-galactosidic, alpha-fucosidic, alpha-mannosidic, beta-glucuronic acid, and beta-glucosamine linkages upon activation with trimethylsilyl trifluoromethanesulfonate (TMSOTf). In addition to serving as efficient donors for O-glycosylations, glycosyl phosphates are effective in the preparation of S-glycosides and C-glycosides. Furthermore, the acid-catalyzed coupling of glycosyl phosphates with silylated acceptors is also discussed. Glycosyl dithiophosphates are synthesized and are also used as glycosyl donors. This alternate method offers compatibility with acceptors containing glycals to form beta-glycosides. To minimize protecting group manipulations, orthogonal and regioselective glycosylation strategies with glycosyl phosphates are reported. An orthogonal glycosylation method involving the activation of a glycosyl phosphate donor in the presence of a thioglycoside acceptor is described, as is an acceptor-mediated regioselective glycosylation strategy. Additionally, a unique glycosylation strategy exploiting the difference in reactivity of alpha- and beta-glycosyl phosphates is disclosed. The procedures outlined here provide the basis for the assembly of complex oligosaccharides in solution and by automated solid-phase synthesis with glycosyl phosphate building blocks exclusively or in concert with other donors.     

Efficient synthesis of β‐(1,6)‐linked oligosaccharides through microwave‐assisted glycosylation

A microwave‐assisted glycosylation method was developed for efficient synthesis of oligosaccharides. Di‐functional AB monomers, 2,3,4‐tri‐O‐acetyl‐α‐d ‐galactopyranosyl bromide ( 3a ) and 2,3,4‐tri‐O‐acetyl‐α‐d ‐glucopyranosyl bromide ( 3b ) were designed and synthesized as weakly reactive monomers to avoid unwanted glycosylation or degradation during preparation and storage. The glycosylations of these monomers gave low conversions and low molecular weight oligosaccharides at rt, reflux, and under low microwave energy irradiation. However, the glycosylation became very effective when high microwave energy was applied, giving 100% conversion and producing oligosaccharides with M_n = 4.76 kDa for 3a and M_n = 4.05 kDa for 3b. The acetylated oligosaccharides were further subjected to deprotection for structural analysis, which indicated the oligosaccharides contain predominantly linear β‐(1,6)‐glycosyl linkages. © 2013 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2013, 51, 3693–3699     

Combinatorial synthesis of an oligosaccharide library by using beta-bromoglycoside-mediated iterative glycosylation of selenoglycosides: rapid expansion of molecular diversity with simple building blocks

A new method for constructing an oligosaccharide library composed of structurally defined oligosaccharides is presented based on an iterative glycosylation of selenoglycosides. Treatment of 2-acyl-protected selenoglycosides with bromine selectively generates beta-bromoglycosides, which serve as glycosyl cation equivalents in the oligosaccharide synthesis. Thus, the coupling of the bromoglycosides with another selenoglycoside affords the corresponding glycosylated selenoglycosides, which can be directly used to next glycosylation. The iteration of this sequence allows the synthesis of a variety of oligosaccharides including an elicitor active heptasaccharide. A characteristic feature of the iterative glycosylation is that glycosyl donors and acceptors with the same anomeric reactivity can be selectively coupled by activation of the glycosyl donor prior to coupling with the glycosyl acceptor. Therefore, same selenoglycosides can be used for both the glycosyl donors and the acceptors. This feature has been exemplified by a construction of an oligosaccharide library directed to elicitor-active oligosaccharides. The library composed of stereochemically defined oligoglucosides with considerable structural diversity can be constructed starting from simple selenoglycosides.     

Cyclic phosphate-linked oligosaccharides: synthesis and conformational behavior of novel cyclic oligosaccharide analogues

CyPLOS (cyclic phosphate-linked oligosaccharides), that is, novel cyclic oligosaccharide surrogates, consisting of two, three, and four phenyl-beta-D-glucopyranoside units, 4,6-linked through stable phosphodiester bonds, were prepared by a straightforward and efficient solid-phase protocol. The assembly of the linear precursors was achieved by standard phosphoramidite chemistry on an automated DNA synthesizer, using a suitably protected 4-phosphoramidite derivative of D-glucose as the building block. For the crucial cyclization step a phosphotriester methodology was exploited, followed by a mild basic treatment releasing the desired cyclic molecules in solution in a highly pure form. The cyclic dimer and trimer were also independently prepared by classical solution synthesis, basically following the same approach. The solution structural preferences of the cyclic dimer and trimer, obtained by detailed NMR analysis, are also reported.     

Glycosylation site analysis of human alpha-1-acid glycoprotein (AGP) by capillary liquid chromatography-electrospray mass spectrometry

A new anionic surfactant (RapiGest SF) was successfully used for site-specific analysis of glycosylation in human alpha-1-acid glycoprotein (AGP). By means of this analytical approach combined with capillary HPLC-mass spectrometry (and tandem mass spectrometry), the N-linked glycosylation pattern of AGP was explored. On the basis of mass matching and MS/MS experiments ca 80 different AGP-derived glycopeptides were identified. Glycosylation shows a markedly different pattern for the various glycosylation sites. At sites I and II, triantennary complex-type oligosaccharides predominate and at sites III, IV and V, tetra-antennary complex-type oligosaccharides predominate. Sites IV and V show the presence of additional N-acetyl lactosamine (Gal-GlcNAc) units (even higher degree of branching and/or longer antennae are also present).     

Boronic acid lectin affinity chromatography (BLAC). 2. Affinity micropartitioning-mediated comparative glycosylation profiling

As a continuation of our work on boronic acid lectin affinity chromatography (BLAC), in this paper we introduce an automated affinity micropartitioning approach using combined boronic acid and concanavalin A (BLAC/Con A) resin-filled micropipette tips to isolate and enrich human serum glycoproteins. The N-linked oligosaccharides of the partitioned glycoproteins were removed by PNGase F enzyme digestion, followed by 8-aminopyrene-1,3,6-trisulfonic acid labeling. Capillary gel electrophoresis with blue LED-induced fluorescence detection was applied in a multiplexed format for comparative glycan profiling. The efficiency of BLAC affinity micropartitioning was compared with that of the individual lectin and pseudolectin affinity enrichment. Finally, we report on our findings in glycosylation differences in human serum samples from healthy and prostate cancer patients by applying BLAC/Con A micropipette tip-based enrichment and comparative multicapillary gel electrophoresis analysis of the released and labeled glycans.     

Divergent heparin oligosaccharide synthesis with preinstalled sulfate esters

Traditional chemical synthesis of heparin oligosaccharides first involves assembly of the full length oligosaccharide backbone followed by sulfation. Herein, we report an alternative strategy in which the O-sulfate was introduced onto glycosyl building blocks as a trichloroethyl ester prior to assembly of the full length oligosaccharide. This allowed divergent preparation of both sulfated and non-sulfated building blocks from common advanced intermediates. The O-sulfate esters were found to be stable during glycosylation as well as typical synthetic manipulations encountered during heparin oligosaccharide synthesis. Furthermore, the presence of sulfate esters in both glycosyl donors and acceptors did not adversely affect the glycosylation yields, which enabled us to assemble multiple heparin oligosaccharides with preinstalled 6-O-sulfates.     

Direct and stereoselective synthesis of 1,3-cis-3- arylsulphonaminodeoxydisaccharides and oligosaccharides

The 3-aminoglycosides are ubiquitous in biologically important classes of glycoconjugates and naturally occurring oligosaccharides. Despite the rapid growth in the development of synthetic method of 3-amino glycosides, the current state-of-the art suffers from limited substrate scope, low yields, long reaction times, and anomeric mixtures. This work presents a novel direct method for the synthesis of 1,3-cis-3-arylsulphonaminodeoxydisaccharides and oligosaccharides via α-selective glycosylation and hydroamination of glycal in a one-pot manner. This efficient multicomponent reaction methodology provides ready access to 1,3-cis-3-arylsulphonaminodeoxydisaccharides and oligosaccharides and allows derivatization by variation of each component.     

Glycosyl thioimidates as versatile building blocks for organic synthesis

This review discusses the synthesis and application of glycosyl thioimidates in chemical glycosylation and oligosaccharide assembly. Although glycosyl thioimidates include a broad range of compounds, the discussion herein centers on S-benzothiazolyl (SBaz), S-benzoxazolyl (SBox), S-thiazolinyl (STaz), and S-benzimidazolyl (SBiz) glycosides. These heterocyclic moieties have recently emerged as excellent anomeric leaving groups that express unique characteristics for highly diastereoselective glycosylation and help to provide a streamlined access to oligosaccharides.     

An efficient ionic liquid supported divergent assembly of 3,6-branched glucosamine-containing pentasaccharide

We utilized the glycosyl acceptor tagging method with ionic liquid support for synthesis of the core segment of Clostridium botulinum C2 toxin ligand through a divergent synthetic strategy without chromatographic purification.The total yield was 57.1% and the reaction was completed in 10 h.The efficient ionic liquid supported glycosylation and purification procedure was applied for the synthesis of branched glucosamine-containing oligosaccharides for the first time,which expanded the scope of ionic liquid supported synthesis of biologically important oligosaccharides.     

Defining oxyanion reactivities in base-promoted glycosylations

Saccharide oxyanions obtained by base treatment could be employed in glycosylation to give oligosaccharides with high stereo- and regioselectivities.     

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.     

α-Selective Organocatalytic Synthesis of 2-Deoxygalactosides

Alpha rules: A thiourea acts as an efficient organocatalyst for the glycosylation of protected galactals to form oligosaccharides containing a 2-deoxymonosaccharide moiety. The reaction is highly stereoselective for α-linkages and proceeds by way of a syn-addition mechanism.     

Oligomannan synthesis using ionic liquid supported glycosylation

The synthesis of complex oligosaccharides has been a challenge for researchers. Herein, we describe a strategy for the synthesis of an activated oligomannan 1 that employs ionic liquid (IL) support glycosylation methodology on an IL-tagged mannosyl fluoride donor. This method is capable of rapidly producing linear alpha(1-->6) oligomannan thioglycosides in a convenient and cost-effective manner without the need of column purification after each glycosylation step.     

Carbohydrate analysis of glycoproteins A review

Many of the products prepared by biotechnological approaches, including recombinant genetic engineering, cell tissue culture, and monoclonal technologies, are glycoproteins. As little as five years ago, glycosylation was believed to play no significant role in the function of glycoproteins. Recent large scale testing of glycoprotein-based pharmaceuticals has indicated that both the extent and type of glycosylation can play a central role in glycoprotein activity. Although methods for compositional and sequence analysis of proteins and nucleic acids are generally available, similar methods have yet to be developed for carbohydrate oligomers and polymers. This review focuses on new, developing methods for the analysis and sequencing of the carbohydrate portion of glycoproteins. Included are: (1) the release of oligosaccharides and hydrolysis of carbohydrate chains using enzymatic and chemical methods; (2) fractionation by LPLC, electrophoresis, HPLC, and lectin affinity chromatography; (3) detection through the preparation of derivatives or by new electrochemical methods; (4) analysis by spectroscopic methods, including MS and high-field NMR; and (5) their sequencing through the use of multiple, well-integrated techniques. The ultimate goal of the analytical approaches discussed is to firmly establish structure and, thus, permit the study of structure-function relationships and eventually to allow the intelligent application of carbohydrate remodeling techniques in the preparation of new glycoproteins.     

Oligosaccharide synthesis and library assembly by one-pot sequential glycosylation strategy

The oligosaccharide residue in glycoconjugates located in cell membranes is responsible for intercellular recognition and interaction: it acts as a receptor for proteins, hormones, and microorganisms and governs immune reactions. These significant activities have stimulated great interest in the field of oligosaccharides and glycoconjugates. Although many advances have been made in the synthesis of oligosaccharides, more convenient and efficient methods are still needed. This review describes one of these new methods-the one-pot sequential glycosylation approach as a potent tool for oligosaccharide assembly. The oligosaccharide library construction in a one-pot fashion is also summarized.     

Vinyl Glycosides in Oligosaccharide Synthesis. 2. The Use of Allyl and Vinyl Glycosides in Oligosaccharide Synthesis

A novel latent-active glycosylation strategy has been described that relies on the isomerization of substituted allyl glycosides to give the corresponding vinyl glycosides, which can subsequently be used in Lewis acid-mediated glycosylations. The isomerization reaction was performed by a rhodium catalyst obtained by treating tris(triphenylphosphine)rhodium(I) chloride with n-butyllithium. This catalyst has many advantageous properties over the use of conventional Wilkinson's catalyst. The glycosylation reactions gave high yields for both primary and secondary sugar alcohols, and the anomeric selectivity could be controlled by the constitution of the glycosyl donor and reaction conditions. The new isomerization and glycosylation approach enables complex oligosaccharides of biological importance to be prepared in a highly convergent manner.