Extraction and purification of a High Mannose type oligosaccharide from Phaseolus lunatus beans by oxidative release with sodium hypochlorite.

High Mannose glycans (HM) are intermediates for the eukaryotic glycosylation pathway but are not commonly found on mature glycoproteins. HM, particularly Man9GlcNAc2 (M9), are present on the glycan shield of viral envelopes and have received attention as elements for semi-synthetic vaccines. The most common strategy to produce HM is extensive pronase digestion from soybean agglutinin (SBA) to obtain Asn-M9. Storage proteins from Phaseolus lunatus (butter beans) are abundant in M9 and could also be used for M9 production. Chemical alternatives to enzyme-based protocols such as sodium hypochlorite (NaClO) have been used effectively to release N-glycans. Here we present an application of the NaClO glycan release strategy to produce M9 from Phaseolus lunatus beans as an alternative to SBA and the characterisation of some of the release side products that support the release reaction mechanism.

Egg yolk sialylglycopeptide: purification, isolation and characterization of N-glycans from minor glycopeptide species.

Sialylglycopeptide (SGP) is a readily available naturally occurring glycopeptide obtained from hen egg yolk which is now commercially available. During SGP extraction, other minor glycopeptide species are identified, bearing N-glycan structures that might be of interest, such as asymmetrically branched and triantennary glycans. As the scale of SGP production increases, recovery of minor glycopeptides and their N-glycans can become more feasible. In this paper, we aim to provide structural characterization of the N-glycans derived from these minor glycopeptides.

Automation of Immunoglobulin Glycosylation Analysis.

The development of reliable, affordable, high-resolution glycomics technologies that can be used for many samples in a high-throughput manner are essential for both the optimization of glycosylation in the biopharmaceutical industry as well as for the advancement of clinical diagnostics based on glycosylation biomarkers. We will use this chapter to review the sample preparation processes that have been used on liquid-handling robots to obtain high-quality glycomics data for both biopharmaceutical and clinical antibody samples. This will focus on glycoprotein purification, followed by glycan or glycopeptide generation, derivatization and enrichment. The use of liquid-handling robots for glycomics studies on other sample types beyond antibodies will not be discussed here. We will summarize our thoughts on the current status of the field and explore the benefits and challenges associated with developing and using automated platforms for sample preparation. Finally, the future outlook for the automation of glycomics will be discussed along with a projected impact on the field in general.

Recognition of Dimeric Lewis X by Anti-Dimeric Lex Antibody SH2.

The carbohydrate antigen dimeric Lewis X (DimLex), which accumulates in colonic and liver adenocarcinomas, is a valuable target to develop anti-cancer therapeutics. Using the native DimLex antigen as a vaccine would elicit an autoimmune response against the Lex antigen found on normal, healthy cells. Thus, we aim to study the immunogenic potential of DimLex and search internal epitopes displayed by DimLex that remain to be recognized by anti-DimLex monoclonal antibodies (mAbs) but no longer possess epitopes recognized by anti-Lex mAbs. In this context, we attempted to map the epitope recognized by anti-DimLex mAb SH2 by titrations and competitive inhibition experiments using oligosaccharide fragments of DimLex as well as Lex analogues. We compare our results with that reported for anti-Lex mAb SH1 and anti-polymeric Lex mAbs 1G5F6 and 291-2G3-A. While SH1 recognizes an epitope localized to the non-reducing end Lex trisaccharide, SH2, 1G5F6, and 291-2G3-A have greater affinity for DimLex conjugates than for Lex conjugates. We show, however, that the Lex trisaccharide is still an important recognition element for SH2, which (like 1G5F6 and 291-2G3-A) makes contacts with all three sugar units of Lex. In contrast to mAb SH1, anti-polymeric Lex mAbs make contact with the GlcNAc acetamido group, suggesting that epitopes extend further from the non-reducing end Lex. Results with SH2 show that this epitope is only recognized when DimLex is presented by glycoconjugates. We have reported that DimLex adopts two conformations around the ß-d-GlcNAc-(1→3)-d-Gal bond connecting the Lex trisaccharides. We propose that only one of these conformations is recognized by SH2 and that this conformation is favored when the hexasaccharide is presented as part of a glycoconjugate such as DimLex-bovine serum albumin (DimLex-BSA). Proper presentation of the oligosaccharide candidate via conjugation to a protein or lipid is essential for the design of an anti-cancer vaccine or immunotherapeutic based on DimLex.

Convergent synthesis of tetra- and penta-saccharide fragments of dimeric Lewis X.

The convergent synthesis of tetra- and penta-saccharide fragments of the TACA dimeric Lex is described. The synthetic strategy relied on the preparation of a protected GlcNTCA-(1,3)-Gal-(1,4)-GlcNAc trisaccharide diol free at O-3 of both glucosamine residues. Key steps in the preparation of this diol involved glycosylation at O-4 of N-acetylglucosamine using activation of a trichloroacetimidate with BF3·Et2O at 40 °C, removal of the non-reducing end O-3' chloroacetate with thiourea, and glycosylation with a N-trichloroacetamido glucosamine trichloroacetimidate donor. After conversion to the diol acceptor, the trisaccharide was selectively fucosylated at the nonreducing end under NIS/TMSOTf activation, or di-fucosylated under CuBr2/Bu4NBr activation. The protected tetra- and pentasaccharides were then efficiently deprotected under dissolving metal conditions and the nonreducing end glucosamine residues were N-acetylated during the reaction work up. The deprotected compounds will be used as soluble competitors to characterize the epitopes recognized by anti-polymeric Lex antibodies.

Complex N-glycan breakdown by gut Bacteroides involves an extensive enzymatic apparatus encoded by multiple co-regulated genetic loci.

Glycans are the major carbon sources available to the human colonic microbiota. Numerous N-glycosylated proteins are found in the human gut, from both dietary and host sources, including immunoglobulins such as IgA that are secreted into the intestine at high levels. Here, we show that many mutualistic gut Bacteroides spp. have the capacity to utilize complex N-glycans (CNGs) as nutrients, including those from immunoglobulins. Detailed mechanistic studies using transcriptomic, biochemical, structural and genetic techniques reveal the pathway employed by Bacteroides thetaiotaomicron (Bt) for CNG degradation. The breakdown process involves an extensive enzymatic apparatus encoded by multiple non-adjacent loci and comprises 19 different carbohydrate-active enzymes from different families, including a CNG-specific endo-glycosidase activity. Furthermore, CNG degradation involves the activity of carbohydrate-active enzymes that have previously been implicated in the degradation of other classes of glycan. This complex and diverse apparatus provides Bt with the capacity to access the myriad different structural variants of CNGs likely to be found in the intestinal niche.

HappyTools: A software for high-throughput HPLC data processing and quantitation.

High-performance liquid chromatography (HPLC) is widely used for absolute quantitation. The advent of new columns and HPLC technology has enabled higher sample throughput, and hence, larger scale studies that perform quantitation on different sample types (e.g. healthy controls vs. patients with rheumatoid arthritis) using HPLC are becoming feasible. However, there remains a lack of methods that can analyse the increased number of HPLC samples. To address this in part, the modular toolkit HappyTools has been developed for the high-throughput targeted quantitation of HPLC measurements. HappyTools enables the user to create an automated workflow that includes retention time (tr) calibration, data extraction and the calculation of several quality criteria for data curation. HappyTools has been tested on a biopharmaceutical standard and previously published clinical samples. The results show comparable accuracy between HappyTools, Waters Empower and ThermoFisher Chromeleon. However, HappyTools offered superior precision and throughput when compared with Waters Empower and ThermoFisher Chromeleon. HappyTools is released under the Apache 2.0 license, both the source code and a Windows binary can be freely downloaded from https://github.com/Tarskin/HappyTools.

Convergent syntheses of Le analogues.

The synthesis of three Le(x) derivatives from one common protected trisaccharide is reported. These analogues will be used respectively for competitive binding experiments, conjugation to carrier proteins and immobilization on gold. An N-acetylglucosamine monosaccharide acceptor was first glycosylated at O-4 with a galactosyl imidate. This coupling was performed at 40° C under excess of BF3.OEt2 activation and proceeded best if the acceptor carried a 6-chlorohexyl rather than a 6-azidohexyl aglycon. The 6-chlorohexyl disaccharide was then converted to an acceptor and submitted to fucosylation yielding the corresponding protected 6-chlorohexyl Le(x) trisaccharide. This protected trisaccharide was used as a precursor to the 6-azidohexyl, 6-acetylthiohexyl and 6-benzylthiohexyl trisaccharide analogues which were obtained in excellent yields (70-95%). In turn, we describe the deprotection of these intermediates in one single step using dissolving metal conditions. Under these conditions, the 6-chlorohexyl and 6-azidohexyl intermediates led respectively to the n-hexyl and 6-aminohexyl trisaccharide targets. Unexpectedly, the 6-acetylthiohexyl analogue underwent desulfurization and gave the n-hexyl glycoside product, whereas the 6-benzylthiohexyl analogue gave the desired disulfide trisaccharide dimer. This study constitutes a particularly efficient and convergent preparation of these three Le(x) analogues.

Protein localization in Escherichia coli cells: comparison of the cytoplasmic membrane proteins ProP, LacY, ProW, AqpZ, MscS, and MscL.

Fluorescence microscopy has revealed that the phospholipid cardiolipin (CL) and FlAsH-labeled transporters ProP and LacY are concentrated at the poles of Escherichia coli cells. The proportion of CL among E. coli phospholipids can be varied in vivo as it is decreased by cls mutations and it increases with the osmolality of the growth medium. In this report we compare the localization of CL, ProP, and LacY with that of other cytoplasmic membrane proteins. The proportion of cells in which FlAsH-labeled membrane proteins were concentrated at the cell poles was determined as a function of protein expression level and CL content. Each tagged protein was expressed from a pBAD24-derived plasmid; tagged ProP was also expressed from the chromosome. The osmosensory transporter ProP and the mechanosensitive channel MscS concentrated at the poles at frequencies correlated with the cellular CL content. The lactose transporter LacY was found at the poles at a high and CL-independent frequency. ProW (a component of the osmoregulatory transporter ProU), AqpZ (an aquaporin), and MscL (a mechanosensitive channel) were concentrated at the poles in a minority of cells, and this polar localization was CL independent. The frequency of polar localization was independent of induction (at arabinose concentrations up to 1 mM) for proteins encoded by pBAD24-derived plasmids. Complementation studies showed that ProW, AqpZ, MscS, and MscL remained functional after introduction of the FlAsH tag (CCPGCC). These data suggest that CL-dependent polar localization in E. coli cells is not a general characteristic of transporters, channels, or osmoregulatory proteins. Polar localization can be frequent and CL independent (as observed for LacY), frequent and CL dependent (as observed for ProP and MscS), or infrequent (as observed for AqpZ, ProW, and MscL).

How the substituent at O-3 of N-acetylglucosamine impacts glycosylation at O-4: a comparative study.

An assessment of the relative reactivities of the 4-OH of N-acetylglucosamine acceptors bearing simple protecting groups, beta-linked or alpha-linked D or L sugars at O-3 is presented, using a per-O-acetylated alpha-D-glucosyl trichloroacetimidate donor under activation by BF(3) x OEt(2). The presence of either an acyl or carbonate protecting group at O-3 did not impact the reactivity at O-4 with all glycosylations proceeding successfully. On the other hand, the presence of peracetylated sugars at O-3 of N-acetylglucosamine acceptors did impact the reactivity of the 4-OH. The acceptors with an alpha-D-Man, beta-D-Gal, or beta-D-Glc at O-3 reacted promptly. In comparison, the acceptors bearing a beta-L-Fuc, alpha-L-Fuc, or alpha-L-Rha underwent glucosylation slowly, and unreacted acceptor was recovered from the reaction mixtures. Systematic searches carried out on the disaccharide acceptors and trisaccharide products carrying either a peracetylated beta-D-Gal or beta-L-Fuc at O-3 of the glucosamine residue suggest that, for these two acceptors, a conformational reorientation necessary around the fucosidic linkage contributes to the lower reactivity of the beta-fucosylated acceptor. The acceptors bearing a beta-linked D-Gal, D-Glc, or L-Fuc residue at O-3 each gave trisaccharide products that were mostly stable in the reaction conditions. In contrast, the alpha-linked residues at O-3 were rather unstable in these reaction conditions and the degradation of either the acceptors or trisaccharide products led to low glycosylation yields. In these later reactions, it was impossible to clearly assess which of the acceptor or product underwent degradation as comigration and detection issues prevented us from following these glycosylations by TLC or RP-HPLC. In contrast, the glycosylation of an acceptor carrying an alpha-linked perbenzylated L-Fuc residue at O-3 could be easily monitored by RP-HPLC. The data obtained when monitoring this glycosylation showed that the acceptor underwent prompt glycosylation but a decrease in the absorbance peak corresponding to the trisaccharide along with the appearance of a peak corresponding to a perbenzylated fucose hemiacetal indicated that the trisaccharide product was unstable in the reaction conditions.

Application and limitations of the methyl imidate protection strategy of N-acetylglucosamine for glycosylations at O-4: synthesis of Lewis A and Lewis X trisaccharide analogues.

We describe here the synthesis of the allyl Le(a) trisaccharide antigen as well as that of an analogue of the Le(x) trisaccharide antigen, in which the galactose residue has been replaced by a glucose unit. Although successful fucosylations at O-4 of N-acetylglucosamine acceptors have been reported using perbenzylated thioethyl fucosyl donors under MeOTf activation, such conditions led in our case to the conversion of our acceptor to the corresponding alkyl imidates. Indeed, in this synthesis of the Le(a) analogue, we demonstrate that the temporary protection of the N-acetyl group as a methyl imidate is advantageous to fucosylate at O-4. In contrast, we report here that glucosylation at O-4 of an N-acetylglucosamine monosaccharide acceptor using the alpha-trichloroacetimidate of peracetylated glucopyranose as a donor proceeded in better yields under activation with excess BF(3) x OEt(2) than that of the corresponding methyl imidate. Therefore, we conclude that activation of thioglycoside donors by MeOTf to glycosylate at O-4 of a glucosamine acceptor is best accomplished following the temporary protection of the N-acetyl group as a methyl imidate, especially when the donors are highly reactive and prone to degradation. In contrast, if donor and acceptor can withstand multiple equivalents of BF(3) x OEt(2), glycosylations at O-4 of a glucosamine acceptor with a trichloroacetimidate donor does not benefit from the temporary protection of the N-acetyl group as a methyl imidate.

Convenient temporary methyl imidate protection of N-acetylglucosamine and glycosylation at O-4.

This paper expands on the scope and utility of the temporary conversion of N-acetyl groups to alkyl imidates when attempting to glycosylate at O-4 of N-acetylglucosamine acceptors. The optimized synthesis of alkyl imidate protected glucosamine acceptors at position 4 and carrying various protecting groups at O-3 is described. These imidates were prepared immediately prior to glycosylation by treating the 4-OH acceptors with 0.5 M MeOTf to obtain the corresponding methyl imidates still carrying a free 4-OH group. When preparing these imidates in diethyl ether as the reaction solvent, we observed the unexpected formation of ethyl imidates in addition to the desired methyl imidates. While the 3-O-allyl acceptors were too unstable to be useful in glycosylation reactions, the 3-O-acylated methyl and ethyl imidates of glucosamine were shown to behave well during the glycosylation of the 4-OH with a variety of reaction conditions and various glycosyl donors. Glycosylation of these acceptors was successfully carried out with perbenzylated beta-thioethyl rhamnopyranoside under MeOTf promotion, while activation of this donor under NIS/TMSOTf or NIS/TfOH proved less successful. In contrast, activation of the less reactive perbenzylated alpha-thioethyl and peracetylated beta-thioethyl rhamnopyranosides with NIS/TfOH led to successful glycosylations of the 4-OH. Activation of a peracetylated rhamnosyl trichloroacetimidate by TMSOTf at low temperature also gave a high yield of glycosylation. We also report one-pot glycosylation reactions via alkyl imidate protected acceptor intermediates. In all cases the alkyl imidate products were readily converted to their corresponding N-acetyl derivatives under mild conditions.