Mass spectrometric analysis of carbohydrates labeled with a biotinylated tag

A derivatization method for mass spectrometric analysis of oligosaccharides is presented. Small saccharides, complex, high‐mannose‐type oligosaccharides and oligosaccharides released from hen ovalbumin were converted into their biotin derivatives by incubating them with biotinamidocaproyl hydrazide (BACH). Improved sensitivity of mass spectrometric analysis of labeled glycans in comparison with their natural counterparts was achieved after derivatization. The labeling reagent contains a biotin handle at one end and a hydrazide group at the other. Hence, the key feature of biotinylated sugars is that in addition to their usefulness in functional studies (e.g. analysis of the interaction between lectins and biotin‐derivatized oligosaccharides) they might be utilized also for structural analysis of oligosaccharides. Mass spectrometric studies were performed by matrix‐assisted laser desorption/ionization time‐of‐flight and electrospray ionization mass spectrometry. Copyright © 2009 John Wiley & Sons, Ltd.     

用不保护或少保护的糖基受体合成寡糖*

用不保护或少保护的葡萄糖、甘露糖、鼠李糖作为糖基受体,经由糖原酸酯的中间体,能高区选和立体选地合成寡糖. α-(1→6)-连接的甘露寡糖、β-(1→6)-连接的葡萄寡糖、3,6-支化的甘露寡糖及葡萄寡糖用此方法能用很简单步骤合成,如具有重要生物活性的寡糖植保素激活剂葡萄六糖、具有抗肿瘤活性的香菇多糖的活性片段,以及一些具有重要生理功能的多糖的重复单元等.本文同时简述了用少保护的半乳糖和氨基葡萄糖为糖基受体合成寡糖的进展.     

Biosynthesis and maturation of cellular membrane glycoproteins

The biosynthesis and the processing of asparagine-linked oligosaccharides of cellular membrane glycoproteins were examined in monolayer cultures of BHK21 cells and human diploid fibroblasts after pulse- and pulse-chase labeling with [2-3H]mannose. After pronase digestion, radiolabeled glycopeptides were characterized by high-resolution gel filtration, with or without additional digestion with various exoglycosidases and endoglycosidases. Pulse-labeled glycoproteins contained a relatively homogenous population of neutral oligosaccharides (major species: Man9GlcNAc2ASN). The vast majority of these asparagine-linked oligosaccharides was smaller than the major fraction of lipid-linked oligosaccharides from the cell and was apparently devoid of terminal glucose. After pulse-chase or long labeling periods, a significant fraction of the large oligomannosyl cores was processed by removal of mannose units and addition of branch sugars (NeuNAc-Gal-GlcNAc), resulting in complex acidic structures containing three and possibly five mannoses. In addition, some of the large oligomannosyl cores were processed by the removal of only several mannoses, resulting in a mixture of neutral structures with 5-9 mannoses. This oligomannosyl core heterogeneity in both neutral and acidic oligosaccharides linked to asparagine in cellular membrane glycoproteins was analogous to the heterogeneity reported for the oligosaccharides of avian RNA tumor virus glycoproteins (Hunt LA, Wright SE, Etchison JR, Summers DF: J Virol 29:336, 1979).     

Oxazolidinone protection of N-acetylglucosamine confers high reactivity on the 4-hydroxy group in glycosylation

[reaction: see text] The preparation of a convenient oxazolidinone protected N-acetyl glucosamine 4-OH derivative is reported. This substance exhibits enhanced reactivity as a glycosyl acceptor in a variety of coupling methods, the products of which are converted to the target N-acetylglucosaminyl saccharides under very mild conditions.     

Capillary zone electrophoresis of lipoarabinomannan by multi-layered concentration

The present article describes a capillary zone electrophoresis method which relies on a multilayered water-alkali solvent stacking with online ionization to enhance detection of mannose, arabinose, and their oligosaccharides, which are used as the migration profile standards but are also the distinctive structural components of lipoarabinomannan. Lipoarabinomannan is detected in patients having tuberculosis. The capillary electrophoresis method with ionization of the whole saccharides without degradation in alkaline solution inside the capillary is based on the structural deprotonation of the molecules under ultrahigh pH conditions. The validation of the capillary electrophoresis parameters revealed that the 15-fold electrolyte–water-injection plug allowed detection of one-third lower concentrations than detected without online concentration. For the first time, the better detectability was seen especially for highly polymerized, but otherwise poorly ionized, arabinooctaose. The applicability of the method for detecting whole large biological saccharide complexes was confirmed by the glycolipid lipoarabinomannan. For the first time also, the migration of the indestructible lipoarabinomannan was detected together with oligosaccharides used representing the capping units, namely mannose, mannobiose, and mannotriose. The myo-inositol-phosphate-lipid unit was seen to migrate separately from the arabinomannan, since it was hydrolyzed from one lipoarabinomannan product under alkaline conditions in capillary electrophoresis.     

Derivatization of small oligosaccharides prior to analysis by matrix-assisted laser desorption/ionization using glycidyltrimethylammonium chloride and Girard's reagent T

In matrix-assisted laser desorption/ionization (MALDI) analyses of small oligosaccharides a very large increase in sensitivity (by a factor of 1000) may be obtained by introducing a quaternary ammonium center ('quaternization'). Such a quaternary ammonium center may be introduced into the saccharide by reaction with commercially available glycidyltrimethylammonium chloride (GTMA), or by using Girard's reagent T (Naven and Harvey, Rapid Commun. Mass Spectrom. 1996; 10: 829). GTMA reacts with alcohol functionalities, whereas Girard's reagent T is specific for aldehyde and keto groups. Thus reducing saccharides can be derivatized by both GTMA and Girard's reagent T. For example, glucose or cellobiose having a stock concentration of 3 x 10(-5) M (5 microg/mL) produces no sugar-derived signals in conventional MALDI, but their quaternized derivatives, also at 3 x 10(-5) M, yield intense signals, with the matrix-derived signals only being weak. Similar results were obtained for glucosamine. Non-reducing saccharides as well as sugar alcohols can be derivatized using GTMA; thus, although sucrose, raffinose and sorbitol do not react with Girard's reagent T, they all produce intense signals after derivatization with GTMA. An example of the application of these derivatization reactions is provided by the analysis of oligosaccharides in beer.     

On-column isomerization of sugars during high-performance liquid chromatography: analysis of the elution profile

Four monosaccharides (glucose, galactose, mannose and fructose) and one disaccharide (maltose) were subjected to high-performance liquid chromatography with UV or refractive index detection. Various profiles such as broad, tailed and splitted peaks were produced, depending on column temperature and eluent flow-rate because these saccharides underwent isomerization. In contrast, -methylglucoside, a non-converting derivative, always produced a sharp peak. By analyzing these profiles kinetic constants of the isomerization were obtained and compared with the literature data.     

Efficient route to orthogonally protected precursors of 2-acylamino-2-deoxy-3-O-substituted-β-d-glucopyranose derivatives and use thereof

An efficient route to two 3-O-acyl-2-deoxy-4,6-O-isopropylidene-2-trichloroacetamido-d-glucopyranosyl trichloroacetimidate donors is reported. As demonstrated for the 3-O-acetyl derivative, these building blocks are exquisite β-d-glucosamine donors when reacted either with simple alcohols or with complex oligosaccharides. Besides, their protection pattern is compatible with selective deprotection and subsequent chain elongation at O-3 of the newly incorporated glucosamine residue.     

The preparation of deoxy derivatives of mannose-1-phosphate and their substrate specificity towards recombinant GDP-mannose pyrophosphorylase from Salmonella enterica,group B

2-Deoxy-α-d-glucose-1-phosphate, 3-deoxy-α-d-arabino-hexose-1-phosphate, 4-deoxy-α-d-lyxo-hexose-1-phosphate, and α-d-lyxose-1-phosphate were synthesised chemically, and evaluated as substrates for a recombinant GDP-mannose pyrophosphorylase (Salmonella enterica, group B, cloned in Escherichia coli). The deoxy derivatives were all substrates for the enzyme, with slightly reduced V_max values but significantly higher K_m values than those recorded for the native substrate, mannose-1-phosphate. The pyrophosphorylase was used for the synthesis of GDP-mannose analogues GDP-2-deoxy-glucose and GDP-lyxose on a milligram scale.     

A novel condition for capillary electrophoretic analysis of reductively aminated saccharides without removal of excess reagents

We have identified novel CE conditions for the separation of 7‐amino‐4‐methylcoumarin‐labeled monosaccharides and oligosaccharides from glycoproteins. Using a neutrally coated capillary and alkaline borate buffer containing hydroxypropylcellulose and ACN, saccharide derivatives form anionic borate complexes, which move from the cathode to the anode in an electric field and are detected near the anodic end. Excess labeling reagents and other fluorescent products remain at the cathodic end. Fluorimetric detection using an LED as a light source enables determination of monosaccharide derivatives with good linearity between at least 0.4 and 400 μM, may correspond to 140 amol to 140 fmol. The lower LOD (S/N = 5) is only 80 nM in the sample solution (ca. 28 amol). The results were comparable to reported values using fluorometric detection LC. The method was also applied to the analysis of oligosaccharides that were enzymatically released from glycoproteins. Fine resolution enables profiling of glycans in glycoproteins. The applicability of the method was examined by applying it to other derivatives labeled with nonacidic tags such as ethyl p‐aminobenzoate‐ and 2‐aminoacridone‐labeled saccharides.     

端基含葡氨糖衍生物的聚乳酸的合成与表征

端基含葡氨糖衍生物的聚乳酸的合成与表征张国栋冯新德（北京大学高分子科学与工程系北京１００８７１）顾忠伟（国家计生委科研所药化室北京１０００８１）关键词壳多糖，聚乳酸，生物医用材料，化学修饰自从发现高分子量聚乳酸在人体内能够降解，并把这类材料引入...     

Modular synthesis of heparin oligosaccharides

A general, modular strategy for the first completely stereoselective synthesis of defined heparin oligosaccharides is described. Six monosaccharide building blocks (four differentially protected glucosamines, one glucuronic and one iduronic acid) were utilized to prepare di- and trisaccharide modules in a fully selective fashion. Installation of the alpha-glucosamine linkage was controlled by placing a conformational constraint on the uronic acid glycosyl acceptors thereby establishing a new concept for stereochemical control. Combination of disaccharide modules to form trans-uronic acid linkages was completely selective by virtue of C2 participating groups. Coupling reactions between disaccharide modules exhibited sequence dependence. While the union of many glucosamine uronic acid disaccharide modules did not meet any problems, certain sequences proved not accessible. Elaboration of glucosamine uronic acid disaccharide building blocks to trisaccharide modules by addition of either one additional glucosamine or uronic acid allowed for stereoselective access to oligosaccharides as demonstrated on the example of a hexasaccharide resembling the ATIII-binding sequence. Final deprotection and sulfation yielded the fully synthetic heparin oligosaccharides.     

Mass spectrometric profiling of N-linked oligosaccharides and uncommon glycoform in mouse serum with head and neck tumor

N-linked oligosaccharides obtained from total serum of mice with implanted head and neck tumors were analyzed and compared with those from control samples of healthy mice. Methods used include a combination of a derivatization procedure with phenylhydrazine (PHN) and analysis by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Oligosaccharides were enzymatically released from total serum with PNGaseF and purified by high-performance liquid chromatography (HPLC) on a reversed-phase column. Mass spectra contained ion peaks of labeled oligosaccharides and MS/MS experiments provided useful data for the structural elucidation of these compounds. More than 40 N-glycans with compositions characteristic of high-mannose, hybrid, complex, neutral, and sialylated structures were identified in the serum of tumoral mice. Significant differences between samples were observed with respect to the abundances of high mannose and hybrid glycans. These oligosaccharides showed higher relative intensities in the spectra obtained from the cancer sera. Complex sialylated oligosaccharides had similar abundances in both types of sera, with the exception of fucosylated biantennary disialylated oligosaccharide, which was mostly detected with lower abundance in control samples. In the MALDI spectra, several minor species corresponded to uncommon carbohydrates. These structures have been investigated in detail by MS/MS. Among these novel glycoforms, a few sialylated oligosaccharides without a free reducing end were identified. Also, glycans with an extra 60 u were observed and likely feature the presence of a 2-acetamido-2-deoxyoctose residue attached on antennae of 3- or 6-linked mannose.     

Microchip electrophoresis of oligosaccharides using lectin-immobilized preconcentrator gels fabricated by in situ photopolymerization

A lectin-impregnated gel was fabricated at the channel crossing point in a microfluidic chip made from polymethyl methacrylate (PMMA). The acrylamide containing lectin was photopolymerized to form a round gel (radius 60 μm) by irradiation with an argon laser, which was also used for fluorometric detection. This gel was applied to specific concentration, elution, and electrophoretic separation of fluorescent-labeled oligosaccharides. Because the lectin in the polyacrylamide gel was mechanically immobilized, it maintained its activity. The lectin was used to trap up to a few tens of femtomoles of specific oligosaccharides labeled with 8-aminopyrene-1,3,6-trisulfonic acid with 2 min by a factor >800, and the amount trapped corresponded to ca. 70% of lectin in the gel. The trapped oligosaccharides were released from the gel by lowering the pH with an acidic background electrolyte. The oligosaccharides that eluted as a broad band were concentrated by transient isotachophoresis stacking using concentrated sodium borate buffer (pH 11.0). The stacked sample components were then separated and fluorometrically detected at the end of the separation channel. Under the optimized conditions, resolution of the saccharides was good, and was similar to that obtained by pinched injection. The method was applied to preconcentration and analysis of oligosaccharides derived from some glycoproteins.     

Protecting‐Group‐Controlled Enzymatic Glycosylation of Oligo‐N‐Acetyllactosamine Derivatives

We describe a chemoenzymatic strategy that can give a library of differentially fucosylated and sialylated oligosaccharides starting from a single chemically synthesized tri‐N‐acetyllactosamine derivative. The common precursor could easily be converted into 6 different hexasaccharides in which the glucosamine moieties are either acetylated (GlcNAc) or modified as a free amine (GlcNH₂) or Boc (GlcNHBoc). Fucosylation of the resulting compounds by a recombinant fucosyl transferase resulted in only modification of the natural GlcNAc moieties, providing access to 6 selectively mono‐ and bis‐fucosylated oligosaccharides. Conversion of the GlcNH₂ or GlcNHBoc moieties into the natural GlcNAc, followed by sialylation by sialyl transferases gave 12 differently fucosylated and sialylated compounds. The oligosaccharides were printed as a microarray that was probed by several glycan‐binding proteins, demonstrating that complex patterns of fucosylation can modulate glycan recognition.     

Synthesis of High‐Mannose Oligosaccharide Analogues through Click Chemistry: True Functional Mimics of Their Natural Counterparts Against Lectins?

Terminal “high‐mannose oligosaccharides” are involved in a broad range of biological and pathological processes, from sperm‐egg fusion to influenza and human immunodeficiency virus infections. In spite of many efforts, their synthesis continues to be very challenging and actually represents a major bottleneck in the field. Whereas multivalent presentation of mannopyranosyl motifs onto a variety of scaffolds has proven to be a successful way to interfere in recognition processes involving high‐mannose oligosaccharides, such constructs fail at reproducing the subtle differences in affinity towards the variety of protein receptors (lectins) and antibodies susceptible to binding to the natural ligands. Here we report a family of functional high‐mannose oligosaccharide mimics that reproduce not only the terminal mannopyranosyl display, but also the core structure and the branching pattern, by replacing some inner mannopyranosyl units with triazole rings. Such molecular design can be implemented by exploiting “click” ligation strategies, resulting in a substantial reduction of synthetic cost. The binding affinities of the new “click” high‐mannose oligosaccharide mimics towards two mannose specific lectins, namely the plant lectin concanavalin A (ConA) and the human macrophage mannose receptor (rhMMR), have been studied by enzyme‐linked lectin assays and found to follow identical trends to those observed for the natural oligosaccharide counterparts. Calorimetric determinations against ConA, and X‐ray structural data support the conclusion that these compounds are not just another family of multivalent mannosides, but real “structural mimics” of the high‐mannose oligosaccharides.     

Synthesis of Selectively Protected Chitobiose and Chitotriose Derivatives from one Precursor. Versatile Building Blocks for Oligosaccharide Synthesis

Abstract

The inner core region of cell surface N-glycoproteins consists of a chitobiose substructure², containing β-(1,4)-linked disaccharides of glucosamine. Such carbohydrate structures are also found as repeating units of the bacterial cell wall peptidoglycan³ and in novel tetra- and pentasaccharide plant hormones, which are nodulation factors on legume roots.⁴ Since the first synthesis of a chitobiose derivative in 1966 by Paulsen,⁵ approaches to these compounds have relied mainly on the oxazoline method.⁶ The coupling reactions of aminosugar chlorides,⁷ bromides,⁸ acetates⁹ and trichloroacetimidates¹⁰ to suitable glycosyl acceptors have also been described. Most of these syntheses¹¹ require two completely different coupling partners; only in very few examples could the glycosyl donor and acceptor be obtained from the same starting material.¹² During our investigations into the stereocontrolled synthesis of glucosamine oligosaccharides, we required an economical synthetic route to protected derivatives of chitotriose. For the purpose of easy oligomerization, the anomeric protecting group of every building block had to be exchangeable selectively with the activating group for the next glycosylation. In this paper, we report an efficient approach to chitobiose and chitotriose from a single precursor. Furthermore, the hydroxyl groups at C-1, C-3, C-4, C-6 of these oligosaccharides are differentially protected. This protecting group scenario allows a specific access to any of these functionalities by regioselective deblocking. The N-phthalimide group was chosen out of several possible amino protecting groups to ensure β-selectivity and simultaneous activation in the coupling.¹³     

An integrated strategy of secondary metabolomics and glycomics to investigate multi-component variations in wine-processing of medicinal herbs and functional foods: A case study on Fructus Corni

Fructus Corni (FC), as a promising Chinese medicinal herb, has aroused considerable interest. Generally, FC needs to be processed according to the limited standard policy in China before clinical application, while the investigations on the specific processing methods (such as wine steaming or high-pressure wine steaming) are unclear. A comprehensive metabolomics strategy based on integrated non-targeted metabolomics and targeted glycomics in this paper was implemented to investigate the influences of the different processing technologies such as steaming, wine steaming, high-pressure steaming, high-pressure wine steaming, wine immersion, and wine stir-frying on FC, respectively. UHPLC-Q-TOF-MS/MS was employed for identifying and distinguishing the secondary metabolites. A total of 85 components were identified in all groups. The results of PCA score plots showed that the crude and processed samples had a complete separation, and wine steamed and high-pressure wine steamed samples could be a category, indicating that the two processed products had a similar quality. Multiple chromatography including HPLC (C₁₈)-PDA, HPLC (NH₂)-ELSD, and HPGPC-ELSD was used for determining the molecular weight distributions, the monosaccharide compositions of polysaccharides, and the contents of free monosaccharides and oligosaccharides. The results indicated that the content and composition of saccharides were different in crude and different processed FC. The polysaccharides were composed of fucose, arabinose, galactose, glucose, galacturonic acid, mannose and rhamnose, and the free monosaccharides were composed of fucose, arabinose, galactose, glucose, mannose, rhamnose and fructose in all FC samples. The PCA score plots of the glycomics indicated that the crude and high-pressure wine steamed FC could be a category, showing that the two groups had similar chemical compositions. Ultimately, the simulation processing experiments indicated that the transformation of morroniside, polysaccharides, oligosaccharides, fructose, and glucose to 5-HMF through the reactions of dehydration and deglycosylation was the potential mechanism of enhancing the effects by processing. Conclusionly, the saccharides should be investigated as thoroughly as the secondary metabolites, and the high-pressure wine steamed FC could be an alternative to wine steamed FC.     

Determination of saccharides in fruit juices by capillary electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

A fast method for the detection of cheap sweeteners is presented. Detecting the adulteration of foods rich in carbohydrates is complicated by the presence of variety of commercial sweeteners that are designed to match exactly the major carbohydrate profiles of these foods. Electrophoretic and mass spectrometric assays for the determination of fruit juice authenticity were developed. Capillary zone electrophoresis with indirect detection was employed to detect adulteration of juices demonstrated by the ratio of the concentrations of major low molecular mass saccharides (glucose, fructose and sucrose). Traces of oligosaccharides, which are not present in the sugar profiles of citrus fruits but are present in inexpensive sweeteners, were evaluated as the other group of target compounds. The fast determination of oligomeric starch hydrolysates in a complex matrix was tested by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) and applied to orange juice. MALDI-TOFMS was shown to be a suitable method for the identification of adulteration of fruit juices by starch hydrolysates. The effects of the presence of salts and low molecular mass saccharides on the detection of oligosaccharides by MALDI-TOFMS were studied. Low molecular mass saccharides and organic acids decrease the detectability of oligosaccharides by MALDI-TOFMS, but the concentration of maltooligosaccharides present in juices sweetened with starch hydrolysates is high enough to be detected with good sensitivity.     

Mass spectrometric determination of glycosylation sites and oligosaccharide composition of insect-expressed mouse interleukin-3

The primary structure of Baculovirus-expressed mouse interleukin-3 produced in infected Bombyx mori larvae was characterized by liquid secondary ion mass spectrometry and 252Cf-plasma desorption mass spectrometry in combination with selected protein microchemical reactions. Interleukin-3 was found to consist of at least two glycoprotein species of ca. 17,000 dalton. Characterization of tryptic and S. aureus V8 protease peptides by Edman degradation combined with plasma desorption mass spectrometry showed that two N-glycosylation sites. Asn-16 and Asn-86, were present. N-Glycan residues were shown by liquid secondary ion mass spectrometry and high-performance liquid chromatography to consist of mannose, fucose, and glucosamine. The presence of galactosamine indicated that O-glycosylated residues were present, in addition to the N-glycosylated residues. Glucose was also present, which indicated incomplete processing of the insect-expressed N-linked oligosaccharides.