A surface-based mass spectrometry method for screening glycosidase specificity in environmental samples

A new surface-based MALDI-Tof-MS glycosyl hydrolase assay has been developed in which lipid-tagged oligosaccharides, representing defined fragments of major plant cell wall polysaccharides, are immobilized via hydrophobic interactions on an alkylthiol functionalised gold sample plate and employed in the functional screening of several purified enzymes, environmental samples and saliva.     

寡糖和多糖的合成

本文重点介绍了寡塘及多馆在化学合成方面的进展,述及了寡掂经典合成方法及近年来发展的一些新方法,如三氯乙酸亚胺基作为离去基团的方法,以缩水内醚糖为糖的给体的方法等。对多糖的合成主要介绍了1,6-、1,4-、1,3-、1,2-缩水内醚搪的阳离子聚合制备均聚多塘,及用糖原酸醋衍生物通过缩聚反应制备多塘。     

A shotgun tandem mass spectrometric analysis of phospholipids with normal-phase and/or reverse-phase liquid chromatography/electrospray ionization mass spectrometry

Electrospray ionization mass spectrometry is used in lipidomics studies. The present research established a top-down liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) shotgun analysis method for phospholipids (PLs) using a normal-phase column or a C30 reverse-phase column with the data-dependent MS/MS scanning mode. A normal-phase column can separate most of the major different classes of PLs. By using LC/ESI-MS/MS with a normal-phase column, approximately 50 molecular species were identified in a PL mixture from rat liver. When the reverse-phase column was used, the PLs could be separated depending on their hydrophobicity, essentially the length of their fatty acyl chains and the number of unsaturated bonds in them. The LC/ESI-MS/MS method using a C30 reverse-phase column was applied to phosphatidylcholine (PC) and phosphatidylethanolamine (PE) mixtures as test samples. Molecular species with the same molecular mass but with different pairs of fatty acyl chains were separately identified. As a result, about 60 PC and 50 PE species were identified. PLs from rat liver were subjected to LC/ESI-MS/MS using the C30 reverse-phase column and about 110 molecular species were identified. Off-line two-dimensional LC/ESI-MS/MS with the normal-phase and C30 reverse-phase columns allowed more accurate identification of molecular species by using one-dimensional C30 reverse-phase LC/ESI-MS/MS analysis of the collected fractions.     

Synthesis of Oligosaccharide Structures from the Lipopolysaccharide of Moraxella catarrhalis

The synthesis of the octasaccharide [p-(trifluoroacetamido)phenyl]ethyl 4-O-[2-O-(2-acetamido-2-deoxy-alpha-D-glucopyranosyl)-beta-D-glucopyranosyl]-6-O-[2-O-[4-O-(4-O-alpha-D-galactopyranosyl-beta-D-galactopyranosyl)-alpha-D-glucopyranosyl]-beta-D-glucopyranosyl]-3-O-beta-D-glucopyranosyl-alpha-D-glucopyranoside, representing the outer part of the lipooligosaccharide from Moraxella catarrhalis serotype A, is described, together with a hepta-, a hexa-, and a pentasaccaride, composing parts thereof with shorter oligosaccharide chains substituted in the 6-position of the central 3,4,6-branched glucose moiety. The versatility of the use of thioglycosides in oligosaccharide synthesis is shown, since throughout the synthesis thioglycosides are used as glycosyl donor precursors, either directly in dimethyl(methylthio)sulfonium triflate (DMTST)-promoted coupling reactions or after conversion to the corresponding glycosyl bromide in silver triflate-promoted couplings. The effects of different protecting groups, anomeric leaving groups, and solvents used in the various coupling reactions are often substantial, which necessitates the use of easily convertible intermediates.     

Profiling of N-linked oligosaccharides using phenylhydrazine derivatization and mass spectrometry

N-linked oligosaccharide standards obtained from commercial sources were derivatized with phenylhydrazine (PHN) and analyzed by on-line reversed-phase high performance liquid chromatography (HPLC)/electrospray ionization mass spectrometry (ESI-MS). This procedure was then applied to mixtures of N-glycans enzymatically released from hen ovalbumin. Under ESI-MS conditions, phenylhydrazones of asialylated oligosaccharide standards and ovalbumin glycans produced mainly [M + 2H]2+ molecular ions at low cone voltage values, while minimal fragmentation was observed. Reversed-phase HPLC/ESI-MS total and selected ion chromatograms obtained for derivatized N-glycans from ovalbumin showed partial but useful separation. Overall glycan profiles obtained by ESI-MS were compared with results obtained by matrix-assisted laser desorption/ionization (MALDI)-MS. Qualitatively, profiles were similar from one technique to the other in terms of relative abundance of glycans versus composition. Post-source decay (PSD) analysis of the [M + Na]+ ions of PHN-glycans showed dominant B, C and internal B/Y, C/Y cleavages. These patterns were helpful in relating fragmentation to proposed structures. Cross-ring cleavage fragment ions (A-type) were also observed in most cases. The PHN derivatization method is fast and simple. It produces abundant parent ions in both MALDI-MS and ESI-MS, while avoiding the presence of salt contaminants during the labeling procedure.     

Analysis of phytochelatin-cadmium complexes from plant tissue culture using nano-electrospray ionization tandem mass spectrometry and capillary liquid chromatography/electrospray ionization tandem mass spectrometry.

Phytochelatins (PCs, also known as class III metallothioneins), a family of sulfhydryl-rich peptides with the formula (gamma-GluCys)(n)Gly(Pc(n), n = 2-11), are induced in plants, yeast and fungi exposed to heavy metals, and are thought to detoxify metals by forming PC- metal complexes. Although PCs have been detected, PC- metal complexes have not been well characterized. In this work, nano-electrospray ionization tandem mass spectrometry (nano-ESI-MS/MS) and capillary liquid chromatography/electrospray ionization tandem mass spectrometry (capillary LC/ESI-MS/MS) methods were used to analyze PC - Cd complexes isolated from Datura innoxia, also known as Jimsonweed, cell culture exposed to Cd. With nano-ESI-MS/MS and capillary LC/ESI-MS/MS we could simultaneously detect the presence of PCs and PC - Cd complexes from plant cell extracts, unambiguously identify these species and elucidate the nature of individual PC - Cd complexes. Phytochelatins with n = 3-6 were detected, as were PC - Cd complexes with PC(3), PC(4) and PC(5). This is the first study to report the size and nature of native PC - Cd complexes from plant tissue samples. These results demonstrate that the direct analysis of plant extracts using nano-ESI-MS/MS and capillary LC/ESI-MS/MS methods is simple and sensitive to the range of PCs and PC - Cd complexes in plants. Hence these methods open up new opportunities for further quantitative analysis of PCs and PC - metal complexes in cell culture and plant systems to understand the relationship between the biosynthesis of these compounds and metal tolerance.     

Synthetic 6-O-methylglucose-containing polysaccharides (sMGPs): design and synthesis

With the hope of mimicking the chemical and biological properties of natural 6-O-methyl-D-glucose-containing polysaccharides (MGPs), synthetic 6-O-methyl-D-glucose-containing polysaccharides (sMGPs) were designed and synthesized from alpha-, beta-, and gamma-cyclodextrins (CDs). The synthetic route proved to be flexible and general, to furnish a series of sMGPs ranging from 6-mer to 20-mer. A practical and scalable method was discovered selectively to cleave the CD derivatives and furnish the linear precursors to the glycosyl donors and acceptors. The Mukaiyama glycosidation was adopted to couple the glycosyl donors with the glycosyl acceptors. Unlike in the 3-O-methyl-D-mannose-containing polysaccharide (sMMP) series, the amount of the Mukaiyama acid required in the sMGP series increased with an increase of substrate size; that is, for large oligomers, more than one equivalent of the acid was required.     

Separation and characterization of underivatized oligosaccharides using liquid chromatography and liquid chromatography-electrospray ionization mass spectrometry

Native cyclodextrin-based columns are particularly useful for the analysis of oligosaccharides because the retention of these carbohydrates is based mainly on the hydrogen bonding interactions of oligosaccharide hydroxyl groups with the stationary phase. Thus, the retention time predictably increases with the number of analyte hydroxyl groups, which corresponds to the elongation of the oligosaccharide chain. High-performance liquid chromatography (HPLC) coupled to electrospray ionization (ESI) mass spectrometry (MS) was used for the separation and characterization of underivatized oligosaccharide mixtures. With the limits of detection as low as 50 pg, all individual components of oligosaccharide mixtures (up to 11 glucose units long) were baseline resolved on a Cyclobond I 2000 column and detected using ESI-MS. Low flow rates and narrow I.D. columns increase the ESI-MS sensitivity significantly. The method showed potential usefulness for the sensitive and quick analysis of hydrolysis products of polysaccharides, and for trace levels of individual oligosaccharide or oligosaccharide isomers from biological systems.     

From glycoside hydrolases to thioglycoligases: the synthesis of thioglycosides

The treatment of various glycosyl acceptors, each containing a reactive thiol group, with the appropriate glycosyl donor and a glycoside hydrolase or glycosynthase, failed to yield any thioglycosides––only the products of O-glycosylation were formed. However, thioglycosides were formed when a thioglycoligase was used to mediate the reaction between acceptor and donor. In fact, pyranose acceptors possessing a thiol group at C3, C4 or C6 (but not C2) were all capable of conversion into thioglycosides. Some comment is given regarding the mechanism of the various processes.     

电喷雾质谱在寡糖序列分析中的应用

选取具有不同结构特征的N-糖链、硫酸软骨素寡糖、人乳寡糖以及海洋来源的壳寡糖、褐藻胶寡糖、卡拉胶寡糖和硫酸岩藻寡糖等,对电喷雾质谱在寡糖的主链序列、分支位点、硫酸基取代位置确定、单糖组成和聚合度分析等方面的应用技术及碎片离子的断裂规律进行了总结.根据相邻同类碎片离子之间的质荷比差值可初步判断寡糖的单糖组成类型;通过与色谱分离技术联用或衍生化方法可提高寡糖的分辨率和离子化效率,并测得寡糖的分子量及聚合度;借助串联质谱及对寡糖还原端的特异性标记,可获得寡糖的还原端残基和部分序列信息;根据寡糖产生的特征碎片离子及其丰度大小可判断残基的特定位置和类型.另外,寡糖的分支通常作为一个整体发生糖苷键断裂或产生D离子,据此可判断分支点的位置;根据硫酸寡糖产生的特异性跨环断裂碎片,可以确定硫酸基的连接位置.这些规律和方法的总结为未知寡糖的结构和序列的分析提供了启发和指导.     

Application of glycosyl thioimidates in solid-phase oligosaccharide synthesis

Two stable classes of thioimidoyl derivatives, S-benzoxazolyl (SBox) and S-thiazolinyl (STaz) glycosides, were investigated as glycosyl donors for solid-phase oligosaccharide synthesis. It was demonstrated that these derivatives are suitable for both glycosyl acceptor-bound and glycosyl donor-bound strategies, commonly employed in resin-supported oligosaccharide synthesis.     

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.     

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.     

Enhancement of palmarumycin C12 and C13 production in liquid culture of the endophytic fungus Berkleasmium sp. Dzf12 by oligosaccharides from its host plant Dioscorea zingiberensis

Three crude oligosaccharides were respectively prepared by acid hydrolysis of three polysaccharides, which were water-extracted polysaccharide (WEP), sodium hydroxide-extracted polysaccharide (SEP) and acid-extracted polysaccharide (AEP) from the rhizomes of Dioscorea zingiberensis. Among the three oligosaccharides, the crude oligosaccharide prepared by acid hydrolysis of WEP was found to be the most efficient elicitor to enhance the production of palmarumycins C(12) and C(13) in liquid culture of endophytic fungus Berkleasmium sp. Dzf12. When OW was applied to the medium at 300 mg/L on day 3 of culture, the maximal yields of palmarumycin C(12) (87.96 mg/L) and palmarumycin C(13) (422.28 mg/L) were achieved on day 15 of culture, which were 9.83 and 3.24-fold in comparison with those (8.95 and 130.43 mg/L) of control, respectively. The results indicate that addition of the oligosaccharides from the host plant D. zingiberensis should be an effective strategy for enhancing production of palmarumycins C(12) and C(13) in liquid culture of endophytic fungus Berkleasmium sp. Dzf12.     

Electrospray ionization mass spectrometry fingerprinting of propolis

Crude ethanolic extracts of propolis, a natural resin, have been directly analysed using electrospray ionization mass (ESI-MS) and tandem mass spectrometry (ESI-MS/MS) in the negative ion mode. European, North American and African samples have been analyzed, but emphasis has been given to Brazilian propolis which displays diverse and region-dependent chemical composition. ESI-MS provides characteristic fingerprint mass spectra, with propolis samples being divided into well-defined groups directly related to their geographical origins. Chemometric multivariate analysis statistically demonstrates the reliability of the ESI-MS fingerprinting method for propolis. On-line ESI-MS/MS tandem mass spectrometry of characteristic [M - H](-) ion markers provides an additional dimension of fingerprinting selectivity, while structurally characterizing the ESI-MS marker components of propolis. By comparison with standards, eight such markers have been identified: para-coumaric acid, 3-methoxy-4-hydroxycinnamaldehyde, 2,2-dimethyl-6-carboxyethenyl-2H-1-benzopyran, 3-prenyl-4-hydroxycinnamic acid, chrysin, pinocembrin, 3,5-diprenyl-4-hydroxycinnamic acid and dicaffeoylquinic acid. The negative mode ESI-MS fingerprinting method is capable of discerning distinct composition patterns to typify, to screen the sample origin and to reveal characteristic details of the more polar and acidic chemical components of propolis samples from different regions of the world.     

On-line sheathless capillary electrophoresis/nanoelectrospray ionization-tandem mass spectrometry for the analysis of glycosaminoglycan oligosaccharides

A novel approach in glycosaminoglycomics, based on sheathless on-line capillary electrophoresis/nanoelectrospray ionization-quadrupole time of flight-mass spectrometry (CE/nanoESI-QTOF-MS) and tandem MS of extended chondroitin sulfate/dermatan (CS/DS) oligosaccharide chains is described. The methodology required the construction of a new sheathless CE/nanoESI-QTOF-MS configuration, its implementation and optimization for the high sensitivity analysis of CS/DS oligosaccharide mixtures from conditioned culture medium of decorin transfected human embryonic kidney (HEK) 293 cells. Under newly established sheathless on-line CE/(-)nanoESI conditions for glycosaminoglycan (GAG) ionization and MS detection, single CS/DS oligosaccharide components of extended chain length and increased sulfation degree were identified. Molecular ions corresponding to species carrying 5 and 6 negative charges could be generated for large GAG oligosaccharide species in the negative ion nanoESI-MS. The optimized on-line conditions enabled the detection of molecular ions assigned to oversulfated tetradeca-, octadeca-, and eicosasaccharide CS/DS molecules, which represent the category of largest sulfated GAG-derived oligosaccharides evidenced by CE/ESI-MS. By on-line CE/ESI tandem MS in data-dependent acquisition mode the oversulfated eicosasaccharide species could be sequenced and the localization of the additional sulfate group along the chain could be determined.     

Quantitative determination of endogenous sorbitol and fructose in human nerve tissues by atmospheric-pressure chemical ionization liquid chromatography/tandem mass spectrometry

Attachment of anions to sorbitol and fructose has been shown to enhance sensitivity in both electrospray ionization (ESI) and atmospheric-pressure chemical ionization (APCI) mass spectrometry. The post-column addition of CHCl3 produced Cl-adducts of sorbitol and fructose but their signals were suppressed due to the elevated background. Different chlorinated compounds and different additive methods were systematically investigated to form more abundant Cl-adduct precursor ions and deprotonated product ions. The major causes of the high background were explored and effective methods were developed to improve the signal-to-noise ratios and reproducibility. The compositions of mobile phase, percentages of organic modifiers (MeCN, MeOH and water), columns, oven temperature, flow rates and different gradients were investigated to separate sorbitol from fructose along with their isomers including glucose, galactose, mannose, sorbose, mannitol, and dulcitol. The optimized separation was achieved on a Luna 5 mu NH2 100A column (150 x 4.6 mm) using a mobile phase containing MeCN with 0.1% of CH2Cl2 and 50% MeOH in water at a flow rate of 800 microL/min and an oven temperature of 40 degrees C using a gradient liquid chromatography (LC) system. Human nerve tissue samples were extracted by protein precipitation followed by mixed-mode solid-phase extraction. The LC/ESI-MS/MS method produced higher peak intensities than LC/APCI-MS/MS. However, there were matrix effects from extracted tissues in LC/ESI-MS/MS but not in LC/APCI-MS/MS. Consequently, APCI proved to be the more effective method of ionization. Then the LC/APCI-MS/MS method was fully validated and successfully applied to analysis of clinical samples. The concentrations of endogenous sorbitol and fructose were determined using calibration curves employing sorbitol-13C6 and fructose-13C6 as surrogate analytes. The method has provided excellent intra- and inter-assay precision and accuracy with linear ranges of 0.2-80 ng/mg for sorbitol and 1-400 ng/mg for fructose in human nerve tissues.     

N-benzyl-2,3-oxazolidinone as a glycosyl donor for selective alpha-glycosylation and one-pot oligosaccharide synthesis involving 1,2-cis-glycosylation

Glycosylation reactions using N-benzyl-2,3-trans-oxazolidinones as the glycosyl donors were shown to be highly alpha-selective. Advantages of the donor include facile preparation in gram-scale preparation and simple deprotection procedures. Subsequently, a one-pot oligosaccharide synthesis involving 1,2-cis glycosidic linkages was demonstrated using the novel glycosyl donors.     

Structure elucidation of everninomicin-6, a new oligosaccharide antibiotic, by chemical degradation and FAB-MS methods

The structural characterization of a new oligosaccharide antibiotic, Everninomicin-6 (EV-6), is described. Detailed fast-atom bombardment mass spectrometry (FAB-MS) studies along with NMR and chemical degradation methods were conducted to elucidate the structure of EV-6. The effects of the use of various matrices, including salt addition, on the quality of the FAB-MS were explored. The use of 3-nitro benzyl alcohol, dimethyl sulfoxide (DMSO), and NaCl produced the best results: an intense sodiated molecular ion plus structurely informative fragmentation. FAB-MS yields information providing the complete sugar sequence information for everninomicins, which is quite valuable to the elucidation of the structure of this complex oligosaccharide antibiotic. In addition, the results of accurate mass work with the molecular ion are consistent with the assigned structure. The use of electrospray ionization mass spectrometry (ESI-MS) and ESI-MS/MS for the study of EV-6 was investigated and was found to produce an abundant molecular ion with limited structural information. These results revealed that EV-6 resembled EV-D quite closely except for the absence of the nitrosugar and the replacement on ring g of the -CH₂OCH₃ group with a -CH₂OH group.     

One‐Pot Synthesis of Unprotected Anomeric Glycosyl Thiols in Water for Glycan Ligation Reactions with Highly Functionalized Sugars

Chemical synthesis of oligosaccharide conjugates is essential for studying the functional relevance of carbohydrates, and this task would be facilitated considerably if reliable methods for the anomeric ligation of unprotected sugars in water were available. Here, a method for the preparation of anomeric glycosyl thiols from complex unprotected mono‐, di‐, and oligosaccharides is presented. By exploiting the neighboring‐group effect of the 2‐acetamido‐group, 1,2‐oxazolines are generated and converted into 1‐glycosyl thioesters through treatment with 1‐thioacids. The unprotected anomeric glycosyl thiolates released in situ were conjugated to Michael acceptors, aliphatic halogenides, and aziridines to furnish versatile glycoconjugates. Conjugation of amino acids and proteins was accomplished using the thiol–ene reaction with terminal olefins. This method gives efficient access to anomeric glycosyl thiols and thiolates, which enables anomeric ligations of complex unprotected glycans in water.