Determination of sulfation pattern in brain glycosaminoglycans by chip-based electrospray ionization ion trap mass spectrometry

Chondroitin sulfate (CS) and dermatan sulfate (DS) glycosaminoglycans display variability of sulfation in their constituent disaccharide repeats during chain elongation. Since a large proportion of the extracellular matrix of the central nervous system (CNS) is composed of proteoglycans, CS/DS disaccharide degree and profile of sulfation play important roles in the functional diversity of neurons, brain development, and some of its pathological states. To investigate the sulfation pattern of CS/DS structures expressed in CNS, we introduced here a novel method based on an advanced system encompassing fully automated chip nanoelectrospray ionization (nanoESI) in the negative ion mode and high capacity ion trap multistage mass spectrometry (MS²–MS³) by collision-induced dissociation (CID). This method, introduced here for the first time in glycomics of brain glycosaminoglycans, was particularly applied to structural investigation of disaccharides obtained by β-elimination and digestion with chondroitin B and AC I lyase of hybrid CS/DS chains from wild-type mouse brain. Screening in the chip-MS mode of DS disaccharide fraction resulting after depolymerization with chondroitin B lyase revealed molecular ions assigned to monosulfated disaccharide species having a composition of 4,5-Δ-[IdoA-GalNAc]. By optimized CID MS²–MS³, fragment ions supporting the localization of sulfate ester group at C4 within GalNAc were produced. Chip ESI MS profiling of CS disaccharide fraction obtained by depolymerization of the same CS/DS chain using chondroitin AC I lyase indicated the occurrence of mono- and bisulfated 4,5-Δ-[GlcA-GalNAc]. The site of oversulfation was determined by MS²–MS³, which provided sequence patterns consistent with a rare GlcA-3-sulfate–GalNAc-6-sulfate structural motif.      

Combining size-exclusion chromatography and fully automated chip-based nanoelectrospray quadrupole time-of-flight tandem mass spectrometry for structural analysis of chondroitin/dermatan sulfate in human decorin

Chondroitin/dermatan sulfate (CS/DS) chain of decorin (DCN) from human skin fibroblasts (HSk) was released by reductive β-elimination reaction and digested with chondroitin AC I lyase. Enzymatic hydrolysis mixture of CS/DS chains was separated by size-exclusion chromatography (SEC). Collected octasaccharide fraction was subjected to fully automated chip-based nanoelectrospray (nanoESI) quadrupole time-of-flight (QTOF) MS and tandem MS (MS/MS). MS of human skin fibroblasts DCN CS/DS displayed a high complexity due to the large variety of glycoforms, which under chip-nanoESI MS readily ionized to form multiply charged ions. Except for the regularly tetrasulfated octasaccharide, the investigated fraction contained four additional octasaccharides of atypical sulfation status. Two new oversulfated glycoforms and two undersulfated species were identified. Remarkably, the series of decasaccharides discovered in the same SEC pool was found to encompass a trisulfated and a novel hexasulfated [4,5-Δ-GlcAGalNAc(IdoAGalNAc)?] species. MS/MS by collision-induced dissociation (CID) on the [M-4H]? ion corresponding to the previously not reported [4,5-Δ-GlcAGalNAc(IdoAGalNAc)?](5S) corroborated for a novel motif in which three N-acetylgalactosamine (GalNAc) moieties are monosulfated, 4,5-Δ-GlcA and the first IdoA from the non-reducing end bear one sulfate group each, while the second N-acetylgalactosamine from the reducing end is unsulfated.     

Silylation of Cellulose and Starch – Selectivity, Structure Analysis, and Subsequent Reactions

The silylation of cellulose and starch under different starting conditions is reviewed. The control of the degree of substitution (DS) and regioselectivity in dependence of the reaction pathway are discussed in detail. The synthesis of trimethylsilyl cellulose (TMSC) in the system hexamethyldisilazane (HMDS)/ammonia leads to partially and completely silylated products controlled by the amount of the components. Hydrolytic desilylation of TMSC in tetrahydrofuran (THF)/ammonia gives the partially desilylated products. The desilylation proceeds statistically along the polymer chains. The reaction of cellulose dissolved in N,N-dimethylacetamide (DMA)/LiCl with bulky thexyldimethylchlorosilane (TDSCl) in the presence of imidazole leads to 2,6-di-O-TDS cellulose. The silylation of starch dissolved in dimethylsulfoxide (DMSO) with TDSCl/pyridine results in the formation of regioselectively 2-O and 6-O functionalized silyl ethers with DS values up to 1.8. 6-O Silyl ethers of cellulose and starch were synthesized with TDSCl highly activated in the reaction system N-methylpyrrolidone (NMP)/ammonia. Two- dimensional NMR techniques after subsequent modifications of the remaining OH groups have been established as important methods for the characterization of the substitution pattern of the described silyl ethers. In the case of starch, the distribution of the substituents could be detected not only in the anhydroglucose units (AGU) but also in the non-reducing end groups (NEG).     

Effects of sulfate position on heparin octasaccharide binding to CCL2 examined by tandem mass spectrometry

Chemokine-glycosaminoglycan (GAG) interactions have been shown to be essential for in vivo chemokine signaling, which functions in such diverse processes as inflammation, development, and cancer metastasis. Despite the importance of these interactions, the saccharide sequence dependency of chemokine-GAG interactions is poorly understood. In a recent study, FT-ICR mass spectrometry was used to show that the chemokine CCL2 (monocyte chemoattractant protein 1) binds only to the 11- and 12-sulfated components of a heparin octasaccharide library. Although the exact structure of the fully sulfated, 12-sulfated octasaccharide is known, the 11-sulfated species could have a number of sulfated disaccharide sequences. In the current study, the composition of the 11-sulfated heparin octasaccharides, as well as the composition of CCL2 affinity purified 11-sulfated heparin octasaccharides, were examined by tandem MS. Of the three possible singly desulfated disaccharides, one species, III-S, is enriched by CCL2 affinity purification, indicating that the 11-sulfated heparin octasaccharides containing this disaccharide are preferentially bound to CCL2. These data suggest that 2-O and N sulfation of heparin may be of greater importance to CCL2-heparin binding than 6-O sulfation.     

Structures and Anticoagulant Activities of the Partially Mild Acidic Hydrolysis Products of the Fucosylated Chondroitin Sulfate from Sea Cucumber Pearsonothuria graeffei

Fucosylated chondroitin sulfate from Pearsonothuria graeffei (fCS-Pg) is a unique glycosaminoglycan (GAG), which was reported to have potent antithrombotic and anticoagulant activities. In the present study, the native fCS-Pg was hydrolyzed by mild acid to improve its bioavailability. The structures of the acid-released and acid-resistant sulfated fucose branches of fCS-Pg before and after acidic hydrolysis were characterized by nuclear magnetic resonance spectroscopy (NMR) technology. The results showed the acid-released fucose residues included both α- and β-fucose conformation, containing 2,4-di-O-sulfated fucose (Fuc2,4S), 3,4-di-O-sulfated fucose (Fus3,4S), 3-O-sulfated fucose (Fuc3S), and 4-O-sulfated fucose (Fuc4S), with a mole ratio of 6:26:22:46, respectively. Their difference to the native fCS-Pg in the sulfation pattern of the backbone could be due to selective removal of the sulfated groups during the release of the fucose branches by acid. For the acid-resistant part, the sulfation patterns of fucose were similar to the native polysaccharides, but the signal strength of Fuc3,4S was obviously decreased, whereas Fuc2,4S was kept unchanged, indicating Fuc3,4S is more liable to acid. The anticoagulant activities of the acid-resistant part were tested in vitro, and the results showed that partial degradation could result in significant reduction of anticoagulant activity, which could help to lower the risk of bleeding when developing fCS as an antithrombotic drug.     

Isolation and characterization of an exopolygalacturonase from Fusarium oxysporum f.sp. cubense race 1 and race 4

Background

Previously, we have reported the presence of highly sulfated dermatans in solitary ascidians from the orders Phlebobranchia (Phallusia nigra) and Stolidobranchia (Halocynthia pyriformis and Styela plicata). Despite the identical disaccharide backbone, consisting of [→4IdoA(2S)β-1→3GalNAcβ-1→], those polymers differ in the position of sulfation on the N-Acetyl galactosamine, which can occur at carbon 4 or 6. We have shown that position rather than degree of sulfation is important for heparin cofactor II activity. As a consequence, 2,4- and 2,6-sulfated dermatans have high and low heparin cofactor II activities, respectively. In the present study we extended the disaccharide analysis of ascidian dermatan sulfates to additional species of the orders Stolidobranchia (Herdmania pallida, Halocynthia roretzi) and Phlebobranchia (Ciona intestinalis), aiming to investigate how sulfation evolved within Tunicata. In addition, we analysed how heparin cofactor II activity responds to dermatan sulfates containing different proportions of 2,6- or 2,4-disulfated units.

Results

Disaccharide analyses indicated a high content of disulfated disaccharide units in the dermatan sulfates from both orders. However, the degree of sulfation decreased from Stolidobranchia to Phlebobranchia. While 76% of the disaccharide units in dermatan sulfates from stolidobranch ascidians are disulfated, 53% of disulfated disaccharides are found in dermatan sulfates from phlebobranch ascidians. Besides this notable difference in the sulfation degree, dermatan sulfates from phlebobranch ascidians contain mainly 2,6-sulfated disaccharides whereas dermatan sulfate from the stolidobranch ascidians contain mostly 2,4-sulfated disaccharides, suggesting that the biosynthesis of dermatan sulfates might be differently regulated during tunicates evolution. Changes in the position of sulfation on N-acetylgalactosamine in the disaccharide [→4IdoA(2-Sulfate)β-1→3GalNAcβ-1→] modulate heparin cofactor II activity of dermatan sulfate polymers. Thus, high and low heparin cofactor II stimulating activity is observed in 2,4-sulfated dermatan sulfates and 2,6-sulfated dermatan sulfates, respectively, confirming the clear correlation between the anticoagulant activities of dermatan sulfates and the presence of 2,4-sulfated units.

Conclusions

Our results indicate that in ascidian dermatan sulfates the position of sulfation on the GalNAc in the disaccharide [→4IdoA(2S)β-1→3GalNAcβ-1→] is directly related to the taxon and that the 6-O sulfation is a novelty apparently restricted to the Phlebobranchia. We also show that the increased content of [→4IdoA(2S)β-1→3GalNAc(4S)β-1→] disaccharide units in dermatan sulfates from Stolidobranchia accounts for the increased heparin cofactor II stimulating activity.     

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.     

Elucidation of the molecular structure of lipid A isolated from both a rough mutant and a wild strain of Aeromonas salmonicida lipopolysaccharides using electrospray ionization quadrupole time-of-flight tandem mass spectrometry

The chemical structure of lipid A, isolated by mild acid hydrolysis from a rough mutant and a wild strain of Aeromonas salmonicida lipopolysaccharide, was investigated using electrospray ionization quadrupole time-of-flight (QqToF) hybrid tandem mass spectrometry and showed a great degree of microheterogeneity. The chemical structure of the main constituent of this heterogeneous mixture was identified as a beta-D-(1 --> 6) linked D-glucosamine disaccharide substituted by two phosphate groups, one being bound to the non-reducing end at position O-4' and the other to the position O-1 of the reducing end of the D-glucosamine disaccharide. The location of the fatty acids linked to the disaccharide backbone was established by identifying diagnostic ions in the conventional QqToF-MS scan. Low-energy collision tandem mass spectrometry analysis of the selected precursor diagnostic ions confirmed, unambiguously, their proposed molecular structures. We have established that myristyloxylauric (C14:0(3-O(12:0))) acid residues were both N-2' and O-3' linked to the non-reducing end of the D-GlcN residue, and that two 3-hydroxymyristic (C14:0(3-OH)) acid chains acylated the remaining positions of the reducing end. The MS and MS/MS data obtained allowed us to determine the complex molecular structure of lipid A. The QqToF-MS/MS instrument has shown excellent superiority over a conventional quadrupole-hexapole-quadrupole tandem instrument which failed to fragment the selected precursor ion.     

Multistage Tandem Mass Spectrometry of Chondroitin Sulfate and Dermatan Sulfate

Chondroitin/dermatan sulfate (CS/DS) is a glycosaminoglycan (GAG) found in abundance in extracellular matrices. In connective tissue, CS/DS proteoglycans play structural roles in maintaining viscoelasticity through the large number of immobilized sulfate groups on CS/DS chains. CS/DS chains also bind protein families including growth factors and growth factor receptors. Through such interactions, CS/DS chains play important roles in neurobiochemical processes, connective tissue homeostasis, coagulation, and cell growth regulation. Expression of DS has been observed to increase in cancerous tissue relative to controls. In earlier studies, MS(2) was used to compare the types of CS/DS isomers present in biological samples. The results demonstrated that product ion abundances reflect the types of CS/DS repeats present and can be used quantitatively. It was not clear, however, to which of the CS/DS repeats the product ions abundances were sensitive. The present work explores the utility of MS(3) for structural characterization of CS/DS oligosaccharides. The data show that MS(3) product ion abundances correlate with the presence of DS-like repeats in specific positions on the oligosaccharide chains.     

Apolipoprotein E Recognizes Alzheimer's Disease Associated 3-O Sulfation of Heparan Sulfate

Apolipoprotein E (ApoE)’s ϵ4 alle is the most important genetic risk factor for late onset Alzheimer's Disease (AD). Cell-surface heparan sulfate (HS) is a cofactor for ApoE/LRP1 interaction and the prion-like spread of tau pathology between cells. 3-O-sulfo (3-O-S) modification of HS has been linked to AD through its interaction with tau, and enhanced levels of 3-O-sulfated HS and 3-O-sulfotransferases in the AD brain. In this study, we characterized ApoE/HS interactions in wildtype ApoE3, AD-linked ApoE4, and AD-protective ApoE2 and ApoE3-Christchurch. Glycan microarray and SPR assays revealed that all ApoE isoforms recognized 3-O-S. NMR titration localized ApoE/3-O-S binding to the vicinity of the canonical HS binding motif. In cells, the knockout of HS3ST1-a major 3-O sulfotransferase-reduced cell surface binding and uptake of ApoE. 3-O-S is thus recognized by both tau and ApoE, suggesting that the interplay between 3-O-sulfated HS, tau and ApoE isoforms may modulate AD risk.     

Synthesis of A Novel Sialyl Lewis X Analogue Containing A Pyrrolidine in Place of N-Acetyl Glucosamine

ABSTRACT

The synthesis of the new sialyl Lewis X analogue, 4-O-(α-L-fucopyranosyl)-3-O-(3-O-sodium sulfonato-β-D-galactopyranosyl)-(2S,3R, 4R)-2-ethyl-3,4-dihydroxypyrrolidine 2 has been achieved. The N-acetyl glucosamine unit of natural Lewis X has been replaced by a rigid 3R/4R-dihydroxylated pyrrolidine 12. This one has been synthezised from the methyl 4-O-benzoyl-2,3-di-O-benzyl-6-deoxy-6-iodo-α-D-altropyranoside sugar precursor 10 using the Ganem/Bernotas one-pot elimination-reductive amination ring contraction reaction. The (2S, 3R, 4R)-1-benzyloxycarbonyl-3,4-dihydroxy-2-ethylpyrrolidine 12 obtained was subsequently regioselectively glycosylated, using 2,3,4-tri-O-benzyl-α-L-fucopyranosyl fluoride and 2,3,4,6-tetra-O-benzoyl-β-D-galactopyranosyl bromide as glycosyl donors. Disaccharide containing pyrrolidine 21 was finally transformed into the target O-sulfated analog 2, after regioselective sulfation and usual deprotection.     

Arabinofuranose disaccharide analogs as inhibitors of Mycobacterium tuberculosis

Several octyl 5-O-(α-d-arabinofuranosyl)-α-d-arabinofuranoside disaccharide analogs substituted at the 5-position of the non-reducing end sugar were synthesized and tested in vitro against Mycobacterium tuberculosis (M.tb.), Mycobacterium avium complex (MAC) as well as in a cell free assay system for arabinosyltransferase acceptor/inhibitor activity. A few compounds showed interesting inhibitory activity in the cell free assay as well as against the whole microorganism in vitro.     

Regioselectively Modified Cellulose and Chitosan Derivatives for Mono- and Multilayer Surface Coatings of Hemocompatible Biomaterials

In this study different synthetic strategies were developed and applied to introduce solely or in combination heparin/heparansulfate-like functional groups such as N-sulfo, O-sulfo, N-acetyl, and N-carboxymethyl groups into chitosan and cellulose with highest possible regioselectivity and completeness and defined distribution along the polymer chain. Completely substituted 6-amino-6-deoxycellulose and related derivatives were prepared from tosylcellulose (DS 2.02; C₆ 1.0) by nucleophilic substitution with azido groups only in the 6-position at 50 °C with subsequent reduction to amino groups and completely removing tosyl groups in the 2,3-position. 2,6-Di-O-sulfocellulose was prepared using the reactivity difference between C-2, C-6 and C-3 of cellulose. The reactivity difference between amino groups and hydroxyl groups was used to prepare various N-substituted derivatives. Partially 2,6-di-O-sulfated cellulose was obtained from trimethylsilylcellulose by the insertion of sulfurtrioxide into the Si–O ether linkage. Partially 3-O-sulfocellulose was synthesized by protecting C-2 and C-6 with trifluoroacetyl groups. A copper–chitosan complex was used to synthesize 6-O-sulfochitosan with a DS of 1.0 at C-6 and various partially 6-O-desulfonated products are possible. Using the phthalimido group to increase the solubility of chitosan in DMF, the regioselectivity of 3-O-sulfo groups was improved by regioselective 6-O-desulfonation of nearly complete 3,6-O-disulfochitosan. The platelet adhesion properties of immobilized regioselectively modified water-soluble derivatives on membranes have been tested in vitro. Some regioselectively modified chitosan and cellulose derivatives are potential candidates for the surface coatings of biomaterials if the regioselective reactions are somewhat further optimized.     

Discrimination of GalNAc (4S/6S) sulfation sites in chondroitin sulfate disaccharides by chip-based nanoelectrospray multistage mass spectrometry

Sulfation pattern within chondroitin sulfate (CS) glycosaminoglycan (GAG) chains is an important post-translational modification that regulates their interaction with proteins. In this context, development of highly efficient and reproducible analytical methods for the investigation of CS sulfation patterns is of high necessity. In this study we report a novel method for straightforward determination of N-acetylgalactosamine (GalNAc) sulfation sites in chondroitin sulfate disaccharides. Our protocol involves combining fully automated chip-based nanoelectrospray (nanoESI) for analyte infusion and ionization in negative ion mode with multistage (MSⁿ) collision-induced dissociation (CID) high capacity ion trap (HCT) mass spectrometry for generation of sequence ions diagnostic for identification of sulfate ester group position within GalNAc residues. The feasibility of this approach is here demonstrated on chondroitin 6-O-sulfate and chondroitin 4-O-sulfate disaccharides. Fragmentation patterns obtained by MS² and MS³ sequencing stages provided first mass spectrometric data from which sulfation site(s) within GalNAc monosaccharide ring could be unequivocally deciphered. Hence, the method allowed discriminating 4S/6S sulfation sites solely on the basis of MS and multistage MS evidence.      

LC-MS<Superscript><Emphasis Type="BoldItalic">n</Emphasis></Superscript> Analysis of Isomeric Chondroitin Sulfate Oligosaccharides Using a Chemical Derivatization Strategy

Improved methods for structural analyses of glycosaminoglycans (GAGs) are required to understand their functional roles in various biological processes. Major challenges in structural characterization of complex GAG oligosaccharides using liquid chromatography-mass spectrometry (LC-MS) include the accurate determination of the patterns of sulfation due to gas-phase losses of the sulfate groups upon collisional activation and inefficient on-line separation of positional sulfation isomers prior to MS/MS analyses. Here, a sequential chemical derivatization procedure including permethylation, desulfation, and acetylation was demonstrated to enable both on-line LC separation of isomeric mixtures of chondroitin sulfate (CS) oligosaccharides and accurate determination of sites of sulfation by MS ⁿ . The derivatized oligosaccharides have sulfate groups replaced with acetyl groups, which are sufficiently stable to survive MS ⁿ fragmentation and reflect the original sulfation patterns. A standard reversed-phase LC-MS system with a capillary C18 column was used for separation, and MS ⁿ experiments using collision-induced dissociation (CID) were performed. Our results indicate that the combination of this derivatization strategy and MS ⁿ methodology enables accurate identification of the sulfation isomers of CS hexasaccharides with either saturated or unsaturated nonreducing ends. Moreover, derivatized CS hexasaccharide isomer mixtures become separable by LC-MS method due to different positions of acetyl modifications.     

Synthesis of carboxyl cellulose sulfates with regioselective sulfation and regiospecific oxidation using cellulose trifluoroacetate as intermediates

Synthesis of cellulose sulfates (CSs) and carboxyl cellulose sulfates (COCSs) with regioselectively or regiospecifically distributed functional groups within anhydroglucose units was reported. CS with regioselectively distributed sulfate groups at 2,3-O- or 2,6-O-position were homogeneously synthesized and cellulose trifluoroacetate (CTFA) was used as intermediates. The trifluoroacetyl groups were detected primarily at 6-O-position and their distributions could be altered by changing the amount of trifluoroacetyl anhydride (TFAA). Various sulfating agents were used for further homogeneous sulfation of CTFA. The total degree of sulfation (DS_S) and the distribution of sulfate groups within the repeating units were affected by the amount of TFAA, the type and amount of sulfating agents. Subsequent homogenous 4-acetamide-TEMPO or TEMPO-mediated oxidation of CS led to COCS with carboxyl groups regiospecifically distributed at C6 position, which may be interesting structural mimics for natural occurring heparin.     

Synthesis of three regioisomers of the pentasaccharide part of the Skp1 glycoprotein of Dictyostelium discoideum

Three regioisomers of the linear pentasaccharide part of the Skp1 glycoprotein, found in Dictyostelium discoideum, were prepared in the form of (2-trimethylsilyl)ethyl glycosides by means of 2+3 block syntheses using the disaccharide donor at the non-reducing end, and three different trisaccharide acceptors at the reducing end. Fucosylation of (2-trimethylsilyl)ethyl 3,4,6-tri-O-benzyl-β-d-galactopyranosyl-(1→3)-4,6-O-benzylidene-2-deoxy-2-NPhth-β-d-glucopyranoside with different fucosyl donors carrying an O-(2-naphthyl)methyl ether as a temporary-protecting group at positions C2, C3 or C4 gave rise to the protected core trisaccharides. After selective removal of the (2-naphthyl)methyl group, the resulting acceptors were glycosylated with the α(1→6) linked digalactosyl donor to yield the respective three regioisomeric pentasaccharides. Transformation of the phthalimido moiety into an N-acetyl group, followed by catalytic hydrogenation of the reducible-protecting groups furnished the free target pentasaccharides, which should be able to assist during the elucidation of the exact structure of the natural pentasaccharide.     

Synthesis of Terminal Oligosaccharides from Type Specific Lipooligosaccharide of Mycobacterium Tuberculosis

Abstract

The coupling reaction between 1,3-di-O-acetyl-4-O-benzyl-2-O-methyl-α-L-rhamnopyranose (9) and methyl 4-O-benzyl-2-O-methyl-α-L-rhamno-pyranoside (4) was carried out in the presence of boron trifluoride-etherate followed by deacetylation to give the disaccharide (11) containing a free 3′ position. The second glycosylation reaction with 2,3,4,6-tetra-O-acetyl-α-d-glucopyranosyl bromide in the presence of mercuric salts followed by removal of benzyl and acetyl groups provided the trisaccharide 2. The boron trifluoride catalysed condensation of 1,3,4-tri-O-acetyl-2-O-methyl-L-fucopyranose (14) and methyl 2,4,6-tri-O-benzyl-α-d-glucopyranoside (15) gave the disaccharide (16) from which the protecting groups were removed to form the disaccharide (3).     

New synthesis of idraparinux,the non-glycosaminoglycan analogue of the antithrombin-binding domain of heparin

Idraparinux, the fully O-sulfated, O-methylated, heparin-related pentasaccharide possessing selective factor Xa inhibitory activity, was prepared by a new synthetic pathway. This route was based on a [2+3] block synthesis utilizing a 6-O-silyl-protected l-idose-containing trisaccharide acceptor, which was glycosylated with a disaccharide donor containing a non-oxidized precursor of the glucuronic acid. The unique strategy of multiple functionalizations at pentasaccharide levels, involving triple methylation followed by oxidation of the glucose and the idose precursors into d-glucuronic and l-iduronic acids in one step, proved to be highly efficient, providing the target pentasaccharide through a 39-step synthesis starting from d-glucose and methyl α-d-glucopyranoside.     

Structural characterization of chondroitin/dermatan sulfate oligosaccharides from bovine aorta by capillary electrophoresis and electrospray ionization quadrupole time-of-flight tandem mass spectrometry

An analytical approach based on high-performance capillary electrophoresis (CE) in conjunction with electrospray ionization quadrupole time-of-flight tandem mass spectrometry (ESI-QTOF-MS/MS) has been developed for providing the basis to obtain new insights into the domain structure of the glycosaminoglycan (GAG) moiety of proteoglycans. The feasibility and performance of the off-line CE/ESI-QTOF-MS approach in GAG oligosaccharide analysis were assessed by screening a chondroitin/dermatan sulfate (DS) oligosaccharide mixture obtained from bovine aorta by enzymatic depolymerization by chondroitin B lyase. The CS/DS mixture was analyzed by CE using 50 mM ammonium acetate, pH 12.0, dissolved in aqueous methanol (2:3; v/v), as a CE carrier. Structural identification of the GAG components was achieved using off-line CE/nanoESI-QTOF-MS and-MS/MS experiments. ESI-QTOF instrumental parameters were found to play an important role in the MS screening of the CE-separated GAG species. By optimizing the ESI conditions, oligosaccharides differing in chain length and degree of sulfation could be detected. The building block composition, the size of the carbohydrate chain, as well as structural features of the repeating HexA-GalNAc, HexA-GalNAc(S) units, have been determined using MS/MS by applying collision-induced dissociation at low energies. Cleavage of GAG chains by chondroitin B lyase occurs with formation of structural markers useful for identification of IdoA-containing domains.