An electron-impact,mass-spectrometric fragment-ion that identifies 3-linked d-glucopyranosyl residues in per-O-alkylated,linear β-d-glucopyrano-oligosaccharide-alditols

A previously unreported fragment-ion, designated J₀, was observed in the electron-impact, mass spectra of per-O-alkylated, linear di- and tri-β-d-glucopyranosylalditols containing 3-linked d-glucopyranosyl residues. The J₀ fragment-ion was absent from, or present in very low abundance in, the spectra of per-O-alkylated, linear di- and tri-β-d-glucopyranosylalditols composed of only 2-, 4-, or 6-linked residues. The presence of the J₀ fragment-ion and the absence of the J₁ fragment-ion were found indicative of the presence of 3-linked d-glucopyranosyl residues, and may be indicative of the presence of all 3-linked-glycosyl residues in per-O-alkylated oligosaccharide-alditols. A possible mechanism for the formation of the J₀ fragment-ion is proposed.     

Structural characterization of oligosaccharides isolated from the pectic polysaccharide rhamnogalacturonan II

Rhamnogalacturonan II (RG-II) is a structurally complex pectic (d-galactosyl-uronic acid-rich) polysaccharide that is present in the primary (growing) cell-walls of higher plants. RG-II is composed of ∼60 glycosyl residues. The isolation and structural characterization of 23 oligosaccharide fragments of the residue of RG-II that remained after removal of hepta- and di-saccharides by partial hydrolysis with acid are reported. In order to obtain the oligosaccharide fragments characterized herein, the carboxyl groups of RG-II were dideuterio-reduced, and the carboxyl-reduced polysaccharide was per-O-methylated. The per-O-methylated polysaccharide was fragmented by partial hydrolysis with acid, producing partially O-methylated oligosaccharides. These derivatized oligosaccharides were reduced, to afford a mixture of partially O-methylated oligoglycosyl-alditols, which was then per-O-methylated. The structures of the resulting per-O-methylated oligoglycosylalditols were determined by chemical-ionization mass spectrometry, electron-impact mass spectrometry, fast-atom-bombardment mass spectrometry, ¹H-n.m.r. spectroscopy, and analysis of corresponding, partially O-acetylated, partially O-methylated alditols. Seventeen of the oligosaccharides isolated from RG-II were parts of a single heptasaccharide, namely.     

Thiodisaccharides with galactofuranose or arabinofuranose as terminal units: Synthesis and inhibitory activity of an exo β-d-galactofuranosidase

Thiodisaccharides having β-d-Galf or α-l-Araf units as non-reducing end have been synthesized by the SnCl₄- or MoO₂Cl₂-promoted thioglycosylation of per-O-benzoyl-d-galactofuranose (1), its 1-O-acetyl analogue 4, or per-O-acetyl-α-l-arabinofuranose (16) with 6-thioglucose or 6-thiogalactose derivatives. After convenient removal of the protecting groups, the free thiodisaccharides having the basic structure β-d-Galf(1→6)-6-thio-α-d-Glcp-OMe (5) or β-d-Galf(1→6)-6-thio-α-d-Galp-OMe (15) were obtained. The respective α-l-Araf analogues 18 and 20 were prepared similarly from 16. Alternatively, β-d-Galf(1→4)-4-thio-3-deoxy-α-l-Xylp-OiPr was synthesized by Michael addition to a sugar enone of 1-thio-β-d-Galf derivative, generated in situ from the glycosyl isothiourea derivative of 1. The free S-linked disaccharides were evaluated as inhibitors of the β-galactofuranosidase from Penicillium fellutanum, being 15 and 20 the more active inhibitors against this enzyme.     

Preparation of some (1→6)-linked disaccharides,and their derivatives suitable for protein modification

Synthetic methods for the preparation of per-O-acetylated, (1→6)-linked disaccharides containing either a d-galactose or a d-glucose residue at the reducing end are described. In these methods, 1,2,3,4-tetra-O-acetyl-6-O-trityl-β-d-glucopyranose was first converted into 1,2,3,4-tetra-O-acetyl-β-d-glucopyranose (1) by rapid treatment with 90% trifluoroacetic acid, followed by rapid isolation designed to minimize O-acyl migration. Disaccharides were formed by glycosylation of 1 or 1,2:3,4-di-O-isopropylidene-d-galactopyranose with per-O-acetylglycosyl halides. Isopropylidene groups in the resulting disaccharide, if present, were removed, and the disaccharide was per-O-acetylated. Per-O-acetylated β-Gal-(1→6)-Glc and β-GlcNAc-(1→6)-Gal, and a mixture of per-O-acetylated α-Gal-(1→6)-Gal and β-Gal-(1→6)-Gal (in the ratio of 3:7) were thus obtained. The per-O-acetylated Gal-(1→6)-Gal disaccharides were converted, by a reaction sequence previously reported, into (2,2-dimethoxyethyl)aminocarbonylmethyl 1-thio-β-d-glycosides, which could then be coupled to proteins via reductive alkylation. For the anomeric mixture of per-O-acetylated Gal-(1→6)-Gal, conversion into the corresponding 1-thioglycoside permitted resolution of the isomers by chromatography on silica gel. When disaccharides, as borate complexes, were chromatographed on a column of a strong, anion-exchange resin, all of the (1→6)-linked disaccharides of neutral sugars tested (including melibiose) were eluted later than analogous disaccharides having other linkages, and also later than any neutral monosaccharides.     

Improved preparations of some per-O-acetylated aldohexopyranosyl cyanides

3,4,6-Tri-O-acetyl-1,2-O-[1-(exo-, endo-cyano)ethylidene]-α-d-galacto- (1a/b), -α-d-gluco- (2a/b), and -β-d-manno-pyranose (3a/b) were stereoselectively isomerized to the corresponding per-O-acetylated 1,2-trans-aldohexopyranosyl cyanides in 75, 16, and 62% yield, respectively, by treatment with boron trifluoride etherate in dry nitromethane. The corresponding per-O-acetylated 1,2-cis-aldohexopyranosyl cyanides were obtained concurrently in respective yields of 1.9, 0.9, and 4.8%. The per-O-acetylaldohexopyranosyl cyanide products were found stable to the reaction conditions and were readily isolated following completion of the rearrangement. It had previously been proved that reaction of 2,3,4,6-tetra-O-acetyl-α-d-manno- and -gluco-pyranosyl bromide with mercuric cyanide in nitromethane generates, in the ratio of ∼1:1, the desired 1,2-trans-glycosyl cyanides and the corresponding 1,2-O-(1-cyanoethylidene) isomers (3a/b and 2a/b, respectively). Treatment of these reaction-mixtures with boron trifluoride etherate in nitromethane effected the rearrangement of 3a/b and 2a/b, thereby facilitating the isolation, and increasing the overall yields, of the per-O-acetylated 1,2-trans-d-manno and -gluco-pyranosyl cyanides (58 and 30% total yield, respectively) relative to the earlier procedures. The boron trifluoride etherate-mediated reaction of per-O-acetyl-α- and -β-d-galacto, -α- and -β-d-gluco-, -α-d-manno-, and -2-deoxy-2-phthalimido-β-d-gluco-pyranoses with trimethylsilyl cyanide in nitromethane was also investigated. This reaction provides a “one-flask” synthesis of the corresponding per-O-acetylated 1,2-trans-aldohexopyranosyl cyanides in which 1,2-O-(1-cyanoethylidene) derivatives are isomerized in situ. Finally, improved preparations of the (not readily accessible) per-O-acetylated 1,2-cis-d-manno- and -gluco-pyranosyl cyanides are described. Thus, 2,3,4,6-tetra-O-acetyl-α- and -β-d-mannopyranosyl cyanide (48 and 16% total yield, respectively) and -α- and -β-d-glucopyranosyl cyanide (12 and 39% total yield, respectively) were synthesized by fusion of the corresponding -α-d-glycosyl bromides with mercuric cyanide.     

Studies on the synthesis and the antimicrobial and antioxidant activities of a novel class of fluorescein-based glycosides

Facile glycosylation of a fluorescein diol derivative with per-O-acetyl/benzoyl sugar derivatives using BF₃·Et₂O catalyst resulted in the formation of the expected glycosides in 54–66% yield. The biological screening of the glycosides against different microbes shows good inhibitory activity. The antioxidant activity of the fluorescein-based glycosides shows remarkable inhibition (IC₅₀ ∼80%).     

Enhancement of protonated molecular ions and ammonium·molecular ion complexes in direct-liquid-introduction-mass spectrometry of carbohydrates

Addition of ammonium acetate to the mobile phase in direct-liquid-introduction mass spectrometry enhances the abundance of the protonated molecular ion or ammonium·molecular ion complex for compounds of biological interest. The efficacy of the method was investigated by comparing mass spectra obtained, with and without ammonium acetate, for a variety of underivatized, per-O-acetylated, and per-O-alkylated carbohydrates, and for several underivatized peptides. The mass spectra of the per-O-alkylated carbohydrates obtained by direct-liquid-introduction mass spectrometry with ammonium acetate were also compared to those obtained by thermospray mass spectrometry.     

Regioselective synthesis of novel 3-alkoxy (phenyl) thiophosphorylamido-2-(per-O-acetylglycosyl-1′-imino)thiazolidine-4-one derivatives from O-alkyl N-glycosyl(thiosemicarbazido)phosphonothioates

A series of 3-alkoxy(phenyl)thiophosphorylamido-2-(per-O-acetylglycosyl-1′-imino)thiazolidine-4-one derivatives were prepared by the reaction of 1-alkoxy(phenyl)thiophosphoryl-4-(per-O-acetylglycosyl) thiosemicarbazides with ethyl bromoacetate. ¹H/¹³C HMBC measurements corroborated by X-ray crystallographic results revealed the exclusive regioselectivity of these ring closures toward the N-2 position of the thiosemicarbazide moiety. The bioactivity data of 3a-k suggest that the thiazolidine-4-one ring is critical for the herbicidal and fungicidal activities.     

The mass spectra of some per-O-acetylaldononitriles

The mass spectra of some per-O-acetylaldono- and per-O-acetyldeoxyaldononitriles have been recorded. The major fragmentation-pathways are discussed in terms of the application of electron-impact mass-spectrometry to structural studies of aldoses. Both acetyl and deuterium-labelled acetyl derivatives are included. The spectra are useful in verifying the position of the deoxy group(s) of deoxyaldoses.     

A comparison of some hydrolytic and gas chromatographic procedures used in methylation analysis of the carbohydrate units of glycopeptides

The polysaccharide secreted by Klebsiella aerogenes type 54 strain A3 was isolated, methylated, the ester carboxyl-reduced, and the product partially hydrolyzed. The resulting, partially O-methylated oligosaccharides were reduced and ethylated, and the mixture of products was fractionated by l.c. The l.c. fractions containing per-O-alkylated oligosaccharide-alditols were analyzed by e.i.-m.s. Pure per-O-alkylated oligosaccharide-alditols were also analyzed by ¹H-n.m.r. spectroscopy. The products obtained by base-catalyzed degradation and subsequent ethylation of the per-O-methylated polysaccharide were fractionated by l.c. The main product isolated was analyzed by e.i.-m.s., c.i.-m.s., and ¹H-n.m.r. spectroscopy. The results of these studies, in conjunction with results of analytical methods commonly used in the elucidation of polysaccharide structures, unambiguously characterized the primary glycosyl structure of the polysaccharide. Base-labile substituents, previously reported to be present in the polysaccharide, were not studied. Structure 1 revises, and complements, previously reported structures.

     

A novel synthesis of β-d-mannopyranosyl azide by phase transfer catalysis

A simple stereoselective synthesis of per-O-benzoyl-β-d-mannopyranosyl azide from per-O-benzoyl-α-d-mannopyranosyl bromide using phase transfer catalysis was developed. The stereochemistry at C-1 of the anomeric O-benzoylated α- and β-d-mannopyranosyl azides was unambiguously established using 2D NOESY NMR spectroscopy. Pure deprotected β-d-mannopyranosyl azide was prepared by debenzoylation with sodium methoxide in methanol.     

Conformations of some per-O-acetylaldononitriles and 5-(polyacetoxyalkyl)tetrazoles

The conformations of several per-O-acetylaldononitriles and 5-(polyacetoxyalkyl)tetrazoles in pyridine-d₅ solution have been studied by p.m.r. spectroscopy. For compounds of both types, those having the arabino, galacto, and manno configurations take an extended, planar, zigzag arrangement for the carbon chain, whereas, for those having the xylo, ribo, and gluco configurations, the values of the coupling constants indicate that a bent conformation is favored.     

Synthesis and conformational analysis of 1,2-cis fused bicyclic α-d-galactofuranosyl thiocarbamate from per-O-tert-butyldimethylsilyl-β-d-galactofuranosyl isothiocyanate

Per-O-tert-butyldimethylsilyl-α,β-d-galactofuranosyl isothiocyanate (4) was synthesized by the reaction of per-O-TBS-β-d-galactofuranose (1) with KSCN, promoted by TMSI. Upon O-desilylation (1,2-dideoxy-α-d-galactofuranoso)[1,2d]-1,3-oxazolidine-2-thione (6), the cis-fused bicyclic thiocarbamate was obtained as the only product. Conformational analysis, aided by molecular modelling, showed two stable twist forms (³T₄ and ⁴T_O) for the five-membered sugar ring in 6. In aqueous solution, the equilibrium favours the first conformation (3:1 ratio). On the other hand, this ratio decreases for less polar solvents.     

Cerheptaric Acid, A new lactone-forming acid isolated from cereus peruvianus (L.) mill

A systematic study of the acid-catalyzed fusion-reaction is reported. The influence of the nature and stereochemistry of the d-aldopentofuranose and the effect of the substituent at C-2 have been investigated by using indazole as a model heterocycle. The results obtained show that the nature and stereochemistry of the starting, per-O-acetylated d-aldopentofuranose have no significant effect upon the product distribution of the acid-catalyzed fusion-reaction. The use of a sugar lacking a participating group at C-2 showed, however, that the absence of participation increases the ratio of cis-1′,2′-nucleosides, and the mechanism involved is discussed. In all cases, the results indicated that the distribution of the products is determined by their relative, thermodynamic stabilities.     

Synthesis of the (1→6)-linked thiodisaccharide of galactofuranose: Inhibitory activity against a β-galactofuranosidase

A new (1→6)-linked thiodisaccharide formed by two galactofuranosyl units has been synthesized. Methyl (methyl α,β-d-galactofuranosid)uronate was employed as the starting compound, which was per-O-silylated with TBSCl and reduced with LiAlH₄ to afford methyl 2,3,5-tri-O-tert-butyldimethylsilyl-β-d-galactofuranoside (2β) as a key precursor for the preparation of methyl per-O-tert-butyldimethylsilyl-6-thio-β-d-galactofuranoside (12). The free thiol group of 12 was glycosylated and the product O-deprotected to afford the target β-d-Galf-S-(1→6)-β-d-Galf-OMe (14). The conformations of this thiodisaccharide were preliminarily studied using combined theoretical calculations and NMR data. Furthermore, the glycomimetic 14 showed to be a competitive inhibitor of the β-galactofuranosidase from Penicillum fellutanum (K_i = 3.62 mM).     

Quantitative microanalysis of oligosaccharides by high-performance liquid chromatography

The rapid separation and quantitative determination of per-O-benzoyl oligosaccharides were obtained using high performance, liquid chromatography. Oligosaccharides were completely O-benzoylated without concomitant N-benzoylation of acetamidodeoxyhexoses. Benzoylation prior to analysis allowed a quantitative determination of picomolar amounts because the absorbance at 230 nm of these derivatives is directly proportional to the number of benzoyl groups present. Separation by normal and reversed-phase chromatography was demonstrated, and the best resolution was obtained on an Ultrasphere octyl column. Excellent separations of oligosaccharides containing up to 10 sugar residues present in mannosidosis urine and of malto-oligosaccharides containing up to 15 sugar residues present in Karo syrup were achieved within an analysis time of 30 min. Anomers of maltose, maltotriose, and maltotetraose were separated; for this reason, reduction of complex samples prior to analysis is advisable. The effect of linkage configuration on retention time was tested with reduced, α-linked di- and tri-glucopyranosides. The presence of an acetamidodeoxyhexose residue in an oligosaccharide significantly reduced its retention time, whereas branching had the opposite effect. A linear response was obtained for the injection of 1–600 pmol of raffinose, and the detection limit was 0.5 pmol. Derivatization and analysis of raffinose was shown to yield reproducible results within the range 0.01–1 μmol, and, with special precautions to minimize losses, as little as 100 pmol could be analyzed successfully.     

Mechanism of O2−- and H2O2-induced stimulation of sugar transport in mouse fibroblast BALB/3T3 cells

Xanthine/xanthine oxidase and H₂O₂ stimulated sugar transport. Application of superoxide dismutase and catalase to the cells showed an inhibitory effect on these agent-stimulated sugar transports. Addition of amiloride and 4-acetamide-4′-isothiocyanostilbene-2,2′-disulfonic acid (SITS), which abolish the cytoplasmic alkalinization, inhibited the stimulation of sugar transport by xanthine/xanthine oxidase in the presence of catalase. The calmodulin antagonists, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) and trifluoperazine inhibited H₂O₂-stimulated sugar transport. These results suggest that O₂⁻ stimulates sugar transport in an intracellular pH-dependent manner and that H₂O₂ stimulates sugar transport in a calcium-calmodulin-dependent manner. These mechanisms may be involved in sugar-transport stimulation in mouse fibroblast BALB/3T3 cells by the tumor-promoting phorbol ester phorbol-12,13-dibutyrate and insulin, since the stimulatory effects of these agents were inhibited by scavengers of oxygen radicals.     

Nitrous acid deamination of methylated amino-oligosaccharide glycosides

G.l.c.-mass spectrometry has been used to characterize the products of N-deacetylation-nitrous acid deamination of per-O-methylated derivatives (8–11) of methyl 2-acetamido-2-deoxy-3-O-β-D-galactopyranosyl-α-D-glucopyranoside(1), methyl (2) and benzyl (3) 2-acetamido-2-deoxy-4-O-β-D-galactopyranosyl-β-D-glucopyranosides, and methyl 2-acetamido-2-deoxy-6-O-β-D-galactopyranosyl-α-D-glucopyranoside (4). 2,5-Anhydrohexoses have been converted into alditol trideuteriomethyl ethers, alditol acetates, and aldononitriles. The importance of side reactions that lead to the formation of 2-deoxy-2-C-formylpentofuranosides is discussed.     

Determination of the position of linkage of 2-acetamido-2deoxy-D-galactose and 2-acetamido-2-deoxy-D-glucose residues in oligosaccharides and glycoproteins. Synthesis of 2-acetamido-2deoxy-D-xylitol and 2-acetamido-2-deoxy-L-threitol

A method has been studied for the determination of the position of the linkage of the 2-acetamido-2-deoxy-D-galactose and 2-acetamido-2-deoxy-D-glucose residues in oligosaccharides and glycoproteins that is based on the borohydride reduction of the reducing terminal residues to the corresponding alditol derivatives periodate oxidation, borohydride reduction, hydrolysis (eventually followed by borohydride reduction), separation of the fragments as per-O-(trimethylsilyl) or per-O-(trifluoroacetyl) derivatives, and identification of the fragments as derivatives of 2-acetamido-2-deoxyglycerol, 2-acetamido-2-deoxy-L-threitol, 2-acetamido-2-deoxy-L-arabinitol, 2-acetamido-2-deoxy-D-xylitol, 2-acetamido-2-deoxy-D-galactitol, and 2-acetamido-2-deoxy-D-glucitol by gas-liquid chromatography-mass spectrometry. New syntheses for the standard compounds 2-acetamido-2-deoxy-L-threitol and 2-acetamido-2-deoxy-D-xylitol are described.     

Functional Characterisation of Three O-methyltransferases Involved in the Biosynthesis of Phenolglycolipids in Mycobacterium tuberculosis

Phenolic glycolipids are produced by a very limited number of slow-growing mycobacterial species, most of which are pathogen for humans. In Mycobacterium tuberculosis, the etiologic agent of tuberculosis, these molecules play a role in the pathogenicity by modulating the host immune response during infection. The major variant of phenolic glycolipids produced by M. tuberculosis, named PGL-tb, consists of a large lipid core terminated by a glycosylated aromatic nucleus. The carbohydrate part is composed of three sugar residues, two rhamnosyl units and a terminal fucosyl residue, which is per-O-methylated, and seems to be important for pathogenicity. While most of the genes responsible for the synthesis of the lipid core domain and the saccharide appendage of PGL-tb have been characterized, the enzymes involved in the O-methylation of the fucosyl residue of PGL-tb remain unknown. In this study we report the identification and characterization of the methyltransferases required for the O-methylation of the terminal fucosyl residue of PGL-tb. These enzymes are encoded by genes Rv2954c, Rv2955c and Rv2956. Mutants of M. tuberculosis harboring deletion within these genes were constructed. Purification and analysis of the phenolglycolipids produced by these strains, using a combination of mass spectrometry and NMR spectroscopy, revealed that Rv2954c, Rv2955c and Rv2956 encode the methyltransferases that respectively catalysed the O-methylation of the hydroxyl groups located at positions 3, 4 and 2 of the terminal fucosyl residue of PGL-tb. Our data also suggest that methylation at these positions is a sequential process, starting with position 2, followed by positions 4 and 3.