Practical Synthesis of the Disaccharide Epitope,D-Galactopyranosyl-α-1,3-D- galactopyranose,by using 1,2;5,6-Di-O-cyclohexylidene-α-D-galactofuranose as the Glycosyl Acceptor

D-Galactosyl-α-1,3-D-galactopyranose (1) was chemically prepared in a good yield by coupling phenyl 2,3,4,6-tetra-O-benzyl-1-thio-β-D-galactopyranoside (5) or 2,3,4,6-tetra-O-benzyl-α-D-galactopyranosyl bromide (8) with 1,2:5,6-di-O-cyclohexylidene-α-D-galactofuranose (3) with subsequent de-O-benzylation and de-O-cyclohexylidenation of the resulting protected α-1,3-disaccharide.     

Toward the synthesis of fine chemicals from lactose: preparation of d-xylo and l-lyxo-aldohexos-5-ulose derivatives

The transformation of (5R)-2,6-di-O-benzyl-5-C-methoxy-β-d-galactopyranosyl-(1→4)-2,3:5,6-di-O-isopropylidene-aldehydo-d-glucose dimethyl acetal (8) into partially protected derivatives of d-xylo- and l-lyxo-aldohexos-5-ulose has been reported, applying appropriate epimerisation methods to its 3′-O- and 4′-O-protected alcoholic derivatives.     

Synthesis of the 6-deoxytalose-containing tetrasaccharide of the glycopeptidolipid from Mycobacterium intracellare serotype 7

An efficient synthesis of 4-methoxyphenyl α-l-Rhap-(1→3)-α-l-Rhap-(1→3)-α-l-Rhap-(1→2)-6-deoxy-α-l-Talp, the tetrasaccharide related to the GPLs of Mycobacterium intracellare serotype 7, was achieved with 4-methoxyphenyl 3,4-di-O-benzoyl-6-deoxy-α-l-talopyranoside (6c) as the key intermediate which was obtained through selective 3-O-benzoylation of 4-O-benzoyl-6-deoxy-α-l-taloside. Coupling of 6c with 3-O-allyloxycarbonyl-2,4-di-O-benzoyl-α-l-rhamnopyranosyl trichloroacetimidate followed by removal of the allyloxycarbonyl protecting group afforded the disaccharide acceptor 11. Condensation of 11 with 2,3,4-tri-O-benzoyl-α-l-rhamnopyranosyl-(1→3)-2,4-di-O-benzoyl-α-l-rhamnopyranosyl trichloroacetimidate and subsequent deprotection gave the target tetrasaccharide.     

Iridoid glycosides from Gardeniae Fructus for treatment of ankle sprain

The iridoid glycosides, genipin 1-O-β-d-isomaltoside (1) and genipin 1,10-di-O-β-d-glucopyranoside (2), together with six known iridoid glycosides, genipin 1-O-β-d-gentiobioside (3), geniposide (4), scandoside methyl ester (5), deacetylasperulosidic acid methyl ester (6), 6-O-methyldeacetylasperulosidic acid methyl ester (7), and gardenoside (8) were isolated from an EtOH extract of Gardeniae Fructus. The structures and relative stereochemistries of the metabolites were elucidated on the basis of 1D- and 2D-NMR spectroscopic techniques, high-resolution mass spectrometry, and chemical evidence. Geniposide (4), one of the main compounds of Gardeniae Fructus, was tested for treatment of ankle sprain using an ankle sprain model in rats. From the second to fifth day, the geniposide (4) (100 mg/ml) treated group exhibited significant differences (p < 0.01) with ∼21-34% reduction in swelling ratio compared with those of the vehicle treated control group. This indicated the potential effect of geniposide (4) for the treatment of disorders such as ankle sprain.     

Regioselective [3+2] cycloaddition of chalcones with a sugar azide: easy access to 1-(5-deoxy-d-xylofuranos-5-yl)-4,5-disubstituted-1H-1,2,3-triazoles

[3+2] Cycloaddition of 5-azido-5-deoxy-1,2-O-isopropylidene-α-d-xylofuranose with 1,3-diphenyl-prop-3-enones, followed by oxidation of the intermediate triazolines in a tandem manner, led to the regioselective formation of 4-benzoyl-1-(5-deoxy-1,2-O-isopropylidene-α-d-xylofuranos-5-yl)-5-phenyl-1H-1,2,3-triazoles in moderate to good yields.     

Stereoselective entry into the d-GalNAc series starting from the d-Gal one: a new access to N-acetyl-d-galactosamine and derivatives thereof

A new stereoselective preparation of N-aceyl-d-galactosamine (1b) starting from the known p-methoxyphenyl 3,4-O-isopropylidene-6-O-(1-methoxy-1-methylethyl)-β-d-galactopyranoside (10) is described using a simple strategy based on (a) epimerization at C-2 of 10 via oxidation-reduction to give the talo derivative 11, (b) amination with configurational inversion at C-2 of 11 via a S_N2-type reaction on its 2-imidazylate, (c) anomeric deprotection of the p-methoxyphenyl β-d-galactosamine glycoside 14, (d) complete deprotection. Applying the same protocol to 2,3:5,6:3′,4′-tri-O-isopropylidene-6′-O-(1-methoxy-1-methylethyl)-lactose dimethyl acetal (4), directly obtained through acetonation of lactose, the disaccharide β-d-GalNAcp-(1→4)-d-Glcp (1a) was obtained with complete stereoselectivity in good (40%) overall yield from lactose.     

Synthesis of phenyl β-acobioside,a derivative of the disaccharide component of actinoidins

The title glycoside [phenyl 2-O-(3-amino-2,3,6-trideoxy-α-l-arabino-hexopyranosyl-β-d-glucopyranoside] was prepared from phenyl 3-O-benzyl-4,6-O-benzylidene-β-d-glucopyranoside and 3-azido-2,3,6-trideoxy-1,4-di-O-p-nitrobenzoyl-l-arabino-hexopyranose or 3-azido-2,3,6-trideoxy-4-O-p-nitrobenzoyl-l-arabino-hexopyranosyl chloride.     

Monoacylation of 2-O-α-d-glucopyranosyl-l-ascorbic acid by protease in N,N-dimethylformamide with low water content

2-O-α-d-Glucopyranosyl-l-ascorbic acid (AA-2G) laurate was synthesized from AA-2G and vinyl laurate with a protease from Bacillus subtilis in N,N-dimethylformamide (DMF) with low water content. Addition of water to DMF dramatically enhanced monoacyl AA-2G synthesis. Maximum synthetic activity was observed when 3% (v/v) water was added to the reaction medium. Under the optimal reaction conditions, 5-O-dodecanoyl-2-O-α-d-glucopyranosyl-l-ascorbic acid, 2-O-(6′-O-dodecanoyl-α-d-glucopyranosyl)-l-ascorbic acid, and 6-O-dodecanoyl-2-O-α-d-glucopyranosyl-l-ascorbic acid were synthesized in yields of 5.5%, 3.2%, and 20.4%, respectively.     

Isolation and structure elucidation of 5′-O-β-d-glucopyranosyl-dihydroascorbigen from Cardamine diphylla rhizome

From the methanol extract of Cardamine diphylla rhizome, 5′-O-β-d-glucopyranosyl-dihydroascorbigen (1) and 6-hydroxyindole-3-carboxylic acid 6-O-β-d-glucopyranoside (2) were isolated. The structures of the compounds were elucidated using spectroscopic methods. This is the second report on the presence of a glucosylated indole ascorbigen in plants.     

The influence of selected O-alkyl derivatives of cyclodextrins on the enzymatic decomposition of l-tryptophan by l-tryptophan indole-lyase

A series of O-alkyl derivatives of cyclodextrin: heksakis[2,3,6-tri-O-(2′-methoxyethyl)]-α-cyclodextrin; heksakis(2,3-di-O-methyl)-α-cyclodextrin; heptakis(2,3-di-O-methyl)-β-cyclodextrin; heksakis[2,3-di-O-methyl-6-O-(2′-methoxyethyl)]-α-cyclodextrin; heptakis[2,3-di-O-methyl-6-O-(2′-methoxyethyl)]-β-cyclodextrin; heksakis[2,3-di-O-(2′-methoxyethyl)]-α-cyclodextrin and heptakis[2,3-di-O-(2′-methoxyethyl)]-β-cyclodextrin have been synthesized. Purity and composition of the obtained substances were examined. The cyclodextrin derivatives listed above as well as (2-hydroxypropyl)-α-cyclodextrin and (2-hydroxypropyl)-β-cyclodextrin, the two commercially available ones, have been investigated as the additives in the course of enzymatic decomposition of l-tryptophan by l-tryptophan indole-lyase. It has been found that each of cyclodextrin derivatives causes the inhibition of enzymatic process, both competitive and non-competitive. The competitive inhibition is connected with the formation of inclusion complexes between cyclodextrins and l-tryptophan, related to the geometry of these complexes. The mechanism of the non-competitive inhibition is not so evident; it could be related to the formation of the cyclodextrin complexes on the surface of the enzyme, leading to the change in the flexibility of the enzyme molecule.     

Aufbau von oligosacchariden mit glycosylfluoriden unter lewissäure-katalyse

Glycosylation of 1,2:3,4-di-O-isopropylidene-α-d-galactopyranose (6), as well as its 6-trimethylsilyl ether 7 with 2,3,4,6-tetra-O-acetyl-β-d-glucopyranosyl fluoride (5) was achieved stereospecifically in a mild and fast manner in the presence of Lewis acids like, e.g., titanium tetrafluoride, to give the β-(1→6)-linked disaccharide derivative 1. By use of 2,3,4,6-tetra-O-benzyl-β-d-glucopyranosyl fluoride (8) or its α anomer 10 and titanium tetrafluoride in acetonitrile with 6 or 7, a fast reaction proceeds preponderantly to yield 1,2:3,4-di-O-isopropylidene 6-O-(2,3,4,6-tetra-O-benzyl-β-d-glucopyranosyl)-α-d-galactopyranose (2). In ether, however, mainly the α-(1→6) anomer was formed. These model systems were used to elucidate the limiting conditions for this procedure, and mechanistic conceptions are discussed. By glycosylation at O-4 of 1,6:2,3-dianhydro-β-d-mannopyranose (11) with the perbenzylated α-fluoride 10 both the α- and the β-d-(1→4) disaccharide derivatives 12 and 14 were obtained, but 5 gave exclusively the β-d-(1→4) compound 16. Opening of the anhydro rings of 12 led to the synthesis of N-acetyl-maltosamine (22). 1,6-Anhydro-2-azido-4-O-benzyl-2-deoxy-β-d-glucopyranose was glycosylated with methyl (2,3,4-tri-O-acetyl-β-d-galactopyranosyl fluoride)uronate under titanium tetrafluoride catalysis to give the β-d-(1→3)-linked disaccharide 16, subsequently transformed into 29.     

Synthesis of a branched d-mannopentaoside and a branched d-mannohexaoside: Models of the inner core of cell-wall glycoproteins of Saccharomyces cerevisiae

Synthetic routes are discussed to the branched d-mannopentaoside methyl 6-O-(2,6-di-O-α-d-mannopyranosyl-α-d-mannopyranosyl)-3-O-α-d-mannopyranosyl-α-d-mannopyranoside and d-mannohexaoside methyl 6-O-(2,6-di-O-α-d-mannopyranosyl-α-d-mannopyranosyl)-3-O-(2-O-α-d-mannopyranosyl-α-d-mannopyranosyl)- α-d-mannopyranoside, employing the properly benzylated d-mannobioside methyl 2,4-di-O-benzyl-6-O-(3,4-di-O-benzyl-α-d-mannopyranosyl)-α-d-mannopyranoside and d-mannotrioside methyl 2,4-di-O-benzyl-6-O-(3,4-di-O-benzyl-α-d-mannopyranosyl)-3-O-(3,4,6-tri-O-benzyl-α-d-mannopyranosyl)-α-d- mannopyranoside as key intermediates.     

Synthesis of monodeoxy and mono-O-methyl congeners of methyl β-d-mannopyranosyl-(1→2)-β-d-mannopyranoside for epitope mapping of anti-Candida albicans antibodies

A panel of six complementary monodeoxy and mono-O-methyl congeners of methyl β-d-mannopyranosyl-(1→2)-β-d-mannopyranoside (1) were synthesized by stereoselective glycosylation of monodeoxy and mono-O-methyl monosaccharide acceptors with a 2-O-acetyl-glucosyl trichloroacetimidate donor, followed by a two-step oxidation-reduction sequence at C-2′. The β-manno configuration of the final deprotected congeners 2-7 was confirmed by measurement of ¹J_C1,H1 heteronuclear and ³J_1′,2′ homonuclear coupling constants. These disaccharide derivatives will be used to map the epitope recognized by a protective anti-Candida albicans monoclonal antibody C3.1 (IgG3) and to determine its key polar contacts with the binding site.     

Presence of 1→3-linked 2-O-β-d-xylopyranosyl-α-l-arabinofuranosyl side chains in cereal arabinoxylans

The presence of a fairly uncommon side chain 2-O-β-d-xylopyranosyl-α-l-arabinofuranosyl in arabinoxylans (AX) from eight different cereal by-products was investigated, using ¹H NMR spectroscopy and high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) after Shearzyme^® (GH10 endo-1,4-β-d-xylanase) hydrolysis. This disaccharide side group was present in significant amounts in AX extracted from corn cobs and barley husks. For the first time, it was also detected in AX from oat spelts and rice husks, and in lesser amounts in wheat straw AX. Arabinoxylo-oligosaccharide (AXOS) containing the 2-O-β-d-Xylp-α-l-Araf side chain was purified from the oat spelt AX hydrolysate and the structure was fully analyzed using 1D and 2D NMR spectroscopy. The AXOS was identified as β-d-Xylp-(1→2)-α-l-Araf-(1→3)-β-d-Xylp-(1→4)-d-Xyl. To our knowledge, such a structure with 2-O-β-d-Xylp-α-l-Araf attached to the O-3 of the nonreducing end of xylobiose has not been described previously. New information on substitution of AX from various cereal by-products was obtained by combining NMR and enzyme-assisted HPAEC-PAD analysis.     

Synthesis of 4-O-glycosylated 1,5-anhydro-d-fructose and of 1,5-anhydro-d-tagatose from a common intermediate 2,3-O-isopropylidene-d-fructose

Four novel disaccharides of glycosylated 1,5-anhydro-d-ketoses have been prepared: 1,5-anhydro-4-O-β-d-glucopyranosyl-d-fructose, 1,5-anhydro-4-O-β-d-galactopyranosyl-d-fructose, 1,5-anhydro-4-O-β-d-glucopyranosyl-d-tagatose, and 1,5-anhydro-4-O-β-d-galactopyranosyl-d-tagatose. The common intermediate, 1,5-anhydro-2,3-O-isopropylidene-β-d-fructopyranose, was prepared from d-fructose and was converted into the d-tagatose derivative by oxidation followed by stereoselective reduction to the 4-epimer. The anhydroketoses thus prepared were glycosylated and deprotected to give the disaccharides.     

Studies on the stereoselective synthesis of a protected α-d-Gal-(1→2)-d-Glc fragment

A novel 1,2-cis stereoselective synthesis of protected α-d-Gal-(1→2)-d-Glc fragments was developed. Methyl 2-O-acetyl-3-O-allyl-4,6-O-benzylidene-α-d-galactopyranosyl-(1→2)-3-O-benzoyl-4,6-O-benzylidene-α-d-glucopyranoside (13), methyl 2-O-acetyl-3-O-allyl-4,6-O-benzylidene-α-d-galactopyranosyl-(1→2)-3,4,6-tri-O-benzoyl-α-d-glucopyranoside (15), methyl 2-O-acetyl-3-O-allyl-4,6-O-benzylidene-α-d-galactopyranosyl-(1→2)-3-O-benzoyl-4,6-O-benzylidene-β-d-glucopyranoside (17), and methyl 2-O-acetyl-3-O-allyl-4,6-O-benzylidene-α-d-galactopyranosyl-(1→2)-3,4,6-tri-O-benzoyl-β-d-glucopyranoside (19) were favorably obtained by coupling a new donor, isopropyl 2-O-acetyl-3-O-allyl-4,6-O-benzylidene-1-thio-β-d-galactopyranoside (2), with acceptors, methyl 3-O-benzoyl-4,6-O-benzylidene-α-d-glucopyranoside (4), methyl 3,4,6-tri-O-benzoyl-α-d-glucopyranoside (5), methyl 3-O-benzoyl-4,6-O-benzylidene-β-d-glucopyranoside (8), and methyl 3,4,6-tri-O-benzoyl-β-d-glucopyranoside (12), respectively. By virtue of the concerted 1,2-cis α-directing action induced by the 3-O-allyl and 4,6-O-benzylidene groups in donor 2 with a C-2 acetyl group capable of neighboring-group participation, the couplings were achieved with a high degree of α selectivity. In particular, higher α/β stereoselective galactosylation (5.0:1.0) was noted in the case of the coupling of donor 2 with acceptor 12 having a β-CH₃ at C-1 and benzoyl groups at C-4 and C-6.     

Oxidation of methyl α-d-galactopyranoside by galactose oxidase: products formed and optimization of reaction conditions for production of aldehyde

The reaction conditions of galactose oxidase-catalyzed, targeted C-6 oxidation of galactose derivatives were optimized for aldehyde production and to minimize the formation of secondary products. Galactose oxidase, produced in transgenic Pichia pastoris carrying the galactose oxidase gene from Fusarium spp., was used as catalyst, methyl α-d-galactopyranoside as substrate, and reaction medium, temperature, concentration, and combinations of galactose oxidase, catalase, and horseradish peroxidase were used as variables. The reactions were followed by ¹H NMR spectroscopy and the main products isolated, characterized, and identified. An optimal combination of all the three enzymes gave aldehyde (methyl α-d-galacto-hexodialdo-1,5-pyranoside) in approximately 90% yield with a substrate concentration of 70 mM in water at 4 °C using air as oxygen source. Oxygen flushing of the reaction mixture was not necessary. The aldehyde existed as a hydrate in water. The main secondary products, a uronic acid (methyl α-d-galactopyranosiduronic acid) and an α,β-unsaturated aldehyde (methyl 4-deoxy-α-d-threo-hex-4-enodialdo-1,5-pyranoside), were observed for the first time to form in parallel. Formation of uronic acid seemed to be the result of impurities in the galactose oxidase preparation. ¹H and ¹³C NMR data of the products are reported for the α,β-unsaturated aldehyde for the first time, and chemical shifts in DMSO-d₆ for all the products for the first time. Oxidation of d-raffinose (α-d-galactopyranosyl-(1-6)-α-d-glucopyranosyl-(1-2)-β-d-fructofuranoside) in the same optimum conditions also proceeded well, resulting in approximately 90% yield of the corresponding aldehyde.     

Synthesis of 3- and 2′-fucosyl-lactose and 3,2′-difucosyl-lactose from partially benzylated lactose derivatives

O-β-d-Galactopyranosyl-(1→4)-O-[α-l-fucopyranosyl-(1→3)]-d-glucose has been synthesised by reaction of benzyl 2,6-di-O-benzyl-4-O-(2,3,4,6-tetra-O-benzyl-β-d-galactopyranosyl)-β-d-glucopyranoside with 2,3,4-tri-O-benzyl-α-l-fucopyranosyl bromide in the presence of mercuric bromide, followed by hydrogenolysis. Benzylation of benzyl 3′,4′-O-isopropylidene-β-lactoside, via tributylstannylation, in the presence of tetrabutylammonium bromide or N-methylimidazole, gave benzyl 2,6-di-O-benzyl-4-O-(6-O-benzyl-3,4-O-isopropylidene-β-d-galactopyranosyl)-β-d-glucopyranoside (6). α-Fucosylation of 6 in the presence of tetraethylammonium bromide provided either benzyl 2,6-di-O-benzyl-4-O-[6-O-benzyl-3,4-O-isopropylidene-2-O-(2,3,4-tri-O-benzyl-α-l-fucopyransoyl)-β-d- galactopyranosyl]-β-d-glucopyranoside (13, 73%) or a mixture of 13 (42%) and benzyl 2,6-di-O-benzyl-4-O-[6-O-benzyl-3,4,-O-isopropylidene-2-O-(2,3,4-tri-O-benzyl-α-l-fucopyranosyl)-β-d- galactopyranosyl-3-O-(2,3,4-tri-O-benzyl-α-l-fucopyranosyl)-β-d-glucopyranoside (16, 34%). α-Fucosylation of 13 in the presence of mercuric bromide and 2,6-di-tert-butyl-4-methylpyridine gave 16 (73%). Hydrogenolysis and acid hydrolysis of 13 and 16 afforded O-α-l-fucopyranosyl-(1→2)-O-β-d-galactopyranosyl-(1→4)-d-glucose and O-α-l-fucopyranosyl-(1→2)-O-β-d-galactopyranosyl-(1→4)-O-[α-l-fucopyranosyl-(1→3)]-d-glucose, respectively.     

The Structure of the Carbohydrate Moiety of a Glycopeptide GP-I-b from Rhizopus Saccharogenic Amylase

The structure of the carbohydrate moiety of GP–I–b which is one out of three glycopeptides isolated from a Pronase digest of the saccharogenic amylase of Rhizopus javanicus sp. 3–46, was investigated by enzymatic and chemical techniques.

Nine moles of mannose followed by one mole of N-acetylglucosamine were released per mole of GP–I–b when it was treated sequentially with purified jack bean α-mannosidase and β-N-acetylglucosaminidase.

Methylation of GP–I–b gave 3, 6-di-O-methyl derivative from the N-acetylglucosamine residues, and 2, 3, 4, 6-tetra-O-methyl, 3, 4, 6-tri-O-methyl and 2, 4-di-O-methyl derivatives from the mannose residues in an approximate ratio of 3: 4: 2.

A smaller glycopeptide (F–l) containing two moles each of mannose and N-acetylglucosamine per mole of asparagine was obtained when GP–I–b was subjected to one step of the Smith degradation. Exhaustive methylation of F–l gave 3, 6-di-O-methyl derivative of Nacetylglucosamine, and 2, 3, 4, 6-tetra-O-methyl and 2, 3, 4-tri-O-methyl derivatives of mannose in a ratio of 1.00: 0.85.

Controlled acetolysis of GP–I–b yielded mannose, O-α-mannosyl-(l→2)-O-α-mannosyl-(l→3)-mannose and a smaller glycopeptide which was resistant to the acetolysis.

From these and previous evidences, the following structure was determined for GP–I–b.     

Bioconversion of ginsenosides Rb(1), Rb(2), Rc and Rd by novel β-glucosidase hydrolyzing outer 3-O glycoside from Sphingomonas sp. 2F2: cloning, expression, and enzyme characterization

A new β-glucosidase gene (bglSp) was cloned from the ginsenoside converting Sphingomonas sp. strain 2F2 isolated from the ginseng cultivating filed. The bglSp consisted of 1344 bp (447 amino acid residues) with a predicted molecular mass of 49,399 Da. A BLAST search using the bglSp sequence revealed significant homology to that of glycoside hydrolase superfamily 1. This enzyme was overexpressed in Escherichia coli BL21 (DE3) using a pET21-MBP (TEV) vector system. Overexpressed recombinant enzymes which could convert the ginsenosides Rb₁, Rb₂, Rc and Rd to the more pharmacological active rare ginsenosides gypenoside XVII, ginsenoside C-O, ginsenoside C-Mc₁ and ginsenoside F₂, respectively, were purified by two steps with Amylose-affinity and DEAE-Cellulose chromatography and characterized. The kinetic parameters for β-glucosidase showed the apparent K_m and V_max values of 2.9 ± 0.3 mM and 515.4 ± 38.3 μmol min⁻¹ mg of protein⁻¹ against p-nitrophenyl-β-d-glucopyranoside. The enzyme could hydrolyze the outer C3 glucose moieties of ginsenosides Rb₁, Rb₂, Rc and Rd into the rare ginsenosides Gyp XVII, C-O, C-Mc₁ and F₂ quickly at optimal conditions of pH 5.0 and 37 °C. A little ginsenoside F₂ production from ginsenosides Gyp XVII, C-O, and C-Mc₁ was observed for the lengthy enzyme reaction caused by the side ability of the enzyme.