Synthesis of 4-cyano and 4-nitrophenyl 1,6-dithio-D-manno-, L-ido- and D-glucoseptanosides possessing antithrombotic activity

1,6-Anhydro-3,4-O-isopropylidene-1-thio-D-mannitol was converted into its sulfoxide which after hydrolysis, acetylation and subsequent Pummerer rearrangement gave the penta-O-acetyl-1-thio-D-mannoseptanose anomers in excellent yield. This anomeric mixture was used as donor for the glycosylation of 4-nitro- and 4-cyanobenzenethiol in the presence of boron trifluoride etherate and trimethylsilyl triflate, respectively, to yield the corresponding thioseptanosides in high yield. The same strategy was applied for the synthesis of the corresponding L-idothioseptanosides using 1,6-anhydro-3,4-O-isopropylidene-1-thio-L-iditol as starting material. The penta-O-acetyl-D-glucothioseptanose donors could not be synthesised the same way, as the Pummerer reaction of the corresponding tetra-O-acetyl-1,6-thioanhydro-1-thio-D-glucitol sulfoxides led to an inseparable mixture of the corresponding L-gulo- and D-glucothioseptanose anomers. Therefore, D-glucose diethyl dithioacetal was converted via its 2,3,4,5-tetra-O-acetyl-6-S-acetyl derivative into an anomeric mixture of its 6-thio-septanose and -furanose peracetates which could be separated by column chromatography. Condensation of the 6-thio-glucoseptanose peracetates with 4-cyano- and 4-nitrobenezenethiol in the presence of boron trifluoride etherate afforded anomeric mixtures of the corresponding thioseptanosides. The D-manno-, L-ido- and D-glucothioseptanosides obtained after Zemplén deacetylation of these mixtures were tested for their oral antithrombotic activity.     

Synthesis of polyhydroxy amino acids based on D- and L-alanine from D-glycero-D-gulo-heptono-1,4-lactone

2-Amino-2,3-dideoxy-D-manno-heptonic acid (7) has been synthesized from 2,5,6,7-tetra-O-acetyl-3-deoxy-D-gluco-heptono-1,4-lactone (1), which was readily prepared from D-glycero-D-gulo-heptono-1,4-lactone. O-Deacetylation of 1 followed by treatment with 13:1 (v/v) 2,2-dimethoxypropane/acetone in the presence of p-toluenesulfonic acid gave methyl 3-deoxy-4,5:6,7-di-O-isopropylidene-D-gluco-heptonate (3) as a crystalline product (80% yield). The free hydroxyl group (OH-2) of 3 was mesylated and substituted by azide to give the corresponding azide derivative 5. Hydrogenolysis and further hydrolysis of the ester function of 5 afforded alpha-amino acid 7 (43% overall yield from 1). Compound 7 is an analog of L-alanine having a polyhydroxy chain attached to C-3. The diastereoisomer of 7 at C-2, 2-amino-2,3-dideoxy-D-gluco-heptonic acid (12) was also prepared from 3, by a route that involved 2,3-dideoxy-2-iodo derivative 8 as a key intermediate.     

An improved synthesis of 5-thio-D-ribose from D-ribono-1,4-lactone

5-Thio-D-ribopyranose was synthesized from D-ribono-1,4-lactone (1) by two approaches: (i) 5-bromo-5-deoxy-D-ribono-1,4-lactone (2) was successively transformed into 5-bromo-5-deoxy, 5-S-acetyl-5-thio or 5-thiocyanato-D-ribofuranose derivatives; appropriate treatment then lead to 5-thio-D-ribopyranose (7) in 46-48% overall yield and; (ii) 2 was transformed into the 5-S-acetyl-5-thio-D-ribono-1,4-lactone derivative (11). Reduction and deprotection of 11 afforded 5-thio-D-ribopyranose (7) in 57% overall yield.     

A simple access to the D-mannosidase inhibitor, 1-deoxymannojirimycin

Crystalline 1,3,4,5-tetra-O-acetyl-6-bromo-6-deoxy-keto-D-fructose was prepared by reaction of 1,3,4,5-tetra-O-acetyl-D-fructopyranose with triphenylphosphane dibromide in dichloromethane. Subsequent deprotection followed by reaction of the free 6-bromodeoxyfructofuranose with sodium azide in N,N-dimethylformamide furnished the corresponding 6-azidodeoxyketose. Catalytic hydrogenation led to 1-deoxymannojirimycin in 27% overall yield from 1,3,4,5-tetra-O-acetyl-D-fructopyranose. This access is simple, inexpensive, high-yielding and clearly suitable for multigram preparations.     

Synthesis of a library of allyl alpha-L-arabinofuranosyl-alpha- or beta-D-xylopyranosides; route to higher oligomers

Six isomeric disaccharides allyl 2,3,5-tri-O-benzoyl-alpha-l-arabinofuranosyl-alpha-d-xylopyranosides and beta-d-xylopyranosides were synthetized by the stereoselective glycosylation of pure allyl alpha- or beta-d-xylopyranosides with 1-O-acetyl-2,3,5-tri-O-benzoyl-l-arabinofuranose as donor, catalyzed with BF(3).Et(2)O in DCM. Regio- and stereoselective glycosylation with excess of donor furnished almost exclusively the trisaccharides allyl 2,3-di-O-(2,3,5-tri-O-benzoyl-alpha-l-arabinofuranosyl)-alpha- or beta-d-xylopyranosides. Extension of the reaction to the triol beta-d-xylopyranosyl-(1-->4)-1,2,3-tri-O-acetyl-alpha-d-xylopyranose, obtained from the 4-hydroxyl penta-O-acetyl-alpha-xylobiose, gave in the same manner the tetrasaccharide [2,3-di-O-(2,3,5-tri-O-benzoyl-alpha-l-arabinofuranosyl)-beta-d-xylopyranosyl]-(1-->4)-1,2,3-tri-O-acetyl-alpha-d-xylopyranose. The protocol described herein should offer the possibility to produce branched oligosaccharides with a 2,3-di-O-(alpha-l-Ara(f))-beta-d-Xyl(p) block unit at the terminal non-reducing end.     

Efficient synthesis of 5-thio-D-arabinopyranose and 5-thio-D-xylopyranose from the corresponding D-pentono-1,4-lactones

5-Thio-D-arabinopyranose (5) and 5-thio-D-xylopyranose (10) were synthesized from the corresponding D-pentono-1,4-lactones. After regioselective bromination at C-5, transformation into 5-S-acetyl-5-thio derivatives, reduction into lactols and deprotection afforded the title compounds in 49 and 42% overall yield, respectively.     

Base catalysed isomerisation of aldoses of the arabino and lyxo series in the presence of aluminate

Base-catalysed isomerisation of aldoses of the arabino and lyxo series in aluminate solution has been investigated. L-Arabinose and D-galactose give L-erythro-2-pentulose (L-ribulose) and D-lyxo-2-hexulose (D-tagatose), respectively, in good yields, whereas lower reactivity is observed for 6-deoxy-D-galactose (D-fucose). From D-lyxose, D-mannose and 6-deoxy-L-mannose (L-rhamnose) are obtained mixtures of ketoses and C-2 epimeric aldoses. Small amounts of the 3-epimers of the ketoses were also formed. 6-Deoxy-L-arabino-2-hexulose (6-deoxy-L-fructose) and 6-deoxy-L-glucose (L-quinovose) were formed in low yields from 6-deoxy-L-mannose and isolated as their O-isopropylidene derivatives. Explanations of the differences in reactivity and course of the reaction have been suggested on the basis of steric effects.     

An original chemoenzymatic route for the synthesis of beta-D-galactofuranosides using an alpha-L-arabinofuranosidase

DGalactofuranose is a widespread component of cell wall polysaccharides in bacteria, protozoa and fungi, but is totally absent in mammals. Importantly, galactofuranose is a key constituent of major cell envelope polysaccharides in pathogenic mycobacteria. In this respect, galactofuranose-based glycoconjugates are interesting target molecules for drug design. O-Glycosidases and notably beta-D-galactofuranosidases could be useful tools for the chemoenzymatic synthesis of galactofuranosides, but to date no studies of this type have been reported. Here we report the use of a GH 51 alpha-l-arabinofuranosidase for the synthesis of beta-D-galactofuranosides. We have demonstrated that this enzyme can catalyse both the autocondensation of p-nitrophenyl-beta-D-galactofuranoside and the transgalactofuranosylation of benzyl alpha-D-xylopyranoside, forming p-nitrophenyl beta-D-galactofuranosyl-(1-->2)-beta-D-galactofuranoside and benzyl beta-D-galactofuranosyl-(1-->2)-alpha-D-xylopyranoside, respectively. Both reactions were very regiospecific and the reaction involving benzyl alpha-D-xylopyranoside afforded very high yields (74.8%) of the major product. To our knowledge, this demonstration of chemoenzymatic synthesis of galactofuranosides constitutes the very first use of an O-glycosidase for the synthesis of galactofuranosides.     

Peracetylated 1,6-dibromo-D-glucitol as efficient precursor of 1,6-diiodo and some mono-, disubstituted and heterocyclic D-glucitol derivatives

2,3,4,5-Tetra-O-acetyl-1,6-dibromo-1,6-dideoxy-D-glucitol (1a) obtained from D-glucitol was easily transformed into the 1,6-diiodo derivative in excellent yield (97%) by reaction with an excess of sodium iodide in refluxing butanone in 2 h. When the reaction time was prolonged to 24 h and the crude product was acetylated, 1,2,3,4,5-penta-O-acetyl-6-deoxy-6-iodo-D-glucitol and D-glucitol hexaacetate were isolated in 50 and 26% yields, respectively. The monodehalogenation then took place regioselectively at C-1. This regioselectivity allowed the synthesis of some mono- and disubstituted derivatives of D-glucitol. Thus, the peracetylated derivatives of D-glucitol, 6-bromo, 6-bromo-1-S-butyl, 6-bromo-1-S-octyl, 6-S-butyl, 6-S-butyl-1-S-octyl, 1-S-butyl, 1,6-di-S-octyl and 6-S-phenyl were synthesised in good to excellent yields. With S= as binucleophilic reagent, 1a gave mainly the thiepane derivative (75%) plus the 1-S-acetyl-2,6-anhydro-D-glucitol derivative as a by-product (10%).     

A selective Sc(OTf)3-catalyzed trialkylsilyl ether to acetyl ester exchange reaction with beta-l-idopyranoside and 3,4-O-isopropylidene-beta-D-galactopyranoside derivatives

The selective silylation of monosaccharide building blocks is useful for preparing complex oligosaccharides. We now report that the diol, methyl (dimethylthexylsilyl 3-O-pivaloyl-beta-L-idopyranosyl)uronate, can be selectively silylated at the O-2 position by trialkylsilyl triflates. After protection of O-4, the O-2 silyl group can be selectively replaced by acetate by taking advantage of a trialkylsilyl-acetate exchange reaction catalyzed by Sc(OTf)3 in the presence of acetic anhydride. The high O-2 selectivity is shown for triethylsilyl (TES), tert-butyldimethylsilyl (TBS), and triisopropylsilyl (TIPS). The selective cleavage reaction only worked well for TES and TBS derivatives. A selection of silyl triflates and silyl chlorides were used as silylating reagents with ethyl 3,4-O-isopropylidene-1-thio-beta-D-galactopyranoside. In most cases, silylation afforded 2,6-di-O-silylated products in high yields. Studies on the cleavage reaction showed that only the primary silylated protecting groups were replaced by acetyl groups. This reaction worked with a variety of silyl protecting groups but not the tert-butyldiphenylsilyl (TBDPS) protecting group. Unfortunately, the 1-thioethyl group was also sensitive to the Sc(OTf)3, leading in these conditions to alpha/beta mixtures of the 1-acetates, which compromised the synthetic utility of this reaction for these compounds. The sequence presented here is a useful synthetic route to differentially protected L-iduronic acid building blocks.     

Synthesis of N-bridgehead heterocycles from saccharide benzimidazoles

Treatment of D-arabino-tetritol-1-yl-benzimidazole with P-toluenesulfonyl chloride (1molequiv) in pyridine, afforded the N-bridgehead heterocycles, 2R,3R,4S-trihydroxy-1:2:3:4-tetrahydropyridino[1,2-a]benzimidazole. The structure of the latter compound was determined by acylation, (1)H, and (13)C NMR spectroscopy and mass spectrometry.     

Syntheses of alpha-D-galactosamine neoglycolipids

Several N-acetyl-alpha-d-galactosamine neoglycolipids, as well as hydrophobized T and T(N) antigen analogues, were prepared for embedment onto liposomes. Three different lipidic structures were used for the anchoring, that is cholesterol, 1,3-bis(undecyloxy)propan-2-ol and 1,3-bis(3,7,11,15-tetramethylhexadecyloxy)propan-2-ol. Oligoethyleneglycol spacers were used to link the carbohydrate and the hydrophobic moieties; their lengths were varied in order to obtain model compounds for the selective recognition by sialyl transferases involved in cancer processes. Glycosylation reactions were optimized to sluggish amphiphilic acceptor alcohols, in order to reach good 1,2-cis-stereoselectivities and acceptable yields. This aim was achieved by using 3,4,6-tri-O-acetyl-2-azido-2-deoxy-d-galactopyranosyl trichloroacetimidate as the donor, trimethylsilyl trifluoromethanesulfonate as the promoter and diethyl ether or mixtures of diethyl ether and dichloromethane as solvents.     

Direct epoxidation of D-glucal and D-galactal derivatives with in situ generated DMDO

A multi-gram epoxidation of 3,4,6-tri-O-benzyl-D-glucal and D-galactal with dimethyldioxirane (DMDO) generated in situ from Oxone/acetone in a biphasic system (CH(2)Cl(2)-aqueous NaHCO(3)) resulted in the formation of the corresponding 1,2-anhydrosugars in a 99% yield and 100% selectivity. In a similar way, 3,4,6-tri-O-acetyl-D-glucal afforded a 7:1 mixture of the corresponding gluco and manno derivatives in an 87% overall yield.     

Synthesis of 1-deoxy-L-gulonojirimycin (L-guloDNJ) and 1-deoxy-D-talonojirimycin (D-taloDNJ)

Carbohydrate based syntheses of azasugars with unusual configurations viz. 1,5-dideoxy-1,5-imino-L-gulitol (L-guloDNJ) and 1,5-dideoxy-1,5-imino-L-talitol (L-taloDNJ) are reported, from D-mannose and D-fructose, respectively. The key steps in both syntheses involved reductive aminative cyclizations. Thus, L-guloDNJ was obtained by reduction of 2,3;4,6-di-O-isopropylidene-5-O-p-toluenesulfonyl-D-mannononitrile with LiAlH(4) in DME to give the protected azasugar which upon hydrolysis with HCl afforded crystalline L-guloDNJ as the HCl salt in 29% overall yield. Reduction of 6-azido-1-O-tert-butyldimethylsilyl-2,3-O-isopropylidene-beta-D-ribohexulofuranose obtained from D-fructose in six steps, followed by treatment with HCl, afforded L-taloDNJ as an HCl salt in approximately 10% overall yield.     

Synthesis and intramolecular reactions of Tyr-Gly and Tyr-Gly-Gly related 6-O-glucopyranose esters

6-O-(L-Tyrosylglycyl)- and 6-O-(L-tyrosylglycylglycyl)-D-glucopyranose were synthesized by condensation of the pentachlorophenyl esters of the respective di- and tripeptide with fully unprotected D-glucose. The intramolecular reactivity of the sugar conjugates was studied in pyridine-acetic acid and in dry methanol, at various temperatures and for various incubation times. The composition of the incubation mixtures was monitored by a reversed-phase HPLC method that permits simultaneous analysis of the disappearance of the starting material and the appearance of rearrangement and degradation products. To determine the influence of esterification of the peptide carboxy group on its amino group reactivity, parallel experiments were done in which free peptides were, under identical reaction conditions, incubated with D-glucose (molar ratios 1:1 and 1:5). Depending on the starting compound, different types of Amadori products (cyclic and bicyclic form), methyl ester of peptides, and Tyr-Gly-diketopiperazine were obtained.     

Enzymatic synthesis of tyrosol and hydroxytyrosol β-d-fructofuranosides

Tyrosol β-d-fructofuranoside and hydroxytyrosol β-d-fructofuranoside have been synthesized as new compounds in 27.6 and 19.5% respective yields through transfructosylation of tyrosol and hydroxytyrosol. Yeast β-galactosidase Lactozym 3000?L comprising invertase activity was used as catalyst. Besides the main monofructosides, an equimolar mixture of tyrosol β-d-fructofuranosyl-((2→1)-β-d-fructofuranoside and tyrosol β-d-fructofuranosyl-(2→6)-β-d-fructofuranoside was isolated as additional product fraction in 14.3% yield.     

Metal-ion interactions with sugars. Crystal structures and FT-IR studies of the LaCl3-ribopyranose and CeCl3-ribopyranose complexes

Single crystals of LaCl3.C5H10O5.5H2O (1) and CeCl3.C5H10O5.5H2O (2) were obtained from ethanol-water solutions and their structures determined by X-ray. The two complexes are isomorphous. Two configurations of complex 1 or complex 2, as a pair of isomers, were found in each single crystal in a disordered state. The ligand of one of the isomer is alpha-D-ribopyranose in the 4C1 conformation, the ligand of the other is beta-D-ribopyranose in the 1C4 conformation. For complex 1, the alpha:beta anomeric ratio is 51:49, and for complex 2, the ratio is 52:48. Both ligands of the two isomers provide three hydroxyl groups in ax-eq-ax orientation for coordination. The Ln3+ (Ln = La or Ce) ion is nine-coordinated with five Ln-O bonds from water molecules, three Ln-O bonds from hydroxyl groups of the D-ribopyranose, and one Ln-Cl bond from chloride ion. The hydroxyl groups, water molecules, and chloride ions form an extensive hydrogen-bond network. The IR spectral C-C, O-H, C-O, and C-O-H vibrations were observed to be shifted in both the two complexes and the IR results are in accord with those of X-ray diffraction.     

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.     

Two novel oligosaccharides isolated from a beverage produced by fermentation of a plant extract

An extract from 50 kinds of fruits and vegetables was fermented to produce a new beverage. Natural fermentation of the extract was carried out mainly by lactic acid bacteria (Leuconostoc spp.) and yeast (Zygosaccharomyces spp. and Pichia spp.). Two new saccharides were found in this fermented beverage. The saccharides were isolated using carbon-Celite column chromatography and preparative high performance liquid chromatography. Gas liquid chromatography analysis of methylated derivatives as well as MALDI-TOF MS and NMR measurements were used for structural confirmation. The (1)H and (13)C NMR signals of each saccharide were assigned using 2D-NMR including COSY, HSQC, HSQC-TOCSY, CH(2)-HSQC-TOCSY, and CT-HMBC experiments. The saccharides were identified as beta-D-fructopyranosyl-(2-->6)-beta-D-glucopyranosyl-(1-->3)-D-glucopyranose and beta-D-fructopyranosyl-(2-->6)-[beta-D-glucopyranosyl-(1-->3)]-D-glucopyranose.     

Synthesis and induction of apoptosis in B cell chronic leukemia by diosgenyl 2-amino-2-deoxy-beta-D-glucopyranoside hydrochloride and its derivatives

2-Acetamido-2-deoxy-D-glucose hydrochloride (D-glucosamine hydrochloride) has been used for the preparation of 1,3,4,6-tetra-O-acetyl-2-deoxy-2-trifluoroacetamido-beta- (4) and 2-tetrachlorophthalimido-alpha,beta-D-glucopyranose (6), which have been transformed into the appropriate bromides and the chloride. Both bromo and chloro sugars were used as a glycosyl donors for the glycosylation of diosgenin [(25R)-spirost-5-en-3beta-ol]. These condensations were conducted under mild conditions, using silver triflate as a promoter, and gave diosgenyl glycosides 9 and 12. Each of them was converted into diosgenyl 2-amino-2-deoxy-beta-D-glucopyranoside hydrochloride (11) and N-acylamido derivatives. The structures of all new glycosides were established by 1H and 13C NMR spectroscopy. These diosgenyl glycosides are the first saponins containing the D-glucosamine residue that have been synthesized. These compounds show promising antitumor activities. The synthetic saponins increase the number of apoptotic B cells, in combination with cladribine (2-CdA), that are isolated from chronic lymphotic leukemia (B-CLL) patients.