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.     

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.     

The structure of the antigenic polysaccharide produced by Eubactrium saburreum T15

The antigenic polysaccharide was obtained from the cell wall of Eubacterium saburreum strain T15 by trypsin digestion followed by gel permeation and ion-exchange chromatography. Its structure was determined using acid hydrolysis, methylation analysis, and 1D and 2D NMR spectroscopy. It contained L-threo-pent-2-ulose (Xul), D-fucose (Fuc), and D-glycero-D-galacto-heptose (Hep) in 2:3:3 ratio. Methylation analysis indicated an octasaccharide repeating-unit containing five branches. The 1H and 13C signals in NMR spectra of the sugar residues were assigned by COSY, HOHAHA, and HMQC 2D experiments, and the sequence of sugar residues in the repeating unit was determined by NOESY and HMBC experiments. The polysaccharide also contains two O-acetyl groups in the repeating unit, located on the Hep residue. The repeating structure can be written as: [see text for equation]. This is a novel structure in bacterial cell-wall polysaccharides from Gram-positive bacteria.     

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.     

A novel family of glucosyl 1,5-anhydro-d-fructose derivatives synthesised by transglucosylation with dextransucrase from Leuconostoc mesenteroides NRRL B-512F

1,5-Anhydro-d-fructose (AF), a metabolite of starch/glycogen degradation, is a good antioxidant. With the prospect of increasing its applications and use as a food ingredient, AF glucosylation catalysed by the dextransucrase from Leuconostoc mesenteroides NRRL B-512F was performed in the presence of sucrose. This led to AF glucosylated derivatives containing alpha-(1-->6) linkages named 1,5-anhydro-d-fructo-glucooligosaccharides (AFGOS). LC-MS analyses showed that AFGOS with a degree of polymerisation (DP) of up to 7 were synthesised. The amount of AFGOS produced and the average DP increased by using a high sucrose/AF molar ratio and high total sugar concentration. AFGOS were proved to present antioxidant properties quite similar to AF.     

Synthesis of D- and L-myo-inositol 2,4,5-trisphosphate and trisphosphorothioate: structural analogues of D-myo-inositol 1,4,5-trisphosphate

The preparation of D- and L-myo-inositol 2,4,5-trisphosphate is described, together with the phosphorothioate counterparts. The known chiral diols D- and L-1,4-di-O-benzyl-5,6-bis-O-p-methoxybenzyl-myo-inositol were regioselectively protected at the 3-position using a benzyl group via a 2,3-O-stannylene acetal. Removal of the p-methoxybenzyl groups of each enantiomer gave D- and L-1,3,6-tri-O-benzyl-myo-inositol. Phosphitylation with bis(benzyloxy)diisoproplyaminophosphine and 1H-tetrazole gave the trisphosphite intermediate for each enantiomer. Oxidation with 3-chloroperoxybenzoic acid gave the fully protected D- and L-myo-inositol 2,4,5-trisphosphates. Sulphoxidation of the D- and L-2,4,5-trisphosphite intermediates gave the fully protected D- and L-myo-inositol 2,4,5-trisphosphorothioate compounds. The fully protected trisphosphates were deblocked using hydrogenolysis and the phosphorothioates were deprotected using sodium in liquid ammonia. The individual compounds were then purified using ion exchange chromatography to afford pure D- and L-myo-inositol 2,4,5-trisphosphates together with the corresponding phosphorothioates.     

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.     

Lactose as an inexpensive starting material for the preparation of aldohexos-5-uloses: synthesis of l-ribo and d-lyxo derivatives

Partially protected derivatives of l-ribo- and d-lyxo-aldohexos-5-ulose have been prepared starting from triacetonlactose dimethyl acetal derivatives. Key steps of the synthetic sequences are (a) the synthesis of 4′-deoxy-4′-eno- and 6′-deoxy-5′-eno lactose derivatives, and (b) the epoxidation-methanolysis of the above-mentioned enol ethers to give 1,5-bis-glycopyranosides, masked form of the target 1,5-dicarbonyl hexoses.     

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.     

NMR studies of molybdate complexes of D-erythro-L-manno-octose and D-erythro-L-gluco-octose and their alditols

Different amounts and various types of bis-dinuclear tetradentate molybdate complexes of D-erythro-L-manno-octose, D-erythro-L-gluco-octose, D-erythro-L-manno-octitol and D-erythro-L-gluco-octitol were characterized by 1H and 13C NMR spectroscopy in aqueous solutions. Detailed analysis of 1H-(1)H coupling constants and NOEs, together with chemical shifts, allowed characterization of the different isomers of these complexes.     

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.     

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.     

1,5-Anhydro-d-fructose: biocatalytic and chemical synthetic methods for the preparation, transformation and derivatization

1,5-Anhydro-d-fructose (1,5AnFru) is a monoketosaccharide that can be prepared enzymatically from starch by α-1,4-glucan lyase or chemically from d-glucose or d-fructose in a few steps with high yields. The formed 1,5AnFru can be derivatized both enzymatically and chemically to interesting new carbohydrate derivatives, some with biological activities. For example dehydratases, isomerases and reductases can convert 1,5AnFru to enolones (as Ascopyrone P) and sugar alcohols with antimicrobial and antioxidant properties, while chemical modifications can give similar compounds as well as natural products like 1-deoxymannonojirimycin and Clavulazine. 1,5AnFru disaccharides (glycosyl 1→4 1,5AnFru) have been prepared as well as glycosyl 1→4 1,5-anhydro-d-tagatose.     

1,5-anhydro-D-fructose from D-fructose

1,5-Anhydro-d-fructose was efficiently prepared from d-fructose via regiospecific 1,5-anhydro ring formation of 2,3-O-isopropylidene-1-O-methyl(tolyl)sulfonyl-d-fructopyranose and subsequent deprotection.     

Glycosylation of l,4:3,6-dianhydro-D-glucitol (isosorbide)

Condensation of 2,3,4,6-tetra-O-acetyl-alpha-D-glucopyranosyl-, 2,3,4-tri-O-acetyl-alpha-D-xylopyranosyl- and of 2,3,4,6-tetra-O-acetyl-alpha-D-galactopyranosyl bromides with l,4:3,6-dianhydro-D-glucitol under Koenigs-Knorr conditions, and using the Helferich modification of the reaction showed regioselectivity in glysosylation at C-5 of isosorbide.     

Crystal structures of D-tagatose 3-epimerase from Pseudomonas cichorii and its complexes with D-tagatose and D-fructose

Pseudomonas cichoriiid-tagatose 3-epimerase (P. cichoriid-TE) can efficiently catalyze the epimerization of not only d-tagatose to d-sorbose, but also d-fructose to d-psicose, and is used for the production of d-psicose from d-fructose. The crystal structures of P. cichoriid-TE alone and in complexes with d-tagatose and d-fructose were determined at resolutions of 1.79, 2.28, and 2.06 Å, respectively. A subunit of P. cichoriid-TE adopts a (β/α)₈ barrel structure, and a metal ion (Mn²⁺) found in the active site is coordinated by Glu152, Asp185, His211, and Glu246 at the end of the β-barrel. P. cichoriid-TE forms a stable dimer to give a favorable accessible surface for substrate binding on the front side of the dimer. The simulated omit map indicates that O2 and O3 of d-tagatose and/or d-fructose coordinate Mn²⁺, and that C3-O3 is located between carboxyl groups of Glu152 and Glu246, supporting the previously proposed mechanism of deprotonation/protonation at C3 by two Glu residues. Although the electron density is poor at the 4-, 5-, and 6-positions of the substrates, substrate-enzyme interactions can be deduced from the significant electron density at O6. The O6 possibly interacts with Cys66 via hydrogen bonding, whereas O4 and O5 in d-tagatose and O4 in d-fructose do not undergo hydrogen bonding to the enzyme and are in a hydrophobic environment created by Phe7, Trp15, Trp113, and Phe248. Due to the lack of specific interactions between the enzyme and its substrates at the 4- and 5-positions, P. cichoriid-TE loosely recognizes substrates in this region, allowing it to efficiently catalyze the epimerization of d-tagatose and d-fructose (C4 epimer of d-tagatose) as well. Furthermore, a C3-O3 proton-exchange mechanism for P. cichoriid-TE is suggested by X-ray structural analysis, providing a clear explanation for the regulation of the ionization state of Glu152 and Glu246.     

Melting behaviour of D-sucrose, D-glucose and D-fructose

The melting behaviour of d-sucrose, d-glucose and d-fructose was studied. The melting peaks were determined with DSC and the start of decomposition was studied with TG at different rates of heating. In addition, melting points were determined with a melting point apparatus. The samples were identified as d-sucrose, alpha-d-glucopyranose and beta-d-fructopyranose by powder diffraction measurements. There were differences in melting between the different samples of the same sugar and the rate of heating had a remarkable effect on the melting behaviour. For example, T(o), DeltaH(f) and T(i) (initial temperature of decomposition) at a 1 degrees Cmin(-1) rate of heating were 184.5 degrees C, 126.6Jg(-1) and 171.3 degrees C for d-sucrose, 146.5 degrees C, 185.4Jg(-1) and 152.0 degrees C for d-glucose and 112.7 degrees C, 154.1Jg(-1) and 113.9 degrees C for d-fructose. The same parameters at 10 degrees Cmin(-1) rate of heating were 188.9 degrees C, 134.4Jg(-1) and 189.2 degrees C for d-sucrose, 155.2 degrees C, 194.3Jg(-1) and 170.3 degrees C for d-glucose and 125.7 degrees C, 176.7Jg(-1) and 136.8 degrees C d-fructose. At slow rates of heating, there were substantial differences between the different samples of the same sugar. The melting point determination is a sensitive method for the characterization of crystal quality but it cannot be used alone for the identification of sugar samples in all cases. Therefore, the melting point method should be validated for different sugars.     

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.     

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.     

Theoretical study of the experimental coordination behavior of N-(2-aminophenyl)-D-glycero-D-gulo-heptonamide to Hg(II) ion

The reactivity of N-(2-aminophenyl)-d-glycero-d-gulo-heptonamide (adgha), with the group 12 cations, Zn(II), Cd(II), and Hg(II), was studied in DMSO-d₆ solution. The studied system showed a selective coordination to Hg(II), and the products formed were characterized by ¹H and ¹³C NMR in DMSO-d₆ solution and fast atom bombardment (FAB⁺) mass spectra. The expected coordination compounds, [Hg(adgha)](NO₃)₂ and [Hg(adgha)₂](NO₃)₂, were observed as unstable intermediates that decompose to bis-[2-(d-glycero-d-gulo-hexahydroxyhexyl)-benzimidazole-κN]mercury(II) dinitrate, [Hg(ghbz)₂](NO₃)₂. The chemical transformation of the complexes was followed by NMR experiments, and the nature of the species formed is sustained by a theoretical study done using DFT methodology. From this study, we propose the structure of the complexes formed in solution, the relative stability of the species formed, and the possible role of the solvent in the observed transformations.