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 2,5-anhydro-(beta-d-glucopyranosyluronate)- and (alpha-l-idopyranosyluronate)-d-mannitol hexa-O-sulfonate hepta sodium salt

Glycosidation of 2,5-anhydro-1,6-di-O-benzoyl-D-mannitol with methyl(2,3,4-tri-O-acetyl-alpha-d-glucopyranosyl-1-O-trichloroacetimidate)uronate in the presence of trimethylsilyl triflate afforded the corresponding 3-O-beta-glycoside, which after deprotection was converted into its hexa-O-sulfate with DMF x SO3 to give after treatment with sodium acetate and subsequent saponification of the methyl ester with sodium hydroxide the hepta sodium salt of 2,5-anhydro-3-O-(beta-d-glucopyranosyl uronate)-D-mannitol hexa-O-sulfate. Glycosidation of the same acceptor with the alpha-thiophenylglycoside of methyl 2,4-di-O-acetyl-3-O-benzyl-L-idopyranosyl uronate in the presence of NIS/TfOH afforded the corresponding 3-O-alpha-glycoside in very low yield, therefore the alpha-thiophenylglycoside of 2-O-acetyl-2,4-O-benzylidene-3-O-benzyl-L-idopyranose was used as donor. The terminal hydroxymethyl group of the obtained disaccharide was subsequently oxidised with NaOCl/TEMPO and the obtained iduronic acid derivative was converted into the hepta sodium salt of 2,5-anhydro-3-O-(-alpha-L-idopyranosyluronate)-D-mannitol hexa-O-sulfonate with DMF x SO3 and subsequent treatment with sodium acetate.     

Evaluation of carbohydrate molecular mechanical force fields by quantum mechanical calculations

A quantitative evaluation of 20 second-generation carbohydrate force fields was carried out using ab initio and density functional methods. Geometry-optimized structures (B3LYP/6-31G(d)) and relative energies using augmented correlation consistent basis sets were calculated in gas phase for monosaccharide carbohydrate benchmark systems. Selected results are: (i). The interaction energy of the alpha-d-glucopyranose.H(2)O heterodimer is estimated to be 4.9 kcal/mol, using a composite method including terms at highly correlated (CCSD(T)) level. Most molecular mechanics force fields are in error in this respect; (ii). The (3)E envelope (south) pseudorotational conformer of methyl 5-deoxy-beta-d-xylofuranoside is 0.66 kcal/mol more stable than the (3)E envelope (north) conformer and the alpha-anomer of methyl d-glucopyranoside is 0.82 kcal/mol more stable than the beta-anomer; (iii). The relative energies of the (gg, gt and tg) rotamers of methyl alpha-d-glucopyranoside and methyl alpha-d-galactopyranoside are (0.13, 0.00, 0.15) and (0.64, 0.00, 0.77) kcal/mol, respectively. The results of the quantum mechanical calculations are compared with the results of calculations using the 20 second-generation carbohydrate force fields. No single force field is consistently better than the others for all the test cases. A statistical assessment of the performance of the force fields indicates that CHEAT(95), CFF, certain versions of Amber and of MM3 have the best overall performance, for these gas phase monosaccharide systems.     

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.     

Inhibition of the D-fructose transporter protein GLUT5 by fused-ring glyco-1,3-oxazolidin-2-thiones and -oxazolidin-2-ones

The glucose transporter 5 (GLUT5)-a specific D-fructose transporter-belongs to a family of facilitating sugar transporters recently enlarged by the human genome sequencing. Prompted by the need to develop specific photolabels of these isoforms, we have studied the interaction of conformationally locked D-fructose and L-sorbose derived 1,3-oxazolidin-2-thiones and 1,3-oxazolidin-2-ones to provide a rational basis for an interaction model. The inhibition properties of the D-fructose transporter GLUT5 by glyco-1,3-oxazolidin-2-thiones and glyco-1,3-oxazolidin-2-ones is now reported. In vitro, the fused-rings systems tested showed an efficient inhibition of GLUT5, thus bringing new insights on the interaction of D-fructose with GLUT5.     

Esterification of select polyols with D-glucaric acid as model reactions for esterification of starch

Aqueous solutions of D-glucaric acid and model polyols xylitol, methyl alpha-D-glucopyranoside or beta-cyclodextrin were freeze dried, then heated, and the product mixtures analyzed by instrumental methods that included GC-MS, electrospray ionization-mass spectrometry (ESIMS), and NMR. The thermal process and analyses were carried out with these polyols in order to determine to what extent multiple acylations of the alcohol functions occurred with D-glucaric acid lactones serving as acylating agents, the extent to which acylations occurred at the 1 degrees alcohol sites, and the relative tendency for acylations to occur at the C1 or C6 end of the glucaryl unit. The results of these studies showed an overwhelming preference for 1 degrees alcohol acylation and preferred acylation occurring at the C1 end of the glucaryl unit.     

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.     

Facile synthesis of sulfonium ion derivatives of 1,5-anhydro-5-thio-L-fucitol as potential alpha-L-fucosidase inhibitors

Five sulfonium ion derivatives with 1,5-anhydro-5-thio-L-fucitol as a core structure were efficiently synthesized as potential alpha-L-fucosidase inhibitors. The key unit, the tri-O-benzyl derivative of L-fucitol, was readily synthesized from methyl alpha-D-mannopyranoside. Alkylation with methyl iodide or 5-methoxycarbonyl-1-pentyl iodide in acetonitrile containing AgBF4 afforded the corresponding alkylated sulfonium tetrafluoroborates. Alternatively, ring opening of three 1,3-cyclic sulfates in 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) containing K2CO3 afforded the corresponding zwitterionic sulfonium sulfates.     

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.     

Preparation of DL-2,3,4-trihydroxybutylarsonic acid and dl-2,3-dihydroxybutane-1,4-bis(arsonic acid): starting compounds for novel arsonolipids

The reaction of DL-1,3-butadiene diepoxide and of DL-1,4-dibromo-2,3-butanediol with aqueous alkaline sodium arsenite, "Na(3)AsO(3)", gave mixtures of the title arsonic acids which can be separated by anion exchange resin. Characterization of by-products leads to a better understanding of these reactions. These compounds are valuable intermediates for the preparation of novel arsonic acids and bis(arsonic acids).     

Enzymatic synthesis of D-glucosaminic acid from D-glucosamine

D-glucosaminic acid (2-amino-2-deoxy-D-gluconic acid), a component of bacterial lipopolysaccharides and a chiral synthon, is easily prepared on a multigram scale by air oxidation of D-glucosamine (2-amino-2-deoxy-D-glucose) catalysed by glucose oxidase.     

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.     

Copper-induced oxidation of epinephrine: protective effect of D-DAHK,a synthetic analogue of the high affinity copper binding site of human albumin

Epinephrine is known to be rapidly oxidized during sepsis. Ischemia and acidosis, which often accompany sepsis, are associated with the release of weakly bound cupric ions from plasma proteins. We investigated whether copper promotes oxidation of epinephrine at both physiological and acidic pH and whether D-Asp-D-Ala-D-His-D-Lys (D-DAHK), a human albumin (HSA) N-terminus synthetic peptide with a high affinity for cupric ions, attenuates this oxidation. Epinephrine alone [100 microM] or with CuCl(2) [10 microM], and with CuCl(2) [10 microM] and D-DAHK [20 microM] at pH 7.4, 7.0, 6.5, and 6.0 were incubated for 1h at 37 degrees C. Epinephrine oxidation was measured by the spectrophotometric quantification of its oxidation product, adrenochrome. We found that adrenochrome increased, suggesting copper-induced oxidation of epinephrine. At pH 7.4, 7.0, 6.5, and 6.0, adrenochrome increased by 47%, 53%, 24%, and 6% above baseline, respectively. D-DAHK attenuated the copper-induced oxidation of epinephrine to baseline levels. These in vitro results indicate that copper-induced epinephrine oxidation is greatest at the physiological pH 7.4 as well as in severe acidosis, pH 7.0, and that D-DAHK completely inhibits this oxidation.     

Structural analysis of a novel saccharide isolated from fermented beverage of plant extract

Fermented beverage of plant extract was prepared from about 50 kinds of vegetables and fruits. Natural fermentation was carried out mainly by lactic acid bacteria (Leuconostoc spp.) and yeast (Zygosaccharomyces spp. and Pichia spp.). Three kinds of saccharides have been found in this beverage and produced by fermentation. The saccharides isolated from the beverage using carbon-Celite column chromatography and preparative HPLC, were identified as a new saccharide, beta-d-fructopyranosyl-(2-->6)-d-glucopyranose, laminaribiose and maltose by examination of constituted sugars, GLC and GC-MS analyses of methyl derivatives and MALDI-TOF-MS and NMR measurements of the saccharides.     

L-Glutamate in the extracellular space regulates endogenous D-aspartate homeostasis in rat pheochromocytoma MPT1 cells

In previous studies [FEBS Lett. 434 (1998) 231, Arch. Biochem. Biophys. 404 (2002) 92], we demonstrated for the first time that D-aspartate (D-Asp) is synthesized in cultured mammalian cell lines, such as pheochromocytoma 12 (PC12) and its subclone, MPT1. Our current focus is analysis of the dynamics of D-Asp homeostasis in these cells. In this communication, we show that L-glutamate (Glu) and L-Glu transporter substrates in the extracellular space regulate the homeostasis of endogenous D-Asp in MPT1 cells. D-Asp is apparently in dynamic homeostasis, whereby endogenous D-Asp is constantly released into the extracellular space by an undefined mechanism, and continuously and intensively taken up into cells by an L-Glu transporter. Under these conditions, L-Glu and its transporter substrates in the medium may competitively inhibit the uptake of D-Asp via the transporter, resulting in accumulation of the amino acid in the extracellular space. We additionally demonstrate that DL-TBOA, a well-established L-Glu transporter inhibitor, is taken up by the transporter during long time intervals, but not on a short time-scale.     

Synthesis, crystal structure, and reactivity of a D-xylose based oxepine

The synthesis and X-ray crystal structure of a D-xylose-based oxepine are reported. The oxepine was prepared from 2,3,4-tri-O-benzyl-D-xylose by the three-step sequence (Wittig olefination, vinyl ether formation, and ring closing metathesis) we recently reported. Epoxidation of this cyclic enol ether using dimethyldioxirane (DMDO) gave 1,2-anhydro-beta-D-idoseptanose, which was trapped by a number of nucleophiles to give alpha-idoseptanosides. The stereochemistry of epoxidation was assigned based on product analysis. Spectroscopic data of methyl 2,3,4,5-tetra-O-acetyl-alpha-D-idoseptanoside, derived from the methanolysis product 11, was compared to data of its enantiomer, the known methyl 2,3,4,5-tetra-O-acetyl-alpha-L-idoseptanoside.     

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.     

Enzymatic synthesis of D-glucosone 6-phosphate (D-arabino-hexos-2-ulose 6-(dihydrogen phosphate)) and NMR analysis of its isomeric forms

D-Glucosone 6-phosphate (D-arabino-hexos-2-ulose 6-(dihydrogen phosphate)) was prepared from D-glucosone (D-arabino-hexos-2-ulose) by enzymatic conversion with hexokinase. The isomeric composition of D-glucosone 6-phosphate in aqueous solution was quantitatively determined by NMR spectroscopy and compared to D-glucosone. The main isomers are the alpha-anomer (58%) and the beta-anomer (28%) of the hydrated pyranose form, and the beta-D-fructofuranose form (14%).     

Synthesis and structural analysis of a series of d-glucose derivatives as low molecular weight gelators

Low molecular weight gelators are an interesting new type of compounds that are important in supramolecular chemistry and advanced materials. Previously, we had synthesized several acyl derivatives of methyl 4,6-O-benzylidene-α-d-glucopyranoside and found that a number of terminal acetylene-containing esters are good gelators. To understand the structure requirement of the acyl chains, we synthesized a series of analogs containing different functional groups including aryl, alkenyl, and halogen derivatives. X-ray crystal structures of a monoester and a diester derivative were also obtained to help understand the relationship between structure and gelation. For good gelation properties, the carboxyl derivatives should possess alkyl groups containing a terminal acetylene group and aryl derivatives.     

Simultaneous measurement of D-serine dehydratase and d-amino acid oxidase activities by the detection of 2-oxo-acid formation with reverse-phase high-performance liquid chromatography

N-methyl-D-aspartate receptors (NMDARs) play critical roles in excitatory synaptic transmission in the vertebrate central nervous system. NMDARs need D-serine for their channel activities in various brain regions. In mammalian brains, D-serine is produced from L-serine by serine racemase and degraded by D-amino acid oxidase (DAO) to 3-hydroxypyruvate. In avian organs, such as the kidney, in addition to DAO, D-serine is also degraded to pyruvate by D-serine dehydratase (DSD). To examine the roles of these two enzymes in avian brains, we developed a method to simultaneously measure DAO and DSD activities. First, the keto acids produced from D-serine were derivatized with 3-methyl-2-benzothiazolinone hydrazone to stable azines. Second, the azine derivatives were quantified by means of reverse-phase high-performance liquid chromatography using 2-oxoglutarate as an internal standard. This method allowed the simultaneous detection of DAO and DSD activities as low as 100 pmol/min/mg protein. Chicken brain showed only DSD activities (0.4+/-0.2 nmol/min/mg protein) whereas rat brain exhibited only DAO activities (0.7+/-0.1 nmol/min/mg protein). This result strongly suggests that DSD plays the same role in avian brains, as DAO plays in mammalian brains. The present method is applicable to other keto acids producing enzymes with minor modifications.