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.     

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.     

Synthesis and antitumor activity of new D-galactose-containing derivatives of doxorubicin

A general scheme of synthesis of antibiotic doxorubicin derivatives is based on the 13-dimethyl ketal of 14-bromodaunorubicin (4). The interaction of 4 with melibiose (5), lactose (6), 3-methoxy-4-O-(2,3,4,6-tetra-O-acetyl-beta-D-galactopyranosyl)-4-oxybenzaldehyde (12) or 4-O-(2,3,4,6-tetra-O-acetyl-beta-D-galactopyranosyl)-4-oxybenzaldehyde (13) by reductive alkylation followed by hydrolysis of the corresponding intermediate bromoketals produced 3'-N-[alpha-D-galactopyranosyl-(1-->6)-O-1-deoxy-D-glucit-1-yl]doxorubicin (7), 3'-N-[beta-D-galactopyranosyl-(1-->4)-O-1-deoxy-D-glucit-1-yl]doxorubicin (8), 3'-N-[3"-methoxy-4"-O-(beta-D-galactopyranosyl)-4"-oxybenzyl]doxorubicin (16), and 3'-N-[4"-O-(beta-D-galactopyranosyl)-4"-oxybenzyl]doxorubicin (17). Cytotoxic and antitumor activity of the synthesized drug candidates compared to the parent doxorubicin was studied using various experimental models, in particular, on mice bearing lymphocyte leukemia P-388 at single and multiple i.v. injection regimens.     

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.     

Ni(II) complexes with Schiff bases derived from amino sugars

It was found by 1H and 13C NMR spectroscopy that the Schiff base, 2-deoxy-2-(2-hydroxybenzaldimino)-D-glucopyranose exhibits enol-imine-keto-amine and anomeric equilibria in methanolic, and in dimethyl sulfoxide solutions. The reaction of the Schiff base with nickel acetate gave the bidentate, mononuclear Ni(II) complex that was characterized by spectroscopic methods and by cyclic voltammetry. The coordination of the Schiff base to the metal is through the enol-imine tautomeric form, and the anomeric equilibrium remains in dimethyl sulfoxide solutions. This complex was also obtained by reaction of D-glucosamine with Ni(II) salicylaldehydate. The same reaction was employed for the synthesis of bis-N-[2-deoxy-D-galactopyranosyl-2-(2-hydroxybenzaldiminate)]Ni(II). The small paramagnetic shifts of the 1H NMR resonances of the complexes suggest that paramagnetic species are present in low proportions.     

2,5-Deoxyfructosazine, a D-glucosamine derivative, inhibits T-cell interleukin-2 production better than D-glucosamine

D-Glucosamine has been widely reported to have immunosuppressive actions on neutrophils, lymphocytes, and other cells of the immune system. However, under conditions used in biological experiments (e.g., neutral pH, and phosphate buffers), we have found that D-glucosamine self-reacts to form 2,5-deoxyfructosazine [2-(D-arabino-tetrahydroxybutyl)-5-(D-erythro-2,3,4-trihydroxybutyl)pyrazine] (1) and 2,5-fructosazine [2,5-bis(D-arabino-tetrahydroxybutyl)pyrazine] (2). When tested for bioactivity at nontoxic concentrations, these D-glucosamine derivatives were more effective inhibitors of IL-2 release from PHA-activated T cells than d-glucosamine. Hence, fructosazines constitute a novel class of immunomodulators.     

High yield production of monomer-free chitosan oligosaccharides by pepsin catalyzed hydrolysis of a high deacetylation degree chitosan

The high molecular weight of chitosan, which results in a poor solubility at neutral pH values and high viscosity aqueous solutions, limits its potential uses in the fields of food, health and agriculture. However, most of these limitations are overcome by chitosan oligosaccharides obtained by enzymatic hydrolysis of the polymer. Several commercial enzymes with different original specificities were assayed for their ability to hydrolyze a 93% deacetylation degree chitosan and compared with a chitosanase. According to the patterns of viscosity decrease and reducing end formation, three enzymes--cellulase, pepsin and lipase A--were found to be particularly suitable for hydrolyzing chitosan at a level comparable to that achieved by chitosanase. Unlike the appreciable levels of both 2-amino-2-deoxy-D-glucose and 2-acetamido-2-deoxy-D-glucose monomers released from chitosan by the other enzymes after a 20h-hydrolysis (4.6-9.1% of the total product weight), no monomer could be detected following pepsin cleavage. As a result, pepsin produced a higher yield of chitosan oligosaccharides than the other enzymes: 52% versus as much as 46%, respectively. Low molecular weight chitosans accounted for the remaining 48% of hydrolysis products. The calculated average polymerization degree of the products released by pepsin was around 16 units after 20h of hydrolysis. This product pattern and yield are proposed to be related to the bond cleavage specificity of pepsin and the high deacetylation degree of chitosan used as substrate. The optimal reaction conditions for hydrolysis of chitosan by pepsin were 40 degrees C and pH 4.5, and an enzyme/substrate ratio of 1:100 (w/w) for reactions longer than 1h.     

An efficient synthesis of methyl 1,3-O-isopropylidene-alpha-D-fructofuranoside and 2,3:5,6-di-O-isopropylidene-D-glucose dimethyl acetal derivatives from sucrose

Acetalation of sucrose with 2,2-dimethoxypropane in 1,4-dioxane in the presence of p-toluenesulfonic acid, followed by acetylation, afforded methyl 4,6-di-O-acetyl-1,3-O-isopropylidene-alpha-D-fructofuranoside and 4-O-acetyl-2,3:5,6-di-O-isopropylidene-D-glucose dimethyl acetal as major products, while tosylation of the intermediate acetals provided methyl 6-O-tosyl-1,3-O-isopropylidene-alpha-D-fructofuranose.     

A concise synthesis of 5-amino-5-deoxyaldonic acids as monomers for the preparation of nylon 5

Saponification of 5-azido-5-deoxy-D-pentonolactones (ribo-, arabino-, xylo-) with NaOH gave the corresponding 5-azido-5-deoxyaldonic acids sodium salts which, after regeneration of the acid form followed by catalytic reduction, led to the target compounds in 98% overall yields.     

Quantitative production of 2-acetamido-2-deoxy-D-glucose from crystalline chitin by bacterial chitinase

Finely powdered alpha- and beta-chitin can be completely hydrolyzed with chitinase (EC 3.2.1.14) and beta-N-acetylhexosaminidase (EC 3.2.1.52) for the production of 2-acetamido-2-deoxy-D-glucose (GlcNAc). Crude chitinase from Burkholderia cepacia TU09 and Bacillus licheniformis SK-1 were used to digest alpha- and beta-chitin powder. Chitinase from B. cepacia TU09 produced GlcNAc in greater than 85% yield from beta- and alpha-chitin within 1 and 7 days, respectively. B. licheniformis SK-1 chitinase completely hydrolyzed beta-chitin within 6 days, giving a final GlcNAc yield of 75%, along with 20% of chitobiose. However, only a 41% yield of GlcNAc was achieved from digesting alpha-chitin with B. licheniformis SK-1 chitinase.     

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.     

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%).     

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.     

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.     

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.     

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.     

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.     

Synthesis of D-mannitol and L-iditol derivatives as monomers for the preparation of new regioregular AABB-type polyamides

1,6-Diamino-1,6-dideoxy-2,3,4,5-tetra-O-methyl-D-mannitol (and its L-iditol analogue) suitable for their utilization as monomers in the preparation of linear polyamides are described. Regio- and stereoregular polyamides of the AABB-type have been prepared by the active ester polycondensation method from these C(2) symmetric monomers and suberic and dodecanedioic acids. The resulting polyamides were obtained in fair yields (70-60%) and were characterized by elemental analyses and infrared and 1H and 13C NMR spectroscopies. Their M(w) and M(w)/M(n) were determined by GPC relative to polystyrene standards. All of them were gummy non-crystalline solids.     

Synthesis of methyl 2-acetamido-2,6-dideoxy-alpha- and beta-d-xylo-hexopyranosid-4-ulose, a keto sugar which misled the analytical chemists

To understand the contradictory results on the structure of the lipopolysaccharide isolated from a Yersinia enterocolitica O:3, both anomers of methyl 2-acetamido-2,6-dideoxy-d-xylo-hexopyranosid-4-ulose were prepared. The key steps of the synthetic pathway were the selective acetylation of the methyl 2-acetamido-2,6-dideoxy-alpha,beta-d-glucopyranosides, the oxidation of the 4-position to form the keto-sugars, and deacetylation to provide the target compound. Surprisingly, the last step was accompanied by a disproportionation to give methyl 2-acetamido-2,6-dideoxy-alpha- and beta-d-glucopyranosides and N-(5-hydroxy-6-methyl-4-oxo-4H-pyran-3-yl)acetamide as side-products.