Purification and properties of d-4-deoxy-5-oxoglucarate hydro-lyase (decarboxylating)

1. An enzyme extracted from Pseudomonas acidovorans was purified and shown to catalyse the simultaneous dehydration and decarboxylation of d-4-deoxy-5-oxoglucarate. It is proposed to name the enzyme d-4-deoxy-5-oxoglucarate hydro-lyase (decarboxylating), trivial name ;deoxyoxoglucarate dehydratase'. 2. No added cofactors were required, and the enzyme was inactivated when incubated with its substrate in the presence of sodium borohydride. Under these conditions the substrate and enzyme appeared to be bound covalently. 3. The action of the enzyme is readily explained if it is assumed that d-4-deoxy-5-oxoglucarate forms a Schiff base with a lysine residue in the enzyme.     

D-glucaric acid and galactaric acid catabolism by Agrobacterium tumefaciens

Cell-free extract (crude extract) of Agrobacterium tumefaciens grown on d-glucuronate or d-glucarate converts d-glucarate and galactarate to a mixture of 2-keto-3-deoxy- and 4-deoxy-5-keto-d-glucarate. These compounds are then converted by partially purified crude extract to an intermediate tentatively identified as 2,5-diketoadipate. The same enzyme preparation further decarboxylates this intermediate to alpha-ketoglutarate semialdehyde, which is subsequently oxidized in a nicotinamide adenine dinucleotide-dependent reaction to alpha-ketoglutaric acid. Since A. tumefaciens converts d-glucuronic acid to d-glucarate, a pathway from d-glucuronate to alpha-ketoglutarate in A. tumefaciens was determined.     

A substrate for the fluorogenic assay of exo-beta-N-acetylmuramidase: synthesis and purification of 4-methylumbelliferyl N-acetylmuramide.

A fluorogenic substrate for exo-β-N-acetylmuramidase from Bacillus subtilis B was synthesized. 4-Methyl-2-oxo-1,2-benzopyran-7-yl 2-acetamido-4,6-O-benzylidene-2-deoxy-β-d-glucopyranoside was prepared from 4-methyl-2-oxo-1,2-benzopyran-7-yl 2-acetamido-2-deoxy-β-d-glucopyranoside, condensed with dl-2-chloropropionic acid, the benzylidene residue removed by acetolysis and the 4-methyl-2-oxo-1,2-benzopyran-7-yl 2-amino-3-O-(d-1-carboxyethyl)-2-deoxy-β-d-glucopyranoside purified by chromatography on silica gel and Sephadex G-10 and by high-voltage paper electrophoresis. The identity of the product was confirmed by pmr studies, acid hydrolysis followed by chromatography of the products, and enzymic digestion.     

Reactions of enol sugar derivatives. 8. Action of -D-glucosidase and -D-galactosidase on D-glucal and D-galactal

D-Glucal and D-galactal were converted into the corresponding 2-deoxy-D-hexoses by β-D-glucosidase and β-D-galactosidase, respectively. The enzymic hydration of D-glucal compared to that of D-galactal occured at a faster rate and also yielded a byproduct of yet unknown structure. In the presence of glycerol as acceptor, D-glucal as well as D-galactal formed glyceryl 2-deoxy-β-D-glycosides. In this case also D-glucal yielded two byproducts which, according to preliminary investigations, seem to be glyceryl pseudoglucal derivatives. The enzymic hydration is irreversible. Glyceryl 2-deoxy-β-D-lyxo-hexopyranoside was hydrolyzed by β-D-galactosidase to give glycerol and 2-deoxy-D-lyxo-hexose. The mechanism of the enzymic hydration and glycosylation of glycals is discussed.     

Studies of polysaccharide fractions from the lipopolysaccharide of Pseudomonas aeruginosa N.C.T.C. 1999.

Two polymeric water-soluble fractions were isolated by gel filtration after mild acid hydrolysis of the lipopolysaccharide from Pseudomonas aeruginosa N.C.T.C. 1999. The fraction of higher molecular weight retained the O-antigenic specificity of the lipopolysaccharide and may be 'side-chain' material. This fraction was rich in N (about 10%) and gave several basic amino compounds on acid hydrolysis; fucosamine (at least 2.8% w/w) was the only specifc component identified. The fraction of lower molecular weight was a phosphorylated polysaccharide apparently corresponding to 'core' material. The major components of this fraction and their approximate molar proportions were: glucose (3-4); rhamnose (1); heptose (2); 3-deoxy-2-octulonic acid (1); galactosamine (1); alanine (1-1.5); phosphorus (6-7). In the intact lipopolysaccharide this fraction was probably linked to lipid A via a second residue of 3-deoxy-2-octulonic acid, and probably also contained additional phosphate residues and ethanolamine. The residues of 3-deoxy-2-octulonic acid were apparently substituted in the C-4 or C-5 position, and the phosphorylated heptose residues in the C-3 position. The rhamnose was mainly 2-substituted, though a little 3-substitution was detected. The glucose residues were either unsubstituted or 6-substituted. Four neutral oligosaccharides were produced by partial acid hydrolysis and were characterized by chemical, enzymic, chromatographic and mass-spectrometric methods of analysis. The structures assigned were: Glcpalpha1-6Glc; Glcpbeta1-2Rha; Rhapalpha1-6Glc; Glcpbeta1-2Rhapalpha1-6Glc. The galactosamine was substituted in the C-3 or C-4 position, the attachment of alanine was indicated, and evidence that the amino sugar linked the glucose-rhamnose region to the 'inner core' was obtained.     

Sur la stéréospécificité de la ribonucléoside-diphosphate-réductase: préparation de désoxyribonucléosides pyrimidiques deutérés

Methyl 2-deoxy-3,5,6-tri-O-p-nitrobenzoyl- -ribo-hexofuranoside was converted into the glycosyl halide which was then condensed with 2,4-bis(trimethylsilyloxy)-pyrimidine in the presence of mercuric chloride to give, after alkaline methanolysis, 1-(2-deoxy-β- -ribo-hexofuranosyl)uracil, in a yield too low for the reaction to be applied to deuterated compounds. Methyl 2-deoxy- -allofuranoside-2-d was degraded into methyl 2-deoxy- -arabinofuranoside-2-d. Its di-p-nitrobenzoate was converted into the glycosyl halide which was coupled with 2,4-bis(trimethylsilyloxy)-pyrimidine to give, after alkaline hydrolysis, deuterated deoxyuridine. Thiationammonolysis of a mixture of the 3′,5′-di-p-nitrobenzoates of the latter compound and its anomer gave the corresponding deoxycytidines. Comparison of the n.m.r. spectra of these compounds to that of deuterated deoxycytidine, prepared by the enzymic reduction of cytidine 5′-pyrophosphate with the Escherichia coli system in the presence of deuterium oxide confirmed that the substitution of a hydroxyl group by a hydrogen atom proceeds with retention of configuration.     

Fluorine as a hydroxy analogue. Stereospecific phosphorylation of 2-deosy-2-fluoroglycerol by glucerol kinase.

Glycerol kinase catalyses the phosphorylation of the symmetrical substrate, 2-dexoy-2-flurooglycerol, by ATP to an asymmetric product, 2-deoxy-2-fluoro-sn-glycerol 3-phosphate. The stereospecificity of the enzymic reaction was extablished by unambiguous chemical synthesis of 2-deoxy-2-fluoro-sn-glycerol labelled with 2H at C-1, followed by glycerol kinase-catalysed phosphorylation and isolation of the labelled phosphate. The configuration of the 2H-labelled phosphate was determined by n.m.r. spectroscopy. This enzymic phosphorylation of 2-dexoy-2-fluoroglycerol is absolutely stereospecific in the same sence as that of glycerol, with fluorine replacing the C-2 hydroxy group. The behaviour of fluorine as a hydroxy analogue in directing the stereospecific course of the enzyme reaction is relevant to the use of the fluorine atom of fluoro analogues of substrate as a reporter group for hydroxy-binding sites of enzymes.     

Inhibition of some hepatic lysosomal glycosidases by ethanolamines and phenyl 6-deoxy-6-(morpholin-4-yl)-beta-D-glucopyranoside.

The hepatic lysosomal glycosidases alpha-glucosidase and beta-glucuronidase were inhibited in vitro and in vivo by mono- and diethanolamines. The in vivo inhibition is dose dependent and occurs at a value less than LD50. Phenyl 6-deoxy-6-(morpholin-4-yl)-beta-D-glucopyranoside inhibited alpha-glucosidase both in vitro and in vivo. The treatment of the enzymes in vitro by ethanolamine exhibited a reversible inhibition of the mixed and competitive types for alpha-glucosidase and beta-glucuronidase, respectively. Diethanolamine showed a reversible inhibition of the competitive type for both enzymes. It is a potent inhibitor for beta-glucuronidase, in vitro, whose inhibition constant (Ki) is 5 x 10(-5) M. Phenyl 6-deoxy-6-(morpholin-4-yl)-beta-D-glucopyranoside is a potent inhibitor only for hepatic alpha-glucosidase with a Ki value of 1.6 x 10(-5) M. The pattern of the pH dependence of enzymic activity was not affected by ethanolamine inhibition. The magnitude of the inhibition of enzymes is dependent on the structure of the inhibitor.     

Catabolism of D-gluaric acid to alpha-ketoglutarate in Bacillus megaterium

Crude cell-free extracts of d-glucarate-grown cells of Bacillus megaterium converted d-glucarate to alpha-keto-beta-deoxy-d-glucarate (KDG). Charcoal-treated cell-free extracts or partially purified enzyme preparations converted KDG to an intermediate which was isolated and identified as 2,5-diketoadipate (DKA). This compound was synthesized, and the cell-free extracts of d-glucarate grown cells were found to catalyze the reduction of nicotinamide adenine dinucleotide (NAD) in its presence. In the absence of NAD, the same enzyme preparation catalyzed the decarboxylation of the DKA to alpha-ketoglutarate semialdehyde (KGS), whereas in the presence of NAD the KGS was subsequently oxidized to alpha-ketoglutarate by alpha-ketoglutarate semialdehyde dehydrogenase. Since galactarate-grown B. megaterium contains a galactarate dehydrase forming KDG, the complete pathway for the metabolism of d-glucarate or galactarate to alpha-ketoglutarate and CO(2) is now known in a gram-positive bacterium.     

Crystallization and some properties of chondroitinase from Arthrobacter aurescens.

A strain of Arthrobacter aurescens which secretes a large amount of chondroitinase into a culture broth, was isolated from soil. The chondroitinase was purified 380-fold over culture broth in 24% yield and crystallized. Some properties of the purified enzyme were studied and described: thermal stability (below 45 degrees), pH stability (pH 4.9 to 7.4), optimum temperature (50 degrees), and optimum pH (pH 6.0). Chrondroitin sulfate A and C, chondroitin, and hyaluronic acid were split by the enzyme but dermatan sulfate could not be. The initial rates of enzymic degradation of chondroitin sulfate C, chondroitin, and hyaluronic acid were 1.1, 1.95, and 3.2, respectively, compared to that of chondroitin sulfate A. When the enzyme was allowed to act on chondroitin sulfate A and C, the reducing power and the ultraviolet absorption at 232 nm increased proportionally to the decrease in viscosity of the substrate solution. Finally these substrates were degraded to the extent of 100% to disaccharides. By the enzyme action the main products from chondroitin sulfate A and C were deta 4,5-unsaturated disaccharides, which were identified as 2-acetamido-2-deoxy-3-O-(Beta-D-gluco-4-enepyranosyluronic acid)-4-O-sulfo-D-galactose and 2-acet-amido-2-deoxy-3-O-(Beta-D-gluco-4-enepyranosyluronic acid)-6-O-sulfo-D-galactose by paper chromatography, ultraviolet absorption spectroscophy, and infrared spectroscopy. Thus it is suggested that the chondroitinase is a chondroitin sulfate A and C lyase, one of the hyaluronate lyases (EC 4.2.99.1).     

Synthesis of 4-cyanophenyl and 4-nitrophenyl 2-azido-2-deoxy-1,5-dithio-beta-D-arabino- and -beta-D-lyxopyranosides possessing antithrombotic activity

Tri-O-acetyl-5-thio-D-ribopyranosyl bromide was converted into 3,4-di-O-benzoyl-1,5-anhydro-5-thio-D-erythro-pent-1-enitol (3,4-di-O-benzoyl-5-thio-D-ribal), the azidonitration of which afforded an unstable mixture of 2-azido-3,4-di-O-benzoyl-2-deoxy-1-O-nitro-5-thio-D-pentopyranoside++ + isomers. This was converted without separation into the corresponding 1-O-acetyl derivatives from which an alpha,beta anomeric mixture of the 1-O-acetyl-2-azido-3,4-di-O-benzoyl-2-deoxy-5-thio-D-arabinopyranose+ ++ isomers could be isolated in high yield. Glycosidation of this mixture with 4-cyano- or 4-nitrobenzenethiol, using trimethylsilyl triflate or boron trifluoride etherate, respectively, as promoters gave the corresponding D anomers exclusively. Zemplén debenzoylation afforded 4-cyanophenyl as well as 4-nitrophenyl 2-azido-2-deoxy-1,5-dithio-beta-D-arabinopyranoside, respectively. When 1-O-acetyl-2-azido-3,4-di-O-benzoyl-2-deoxy-5-thio-D-lyxopyranose was used as glycosyl donor only the corresponding 1 anomers, i.e., 4-cyanophenyl as well as 4-nitrophenyl 2-azido-2-deoxy-1,5-dithio-beta-D-lyxopyranosides, could be isolated after Zemplén debenzoylation in high yield. All four 1,5-dithioglycosides possess significant oral antithrombotic activity.     

Access to tetrachlorophthalimide-protected ethyl 2-amino-2-deoxy-1-thio-beta-D-glucopyranosides.

Ethyl 2-deoxy-2-tetrachlorophthalimido-1-thio-beta-D-glucopyranoside (7) was prepared from glucosamine hydrochloride in four steps with a 20-25% overall yield. Formation of 1,3,4,6-tetra-O-acetyl-2-deoxy-2-tetrachlorophthalimido-beta-D- glucopyranoside (5) was found to be crucial for this reaction sequence since the corresponding alpha-1-acetate did not react in Lewis-acid-catalyzed ethylthio glycosidations. Formation of the beta-1-acetate (5) was achieved by treatment of 3,4,6-tri-O-acetyl-2-deoxy-2-tetrachlorophthalimido-alpha-D-glucop yranosyl bromide (4) with acetic acid under silver zeolite promotion. This was necessary because conditions normally used for beta-1-acetate formation were not tolerated by the tetrachlorophthalimido (TCP) group.     

Fluorinated carbohydrates as potential plasma membrane modifiers. Synthesis of 4- and 6-fluoro derivatives of 2-acetamido-2-deoxy-D-hexopyranoses

2-Amino-2,4-dideoxy-4-fluoro- and 2-amino-2,4,6-trideoxy-4, 6-difluoro-D-galactose, and 2-amino-2,4-dideoxy-4-fluoro- and 2-amino-4-deoxy-4, 4-difluoro-D-xylo-hexose were synthesized, as potential modifiers of tumor cell-surface glyco-conjugate, from benzyl 2-acetamido-3-O-benzyl-2-deoxy-4, 6-di-O-mesyl-alpha-D-glucopyranoside and benzyl 2-acetamido-3, 6-di-O-benzyl-2-deoxy-4-O-mesyl-alpha-D-glucopyranoside, which were converted into the corresponding 4,6-difluoro-2,4, 6-trideoxy and 2,4-dideoxy-4-fluoro derivatives. Benzyl 2-acetamido-2-deoxy-4-O-mesyl-alpha-D-galactopyranoside and benzyl 2-acetamido-3,6-di-O-benzyl-2-deoxy-alpha-D-xylo-hexo-4-ulopyra noside were treated with diethylaminosulfur trifluoride to give 2-amino-2,4-dideoxy-4-fluoro-D-glucose and 2-amino-2,4-dideoxy-4, 4-di-fluoro-D-xylo-hexose derivatives, respectively, to give after deprotection the target compounds. Several of the peracetylated sugar derivatives inhibited L1210 tumor-cell growth in vitro at concentrations of 1-5 10(-5) M. The peracetylated derivative of 2-amino-2,4-dideoxy-4-fluoro-D-galactose inhibited protein and glycoconjugate biosynthesis, and also exhibited antitumor activity in mice with L1210 leukemia.     

Synthesis and structure determination of some sugar amino acids related to alanine and 6-deoxymannojirimycin.

(5'R)-5'-Methyl-5'-[methyl (4S)-2,3-O-isopropylidene-beta-L-erythrofuranosid-4-C-yl]-imidazolidin-2',4'-dione was synthesised starting from methyl 6-deoxy-2,3-O-isopropylidene-alpha-D-lyxo-hexofuranosid-5-ulose applying the Bucherer-Bergs reaction. Its 5'-R configuration was confirmed by X-ray crystallography. Corresponding alpha-amino acid-methyl (5R)-5-amino-5-C-carboxy-5,6-dideoxy-alpha-D-lyxo-hexofuranoside (alternative name: 2-[methyl (4S)-2,3-O-isopropylidene-beta-L-erythrofuranosid-4-C-yl]-D-alanine) was obtained from the above hydantoin by acid hydrolysis of the isopropylidene group followed by basic hydrolysis of the hydantoin ring. Total deprotection afforded 5-C-carboxy-6-deoxymannojirimycin. Analogously, methyl (5S)-5-amino-5-C-carboxy-5,6-dideoxy-alpha-L-lyxo-hexofuranoside and 5-C-carboxy-6-deoxy-L-mannojirimycin were prepared from the corresponding (5'S)-5'-methyl-5'-[methyl (4R)-2,3-O-isopropylidene-beta-D-erythrofuranosid-4-C-yl]-imidazolidin-2',4'-dione starting from methyl 6-deoxy-2,3-O-isopropylidene-alpha-L-lyxo-hexofuranosid-5-ulose.     

Concentrated hydriodic acid in simultaneous deprotections of multifunctional inositols

l-1-Deoxy-1-fluoro-6-O-methyl-myo-inositol was epimerized by chloral/DCC in boiling 1,2-dichloroethane yielding D-1-O-cyclohexylcarbamoyl-2-deoxy-2-fluoro-3-O-methyl-5,6-O-[(R/S)-2,2,2-trichloroethylidene]-chiro-inositol. The latter and l-4-O-benzyl-3-O-cyclohexylcarbamoyl-5-O-methyl-1,2-O-(2,2,2-trichloroethylidene)-muco-inositol, l-4-O-benzyl-3-O-cyclohexylcarbamoyl-1,2-O-ethylidene-5-O-methyl-muco-inositol, d-1-O-cyclohexylcarbamoyl-2-deoxy-5,6-O-ethylidene-2-fluoro-3-O-methyl-chiro-inositol, as well as D-5-O-benzyl-4-O-cyclohexylcarbamoyl-3-deoxy-3-(N,N'-dicyclohexylureido)-6-O-methyl-1,2-O-(2,2,2-trichloroethylidene)-chiro-inositol were deprotected with boiling 57% aq hydrogen iodide. Ether, urethane and ethylidene acetal functions were simultaneously cleaved by the reagent, whereas the trichloroethylidene groups were still intact or were only removed in small quantities. Especially, the urea function of D-5-O-benzyl-4-O-cyclohexylcarbamoyl-3-deoxy-3-(N,N'-dicyclohexylureido)-6-O-methyl-1,2-O-(2,2,2-trichloroethylidene)-chiro-inositol was decomposed to a cyclohexylamino group. The hydrodechlorination of D-1-O-cyclohexylcarbamoyl-2-deoxy-2-fluoro-3-O-methyl-5,6-O-[(R/S)-2,2,2-trichloroethylidene]-chiro-inositol using Raney-Nickel yielded a mixture of the corresponding 5,6-O-ethylidene- and 5,6-O-chloroethylidene derivatives. The three synthetic steps-hydrodehalogenation, HI-deprotection and peracylation- were combined without purification of the intermediates.     

The use of deoxyfluoro-d-galactopyranoses in a study of yeast galactokinase specificity

1. 2-Deoxy-2-fluoro-d-galactose, 3-deoxy-3-fluoro-d-galactose, 4-deoxy-4-fluoro-d-galactose, 6-deoxy-6-fluoro-d-galactose and 2-deoxy-d-lyxo-hexose are substrates for yeast galactokinase. 2. The variation in K(m) values for the d-hexose derivatives was not associated with a variation in the value of K(m) for MgATP(2-) indicating that the binding of MgATP(2-) is not modified by the binding of the sugar substrate. 3. Donated H bonds from OH-3, OH-4 and OH-6 and an accepted H bond to OH-2 of the d-hexose are important for the binding of the sugar substrate to galactokinase. 4. Yeast galactokinase exhibits similar kinetics to the galactokinase from Escherichia coli and operates by a similar random sequential mechanism. 5. 4-Deoxy-4-fluoro-d-glucose was neither a substrate for nor an inhibitor of yeast galactokinase.     

Mechanism of enzymic isomerization and epimerization of D-erythrose 4-phosphate.

The mechanism of the enzymic isomerization and epimerization of D-erythrose 4-phosphate (Ery4P) by an enzyme preparation from bovine liver was investigated with the use of 2H2O. The incorporation of 2H was quantitatively determined by a procedure using gas chromatography-mass spectrometry. About one atom of 2H was incorporated per molecule of the enzymic epimerization reaction product of Ery4P (D-threose 4-phosphate) or that of D-ribulose 5-phosphate. Computer simulation of the Ery4P isomerization reaction indicated that the 2H of 2H2O was not directly incorporated into the enzymic reaction product (D-erythrulose 4-phosphate). Instead, intramolecular transfer of hydrogen atoms had occurred.     

SYNTHESIS AND IN VITRO ANTI-HCV ACTIVITY OF β-D- and L-2′-DEOXY-2′-FLUORORIBONUCLEOSIDES

Based on the discovery of β-D-2′-deoxy-2′-fluorocytidine as a potent anti-hepatitis C virus (HCV) agent, a series of β-D- and l-2′-deoxy-2′-fluororibonucleosides with modifications at 5 and/or 4 positions were synthesized and evaluated for their in vitro activity against HCV and bovine viral diarrhea virus (BVDV). The introduction of the 2′-fluoro group was achieved by either fluorination of 2,2′-anhydronucleosides with hydrogen fluoride-pyridine or potassium fluoride, or a fluorination of arabinonucleosides with DAST. Among the 27 analogues synthesized, only the 5-fluoro compounds, namely β-D-2′-deoxy-2′,5-difluorocytidine (5), had anti-HCV activity in the subgenomic HCV replicon cell line, and inhibitory activity against ribosomal RNA. As β-D-N4-hydroxycytidine (NHC) had previously shown potent anti-HCV activity, the two functionalities of the N4-hydroxyl and the 2′-fluoro were combined into one molecule, yielding β-D-2′-deoxy-2′-fluoro-N4-hydroxycytidine (12). However, this nucleoside showed neither anti-HCV activity nor toxicity. All the l-forms of the analogues were devoid of anti-HCV activity. None of the compounds showed anti-BVDV activity, suggesting that the BVDV system cannot reliably predict anti-HCV activity in vitro.     

Inspection of active sites of human salivary alpha-amylase isozymes by means of non-reducing-end substituted maltooligosaccharides with 2-pyridylamino residue

The modes of action of four alpha-amylase isozymes, which were purified from human saliva, on p-nitrophenyl alpha-maltopentaoside (G5P), maltohexaitol (G6R), and their 2-pyridylamino derivatives, p-nitrophenyl O-6-deoxy-6-[(2-pyridyl)amino]-alpha-D-glucopyranosyl-(1----4)-O-alpha- D-glucopyranosyl-(1----4)-O-alpha-D-glucopyranosyl-(1----4)-O-alpha-D- glucopyranosyl-(1----4)-alpha-D-glucopyranoside (FG5P) and O-6-deoxy-6-[(2-pyridyl)amino]-alpha-D-glucopyranosyl-(1----4)- O-alpha-D-glucopyranosyl-(1----4)-O-alpha-D-glucopyranosyl-(1----4)-O- alpha-D-glucopyranosyl-(1----4)-O-alpha-D-glucopyranosyl-(1----4)-D- glucitol (FG6R) were examined at various pH values. No differences in their modes of action on the substrates was found. Irrespective of which enzyme was used, the molar ratio of the hydrolysis products of G5P or G6R was almost constant at any pH examined. On the other hand, those of FG5P and FG6R varied with pH such that predominantly O-6-deoxy-6-[(2-pyridyl)amino]-alpha-D-glucopyranosyl- (1----4)-O-alpha-D-glucopyranosyl-(1----4)-D-glucose (FG3) was formed at high pH ranges, while the formation of O-6-deoxy-6-[(2-pyridyl)amino]-alpha-D-glucopyranosyl-(1----4)- O-alpha-D-glucopyranosyl-(1----4)-O-alpha-D-glucopyranosyl-(1----4)-D-gl ucose (FG4) increased at lower pH. The result indicates that the binding mode of FG5P or FG6R to the active sites of the enzymes changed with pH; namely, interactions between the 2-pyridylamino residue of the substrates and some amino acid residue(s) located in the active sites were influenced by pH.(ABSTRACT TRUNCATED AT 250 WORDS)     

Synthesis and anti-cancer activity of some novel 5-azacytosine nucleosides

1-O-Acetyl-2-deoxy-3,5-di-O-toluoyl-4-thio-D-erythro-pentofuranose and 2-deoxy-1,3,5-tri-O-acetyl-4-thio-L-threo-pentofuranose were coupled with 5-azacytosine to obtain alpha and beta anomers of nucleosides. All four nucleosides were reduced to the corresponding dihydro derivatives and deblocked to give target compounds. All eight target compounds were evaluated in a series of human cancer cell lines in culture. Only 2'-deoxy-4'-thio-5-azacytidine (3beta) was found to be cytotoxic in all the cell lines and was further evaluated in vivo. Details of the synthesis and biological activity are reported.