Ramesh H. Shah

Glycosidases. Ligands for affinity chromatography: I. Syntheses of 1,2-transp-aminophenyl 1-thio-D-glycopyranosides

Abstract 1,2-transp-Aminophenyl 1-thioglycosides derived from β- D -galactose, β- D -fucose, and α- D -mannose were synthesized as potential ligands for the purification of β- D -galactosidase and α- D -mannosidase by affinity chromatography. The appropriate acetylglycosyl bromides were condensed with p-nitrothiophenol in the presence of potassium hydroxide. Deacylation of the 1,2-transp-nitrophenyl O-acetyl-1-thio- D -glycopyranosides thus obtained, followed by reduction with hydrogen over palladium on barium sulfate, afforded the desired p-aminophenyl 1-thioglycopyranosides.

Glycosidases. Ligands for affinity chromatography: : III. Syntheses of p-aminophenyl 2-acetamido-2-deoxy-1-thio-β-D-glucopyranoside and -galactopyranoside☆

Abstract p -Aminophenyl 2-acetamido-2-deoxy-1-thio-β- D -glucopyranoside and -galactopyranoside were synthesized for use as ligands in the purification of 2-acetamido-2-deoxy-β- D -glucosidase, from Aspergillus niger and Phaseolus vulgaris , by affinity chromatography. The condensation of 2-acetamido-1,3,4,6-tetra- O -acetyl-2-deoxy-α- D -glucose and -β- D -galactose with p -nitrothiophenol in the presence of zinc chloride afforded p -nitrophenyl 2-acetamido-3,4,6-tri- O -acetyl-2-deoxy-1-thio-β- D -glucopyranoside and -galactopyranoside, respectively. The former was also obtained by the reaction of 2-acetamido-3,4,6-tri- O -acetyl-2-deoxy-α- D -glucopyranosyl chloride with p -nitrothiophenol in the presence of potassium hydroxide. O -Deacetylation followed by reduction with hydrogen over palladium on barium sulfate gave the p -aminophenyl 2-acetamido-2-deoxy-1-thio-β- D -glucopyranoside and -galactopyranoside. Inhibition constants ( K i ) of the p -nitrophenyl and p -aminophenyl 2-acetamido-2-deoxy-1-thio-β- D -glucosides and -galactosides for A. niger 2-acetamido-2-deoxy-β- D -glucosidase were determined by using p -nitrophenyl 2-acetamido-2-deoxy-β- D glucopyranoside and -galactopyranoside as substrates.

Reaction of tetra-O-acetyl-α-d-hexopyranosyl bromides with sodium p-nitrophenoxide in N,N-dimethylformamide. Formation of p-nitrophenyl 2,3,4,6-tetra-O-acetyl-β-d-hexopyranosides versus 2,3,4,6-tetra-O-acetyl-1,5-anhydro-d-hex-1-enitols

Abstract The reaction of the four acetylated hexopyranosyl bromides ( d -allo, d -gluco, d -gulo, and d -galacto) with sodium p-nitrophenoxide in N,N-dimethylformamide was investigated in order to determine their tendency for displacement at C-1 (to form the p-nitrophenyl 2,3,4,6-tetra-O-acetyl-β- d -hexopyranoside) or β-elimination (to give the 2,3,4,6-tetra-O-acetyl-1,5-anhydro- d -hex-l-enitol). The tendency for the formation of tetra-O-acetyl-1,5-anhydro- d -hex-l-enitols decreased in the following order: d -allo > d -gulo ≳ d -gluco > d -galacto, whereas the preference for the formation of p-nitrophenyl tetra-O-acetyl-β- d -hexopyranosides was in the reverse order ( d -allo d -gulo ≲ d -gluco d -galacto). The conformations of the four tetra-O-acetyl-1,5-anhydro- d -hex-l-enitols ( d -ribo, d -arabino, d -xylo, and d -lyxo) were determined from their 250-MHz >1H-n.m.r. data; the d -lyxo derivative was determined to exist in the H54( d ) conformation, whereas the H45( d ) conformation was demonstrated for the other three isomers. The yields of the four tetra-O-acetyl-1,5-anhydro- d -hex-l-enitols were found to be directly related to factors governing the stability of their conformations.

Preparation of methyl-p14Cd-glucuronate and nuclear magnetic resonance studies of its mono- and di-o-benzylidene intermediates

Abstract A procedure suitable for the synthesis of methyl-labeled methyl d -glucuronate from d -glucuronic acid was devised. The intermediates prepared during the synthesis, 1,2:3,5-di- O -benzylidene-α- d -glucofuranuronic acid and its methyl ester, were determined to exist in an “O-inside” conformation by examination of their n.m.r. spectra. Evidence obtained by n.m.r. and g.l.c. indicated that when a reaction of d -glucuronic acid with benzaldehyde in the presence of anhydrous zinc chloride proceeded for 48 h, the major product was ( 1,2-exo-phenyl )-1,2- O -benzylidene-α- d -glucofuranurono-6,3-lactone, along with a small amount of the (1,2- endo -phenyl)-isomer.

Synthesis of 4-thio-dD-mannose

Abstract 1,6-Anhydro-4- S -benzoyl-4-thio-β- D -mannopyranose, obtained by treatment of 1,6:3,4-dianhydro-β- D -talopyranose with pyridinium thiolbenzoate in N , N -di-methylformamide, was converted into its 2,3-di- O -acetyl derivative, which was acetolyzed to give 1,2,3,6-tetra- O -acetyl-4- S -benzoyl-4-thio- D -mannopyranose. Deacylation of the last-named compound with sodium methoxide in methanol gave syrupy 4-thio- D -mannose, which was characterized as 1,2,3,5,6-penta- O -acetyl-4-thio-α- and -β- D -mannofuranose.

Studies on Turbatrix aceti β-N-acetylglucosaminidase: 1. Purification and physicochemical characterization

N-Acetyl-beta-D-glucosaminidase was purified, from the culture medium of the nematode Turbatrix aceti, to homogeneity, as judged by electrophoresis in polyacrylamide gel and ultracentrifugation. The purification scheme involved the following steps: (i) concentration of the culture medium by ultra-filtration by an Amicon PM-30 membrane; (ii) ammonium sulfate precipitation; (iii) DEAE-Sephadex and (iv) Sephadex G-200 chromatography; and (v) affinity chromatography on succinyldiaminopropyl amino-Sepharose bearing the ligand p-aminophenyl 2-acetamido-2-deoxy-1-thio-beta-D-glucopyranoside. The molecular weight of the enzyme was 112,000 +/- 4800 and 124,000 as determined by polyacrylamide gel electrophoresis and by gel filtration through Sephacryl S-200, respectively. The enzyme showed a pH optimum of 4.8 for N-acetylglucosaminidase and 5.4 for N-acetylgalactosaminidase. The detailed substrate specificity studies were carried out on both synthetic and natural oligosaccharides and glycopeptides. The chitin oligosaccharides and asialo-agalacto complex type as well as high mannose-type glycoproteins such as fetuin and ovalbumin, respectively, were good substrates for the enzyme. Substrate analogs in which the oxygen atom of the acetamido group was replaced by sulfur atom proved to be poor substrates.

A convenient synthesis of p-nitrophenyl 2-deoxy-2-(thio-acetamido)-β-d-glucopyranoside, -galactopyranoside, and their 1-thio analogs as inhibitors of 2-acetamido-2-deoxy-β-d-glucosidase

Abstract The acetamido group of p -nitrophenyl 2-acetamido-2-deoxy-β- d -glucopyranoside, -β- d -galactopyranoside, and their 1-thio analogs was modified by replacement of the amide-carbonyl oxygen atom with sulfur by treatment of their fully acetylated derivatives with phosphorus pentasulfide in pyridine. The resulting p -nitrophenyl 2-deoxy-2-thioacetamido-β- d -hexopyranoside triacetates were O -deacetylated with catalytic amounts of sodium methoxide in methanol to obtain p -nitrophenyl 2-deoxy-2-thioacetamido-β- d -glucopyranoside, -β- d -galactopyranoside, and their 1-thio analogs. These derivatives inhibited 2-acetamido-2-deoxy-β- d -glucosidase from Turbatrix aceti to various extents. Also obtained in significant yields in the aforementioned reaction with phosphorus pentasulfide in pyridine were the two hitherto unreported thiazolines, namely, 2-methyl(2-acetamido-3,4,6-tri- O -acetyl-α- d -glucopyrano)[2′,1′:4,5]-2-thiazoline and 2-methyl(2-acetamido-3,4,6-tri- O -acetyl-α- d -galactopyrano)[2′,1′:4,5]-2-thiazoline.

Studies on Turbatrix aceti β-N-acetylglucosaminidase: 2. Kinetic studies on the active site

The purified beta-N-acetylglucosaminidase isolated from Turbatrix aceti hydrolyzes both p-nitrophenyl 2-acetamido-2-deoxy-beta-D-gluco- and beta-D-galactopyranosides. The enzyme had Km values of 0.28 and 0.23 mM, Vmax values of 104 and 69 mumol min-1 mg protein-1, and activation energies of 11.7 and 9.9 kcal/mol for the two substrates, respectively. Several lines of experimental evidence show that both beta-N-acetylglucosaminidase and beta-N-acetylgalactosaminidase activities reside in the same molecule at a single catalytic site. Substrate analogs were synthesized in which the acetamido group of p-nitrophenyl 2-acetamido-2-deoxy-beta-D-gluco- and galactopyranoside, and their 1-thio analogs was modified by replacement of the amido-carbonyl oxygen with sulfur. These substrate analogs competitively inhibited both enzymatic activities. Analysis of the inhibition data indicates that a single catalytic site of the enzyme is responsible for both beta-N-acetylglucosaminidase and beta-N-acetylgalactosaminidase activities. Competition kinetics between the two substrates further confirm the presence of a single active site for both activities. The pH dependence of the hydrolysis of p-nitrophenyl 2-acetamido-2-deoxy-beta-D-gluco- and beta-D-galactopyranosides has been determined. pKe1 and pKe2 values of 4.7 and 5.2, determined from the dependence of log Vmax/Km on pH, suggest that two carboxyl groups are involved in the reaction mechanism. The heats of ionization of the groups further confirm the above results.

Syntheses of p-aminophenyl α-d-talopyranoside and 1-thio α-d-talopyranoside as analogs of glycosidase substrates☆

Abstract p -Nitrophenyl and p -aminophenyl α- d -talopyranoside and 1-thio-α- d -talopyranosides were prepared for studies on specificity of glycosidases. Reaction of α- d -talopyranose pentaacetate with p -nitrophenol gave exclusively p -nitrophenyl 2,3,4,6-tetra- O -acetyl-α- d -talopyranoside ( 2 ) in 63% yield. A similar reaction with p -nitrobenzenethiol afforded the 1-thio analog ( 3 ) of 2 in 41.8% yield; the p -nitrophenyl 2,3,4,6-tetra- O -acetyl-1-thio-β- d -talopyranoside ( 6 ) was also obtained in low yield (6.7%). The two α- d -talosides 2 and 3 were catalytically deacetylated in near-quantitative yields by methanolic sodium methoxide. The p -nitrophenyl α- d -talopyranoside ( 4 ) and 1-thio-α- d -talopyranoside ( 5 ) were reduced with palladium on barium sulfate catalyst to the corresponding p -aminophenyl talosides. The acetylated p -nitrophenyl d -talosides 2 , 3 , and 6 were determined, from their 250-MHz n.m.r. spectra, to exist in the 4 C 1 ( d ) conformation in chloroform solution.

Syntheses of p-aminophenyl 1-thio-α- and -β-d-idopyranosides as analogs of glycosidase substrates

Abstract The p -nitrophenyl and p -aminophenyl 1-thio-α- and -β- d -idopyranosides were synthesized for use in structure-activity studies of glycosidases. Zinc chloride-catalyzed fusion of α- d -idopyranose pentaacetate with p -nitrobenzenethiol gave p -nitrophenyl 2,3,4,6-tetra- O -acetyl-1-thio-α- d -idopyranoside as an amorphous glass in 67% yield, and the crystalline β anomer in 13% yield. Deacetylation with catalytic amounts of sodium methoxide in methanol, followed by hydrogenation under pressure over palladium-on-barium sulfate catalyst, afforded p -aminophenyl 1-thio-α- and -β- d -idopyranosides.