Biomedical subjects
Publications and source records attributed to K L Matta.
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Benzyl 2-acetamido-2-deoxy-3-O-methyl-alpha-D-glucopyranoside (3) was obtained by deacetalation of its 4,6-O-benzylidene derivative (2). Compound 2 was prepared by methylation of benzyl 2-acetamido-4,6-O-benzylidene-2-deoxy-alpha-D-glucopyranoside with methyl iodide-silver oxide in N,N-dimethylformamide. Diol 3 was selectively benzoylated and p-toluenesulfonylated, to give the 6-benzoic and 6-p-toluenesulfonic esters (4 and 5, respectively). Displacement of the sulfonyl group of 5 with sodium benzoxide in benzyl alcohol afforded the 6-O-benzyl derivative (6). Glycosylation of 4 with 2,3,4,6-tetra-O-acetyl-alpha-D-galactopyranosyl bromide (7) in dichloromethane, in the presence of 1,1,3,3-tetramethylurea, furnished the disaccharide derivative 8. Similar glycosylation of compound 6 with bromide 7 gave the disaccharide derivative 10. O-Deacetylation of 8 and 10 afforded disaccharides 9 and 11. The structure of compound 9 was established by 13C-n.m.r. spectroscopy. Hydrogenolysis of the benzyl groups of 11 furnished the disaccharide 2-acetamido-2-deoxy-4-O-beta-D-galactopyranosyl-3-O-methyl-D-glucopyr ano se (N-acetyl-3-O-methyllactosamine).
Methyl 2,4,6-tri-O-benzyl-beta-D-galactopyranoside (5) was obtained crystalline by way of its 3-O-allyl derivative, which was in turn obtained by ring-opening of a presumed 3,4-O-stannylene derivative of methyl beta-D-galactopyranoside, followed by benzylation. Condensation of 5 with 2-methyl-(2-acetamido-3,4,6-tri-O-acetyl-1,2-dideoxy-beta-D-glucopyra no)-[2,1-d]-2-oxazoline in 1,2-dichloroethane in the presence of p-toluenesulfonic acid afforded the disaccharide derivative methyl 3-O-(2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-beta-D-glucopyranosyl)-2, 4,6-tri-O-benzyl-beta-D-galactopyranoside (6) Deacetylation of 6 in methanolic sodium methoxide afforded the disaccharide derivative 7, which was acetalated with alpha, alpha-dimethoxytoluene to afford the 4',6'-O-benzylidene acetal (10). Catalytic hydrogenolysis of the benzyl groups of 7 afforded the title disaccharide 8. Glycosylation of 10 with 2,3,4,6-tetra-O-acetyl-alpha-D-galactopyranosyl bromide in 1:1 benzene-nitromethane in the presence of mercuric cyanide gave the fully protected trisaccharide derivative 12. Systematic removal of the protecting groups of 12 then furnished the title trisaccharide 14. The structures of 5, 8, and 14 were all confirmed by 13C-n.m.r. spectroscopy. The 13C-n.m.r. chemical shifts for methyl alpha- and beta-D-galactopyranoside, and also those of their 3-O-allyl derivatives, are recorded, for the sake of comparison, in conjunction with those of compound 5.
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A practical approach for the synthesis of oligosaccharides containing the O-alpha-L-fucopyranosyl-(1----2)-D-galactopyranosyl unit has been developed by utilizing the readily accessible 3,4,6-tri-O-acetyl-2-O-(2,3,4-tri-O-acetyl-alpha-L- fucopyranosyl)-alpha-D-galactopyranosyl bromide (1). Thus, condensation of bromide 1 with 8-(methoxycarbonyl)octanol in benzene, in the presence of mercuric cyanide, afforded, after column-chromatographic separation, the anomers (3 and 4) of 8-(methoxycarbonyl)octyl 3,4,6-tri-O-acetyl-2-O-(2,3,4-tri-O-acetyl-alpha-L-fucopyranosyl)-D- galactopyranoside in the ratio of 13:4. On the basis of their specific rotations, compounds 3 and 4 were tentatively assigned the alpha and beta configuration, respectively. Similarly, bromide 1 was allowed to react with some other, appropriately protected sugars having a free alcohol group, to afford the corresponding alpha and beta anomers of a galactopyranosyl residue having an alpha-L-fucopyranosyl substituent at O-2. Column-chromatographic separation of the anomeric mixtures, followed by systematic removal of the protecting groups, then provided the final oligosaccharides desired. In this manner, the synthesis of the following oligosaccharides has been accomplished: alpha-L-Fuc-(1----2)-alpha-D-Gal-1----O(CH2)8CO2Me (5), alpha-L-Fuc-(1----2)-beta-D-Gal-1----O(CH2)8CO2Me (6), alpha-L-Fuc-(1----2)-alpha-D-Gal-(1----3)-beta-D-GlcNAc-1----OC6H4NO2 -p (11), alpha-L-Fuc-(1----2)-beta-D-Gal-(1----3)-beta-D-GlcNAc-1----OC6H4NO2+ ++-p (13), alpha-L-Fuc-(1----2)-alpha-D-Gal-(1----3)-alpha-D-GalNAc-1----OC6H4NO 2-o, and alpha-L-Fuc-(1----2)-beta-D-Gal-(1----3)-alpha-D-GalNAc-1----OC6H4NO2 -o (18).(ABSTRACT TRUNCATED AT 250 WORDS)
Condensation of benzyl 2-acetamido-4,6-O-benzylidene-2-deoxy-alpha-D- galactopyranoside with 2,3,4-tri-O-acetyl-alpha-D-fucopyranosyl bromide in 1:1 nitromethane-benzene, in the presence of powdered mercuric cyanide, afforded benzyl 2-acetamido-4,6-O-benzylidene-2-deoxy-3-O-(2,3,4-tri-O-acetyl-beta-D- fucopyranosyl)-alpha-D-galactopyranoside (3). Cleavage of the benzylidene group of 3 with hot, 60% aqueous acetic acid afforded diol 4, which, on deacetylation, furnished the disaccharide 5. Condensation of diol 4 with 2-methyl-(3,4,6-tri-O-acetyl-1,2-di-deoxy-alpha-D-glucopyrano)-[2, 1-d]-2- oxazoline in 1,2-dichloroethane afforded the trisaccharide derivative (7). Deacetylation of 7 with Amberlyst A-26 (OH-) anion-exchange resin in methanol gave the title trisaccharide (8). The structures of 5 and 8 were confirmed by 13C-n.m.r. spectroscopy.
Acetalation of o-nitrophenyl 2-acetamido-2-deoxy-alpha-D-galactopyranoside with p-methoxybenzaldehyde-zinc chloride complex afforded the 4,6-O-(p-methoxybenzylidene) derivative (1). Acetylation of 1 with pyridine-acetic anhydride gave the crystalline monoacetate (2), the acetal group of which was cleaved by brief treatment with hot, 80% aqueous acetic acid to furnish the diol (3). The 1H-n.m.r. spectra of both 2 and 3 supported their overall structures. Glycosylation of 3 with 2,3,4,6-tetra-O-acetyl-alpha-D-galactopyranosyl bromide (5) in 1:1 nitromethane-benzene, in the presence of mercuric cyanide, afforded the disaccharide derivative (6). O-Deacetylation of 6 in methanolic sodium methoxide gave the title disaccharide (8). A similar glycosylation of benzyl 2-acetamido-3-O-acetyl-2-deoxy-alpha-D-galactopyranoside with bromide 5, followed by O-deacetylation of the resulting intermediate, produced disaccharide 9. The structures of 8 and 9 were confirmed by 13C-n.m.r. spectroscopy. Hydrogenolysis of the benzyl aglycon of 9 gave the free disaccharide 2-acetamido-2-deoxy-6-O-beta-D-galactopyranosyl-D-galactopyranose (13). The 13C-n.m.r. spectrum of 13 is recorded, and discussed in conjunction with those of the beta-(1----3)- and the beta-(1----4)-linked isomers.
The spectral properties of 4-methylumbelliferyl-glycosides (MeUmb-glycosides) were investigated in order to assess their usefulness as probes of the microenvironment of sugar binding sites on lectin molecules. It was shown that the abnormally high values for fluorescence polarization of free MeUmb-glycosides (from 0.07 to 0.251) were due neither to their molecular size nor to the blockade of their movement, but to the short lifetimes (less than 0.55 ns) of the excited state of these compounds. Working essentially with two MeUmb-monosaccharides and one MeUmb-disaccharide (MeUmb-alpha-D-galactopyranoside, MeUmb-beta-D-galactopyranoside, and MeUmb-2-acetamido-2-deoxy-3-O-(beta-D-galactopyranosyl)-beta-D- galactopyranoside) which were solubilized in various solvents, it was demonstrated that solvent polarity and viscosity definitely affected the fluorescence intensity of MeUmb-glycosides. A low-polarity medium reduced this intensity, and high viscosity enhanced it. The implications of these findings are discussed in relation to the variations in the fluorescence intensity of MeUmb-glycosides when these compounds were bound to lectins.
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A beta-D-galactosidase from bovine liver was purified to apparent homogeneity. The major purification step was affinity chromatography on a column of D-galactose attached to a Sepharose support activated with divinyl sulfone. Affinity media prepared by binding ligands to Sepharose activated with cyanogen bromide were unsuitable for purification of the enzyme, even though such media have been used to purify beta-D-galactosidases from other sources. The molecular weight of the denatured enzyme was 67,000. The molecular weight of the native enzyme at pH 7.0 was 68,000, and at pH 4.5 or 5.0, was 141,000. These data suggest that the enzyme has a single, fundamental subunit with a molecular weight of 67,000, and that the enzyme exists as a monomer at pH 7.0, and a dimer at pH 4.5 or 5.0. The Vmax values of the enzyme with p-nitrophenyl beta-D-galactoside, p-nitrophenyl beta-D-fucoside, lactose, and beta-Gal-(1----4)-beta-GlcNAc-1---- OC6H4NO2 -p were 10,204, 11,550, 9,479, and 8,859 nmol/min/mg of protein, respectively, and the Km values for these substrates were 0.08, 14.9, 14.2, and 1.6mM, respectively. D-Galactose, beta-D- galactosylamine , p-aminophenyl 1-thio-beta-D-galactoside, and D- galactono -1,4-lactone were competitive inhibitors of the enzyme, with Ki values of 0.9, 0.6, 0.6, and 0.8mM, respectively. The enzyme catalyzed the transfer of the D-galactosyl group from p-nitrophenyl beta-D-galactoside to D-glucose. The pH optimum of the enzyme was 4.5, and the pI was 4.7.
Glycosylation of benzyl 2-acetamido-4,6-O-benzylidene-2-deoxy-alpha-D- galactopyranoside with 2,3,4,6-tetra-O-acetyl-alpha-D-galactopyranosyl bromide, catalyzed by mercuric cyanide, afforded benzyl-2-acetamido-4,6-O-benzylidene-2-deoxy-3- O-(2,3,4,6-tetra-O-acetyl-beta-D-galactopyranosyl)-alpha-D- galactopyranoside (3). O-Deacetylation of 3 gave benzyl 2-acetamido-4,6-O-benzylidene-2-deoxy-3-O-beta- D-galactopyranosyl-alpha-D-galactopyranoside which, on acetalation with benzaldehyde-zinc chloride complex followed by acetylation of the resulting dibenzylidene acetal, gave the disaccharide diacetate (7). Cleavage of the acetal groups of 3 and 7 in hot, 80% aqueous acetic acid furnished, respectively, the disaccharide tetraacetate (4) and diacetate (8). O-Deacetylation of 8 in methanolic sodium methoxide gave the disaccharide (9). Condensation of 4 or 8 with 2-methyl-(3,4,6-tri-O-acetyl-1,2- dideoxy-alpha-D-glucopyrano)-[2,1-d]-2-oxazoline, followed by O-deacetylation, afforded the title tri- and tetra-saccharides, 12 and 14, respectively. The structures of compounds 9, 12, and 14 were established by 13C-n.m.r. spectroscopy.
Condensation of methyl 2-O-benzoyl-4,6-O-benzylidene-alpha-D-galactopyranoside with 3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-beta-D-glucopyranosyl bromide (1) in dichloromethane, in the presence of silver trifluoromethanesulfonate, 2,4,6-trimethylpyridine, and molecular sieves, afforded methyl 2-O-benzoyl-4,6-O-benzylidene-3-O-(3,4,6-tri-O-acetyl-2-deoxy-2-phthalim ido- beta-D-glucopyranosyl)-alpha-D-galactopyranoside (4). Deacetalation of 4 in hot, 80% aqueous acetic acid gave methyl 2-O-benzoyl-3-O-(3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido- beta-D-glucopyranosyl)-alpha-D-galactopyranoside (5), which, on deacylation, followed by peracetylation, furnished the peracetylated disaccharide derivative (6). The structures of 5 and 6 were established by 1H-n.m.r. spectroscopy. O-Deacetylation of 6 afforded the title beta-(1 leads to 3)-linked disaccharide 7. For the synthesis of the beta-(1 leads to 2)-linked isomer, methyl 3-O-benzoyl-4,6-O-benzylidene-alpha-D-galactopyranoside was similarly condensed with bromide 1 to give the fully protected disaccharide derivative (8). Cleavage of the benzylidene group of 8 gave methyl 3-O-benzoyl-2-O-(3,4,6- tri-O-acetyl-2-deoxy-2-phthalimido-beta-D-glucopyranosyl)-alpha-D- galactopyranoside (9). Deacetylation of 9, followed by peracetylation, afforded the peracetate (10). O-Deacetylation of 10 gave the beta-(1 leads to 2)-linked disaccharide (11). The structures of the disaccharides 7 and 11 were confirmed by 13C-n.m.r. spectroscopy.
Liquid chromatography under elevated pressure (h.p.l.c.) has been applied to the separation of the phenyl, benzyl, and O-nitrophenyl glycosides of 2-acetamido-2-deoxy-D-galactopyranose and of various mucin-type, di-, tri-, and tetra-saccharides. The separations were carried out with a Whatman Partisil PXS 5/25 PAC column and various proportions of acetonitrile and water in the mobile phase. These methods were subsequently used to separate the substrates and products of the following N-acetylglucosaminyltransferase reactions: UDP-GlcNAc + beta-Gal-(1 leads to 3)-GalNAc-R leads to beta-Gal-(1 leads to 3)-[beta-GlcNAc-(1 leads to 6)]-GalNAc-R + UDP (1); UDP-GlcNAc + beta-Gal-(1 leads to 3)-[beta-GlcNAc-(1 leads to 6)]-GalNAc-R leads to beta-GlcNAc-(1 leads to 3)-beta-Gal-(1 leads to 3)-[beta-GlcNAc-(1 leads to 6)]-GalNAc-R + UDP (2); UDP-GlcNAc + GalNAc-R' leads to beta-GlcNAc-(1 leads to 3)-GalNAc-R' + UDP (3); and UDP-GlcNAc + beta-GlcNAc-(1 leads to 3)-GalNAc-R' leads to beta-GlcNAc-(1 leads to 6)-[beta-GlcNAc-(1 leads to 3)]-GalNAc-R' + UDP (4), where R is = benzyl or o-nitrophenyl, and R' = benzyl or phenyl alpha-D-glycoside. Reaction 1 is catalyzed by a transferase in canine submaxillary glands and porcine gastric mucosa, and reaction 2 by an enzyme in porcine gastric mucosa. Enzyme activities catalyzing reactions 3 and 4 have recently been demonstrated in rat colonic mucosa. Liquid chromatography can be used at the preparative level for the purification and identification of the transferase products, and at the analytical level in the assay of glycosyltransferases.
beta-D-Galactosidase from Aspergillus niger was purified by conventional techniques, including the repeated use of chromatography on hydroxylapatite. The final preparation represented a 112-fold purification, with a 22% yield. The specific activity of the purified enzyme was 72 mumol of D-galactose released/min/mg of protein, using p-nitrophenyl beta-D-galactopyranoside as the substrate. The substrate specificity of the enzyme was studied by using saccharides having structural linkages similar to those found in naturally occurring glycoconjugates. At substrate concentrations of 5mM, the beta-D-galactosidase efficiently hydrolyzed beta-Gal-1 leads to OC6H4NO2-p, beta-Gal-(1 leads to 3)-Gal, beta-Gal-(1 leads to 3)-beta-Gal-1 leads to OC6H4NO2-p, and beta-Gal-(1 leads to 3)-alpha-Gal-1 leads to OC6H4NO2-p, at rates of 63, 53, 65, and 29 mumol/min/mg of protein, respectively. Slower hydrolysis was observed for beta-Gal-(1 leads to 4)-beta-Glc, beta-Gal-(1 leads to 4)-beta-GlcNAc-1 leads to OC6H4NO2-p, and beta-Gal-(1 leads to 6)-beta-GlcNAc-1 leads to OC6H4NO2-p, with rates of 10, 13 and 9 mumol/min/mg of protein, respectively. Poorly hydrolyzed, at rates 1/300th of that of beta-Gal-1 leads to OC6H4NO2-p, were synthetic substrates having D-galactose attached beta-(1 leads to 3)- to either GalNAc or GlcNAc. The Km value for beta-D-galactosidase with beta-Gal-(1 leads to 4)-beta-GlNAc-1 leads to OC6H4NO2-p was approximately 20 times that with beta-Gal-1 leads to OC6H4NO2-p. The beta-D-galactosidase of A. niger has a molecular weight of 300,000, as demonstrated by gel-filtration chromatography. Sodium dodecyl sulfate-poly(acrylamide)-gel electrophoresis indicated a single subunit having a molecular weight of 130,000.
Binding of 4-methylumbelliferyl-2-acetamido-2-deoxy-3-O-(beta-D-galactopyranosyl) beta-D-galactopyranoside [MeUmb beta Gal(beta 1 leads to 3)GalNAc] to peanut agglutinin was characterized by equilibrium dialysis and by measurement of the increase in ultraviolet absorption or fluorescence of the chromophoric glycoside upon continuous titration with excess of the lectin. All data in the 4-30 degrees C range correspond to delta G = -(26.5 +/- 0.1) kJ mol-1, delta H = -(58.4 +/- 2) kJ mol-1 and delta S = -(107 +/- 8)J mol-1 K-1. Values of the association constants are e.g. K = 2.5 X 10(5) M-1 at 4 degrees C and K = 4.5 X 10(4) M-1 at 25 degrees C. MeUmb beta Gal(beta 1 leads to 3)GalNAc was used as an indicator ligand to determine K values for nonchromophoric carbohydrates by continuous displacement titrations, measuring either fluorescence or difference in absorption of the indicator. The data were analyzed in terms of the general expression for a non-ideal indicator system (as detailed in the appendix). Thus, the values of K are not underestimated. They are K = 4.8 X 10(3) M-1 for methyl alpha-D-galactopyranoside [Me alpha Gal], 2.0 X 10(3) M-1 for methyl beta-D-galactopyranoside [Me beta Gal] and 4.7 X 10(3) M-1 for lactose [Gal(beta 1 leads to 4)Glc], all at 14.5 degrees C. The MeUmb difference absorption spectra resulting from binding of the lectin with MeUmb beta Gal(beta 1 leads to 3)GalNAc and MeUmb beta Gal(beta 1 leads to 4)Glc are larger than for MeUmb beta Gal and MeUmb alpha Gal. These observations are consistent with the extended nature of the combining site of peanut agglutinin.
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Starting from phenyl 2-acetamido-2-deoxy-4,6-O-(p-methoxybenzylidene)-beta-D-glucopyranoside (1), chemical syntheses were developed for phenyl 2-acetamido-2-deoxy-3-O-beta-D-galactopyranosyl-beta-D-glucopyranoside (4) and phenyl 2-acetamido-2-deoxy-4-O-alpha-L-fucopyranosyl-3-O-beta-D-galactopyranosyl -beta-D-glucopyranoside (8). Thin-layer chromatography in the solvent system 6:4:1:5 (v/v) 2-propanol-ethyl acetate-ammonium hydroxide-water clearly separated the synthetic trisaccharide 8 (RF 0.69) from synthetic disaccharide 4 (RF 0.78), fucose (RF 0.56), and GDP-fucose (which remained at the origin). Based upon this observation, a modified method for the determination of GDP-L-fucose: N-acetylglucosaminide-(1 leads to 4)-alpha-L-fucosyltransferase was developed that employed the synthetic disaccharide 4 as an acceptor, and compound 8 as an authentic reference-compound. This modified assay-procedure can simultaneously monitor possible competing reactions which may interfere with determination of alpha-(1 leads to 4)-L-fucosyltransferase activity; these include phosphorylase and alpha-L-fucosidase activities, and incorporation of alpha-L-[14C]-fucose into endogenous acceptors of enzyme preparations. Thus, the modified assay-procedure should facilitate determination of alpha-(1 leads to 4)-L-fucosyltransferase.