Saeed A. Abbas

Synthetic mucin fragments: methyl 3-O-(2-acetamido-2-deoxy-β-d-glucopyranosyl)-β-d-galactopyranoside and methyl 3-O-(2-acetamido-2-deoxy-3-O-β-d-galactopyranosyl-β-d-glucopyranosyl)-β-d-galactopyranoside

Abstract Methyl 2,4,6-tri- O -benzyl-β- 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 β- d -galactopyranoside, followed by benzylation. Condensation of 5 with 2-methyl-(2-acetamido-3,4,6-tri- O -acetyl-1,2-dideoxy-β- d -glucopyrano)-[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-β- d -glucopyranosyl)-2,4,6-tri- O -benzyl-β- d -galactopyranoside ( 6 ). Deacetylation of 6 in methanolic sodium methoxide afforded the disaccharide derivative 7 , which was acetalated with α,α-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-α- d -galactopyranosyl bromide in 1:1 benzenenitromethane 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 13 C-n.m.r. spectroscopy. The 13 C-n.m.r. chemical shifts for methyl α- and β- 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 .

N-acetyl-β-d-glucosaminyltransferases related to the synthesis of mucin-type glycoproteins in human ovarian tissue

The presence of N-acetyl-beta-D-glucosaminyltransferases in microsome preparations from human ovarian tissues was investigated with UDP-GlcNAc and several synthetic oligosaccharides as acceptors. The products were identified by paper chromatography and the linkage of the 2-acetamido-2-deoxy-beta-D-glucopyranosyl group incorporated into oligosaccharides was determined by exoglycosidase digestions, 1H-n.m.r. spectroscopy, and methylation analysis. These results showed that ovarian microsome preparations contain both beta-(1----3)- and beta-(1----6)-N-acetyl-D-glucosaminyltransferase activities which might be involved in the synthesis of mucin-type glycoproteins. Substrate competition tests suggested that both UDP-GlcNAc:-Bn glycoside of beta-D-GlcpNAc-(1----6)-alpha-D-GalpNAc [GlcNAc to GalNAc] and -Bn glycoside of beta-D-Galp-(1----3)-[beta-D-GlcNAc-(1----6)]-alpha-D-GalpNAc [GlcNAc to Gal] beta-(1----3)-N-acetyl-D-glucosaminyltransferase activities reside in a single enzyme species.

Synthesis of benzyl 2-acetamido-3-O- and -6-O-(2-acetamido-2-deoxy-β-d-glucopyranosyl)-2-deoxy-α-d-galactopyranoside

Abstract Glycosylation of benzyl 2-acetamido-4,6-O-benzylidene-2-deoxy-α- d -galactopyranoside with 3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-β- d -glucopyranosyl bromide in dichloromethane, in the presence of silver trifluoromethanesulfonate, 2,4,6-trimethylpyridine, and molecular sieves, afforded benzyl 2-acetamido-4,6-O-benzylidene-2-deoxy-3-O-(3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-β- d -glucopyranosyl)-α- d -galactopyranoside (3). Cleavage of the benzylidene group of 3 gave benzyl 2-acetamido-2-deoxy-3-O-(3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-β- d -glucopyranosyl)-α- d -galactopyranoside (4), which, on deacylation, followed by peracetylation, furnished the peracetylated disaccharide derivative (7). The structures of 3, 4, and 7 were established by 1H-n.m.r. spectroscopy. O-Deacetylation of 7 afforded the title β-(1→3)-linked disaccharide (8). Compound 3 was also deacylated and then peracetylated, to give benzyl 2-acetamido-3-O-(2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β- d -glucopyranosyl)-4,6-O-benzylidene-2-deoxy-α- d -galactopyranoside, which was O-deacetylated to give its 4,6-O-benzylidene derivative (6). For the synthesis of the β-(1→6)-linked disaccharide, the readily accessible benzyl 2-acetamido-3-O-acetyl-2-deoxy-α- d -galactopyranoside was condensed with 2-methyl-(3,4,6-tri-O-acetyl-1,2-dideoxy-α- d -glucopyrano)-[2,1-d]-2-oxozaline, and the product was isolated as its peracetylated derivative, which, on saponification, afforded the title β-(1→6)-linked disaccharide (12). The structures of compounds 6, 8, and 12 were established by 13C-n.m.r. spectroscopy.

Synthesis of some oligosaccharides containing the O-(2-acetamido-2-deoxy-β-d-glucopyranosyl)-(1→2)-O-α-d-mannopyranosyl unit. Potential substrates for UDP-GlcNAc: α-d-mannopyranosyl-(1→6)-N-acetyl-β-d-glucosaminyl-transferase (GnT-V)

Abstract Four different oligosaccharides containing the 2-acetamido-2-deoxy-β- d -glucopyranosyl-(1→2)-α- d -mannopyranosyl sequence as a terminal disaccharide unit were synthesized, namely: 4-nitrophenyl O -(2-acetamido-2-deoxy-β- d -glucopyranosyl)-(1→2)- O -α- d -mannopyranosyl-(1→6)-β- d -mannopyranoside ( 27 ), 4-nitrophenyl O -(2-acetamido-2-deoxy-β- d -glucopyranosyl)-(1→2)- O -α- d -mannopyranosyl-(1→6)-β- d -glucopyranoside ( 29 ), allyl O -(2-acetamido-2-deoxy-β- d -glucopyranosyl-(1→2)-α- d -mannopyranosyl-(1→6)-β- d -glucopyranoside ( 31 ), and allyl O -(2-acetamido-2-deoxy-β- d -glucopyranosyl)-(1→2)- O -α- d -mannopyranosyl-(1→6)- O -β- d -glucopyranosyl-(1→4)-β- d - glucopyranoside ( 33 ). A common glycosyl donor, namely, 2- O -(2-acetamido-3,4,6-tri- O -acetyl-2-deoxy-β- d -glucopyranosyl)-3,4,6-tri- O -acetyl-α- d -mannopyranosyl bromide was employed for the synthesis of 27 , 29 , 31, and 33 , the structures of which were all established by 13 C-n.m.r. spectroscopy.

Substrate specificity and other properties of the β-d- galactosidase from Aspergillus niger

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.

Cell surface ligands for rotavirus: Mouse intestinal glycolipids and synthetic carbohydrate analogs

Rotaviral binding to receptors on epithelial cells in the small intestine is thought to be a key event in the infection process and may be carbohydrate-mediated. Strain SA11 of rotavirus bound in vitro both to glycolipids isolated from mouse small intestine and to authentic glycolipids using thin layer chromatography overlay and microtiter well adsorption assays. Neutral mouse intestinal glycolipids which bound rotavirus were GA1 (Gal beta 1----3GalNAc beta 1---4Glc beta 1----4Glc beta 1----1-ceramide) and pentaosylceramides with terminal N-acetylgalactosamine, while acidic lipids which bound rotavirus included cholesterol 3-sulfate and two compounds termed bands 80 and 81. Digestion with ceramide glycanase suggested that bands 80 and 81 have lactosyl ceramide cores and an unidentified acidic moiety(s). No sialic-acid-containing glycolipids tested were active in viral binding. Band 81, which may have a ganglio core, bound rotavirus with greatest avidity, followed by GA1. Of authentic glycolipids assayed, only GA1 and GA2 (GalNAc beta 1----4Gal beta 1----4Glc beta 1----1-ceramide) displayed rotaviral binding. A phosphatidylethanolamide dipalmitoyl-containing neoglycolipid analog of GA2 bound rotavirus with avidity similar to native GA2. Substitution of beta 1----4-linked GlcNAc or beta 1----3-linked GalNAc for terminal GalNAc of GA2 neoglycolipid supported rotaviral binding, while other substitutions abrogated it. These findings suggest that a carbohydrate epitope similar to that of GA2 is sufficient for in vitro rotaviral binding, although binding may be enhanced by galactose and/or an acidic moiety in a secondary epitope.

Synthetic mucin fragments: Benzyl 2-acetamido-6-O-(2-acetamido-2-deoxy-β-d-glucopyranosyl)-2-deoxy-3-O-β-d-galactopyranosyl-α-d-galactopyranoside and benzyl 2-acetamido-6-O-(2-acetamido-2-deoxy-β-d-glucopyranosyl)-3-O-[6-O-(2-acetamido-2-deoxy-β-d-glucopyranosyl)-β- d-galactopyranosyl]-2-deoxy-α-d-galactopyranoside

Abstract Glycosylation of benzyl 2-acetamido-4,6- O -benzylidene-2-deoxy-α- d -galactopyranoside with 2,3,4,6-tetra- O -acetyl-α- 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-β- d -galactopyranosyl)-α- d -galactopyranoside ( 3 ). O -Deacetylation of 3 gave benzyl 2-acetamido-4,6- O -benzylidene-2-deoxy-3- O -β- d -galactopyranosyl-α- 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-α- 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 13 C-n.m.r. spectroscopy.

Serum α(1 3)-L-fucosyltransferase, carcinoembryonic antigen, and sialyl lewis X-i antigen levels in lung cancer

Serum α(1 → 3)‐L‐fucosyltransferase activity was measured in 58 patients with lung cancer, 27 benign diseases, and in 100 healthy controls. The levels of enzyme activity were significantly higher in the sera of patients with cancer when compared to those in benign diseases and healthy controls. The elevation of the enzyme activity correlated with the clinical stages and to the size of the primary tumors. Follow‐up studies with various stages showed that the enzyme activity was useful in tracking the clinical course of disease after surgery. To evaluate the usefulness of this enzyme as a diagnostic marker, carcinoembryonic antigen (CEA) and sialyl Lewis X‐i antigen levels were also measured. The results indicate that α(1 → 3)‐L‐fucosyltransferase could be a more specific tumor marker than such tumor‐associated antigens in lung cancer. Cancer 64:2541–2545, 1989.

Use of 3-O-(2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-d-glucopyranosyl)-2,4,6-tri-O-acetyl-α-d-galactopyranosyl bromide as a glycosyl donor: Synthesis of p-nitrophenyl 3-O-(2-acetamido-2-deoxy-β-d-glucopyranosyl)-β-d-galactopyranoside

Abstract Acetolysis of methyl 3-O-(2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β- d -glucopyranosyl)-2,4,6-tri-O-acetyl-α- d -galactopyranoside afforded 3-O-(2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β- d -glucopyranosyl)-1,2,4,6-tetra-O-acetyl- d -galactopyranose (2). Treatment of 2 in dichloromethane with hydrogen bromide in glacial acetic acid gave 3-O-(2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β- d -glucopyranosyl)- 2,4,6-tri-O-acetyl-α- d -galactopyranosyl bromide (3). The α configuration of 3 was indicated by its high, positive, specific rotation, and supported by its 1H-n.m.r. spectrum. Reaction of 3 with Amberlyst A-26-p-nitrophenoxide resin in 1:4 dichloromethane-2-propanol furnished p-nitrophenyl 3-O-(2-acetamido-3,4,6- tri-O-acetyl-2-deoxy-β- d -glucopyranosyl)-2,4,6-tri-O-acetyl-β- d -galactopyranoside (7). Compound 7 was also obtained by the condensation (catalyzed by silver trifluoromethanesulfonate-2,4,6-trimethylpyridine) of 3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-β- d -glucopyranosyl bromide with p-nitrophenyl 2,4,6-tri-O-acetyl-β- d -galactopyranoside, followed by the usual deacylation-peracetylation procedure. O-Deacetylation of 7 in methanolic sodium methoxide furnished the title disaccharide (8). The structure of 8 was established by 13C-n.m.r. spectroscopy.

Synthesis of p-nitrophenyl 3-O-β-d-galactopyranosyl-β-d-galactopyranoside and p-nitrophenyl 3-O-α-d-galactopyranosyl-β-d-galactopyranoside

Abstract Glycosylation (catalyzed by mercuric cyanide) of p -nitrophenyl 2,4,6-tri- O -acetyl-β- d -galactopyranoside ( 2 ) with 2,3,4,6-tetra- O -acetyl-α- d -galactopyranosyl bromide in acetonitrile afforded the α-(1→3)- and β-(1→3)-linked disaccharide heptaacetates ( 4 and 6 , respectively) in almost equal proportions. Similar glycosylation of p -nitrophenyl 2- O -benzoyl-4,6- O -benzylidene-β- d -galactopyranoside ( 3 ) gave the β-(1→3)- and the α-(1→3)-linked, fully protected, disaccharide derivatives ( 8 and 10 , respectively) in the ratio of 3:1. The structures of 4 , 6 , 8 , and 10 were evidenced by their respective 1 H-n.m.r. spectra. O -Deacetylation of 4 and 6 afforded, respectively, p -nitrophenyl 3- O -α- d -galactopyranosyl-β- d -galactopyranoside ( 5 ) and p -nitrophenyl 3- O -β- d -galactopyranosyl-β- d -galactopyranoside ( 7 ). O -Deacylation of 8 and 10 furnished the disaccharide derivatives ( 9 and 11 ). Cleavage of the benzylidene groups of 9 and 11 gave the disaccharides 7 and 5 , respectively. The structures of 5 , 7 , 9 , and 11 were established by 13 C-n.m.r. spectroscopy. Additionally, the structures of 5 and 7 were confirmed by permethylation, and acid hydrolysis to 2,4,6-tri- O -methyl- d -galactose. The synthesis of triacetate 2 , starting from p -nitrophenyl 3,4- O -isopropylidene-β- d -galactopyranoside ( 1 ), is also described. Compound 1 was obtained as the major product of the isopropylidenation of p -nitrophenyl β- d -galactopyranoside under thermodynamic control, and its structure was likewise established by 13 C-n.m.r. spectroscopy, and confirmed by methylation, and acid hydrolysis to 2,6-di- O -methyl- d -galactose. Compound 3 was obtained from p -nitrophenyl 2- O -benzoyl-4,6- O -isopropylidene-β- d -galactopyranoside by cleavage of the isopropylidene group, and acetalation of the resulting triol with benzaldehyde-zinc chloride.