Binding of D-galactose-terminated ligands to rabbit asialoglycoprotein receptor.

The binding affinities of a series of D-galactose-terminated glycerol glycosides and oligosaccharides for the asialoglycoprotein receptor isolated from rabbit liver were determined in vitro using a radioreceptor-inhibition assay with 125I-asialoorosomucoid. The relative affinities of the synthetic ligands increased with the number of exposed D-galactose termini. Of the compounds examined, 1,2,3-tri-O-beta-lactosylglycerol associated with the greatest affinity (estimated Kd = 7.97 x 10(-5) M). Examination of the affinities of the synthetic series indicated that both the number and propinquity of the D-galactose termini influenced the strength of the binding interactions.

Synthesis and characterization of 6-O-beta-lactosyl-alpha,beta-lactoses, 1-O-(6-O-beta-lactosyl-beta-lactosyl)-(R,S)-glycerols, and 4,6-di-O-beta-D-galactopyranosyl-alpha,beta-D-glucoses.

1,2,3,2',3',4',6'-Hepta-O-acetyl-beta-lactose (4) was coupled with 2,3,6,2',3',4',6'-hepta-O-acetyl-alpha-lactosyl bromide (7) in the presence of Hg(CN)2 to afford 1,2,3,2',3',4',6'-hepta-O-acetyl-6-O-(2,3,6,2',3',4',6'-hepta-O-acetyl-b eta- lactosyl)-beta-lactose (11) which, upon O-deacetylation, gave 6-O-beta-lactosyl-alpha,beta-lactoses (64% from 4). In contrast, the reaction of 7 with benzyl 2,3,2',3',4',6'-hexa-O-acetyl-beta-lactoside in the presence of Hg(CN)2 produced 3,6,2',3',4',6'-hexa-O-acetyl-1,2-O- (2,3,2',3',4',6'-hexa-O-acetyl-1-O-benzyl-beta-lactos-6-yl orthoacetyl)-alpha-lactose (63%) and 3,6,2',3',4',6'-hexa-O-acetyl-1,2-O-(1- cyanoethylidene)-alpha-lactose (27%). The glycosidation of 4 using 2,3,4,6-tetra-O-acetyl-alpha-D-galactopyranosyl bromide in the presence of Hg(CN)2 afforded, after deprotection, 4,6-di-O-beta-D-galactopyranosyl-alpha,beta-D-glucoses (66%). The reaction of 11 with 1,2-di-O-benzyl-(R,S)-glycerols and trimethylsilyl trifluoromethanesulfonate yielded, after deprotection, 1-O-(6-O-beta-lactosyl-beta-lactosyl)-(R,S)-glycerols (18%). Under the same coupling conditions 11 reacted with 2-O-benzylglycerol to form 3-O-acetyl-2-O-benzyl-1-O-[2',3',4',6'-hexa-O-acetyl-6-O-(2,3,6,2',3',4' ,6'- hepta-O-acetyl-beta-lactosyl)-beta-lactosyl]-(R,S)-glycerols (16%).

Synthesis and characterization of 6-O-beta-lactosyl-alpha,beta-D-mannopyranoses and 2,6-di-O-beta-lactosyl-alpha,beta-D-mannopyranoses.

The reaction of 2,3,6,2',3',4',6'-hepta-O-acetyl-alpha-lactosyl bromide (4) and benzyl 3,4-di-O-benzyl-alpha-D-mannopyranoside (3) in the presence of mercury(II) cyanide in benzene-nitromethane produced benzyl 3,4-di-O-benzyl-2,6-bis-O-(2,3,6,2',3',4',6'-hepta-O-acetyl-beta-lact osy l)-alph a D-mannopyranoside (5) and benzyl 3,4-di-O-benzyl-6-O-(2,3,6,2',3',4',6'-hepta-O-acetyl-beta-lactosyl)-alp ha-D- mannopyranoside (6), as part of a complex mixture. Column chromatography, followed by acetylation of the fraction containing 5 and 6, gave a sample of 5 and benzyl 2-O-acetyl-3,4-di-O-benzyl-6-O (2,3,6,2',3',4',6'-hepta-O-acetyl-beta-lactosyl)-alpha-D-mannopyranoside (7) in approximately 35% and 17% yields (based on 4), respectively. Deprotection of 5 and 7 afforded the target compounds, namely 2,6-di-O-beta-lactosyl-alpha,beta-D-mannopyranoses and 6-O-beta-lactosyl-alpha,beta-D-mannopyranoses, respectively. If the coupling of 4 with 3 were performed in the presence of silver trifluoromethanesulfonate and 2,4,6-trimethylpyridine, only a mixture of 3,6,2',3',4',6'-hexa-O-acetyl- alpha-lactose-1,2-[( 3,6,2',3',4',6'-hexa-O-acetyl-alpha-lactose 1,2-(benzyl 3,4-di-O-benzyl-alpha-D-mannopyranosid-6-yl orthoacetyl)-2-yl]orthoacetate) and 3,6,2',3',4',6'-hexa-O-acetyl-alpha-lactose 1,2-(benzyl 3,4-di-O-benzyl-alpha-D-mannopyranosid-6-yl orthoacetate) was obtained. The orthoacetates were characterized by n.m.r. spectroscopy. The two target materials are useful in the assessment of the binding properties of galactose-terminated ligands to the asialoglycoprotein receptor of normal rabbit and human hepatocytes.

Synthesis and characterization of 2-O-beta-lactosylglycerol, 1,2-di-O-beta-lactosyl-(R,S)-glycerols, and 1,2,3,-tri-O-beta-lactosylglycerol.

The reaction of 2,3,6,2',3',4',6'-hepta-O-acetyl-alpha-lactosyl bromide (5) and 1,3-di-O-benzylglycerol in the presence of mercury(II) cyanide in benzene-nitromethane afforded 1,3-di-O-benzyl-2-O-(2,3,6,2',3',4',6'-hepta-O-acetyl-beta-lactosyl)glyc erol (70%), which was converted into 2-O-beta-lactosylglycerol. 1,2-Di-O-beta-lactosyl-(R,S)-glycerols were obtained by way of the coupling of 5 to either 1-O-benzyl-(R,S)-glycerol or 1-O-benzyl-2-O-(2,3,6,2',3',4',6'-hepta-O-acetyl-beta-lactosyl)-(R,S)-gl ycerols. The most efficient route to 1,2, 3-tri-O-beta-lactosylglycerol (17) involved treatment of 2-O-(2,3,6,2',3',4',6'-hepta-O-acetyl-beta-lactosyl)glycerol with 3 mol. equiv. of 5 followed by removal of the blocking groups, to give 17 (47%).

Evidence that L-sucrose is resistant to hydrolysis catalyzed by jejunal brush border enzymes.

The sucrase-isomaltase complex of the intestinal brush border membrane (BBM) catalyzes the hydrolysis of sucrose. The stereospecificity of this enzyme, however, is not known. To investigate this, BBM of hamster jejunum was incubated with D-sucrose or L-sucrose, and the reaction mixture was analyzed using a gas-liquid chromatograph. It was found that D-sucrose was hydrolyzed to its monomers, but L-sucrose remained unhydrolyzed. It is concluded that the sucrase-isomaltase of intestinal BBM of hamster jejunum does not hydrolyze L-sucrose and therefore this enzyme is stereospecific.