Cyanomethyl 1-thioglycosides ofD-galactose, D-glucose, 2-acetamido-2-deoxy-D-glucose, and D-mannose were prepared from the respective pseudothiourea derivatives and chloroacetonitrile. The nitrile group in these cyanomethyl thioglycosides can be converted to a methyl imidate group by treatment with sodium methoxide or HC1 in dry methanol to yield 2-imino-2-methoxyethyl 1-thioglycosides (IME-thioglycosides). The factors influencing the yield of IME-thioglycosides were investigated. The most convenient method of preparing IME-thioglycosides was treating 0.1 M cyanomethyl thioglycoside peracetate in dry methanol with 0.01 M sodium methoxide at room temperature for 24-48 h (50-60% yield). These IME-thioglycosides reacted readily with simple amines, amino acids, and proteins in mildly alkaline buffer solutions. Alpha-amylase and lysozyme modified with these reagents under appropriate conditions retained full activities. Thus the IME-thioglycosides constitute a new group of reagents for attaching sugars to proteins.

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Synthetic ligands containing up to six residues of nonreducing terminal galactose were prepared. The synthesis involved coupling of carboxyl groups of N-benzyloxy-carbonylaspartic acid or of N-benzyloxycarbonyltyrosyl-gamma-glutamylglutamic acid to the omega-amino group of the aglycon of a glycoside that contained up to three lactosyl residues. The benzyloxycarbonyl group was removed by hydrogenolysis before these ligands were tested as inhibitors to the binding of 125I-asialoorosomucoid to the galactose/N-acetylgalactosamine lectin, both soluble and on the surface of freshly isolated mammalian hepatocytes. Each addition of a galactosyl residue to an existing ligand structure invariably increased the binding affinity of such a ligand. However, at each level of galactose valency, the binding constant varied as much as 1000-fold depending on the structure of the ligand. At a given level of valency, the binding strength of a cluster ligand depended mainly on two factors: (1) the maximum spatial inter-galactose distances and (2) the flexibility of the arm connecting galactosyl residues and the branch points. It has been postulated that the three galactose-combining sites of the lectin are arranged in space at the vertexes of a triangle whose sides are 15, 22, and 25 A [Lee, Y. C., Townsend, R. R., Hardy, M. R., Lönngren, J., & Bock, K. (1984) in Biochemical and Biophysical Studies of Proteins and Nucleic Acids (Lo, T. B., Liu, T. Y., & Li, C. H., Eds.) pp 349-360, Elsevier, New York]. Ligands having inter-galactose distances shorter than these lengths were invariably poor ligands at their respective level of valency.(ABSTRACT TRUNCATED AT 250 WORDS)

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