p-Isothiocyanatophenyl 6-phospho-.alpha.-D-mannopyranoside coupled to albumin. A model compound recognized by the fibroblast lysosomal enzyme uptake system. 1. Chemical synthesis and characterization

We have developed a simple synthesis for a conjugate of albumin and isothio-nitrophenyl alpha-D-mannopyranoside to study the requirements of the fibroblast lysosomal enzyme recognition system. p-aminophenyl 6-phospho-alpha-D-mannopyranoside was prepared in two ways: (1) phosphorylation of isothio-nitrophenyl alpha-D-mannopyranoside and subsequent reduction of the nitro group by catalytic hydrogenation and (2) direct phosphorylation of p-aminophenyl alpha-D-mannopyranoside. Mannosides were phosphorylated in a reaction with phosphoryl chloride, pyridine, and water at 0 degrees C for 1 h, by a procedure selective for primary hydroxyl groups. Purified p-a minophenyl 6-phospho-alpha-D-mannopyranoside was characterized by chromatographic, enzymatic, and 13C nuclear magnetic resonance spectroscopic methods. Isothio-Isothiocyanatophenyl 6-phospho-alpha-D-mannopyranoside and the p-isothiocyanatophenyl glycosides of alpha-mannose, alpha-glucose, alpha- and beta-galactose, and alpha-L-fucose were formed by reaction of the respective p-aminophenyl glycosides with thiophosgene. Incubation of the p-isothiocyanatophenyl glycosides with bovine serum albumin at pH 8.5, 25 degrees C, for 18 h generally resulted in the coupling, primarily through lysine residues, of up to 20-30 mol of glycoside per mol of protein. Biological properties of the conjugates in the fibroblast lysosomal enzyme recognition system are described in the accompanying paper.