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.
D000469:Alkaline Phosphatase; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001692:Biological Transport; D002417:Cattle; D005347:Fibroblasts / Q000378:metabolism; D006027:Glycosides / Q000138:chemical synthesis*; D017879:Isothiocyanates; D008247:Lysosomes / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D008361:Mannosidases; D008362:Mannosides / Q000138:chemical synthesis*; D008722:Methods; D011485:Protein Binding; D012710:Serum Albumin, Bovine
D000596:Amino Acids; D006027:Glycosides; D017879:Isothiocyanates; D008362:Mannosides; D012710:Serum Albumin, Bovine; C026808:4-isothiocyanatophenyl-6-phospho alpha-D-mannopyranoside; C026809:4-aminophenyl 6-phospho-alpha-mannopyranoside; D000469:Alkaline Phosphatase; D008361:Mannosidases