Chemical synthesis of the 3-sulfooxy-7-N-acetylglucosaminyl-24-amidated conjugates of 3β,7β-dihydroxy-5-cholen-24-oic acid, and related compounds: Unusual, major metabolites of bile acid in a patient with Niemann-Pick disease type C1

The chemical synthesis of 3β,7β-dihydroxy-5-cholen-24-oic acid, triply conjugated by sulfuric acid at C-3, by N-acetylglucosamine (GlcNAc) at C-7, and by glycine or taurine at C-24, is described. These are unusual, major metabolites of bile acid found to be excreted in the urine of a patient with Niemann-Pick disease type C1. Analogous double-conjugates of 3β-hydroxy-7-oxo-5-cholen-24-oic acid were also prepared. The principal reactions involved were: (1) β-d-N-acetylglucosaminidation at C-7 of methyl 3β-tert-butyldimethylsilyloxy (TBDMSi)-7β-hydroxy-5-cholen-24-oate with 2-acetamido-1α-chloro-1,2-dideoxy-3,4,6-tri-O-acetyl-d-glucopyranose in the presence of CdCO3 in boiling toluene; (2) sulfation at C-3 of the resulting 3β-TBDMSi-7β-GlcNAc with sulfur trioxide-trimethylamine complex in pyridine; and (3) direct amidation at C-24 of the 3β-sulfooxy-7β-GlcNAc conjugate with glycine methyl ester hydrochloride (or taurine) using 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride as a coupling agent in DMF. The structures of the multi-conjugated bile acids were characterized by liquid chromatography-mass spectrometry with an electrospray ionization probe under the positive and negative ionization modes.