Biosynthesis and chemical synthesis of carboxyl-linked glucuronide of lithocholic acid.
The glucuronidation of lithocholic acid (LA) by phenobarbital-induced male Fischer 344 rat liver microsomes supplemented with UDP-glucuronic acid was studied. A single radioactive metabolite was formed and its structure was determined by high pressure liquid chromatography/particle beam/mass spectrometry (HPLC/PB/MS), both with and without prior methylation and acetylation of the sample. The reaction product was rigorously identified as the 1-O-acyl-beta-D-glucuronide of LA by comparison with a chemically synthesized standard. The chemical synthesis of the acyl glucuronide of LA was accomplished via a condensation reaction using benzyl 2,3,4-tri-O-benzyl-D-glucopyranuronate. The latter compound was prepared in two steps from benzyl 2,3,4-tri-O-benzyl-1-O-methyl-alpha-D-glucopyranuronate via the 1-O-acetyl derivative. The stereoselective beta coupling of LA with 2,3,4-tri-O-benzyl-D-glucopyranuronate was achieved by the Mitsunobu reaction, in the presence of the free hydroxyl function of LA, using triphenylphosphine and diisopropyl azodicarboxylate in THF followed by preparative TLC. The benzylic ester and ether groups were cleaved by hydrogenation with Pd on charcoal as the catalyst. Positive identification of the glucuronide was established by HPLC/PB/MS and 1H-NMR spectra. No side products formed by acyl migration were detected, but the free acyl glucuronide underwent rapid transesterification in methanol.
Panfil
I
I
Department of Internal Medicine, University of Arkansas for Medical Sciences, Little Rock 72205.
Lehman
P A
PA
Zimniak
P
P
Ernst
B
B
Franz
T
T
Lester
R
R
Radominska
A
A
D000107:Acetylation; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D004951:Esterification; D005965:Glucuronates / Q000138:chemical synthesis* / Q000378:metabolism*; D006868:Hydrolysis; D008095:Lithocholic Acid / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D008297:Male; D013058:Mass Spectrometry; D008745:Methylation; D008862:Microsomes, Liver / Q000378:metabolism; D051381:Rats; D011916:Rats, Inbred F344; D011919:Rats, Inbred Strains
D005965:Glucuronates; D008095:Lithocholic Acid