Synthesis of [3,4-13C2]-Enriched Bile Salts as NMR Probes of Protein−Ligand Interactions

Abstract

Synthetic methodology that allows for incorporation of isotopic carbon at the C-3 and C-4 positions of bile salts is reported. Three [3,4-(13)C(2)]-enriched bile salts were synthesized from either deoxycholic or lithocholic acid. The steroid 3alpha-OH group was oxidized and the A-ring was converted into the Delta(4)-3-ketone. The C-24 carboxylic acid was next converted into the carbonate group and selectively reduced to the alcohol in the presence of the A-ring enone. Following protection of the 24-OH group, the Delta(4)-3-ketone was converted into the A-ring enol lactone. Condensation of the enol lactone with [1,2-(13)C(2)]-enriched acetyl chloride and subsequent Robinson annulation afforded a [3,4-(13)C(2)]-enriched Delta(4)-3-ketone that was subsequently converted back into a 3alpha-hydroxy-5beta-reduced bile steroid. C-7 hydroxylation, when necessary, was achieved via conversion of the Delta(4)-3-ketone into the corresponding Delta(4,6)-dien-3-one, epoxidation of the Delta(6)-double bond, and hydrogenolysis/hydrogenation of the 5,6-epoxy enone system. The [3,4-(13)C(2)]-enriched bile salts were subsequently complexed to human ileal bile acid binding protein (I-BABP), and (1)H-(13)C HSQC spectra were recorded to show the utility of the compounds for investigating the interactions of bile acids with I-BABP.