A C₆-flavin adduct is the major product of irreversible inactivation of cholesterol oxidase by 2α,3α-cyclopropano-5α-cholestan-3β-ol

We have synthesized 2α,3α-cyclopropano-5α-cholestan-3β-ol and tested it as a substrate and inhibitor of cholesterol oxidase. The cyclopropylsterol irreversibly inhibits cholesterol oxidase with a k(inact) = 0.010 min^-1 and K(i) = 36 μM. Efficient inactivation requires the general base His⁴⁴⁷. Two FAD-steroid adducts were isolated by reversed-phase HPLC. The UV/vis, fluorescence and mass spectra of the adducts suggest that the FAD cofactor acts as an electrophile in a cyclopropoxide ring-opening reaction to form a C₆-alkylated flavin (68%) and either an N₅ flavin adduct or a cyclic N₅- C(4a) flavin adduct (32%). Cyclopropoxide ring-opening to form a C₆-FAD adduct represents a new approach to flavoenzyme inhibition.