Syntheses of (Z)- and (E)-4-amino-2-(trifluoromethyl)-2-butenoic acid and their inactivation of gamma-aminobutyric acid aminotransferase

(Z)- and (E)-4-amino-2-(trifluoromethyl)-2-butenoic acid (4 and 5, respecti vely) were synthesized and investigated as potential mechanism-based inacti vators of gamma-aminobutyric acid aminotransferase (GABA-AT) in a continuin g effort to map the active site of this enzyme. The core alpha-trifluoromet hyl-alpha,beta-unsaturated ester moiety was prepared via a Reformatsky/redu ctive elimination coupling of the key intermediates tert-butyl 2,2-dichloro -3,3,3-trifluoropropionate and N,N-bis(tert-butoxycarbonyl)glycinal. Both 4 and 5 inhibited GABA-AT in a time-dependent manner, but displayed non-pseu do-first-order inactivation kinetics; initially, the inactivation rate incr eased with time. Further investigation demonstrated that the actual inactiv ator is generated enzymatically from 4 or 5. This inactivating species is r eleased from the active site prior to inactivation, and as a result, 4 and 5 cannot be defined as mechanism-based inactivators. Furthermore, 4 and 5 a re alternate substrates for GABA-AT, transaminated by the enzyme with K-m v alues of 0.74 and 20.5 mM, respectively. Transamination occurs approximatel y 276 and 305 times per inactivation event for 4 and 5, respectively. The e nzyme also catalyzes the elimination of the fluoride ion from 4 and 5. A me chanism to account for these observations is proposed. (C) 1999 Elsevier Sc ience Ltd. All rights reserved.