Structure-reactivity probes for active site shapes of cholesterol esteraseby carbamate inhibitors

4.4'-Biphenyl-di-N-butylcarbamate (1), (S)-1,1'-bi-2-naphthyl-2,2'-di-N-but ylcarbama (S-2), (S)-1,1'-bi-2-naphthyl-2-N-butylcarbamate-2'-butyrate (S-3 ), 2,2'-biphenyl-di-N-butylcarbamate (4), 2,2'-biphenyl-2-N-octadecylcarbam ate-2'-N-octylcarbamate (5), 2,2'-biphenyl-2-N-octadecylcarbamate-2'-N-phen ylcarbamate (6), 2,2'-biphenyl-2-N-butylcarbamate-2'-butyrate (7), 2,2'-bip henyl-2-N-butylcarbamate-2'-ol (8), 2,2'-biphnyl-2-N-octylcarbamate-2'-ol ( 9), (R)-1,1'-bi-2-N-naphthyl-2-butylcarbamate-2'-ol (R-10), and glyceryl-1, 2,3-tri-N-butylcarbarnate (11) are prepared and evaluated for their inhibit ion effects on porcine pancreatic cholesterol esterase. All inhibitors are irreversible inhibitors of the enzyme. Carbamates 1-3 and 7-10 are the firs t alkyl chain and esteratic binding site-directed irreversible inhibitors d ue to the fact that the reactivity of the enzyme is protected by the irreve rsible inhibitor, trifluoroacetophenone in the presence of these carbamates . Carbamate 1 is the least potent inhibitor for the enzyme probably due to the fact that the inhibitor molecule adopts a linear conformation and one o f the carbamyl groups of the inhibitor molecule covalently interacts with t he first alkyl chain binding site of the enzyme while the other carbamyl gr oup of the inhibitor molecule exposes outside the active site. With near or thogonal conformations at the pivot bond of biaryl groups, one carbamyl gro up of carbamates S-2, S-3, and R-10 covalently binds to the first alkyl cha in binding site of the enzyme while the other carbamyl, butyryl, or hydroxy group can not bind covalently to the second alkyl chain binding site proba bly due to the orthogonal conformations. Carbamates 4-9 and 11 are very pot ent inhibitors for the enzyme probably due to the fact that all these molec ules freely rotate at the pivot bond of the biphenyl or glyceryl group and therefore can fit well into the bent-shaped first and second alkyl chains b inding sites of the enzyme. Although, carbamates 4-6 and 11 are irreversibl e inhibitors of cholesterol esterase, the enzyme is not protected but furth er inhibited by trifluoroacetophenone in the presence of these carbamates. Therefore, carbamates 4-6 and 11 covalently bind to the first alkyl chain b inding site of the enzyme by one of the carbamyl groups and may also bind t o the second alkyl chain binding site of the enzyme by the second carbamyl group. Besides the bent-shaped conformation, the inhibition by carbamate 6 is probably assisted by a favorable pi-pi interaction between Phe 324 at th e second alkyl chain binding site of the enzyme and the phenyl group of the inhibitor molecule. For cholesterol esterase, carbamates 8-10 are more pot ent than carbamates S-2, 4, and 5 probably due to the fact that the inhibit or molecules interact with the second alkyl chain binding site of the enzym e through a hydrogen bond between the phenol hydroxy group of the inhibitor molecules and the His 435 residue in that site. (C) 1999 Published by Else vier Science B.V. All rights reserved.