Probing stereoselective inhibition of the acyl binding site of cholesterol esterase with four diastereomers of 2'-N-alpha-methylbenzylcarbamyl-1, 1'-bi-2-naphthol.

BACKGROUND: Recently there has been increased interest in pancreatic cholesterol esterase due to correlation between enzymatic activity in vivo and absorption of dietary cholesterol. Cholesterol esterase plays a role in digestive lipid absorption in the upper intestinal tract, though its role in cholesterol absorption in particular is controversial. Serine lipases, acetylcholinesterase, butyrylcholinesterase, and cholesterol esterase belong to a large family of proteins called the alpha/beta-hydrolase fold, and they share the same catalytic machinery as serine proteases in that they have an active site serine residue which, with a histidine and an aspartic or glutamic acid, forms a catalytic triad. The aim of this work is to study the stereoselectivity of the acyl chain binding site of the enzyme for four diastereomers of an inhibitor. RESULTS: Four diastereomers of 2'-N-alpha-methylbenzylcarbamyl-1, 1'-bi-2-naphthol (1) are synthesized from the condensation of R-(+)- or S-(-)-1, 1'-bi-2-naphthanol with R-(+)- or S-(-)-alpha-methylbenzyl isocyanate in the presence of a catalytic amount of pyridine in CH2Cl2. The [alpha]25D values for (1R, alphaR)-1, (1R, alphaS)-1, (1S, alphaR)-1, and (1S, alphaS)-1 are +40, +21, -21, and -41 degrees, respectively. All four diastereomers of inhibitors are characterized as pseudo substrate inhibitors of pancreatic cholesterol esterase. Values of the inhibition constant (Ki), the carbamylation constant (k2), and the bimolecular rate constant (ki) for these four diastereomeric inhibitors are investigated. The inhibitory potencies for these four diastereomers are in the descending order of (1R, alphaR)-1, (1R, alphaS)-1, (1S, alphaR)-1, and (1S, alphaS)-1. The k2 values for these four diastereomers are about the same. The enzyme stereoselectivity for the 1, 1'-bi-2-naphthyl moiety of the inhibitors (R > S, ca. 10 times) is the same as that for 2'-N-butylcarbamyl-1, 1'-bi-2-naphthol (2). The enzyme stereoselectivity for the alpha-methylbenzylcarbamyl moiety of the inhibitors is also R > S (2-3 times) due to the constraints in the acyl binding site. CONCLUSION: We are the first to report that the acyl chain binding site of cholesterol esterase shows stereoselectivity for the four diastereomers of 1.