ANTIBODY-CATALYZED HYDROLYSIS OF ENOL ETHERS

The hydrolysis of alkyl enol ethers to their corresponding carbonyl co mpounds proceeds by rate determining protonation of the beta-carbon to form an activated oxocarbonium ion intermediate and is catalyzed by a cids (Kresge, A. J.; Chiang, Y. J. Chem. Soc. B 1967, 53). It can be c atalyzed by antibodies with very high enantioselectivity of protonatio n at the beta-carbon to form optically pure carbonyl compounds (Reymon d, J.-L.; Janda, K. D.; Lerner, R. A. J. Am. Chem. Soc. 1992, 114, 225 7). In the present study, the pH profile of the antibody 14D9 (anti-1) catalyzed, enantioselective hydrolysis of enol ether 4 between pH = 3 .1 and pH = 7.2 has been measured in both H2O and D2O at 20-degrees-C. The kinetic solvent isotope effect is (k(H)/k(D))cat = 1.75 for the a ntibody catalyzed reaction and (k(H)/k(D))uncat = 1.92 for the backgro und reaction. The Michaelis-Menten constant K(m) for substrate 4 chang es from 35 muM at low pH to 190 muM at high pH. Saturation of the cata lytic activity is observed at low pH. These observations are consisten t with general acid catalysis by an ionizable side chain with pK = 5.2 , presumably a carboxyl group, in the active site. A maximum rate acce leration k(cat)/k(uncat) = 8200 is obtained at the high pH end of the profile, and a maximum turnover number of 9.75 X 10(-5) s-1 is obtaine d at the low pH end. Enol ethers 15-21 are also catalytically hydrolyz ed by 14D9. The maximum turnover numbered measured is 0.39 s-1 with 17 at pH = 6.0 at 20-degrees-C. The catalytic effect k(cat)/k(uncat) is influenced by the structure of the enol ether. Catalysis,increases by a factor of 12 between 15 and its beta-methyl analog 4 and by a factor of 34 between the six-membered ring enol ether 19 and its five-member ed ring analog 17. These rate effects may reflect the principle of str ain in catalysis. They suggest that hydrophobic interactions directly participate in transition-state stabilization, which is unexpected for an acid-base reaction usually discussed in terms of proton relay mech anisms. The implication of these findings for the design and improveme nt of antibody catalysts is discussed.