Antibody catalyzed hydrolysis of enol ethers

Article abstract of DOI:10.1021/ja00063a009

The hydrolysis of alkyl enol ethers to their corresponding carbonyl compounds proceeds by rate determining protonation of the β-carbon to form an activated oxocarbonium ion intermediate and is catalyzed by acids (Kresge, A. J.; Chiang, Y. J. Chem. Soc. B 1967, 53). It can be catalyzed by antibodies with very high enantioselectivity of protonation at the β-carbon to form optically pure carbonyl compounds (Reymond, J.-L.; Janda, K. D.; Lerner, R. A. J. Am. Chem. Soc. 1992, 114, 2257). 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°C. The kinetic solvent isotope effect is (k_H/k_D)_cat = 1.75 for the antibody catalyzed reaction and (k_H/k_D)_uncat = 1.92 for the background reaction. The Michaelis-Menten constant K_m for substrate 4 changes from 35 μM at low pH to 190 μM. at high pH. Saturation of the catalytic activity is observed at low pH. These observations are consistent with general acid catalysis by an ionizable side chain with pK = 5.2, presumably a carboxyl group, in the active site. A maximum rate acceleration k_cat/k_uncat = 8200 is obtained at the high pH end of the profile, and a maximum turnover number of 9.75 × 10^-5 s^-1 is obtained at the low pH end. Enol ethers 15-21 are also catalytically hydrolyzed by 14D9. The maximum turnover numbered measured is 0.39 s^-1 with 17 at pH = 6.0 at 20°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 β-methyl analog 4 and by a factor of 34 between the six-membered ring enol ether 19 and its five-membered ring analog 17. These rate effects may reflect the principle of strain 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 mechanisms. The implication of these findings for the design and improvement of antibody catalysts is discussed.