Oxidative cleavage of carboxylic esters by cytochrome P-450.

F P Guengerich

doi:10.1016/s0021-9258(18)47434-0

Abstract

Cytochrome P-450 was demonstrated to catalyze the oxidative cleavage of carboxylic acid esters to the corresponding carboxylic acids. 2,6-Dimethyl-4-phenyl-3,5-pyridinedicarboxylic acid diethyl ester and related dialkyl esters were shown to serve as substrates in NADPH-fortified rat liver microsomes and reconstituted systems containing purified cytochrome P-450 enzymes. The ethyl group gave rise to acetaldehyde. The reactions proceed with large kinetic deuterium isotope effects, consistent with the view that P-450 abstracts a hydrogen atom in the mechanism. Oxygen rebound to the radical site is then postulated to complete the reaction and lead to a hemiacetal-like structure which collapses to give the products. Rate studies with differing alkyl substituents showed that the reaction was more rapid with removal of an ethyl than a methyl or isopropyl group, consistent with the view that the ethyl optimizes steric and inductive effects. Oxidative cleavage of carboxylic acid esters has little biochemical precedent, due to the difficult character of the reaction, and should be considered as an alternative to direct hydrolysis.

Highlights

Enzymes-P-450uT.~, P-450pB.B,P-450BNF.Ba,nd P-450PcN.~were purified from rat liver microsomes (Sprague-Dawley male rats, 200 g body weight) using the general procedures described elsewhere (7). (For comparison of the properties of these preparations to others reported in the literaturesee Ref. 8.)Rabbit NADPH-P-450reductase was purified using procedures described previously (7,9, 10)
The solvent was removed in uacun and the residue was dissolved in H,O; the starting material precipitated a2n.6dg was recovered (m.p. 131.5134.5 "C, cf. m.p. 135-136 "C (4))
The filtratewas acidified to pH 2 and extracted three times with CHC1

Summary

MATERIAL SA N DM ETHODS

Enzymes-P-450uT.~, P-450pB.B,P-450BNF.Ba,nd P-450PcN.~were purified from rat liver microsomes (Sprague-Dawley male rats, 200 g body weight) using the general procedures described elsewhere (7). (For comparison of the properties of these preparations to others reported in the literaturesee Ref. 8.)Rabbit NADPH-P-450reductase was purified using procedures described previously (7,9, 10). 2,6-Dimethyl-4-phenyl-3,5-pyridinedicarboxylaiccid monomethyl ester was prepared by stirring and heating2,6-dimethyl-3,5-pyridinedicarboxylic acid dimethyl ester (9.0 g, 30 mmol) with 1eq of ground KOH (1.68 g) in 50 ml of CH,OH for 40 h under reflux (11). 1,4-Dihydro-2,6-dimethyl-4-phenyl-3,5-pyridinedicarboxyliaccid sults in the formationof aldehydes and ketonesvia carbinolamines (from amines), hemiacetals (from ethers), and gemmonoethyl ester was prepared by stirring and heating g(0.15 mol) of l,l-dihydro-2,6-dimethyl-3,5-pyridinedicarboxylaiccid diethyl ester with 8.5 g of ground KOH (0.15 mol) in 400 ml of CH,CH,OH halohydrins (from halides). This laboratory demonstrated the roles of P-450s in the samemanner described for 2,6-dimethyl-3,5-pyridinedicarboxylic oxidation of dihydropyridine calcium channel blockers (3-5). The product, obtained in 91% yield, was recrystallized from 50% aqueous

RESULTS

DISCUSSION

Findings

Alkyl substituents