Side chain hydroxylation of aromatic hydrocarbons by fungi. Part 2. Isotope effects and mechanism

Abstract

The benzylic hydroxylation of ethylbenzene, p-diethylbenzene, tetralin, indane, and toluene by the fungi Mortierella isabellina, Cunninghamella echinulata, and Helminthosporium species has been investigated by the use of deuterium-labelled substrates. An intermolecular primary isotope effect k_H/k_D 1.04 ± 0.05 was observed for conversion of ethylbenzene into 1-phenylethanol. The secondary deuterium isotope effect for this conversion was large and positive (k_H/k_D averaging 1.27 ± 0.05). p-Diethylbenzene, tetralim, and indane were hydroxylated at the benzylic position with an intramolecular primary deuterium isotope effect averaging 2.6 ± 0.2. The use of R-(–)- and S-(+)-1-deuterioethylbenzenes as substrates with the fungus M. isabellina show that hydrogen removal is specific for the pro-R position of substrate. Toluene is converted into benzyl alcohol by the fungi Mortierella isabellina and Helminthosporium species; in the latter case, the product is further metabolized. [α-²H]-, [αα-²H₂]-, and [ααα-²H₃]-toluene have been used with M. isabellina in a series of experiments to determine both primary and secondary deuterium kinetic isotope effects for the enzymic benzylic hydroxylation reaction. The values obtained, intermolecular primary k_H/k_D= intramolecular primary k_H/k_D= 1.02 ± 0.05, and secondary k_H/k_D= 1.37 ± 0.05, lead to the suggestion of a mechanism for benzylic hydroxylation involving benzylic proton removal from a radical-cation intermediate in a non-symmetrical transition state.