The fungal enzyme galactose oxidase is a radical copper oxidase that catalyzes the oxidation of a broad range of primary alcohols to aldehydes. Previous mechanistic studies have revealed a large substrate deuterium kinetic isotope effect on galactose oxidase turnover whose magnitude varies systematically over a series of substituted benzyl alcohols, reflecting a change in the character of the transition state for substrate oxidation. In this work, these detailed mechanistic studies have been extended using a series of stereospecifically monodeuterated substrates, including 1-O-methyl-α-D- galactose as well as unsubstituted benzyl alcohol and 3- and 4-methoxy and 4-nitrobenzyl derivatives. Synthesis of all of these substrates was based on oxidation of the α,α′-dideuterated alcohol to the corresponding 2H-labeled aldehyde, followed by asymmetric hydroboration using α-pinene/9-BBN reagents to form the stereoisomeric alcohols. Products from enzymatic oxidation of each of these substrates were characterized by mass spectrometry to quantitatively evaluate the substrate dependence of the stereoselectivity of the catalytic reaction. For all of these substrates, the selectivity for pro-S hydrogen abstraction was at least 95%. This selectivity appears to be a direct consequence of constraints imposed by the enzyme on the orientation of substrates bearing a branched β-carbon. Steady state analysis of kinetic isotope effects on V/K has resolved individual contributions from primary and α-secondary kinetic isotope effects in the reaction, providing a test for the involvement of an electron transfer redox equilibrium in the oxidation process. Multiple isotope effect measurements utilizing simultaneous labeling of the substrate and solvent have contributed to refinement of the relation between proton transfer and hydrogen atom transfer steps in substrate oxidation.
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