Importance of C4a- and N5-covalent adducts in the flavin oxidation of carbanions.

The reaction of nitroalkanes, furoin, and phenacyl alcohol with 7- and 8-cyano-3,10-dimethylisoalloxazine (7-CNFlox, 8-CNFlox), 5-ethyl-3-methyllumiflavinium ion (Flox+Et), and 2.6-dichloroindophenol (DCI) have been studied. Nitromethane anion forms an adduct on reaction with Flox +Et. The condensation reaction has been shown (nuclear magnetic resonance) to occur through addition of the methylene carbon of the nitromethane anion to the 4a position of Flox+Et. This adduct undergoes spontaneous dissociation to its components in acid. It does not undergo conversion in base to reduced flavin (1,5-F1EtH), NO2-, and CH2O. For this reason one may conclude that nitroalkane anion oxidation by flavin does not involve the intermediacy of a 4a-covalent intermediate. Oxidation of nitroalkane anion through formation of an N5-flavin adduct is discussed in terms of the peculiar requirements of the substrate when compared with carbanions derived from normal flavoenzyme substrates. It is concluded that the mechanism of nitroalkane oxidation by flavin must bear no relationship to the flavin oxidation of normal substrates. Furoin and phenacyl alcohol (compounds containing the-CH(OH)--CO-functionality) are oxidized by 8-CNFlOX, 7-CNFlOX, DCI and by the N5-blocked flavin Flox+Et. These reactions are found to be zero order in oxidant at the concentrations of oxidants (approximately 1.0 X 10(-5) M) and reductant (approximately 10(-3) to 10(-4) M) employed. This finding, and that of an 1H/2H kinetic isotope effect of 3.5, as well as certain equalities of the determined rate constants establish the rate-determining step in all these oxidations to be carbanion formation. That the N5-blocked flavin (Flox+Et) serves just as well as an oxidant as do substituted and unsubstituted isoalloxazines provides evidence that the flavin oxidation of carbanions of general structure (-C-(OH)--CO-) does not require the formation of an intermediate N5-flavin adduct. This conclusion when taken with the fact that the N5-flavin adduct formed on reaction of dihydroflavin with -C(=O)--CO-is not an intermediate in the reduction of -C(=O)--CO-to -CH(OH)--CO- by dihydroflavin shows that N5 adducts do not arise as intermediates in these oxidations. Remaining mechanisms (4a addition and radical pair formation) are discussed. 4a addition of carbanions to Flox+Et is shown to be subject to considerable steric strain by the instability of the nitroethane adduct and the inability to observe the formation of the 2-nitropropane adduct.