Chan T W, Bruice T C
Biochemistry. 1978 Oct 31;17(22):4784-93. doi: 10.1021/bi00615a028.
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.
已对硝基烷烃、糠偶姻和苯甲酰甲醇与7-和8-氰基-3,10-二甲基异咯嗪（7-CNFlox、8-CNFlox）、5-乙基-3-甲基荧光黄素离子（Flox+Et）以及2,6-二氯靛酚（DCI）的反应进行了研究。硝基甲烷阴离子与Flox +Et反应形成加合物。核磁共振显示缩合反应是通过硝基甲烷阴离子的亚甲基碳加到Flox+Et的4a位发生的。该加合物在酸性条件下会自发分解为其组分。它在碱性条件下不会转化为还原型黄素（1,5-F1EtH）、NO2-和CH2O。因此可以得出结论，黄素对硝基烷烃阴离子的氧化不涉及4a-共价中间体。与源自正常黄素酶底物的碳负离子相比，根据底物的特殊要求讨论了通过形成N5-黄素加合物对硝基烷烃阴离子的氧化。得出的结论是，黄素氧化硝基烷烃的机制与正常底物的黄素氧化肯定没有关系。糠偶姻和苯甲酰甲醇（含有-CH(OH)--CO-官能团的化合物）可被8-CNFlOX、7-CNFlOX、DCI以及N5-封闭的黄素Flox+Et氧化。发现在所用氧化剂浓度（约1.0×10(-5) M）和还原剂浓度（约10(-3)至10(-4) M）下，这些反应对氧化剂呈零级反应。这一发现、1H/2H动力学同位素效应为3.5以及某些测定速率常数的相等性表明，所有这些氧化反应的速率决定步骤是碳负离子的形成。N5-封闭的黄素（Flox+Et）作为氧化剂与取代和未取代的异咯嗪一样有效，这提供了证据，即一般结构为（-C-(OH)--CO-）的碳负离子的黄素氧化不需要形成中间体N5-黄素加合物。这一结论与二氢黄素与-C(=O)--CO-反应形成的N5-黄素加合物不是二氢黄素将-C(=O)--CO-还原为-CH(OH)--CO-的中间体这一事实相结合，表明N5加合物在这些氧化反应中不作为中间体出现。还讨论了其余的机制（4a加成和自由基对形成）。通过硝基乙烷加合物的不稳定性以及无法观察到2-硝基丙烷加合物的形成，表明碳负离子对Flox+Et的4a加成受到相当大的空间位阻。
