Moravian College, Chemistry Department, Bethlehem, PA 18018, United States.
Moravian College, Chemistry Department, Bethlehem, PA 18018, United States.
Bioorg Chem. 2012 Feb;40(1):57-66. doi: 10.1016/j.bioorg.2011.10.002. Epub 2011 Oct 20.
A study of the reactions of an NADH model, 1,4-di(trimethylsilyl)-1,4-dihydropyridine, 7, with a series of α,β-unsaturated cyano and carbonyl compounds has produced the first direct evidence for an obligatory covalent adduct between a dihydropyridine and substrate in a reduction reaction. The reactions were monitored by NMR spectroscopy. In all reactions studied, the covalent adduct was the first new species detected and its decomposition to form products could be observed. Concentrations of adducts were sufficiently high at steady-state that their structures could be determined directly from NMR spectra of the reaction mixtures; adduct structures are those expected from an Ene reaction between 7 and the substrate. This first reaction step results in transfer of the C(4) hydrogen nucleus of 7 to the substrate and formation of a covalent bond between C(2) of the dihydropyridine ring and the substrate α-atom. Discovery of these Ene-adduct intermediates completes the spectrum of mechanisms observed in NADH model reactions to span those with free radical intermediates, no detectable intermediates and now covalent intermediates. The geometry of the transition state for formation of the Ene adduct is compared with those of theoretical transition state models and crystal structures of enzyme-substrate/inhibitor complexes to suggest a relative orientation for the dihydropyridine ring and the substrate in an initial cyclic transition state that is flexible enough to accommodate all observed mechanistic outcomes.
对 NADH 模型 1,4-二（三甲基硅基）-1,4-二氢吡啶 7 与一系列α,β-不饱和氰基和羰基化合物的反应进行了研究，为还原反应中二氢吡啶与底物之间存在必需的共价加合物提供了第一个直接证据。反应通过 NMR 光谱进行监测。在所研究的所有反应中，共价加合物是第一个检测到的新物种，其分解形成产物的过程可以观察到。在稳态时，加合物的浓度足够高，可以直接从反应混合物的 NMR 光谱中确定其结构；加合物的结构与 7 与底物之间的 Ene 反应所预期的结构一致。这第一步反应导致 7 的 C(4)氢原子核转移到底物上，并在二氢吡啶环的 C(2)和底物的α-原子之间形成共价键。这些 Ene-加合物中间体的发现完成了 NADH 模型反应中观察到的机制范围，涵盖了自由基中间体、不可检测中间体以及现在的共价中间体。Ene 加合物形成的过渡态的几何形状与理论过渡态模型和酶-底物/抑制剂复合物的晶体结构进行了比较，以表明在初始环状过渡态中二氢吡啶环和底物的相对取向具有足够的灵活性以适应所有观察到的机制结果。
