Preparation and spectroscopic studies of 5-deazaflavin isotopologues in isotropic and anisotropic surroundings

去氮黄素是天然黄素的重要类似物：核黄素、黄素单核苷酸(FMN)和黄素腺嘌呤二核苷酸(FAD)。事实证明，使用5-去氮黄素作为一些黄素蛋白中的替代辅酶在解开和操纵它们的反应机制方面特别有价值。人们经常观察到，5-去氮黄素作为辅因子阻碍了黄素蛋白的单电子转移反应。根据这些发现，得出结论，5-去氮黄素自由基与相应的黄素半喹酮相比明显不稳定，并迅速重新氧化或经历歧化。鉴于我们最近成功地(间接)使用光化学诱导动态核极化(Photo-CIDNP)核磁共振观察到单电子还原(顺磁)物种，长期存在的5-去氮黄素仅是两电子/氢化物受体/供体的范例需要重新评估。我们建议使用电子顺磁共振(EPR)衍生的超精细光谱以及光致CIDNP核磁共振来表征该自由基的电子结构。我们将合成一个选择性稳定同位素(13C，15N)标记的5-去氮黄素文库，并将其整合到各种蓝光光感受器结构域(LOV和BLUF)中，这些结构域在光遗传应用中具有重要意义。利用二维核磁共振，我们计划揭示野生型和突变型LOV结构域中以5-deazaFMN为发色团的光加合物形成的结构特征。

Deazaflavins are important analogues of the naturally occurring flavins: riboflavin, flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD). The use of 5-deazaflavins as replacement coenzymes in a number of flavoproteins has proven particularly valuable in unraveling and manipulating their reaction mechanisms. It was frequently observed that one-electron-transfer reactions in flavoproteins are impeded with 5-deazaflavin as cofactor. Based on these findings, it was concluded that the 5-deazaflavin radical is significantly less stable compared to the respective flavin semiquinone, and quickly re-oxidizes or undergoes disproportionation. The long-standing paradigm of 5-deazaflavin being solely a two-electron / hydride acceptor / donor needs to be reevaluated given our recent success in (indirectly) observing a one-electron reduced (paramagnetic) species using photo-chemically induced dynamic nuclear polarization (photo-CIDNP) NMR. We propose characterizing the electronic structure of this radical by using electron paramagnetic resonance (EPR) derived hyperfine spectroscopies as well as photo-CIDNP NMR. A library of selectively stable-isotope (13C, 15N) labeled 5-deazaflavins required for this purpose will be synthesized and incorporated into various blue-light photoreceptor domains (LOV and BLUF) that are of interest in optogenetic applications. Using two-dimensional NMR, we plan to unravel structural traits of photo-adduct formation in wild-type and mutant LOV domains harboring 5-deazaFMN as chromophore.