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

各向同性和各向异性环境中 5-去氮黄素同位素体的制备和光谱研究

• 批准号: 459493567
• 负责人: Professor Dr. Markus Fischer
• 金额: --
• 依托单位: Institut für Physikalische Chemie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/459493567?language=en
• 关键词: Preparation; spectroscopic; studies; deazaflavin; isotopologues

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.

脱氮黄素是天然存在的黄素的重要类似物：核黄素、黄素单核苷酸（FMN）和黄素腺嘌呤二核苷酸（FAD）。在许多黄素蛋白中使用5-脱氮黄素作为替代辅酶已被证明在解开和操纵它们的反应机制方面特别有价值。经常观察到，黄素蛋白中的单电子转移反应被5-脱氮黄素作为辅因子阻碍。基于这些发现，可以得出结论，与相应的黄素半醌相比，5-脱氮黄素自由基的稳定性明显较低，并且快速地再氧化或经历反硝化。长期以来的范式5-deazaflavin仅仅是一个两个电子/氢化物受体/供体需要重新评估我们最近的成功（间接）观察一个电子减少（顺磁性）的物种，使用光化学诱导的动态核极化（光CIDNP）NMR。我们建议使用电子顺磁共振（EPR）衍生的超精细光谱，以及光CIDNP NMR表征这个自由基的电子结构。将合成为此目的所需的选择性稳定同位素（13 C，15 N）标记的5-脱氮黄素的文库，并将其掺入光遗传学应用中感兴趣的各种蓝光光感受器结构域（LOV和BLUF）中。使用二维NMR，我们计划解开野生型和突变体LOV域中的光加合物形成的结构特征窝藏5-deazaFMN作为发色团。