Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA, 92093-0358, USA.
Department of Chemistry, Ludwig-Maximilians-Universität München, Butenandtstr. 5-13, 81377, Munich, Germany.
Chemistry. 2019 Mar 21;25(17):4379-4389. doi: 10.1002/chem.201805520. Epub 2019 Feb 25.
Apart from its vital function as a redox cofactor, nicotinamide adenine dinucleotide (NAD ) has emerged as a crucial substrate for NAD -consuming enzymes, including poly(ADP-ribosyl)transferase 1 (PARP1) and CD38/CD157. Their association with severe diseases, such as cancer, Alzheimer's disease, and depressions, necessitates the development of new analytical tools based on traceable NAD surrogates. Here, the synthesis, photophysics and biochemical utilization of an emissive, thieno[3,4-d]pyrimidine-based NAD surrogate, termed N AD , are described. Its preparation was accomplished by enzymatic conversion of synthetic ATP by nicotinamide mononucleotide adenylyltransferase 1 (NMNAT1). The new NAD analogue possesses useful photophysical features including redshifted absorption and emission maxima as well as a relatively high quantum yield. Serving as a versatile substrate, N AD was reduced by alcohol dehydrogenase (ADH) to N ADH and afforded ADP-ribose ( ADPr) upon hydrolysis by NAD -nucleosidase (NADase). Furthermore, N AD was engaged in cholera toxin A (CTA)-catalyzed mono( ADP-ribosyl)ation, but was found incapable in promoting PARP1-mediated poly( ADP-ribosyl)ation. Due to its high photophysical responsiveness, N AD is suited for spectroscopic real-time monitoring. Intriguingly, and as an N7-lacking NAD surrogate, the thieno-based cofactor showed reduced compatibility (i.e., functional similarity compared to native NAD ) relative to its isothiazolo-based analogue. The distinct tolerance, displayed by diverse NAD producing and consuming enzymes, suggests unique biological recognition features and dependency on the purine N7 moiety, which is found to be of importance, if not essential, for PARP1-mediated reactions.
除了作为氧化还原辅助因子的重要功能外，烟酰胺腺嘌呤二核苷酸（NAD）已成为包括聚（ADP-核糖基）转移酶 1（PARP1）和 CD38/CD157 在内的 NAD 消耗酶的关键底物。它们与癌症、阿尔茨海默病和抑郁症等严重疾病的关联，需要基于可追踪的 NAD 类似物开发新的分析工具。在这里，描述了一种发光的噻吩并[3,4-d]嘧啶基 NAD 类似物，称为 NAD，其合成、光物理和生化利用。它的制备是通过烟酰胺单核核苷酸腺苷酰转移酶 1（NMNAT1）对合成的 ATP 进行酶促转化来完成的。这种新的 NAD 类似物具有有用的光物理特性，包括红移的吸收和发射最大值以及相对较高的量子产率。作为一种多功能的底物，NAD 被醇脱氢酶（ADH）还原为 NADH，并在 NAD-核苷酶（NADase）水解时提供 ADP-核糖（ADPr）。此外，NAD 参与霍乱毒素 A（CTA）催化的单（ADP-核糖基）化，但发现不能促进 PARP1 介导的聚（ADP-核糖基）化。由于其高光物理响应性，NAD 适合用于光谱实时监测。有趣的是，作为一种缺乏 N7 的 NAD 类似物，噻吩基辅因子相对于其异噻唑基类似物显示出较低的兼容性（即与天然 NAD 的功能相似性）。不同的 NAD 产生和消耗酶表现出的独特耐受性表明存在独特的生物学识别特征和对嘌呤 N7 部分的依赖性，如果不是必需的，这对于 PARP1 介导的反应很重要。
