Gaffarogullari Ece Cazibe, Greulich Peter, Kobitski Andrei Yu, Nierth Alexander, Nienhaus G Ulrich, Jäschke Andres
Institute of Pharmacy and Molecular Biotechnology, Heidelberg University, Im Neuenheimer Feld 364, 69120 Heidelberg (Germany), Fax: (+49) 6221-54-6430.
Chemistry. 2015 Apr 7;21(15):5864-71. doi: 10.1002/chem.201406512. Epub 2015 Mar 4.
The Diels-Alder reaction is one of the most important C-C bond-forming reactions in organic chemistry, and much effort has been devoted to controlling its enantio- and diastereoselectivity. The Diels-Alderase ribozyme (DAse) catalyses the reaction between anthracene dienes and maleimide dienophiles with multiple-turnover, stereoselectivity, and up to 1100-fold rate acceleration. Here, a new generation of anthracene-BODIPY-based fluorescent probes was developed to monitor catalysis by the DAse. The brightness of these probes increases up to 93-fold upon reaction with N-pentylmaleimide (NPM), making these useful tools for investigating the stereochemistry of the ribozyme-catalysed reaction. With these probes, we observed that the DAse catalyses the reaction with >91% de and >99% ee. The stereochemistry of the major product was determined unambiguously by rotating-frame nuclear Overhauser NMR spectroscopy (ROESY-NMR) and is in agreement with crystallographic structure information. The pronounced fluorescence change of the probes furthermore allowed a complete kinetic analysis, which revealed an ordered bi uni type reaction mechanism, with the dienophile binding first.
狄尔斯-阿尔德反应是有机化学中最重要的碳-碳键形成反应之一，人们在控制其对映选择性和非对映选择性方面付出了诸多努力。狄尔斯-阿尔德酶核酶（DAse）能催化蒽二烯和亲双烯体马来酰亚胺之间的反应，具有多次周转、立体选择性，且反应速率能加速高达1100倍。在此，开发了新一代基于蒽-硼二吡咯的荧光探针来监测DAse的催化作用。这些探针与N-戊基马来酰亚胺（NPM）反应后，亮度增加高达93倍，使其成为研究核酶催化反应立体化学的有用工具。使用这些探针，我们观察到DAse催化反应的非对映体过量（de）>91%，对映体过量（ee）>99%。通过旋转框架核Overhauser核磁共振光谱（ROESY-NMR）明确确定了主要产物的立体化学，且与晶体结构信息一致。此外，探针明显的荧光变化使得进行完整的动力学分析成为可能，该分析揭示了一种有序的双分子单底物类型反应机制，亲双烯体先结合。
