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Unravelling RNA–Substrate Interactions in a Ribozyme‐Catalysed Reaction Using Fluorescent Turn‐On Probes
Author(s) -
Gaffarogullari Ece Cazibe,
Greulich Peter,
Kobitski Andrei Yu.,
Nierth Alexander,
Nienhaus G. Ulrich,
Jäschke Andres
Publication year - 2015
Publication title -
chemistry – a european journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.687
H-Index - 242
eISSN - 1521-3765
pISSN - 0947-6539
DOI - 10.1002/chem.201406512
Subject(s) - chemistry , anthracene , fluorescence , ribozyme , stereoselectivity , catalysis , cyclopentene , stereochemistry , photochemistry , reaction mechanism , substrate (aquarium) , maleimide , rna , organic chemistry , biochemistry , physics , quantum mechanics , gene , oceanography , geology
Abstract The Diels–Alder reaction is one of the most important CC 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.