Unravelling RNA-substrate interactions in a ribozyme-catalysed reaction using fluorescent turn-on probes.
Chemistry
; 21(15): 5864-71, 2015 Apr 07.
Article
em En
| MEDLINE
| ID: mdl-25753253
ABSTRACT
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%â
de and >99%â
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.
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01-internacional
Base de dados:
MEDLINE
Assunto principal:
Compostos de Boro
/
RNA Catalítico
/
Corantes Fluorescentes
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Antracenos
Idioma:
En
Ano de publicação:
2015
Tipo de documento:
Article