title: Unravelling RNA-substrate interactions in a ribozyme-catalysed reaction using fluorescent turn-on probes.
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% 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.
authors.affiliation: Institute of Pharmacy and Molecular Biotechnology, Heidelberg University, Im Neuenheimer Feld 364, 69120 Heidelberg (Germany), Fax: (+49) 6221-54-6430.
authors.forename: Ece Cazibe
authors.initials: EC
authors.lastname: Gaffarogullari
authors.forename: Peter
authors.initials: P
authors.lastname: Greulich
authors.forename: Andrei Yu
authors.initials: AY
authors.lastname: Kobitski
authors.forename: Alexander
authors.initials: A
authors.lastname: Nierth
authors.forename: G Ulrich
authors.initials: GU
authors.lastname: Nienhaus
authors.forename: Andres
authors.initials: A
authors.lastname: Jäschke
mesh_terms: D000873:Anthracenes / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism*; D001896:Boron Compounds / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism*; D002384:Catalysis; D061565:Cycloaddition Reaction; D005456:Fluorescent Dyes / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D015335:Molecular Probes / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism; D016337:RNA, Catalytic / Q000378:metabolism*; D013237:Stereoisomerism; D013379:Substrate Specificity
chemical_list: C095489:4,4-difluoro-4-bora-3a,4a-diaza-s-indacene; D000873:Anthracenes; D001896:Boron Compounds; D005456:Fluorescent Dyes; D015335:Molecular Probes; D016337:RNA, Catalytic; C034020:anthracene
keywords: cycloaddition; enantioselectivity; fluorescent probes; reaction mechanisms; ribozymes