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A New Robust and Highly Sensitive FRET Donor–Acceptor Pair: Synthesis, Characterization, and Application in a Thrombin Assay
Author(s) -
Kainmüller Eva K.,
Bannwarth Willi
Publication year - 2006
Publication title -
helvetica chimica acta
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.74
H-Index - 82
eISSN - 1522-2675
pISSN - 0018-019X
DOI - 10.1002/hlca.200690275
Subject(s) - chemistry , förster resonance energy transfer , acceptor , fluorescence , photochemistry , chromophore , ruthenium , covalent bond , peptide , catalysis , organic chemistry , biochemistry , physics , quantum mechanics , condensed matter physics
The synthesis of a new, robust fluorescence‐resonance‐energy‐transfer (FRET) system is described. Its donor chromophore is derived from an N ‐allyl‐substituted quinolinone attached to 4‐bromophenylalanine via Heck cross‐coupling. The resulting Fmoc‐protected derivative 11 was used as building block in solid‐phase peptide synthesis (SPPS). As FRET acceptor, a sulfonylated ruthenium(II)–bathophenanthroline complex with a peripheral COOH function was prepared for covalent attachment to target molecules. The UV/VIS absorption and emission spectra of peptides bearing only the donor (D) or acceptor (A) dye showed a good overlap of the emission band of the donor with the absorption band of the acceptor. The fluorescence spectra of a peptide bearing both dyes revealed an additional emission after excitation of the donor, which is due to indirect excitation of the acceptor via FRET. The long fluorescence lifetime of the Ru II complex (0.53 μs) makes it well‐suited for time‐resolved measurements. As a first application of this new FRET system, the peptide 18 , with the recognition sequence for the protease thrombin, flanked by the two dyes, was synthesized and successfully cleaved by the enzyme. The change in the ratio of the fluorescence intensities could be determined.