Synthesis and properties of the red chromophore of the green-to-red photoconvertible fluorescent protein Kaede and its analogs

Green fluorescent protein (GFP) and homologous proteins possess a unique pathway of chromophore formation based on autocatalytic modification of their own amino acid residues. Green-to-red photoconvertible fluorescent protein Kaede carries His–Tyr–Gly chromophore-forming triad. Here, we describe synthesis of Kaede red chromophore (2-[(1E)-2-(5-imidazolyl)ethenyl]-4-(p-hydroxybenzylidene)-5-imidazolone) and its analogs that can be potentially formed by natural amino acid residues. Chromophores corresponding to the following tripeptides were obtained: His–Tyr–Gly, Trp–Tyr–Gly, Phe–Trp–Gly, Tyr–Trp–Gly, Asn–Tyr–Gly, Phe–Tyr–Gly, and Tyr–Tyr–Gly. In basic conditions they fluoresced red with relatively high quantum yield (up to 0.017 for Trp-derived compounds). The most red-shifted absorption peak at 595 nm was found for the chromophore Trp–Tyr–Gly in basic DMSO. Surprisingly, in basic DMF non-aromatic Asn-derived chromophore Asn–Tyr–Gly demonstrated the most red-shifted emission maximum at 642 nm. Thus, Asn residue may be a promising substituent, which can potentially diversify posttranslational chemistry in GFP-like proteins.