Synthesis and Fluorescence Mechanism of the Aminoimidazolone Analogues of the Green Fluorescent Protein: Towards Advanced Dyes with Enhanced Stokes Shift, Quantum Yield and Two‐Photon Absorption

• Published in: European journal of organic chemistry 2021-11, Vol.2021 (41), p.5649-5660
• Main Authors: Kovács, Ervin, Cseri, Levente, Jancsó, Attila, Terényi, Ferenc, Fülöp, Anna, Rózsa, Balázs, Galbács, Gábor, Mucsi, Zoltán
• Format: Article
• Language: English
• Subjects: Bonding strength; Charge transfer; Chromophores; Emission spectra; Fluorescence; GFP analogues; Hydrogen bonding; Hydroxyl groups; Medical imaging; Photon absorption; Photons; Proteins; Protonation; Red shift; Stokes shift; Two-photon fluorescence
• ISSN: 1434-193X; 1099-0690
• DOI: 10.1002/ejoc.202101173

Novel small‐molecular analogues the green fluorescence protein (GFP) chromophore were synthesised to expand and improve this fluorophore family and to deepen the understanding of their fluorescence mechanism. The introduction of an aminophenyl substituent and the repositioning of the hydroxyl group, to enable strong intramolecular hydrogen bonding, not only enhanced fluorescence emission but also resulted in an increased Stokes shift and a considerable redshift. Experimental and computational results described dual fluorescence involving both excited‐state intramolecular proton transfer and internal charge transfer (ESIPT‐ICT) mechanisms. Screening of the pH and the solvent medium revealed a complicated equilibrium involving hydrogen bonding, protonation and deprotonation that influences the absorption and emission spectra. Further improvement of the photophysical properties via the systematic variation of dialkylamino substituents at a single position of the chromophore led to a two‐orders of magnitude enhancement in the quantum yields. In addition, the novel compounds also have significant two‐photon absorption, which widens the possibilities for applications in the field of bioimaging. Intramolecularly hydrogen‐bonded analogues of the green fluorescence protein chromophore with an aminoimidazole moiety were synthesised and characterised. The fluorescence mechanism and substituent effects were investigated in a combined experimental‐computational approach, and a 279‐fold improvement in the quantum yield was realised. The novel compounds exhibit promising two‐photon fluorescence for bioimaging applications.