Synthesis and photophysical studies of analogues of the GFP chromophore and epicocconone
thesisposted on 2022-03-28, 17:20 authored by Soumit Chatterjee
Epicocconone, isolated from the fungus Epicoccum nigrum, is only weakly green fluorescent in aqueous solution but emits brightly in the orange-red region in presence of amines due to reversible enamine formation. This makes it an attractive fluorescent sensor for proteins as lysine is one of the commonest amino acids. However, epicocconone has a low quantum yield (< 0.01) in water, increasing to a maximum of 0.17 in the presence of excess amine and detergent. Studying the fluorescent decays, an ultrafast component was found responsible for the non-radiative decay of epicocconone, explaining its relatively low quantum yield. This component was found to be only a minor contributor to the relaxation of the butylamine (a mimic of lysine residues of protein) adduct of epicocconone while accompanied by a rise time that extends the fluorescence decay to well over 2 ns, explaining the higher quantum yield in epicocconone-protein adduct. These observations indicated that photoisomerization or excited-state tautomerization to be the origin of the ultrafast nonradiative process. To have a better understanding of the excited state dynamics, four analogues were synthesized which reacted with amines in a similar fashion to epicocconone. Ultrafast dynamics of these analogues established the involvement of photoisomerization of the heptatriene side chain, rather than the tautomerism of the β-diketone, as the major nonradiative process in epicocconone. Two more analogues with much higher quantum yields were synthesized. In this set of compounds the native compounds were found to be an order of magnitude more fluorescent than epicocconone in neat solutions while the quantum yields of the respective butylamine adducts were found to be about the same as epicocconone-butylamine adducts. Our results suggested that both these compounds could be useful as synthetic alternatives to epicocconone for protein staining and as dual-stains in biotechnology. The isolated chromophore of green fluorescence protein (p-HBDI) is virtually non-fluorescent at room temperature, unlike the protein, due to photoisomerization. A series of GFP analogues were synthesized via oxazolone formation, which we found a superior method to published synthetic routes. One of the analogues exhibited dual emission at room temperature in water and was 500 times more fluorescent than p-HBDI. This unprecedented result suggests that GFP analogues of this type could be useful in applications where dual fluorescence or switchable fluorescence can be used such as ultra high resolution microscopy (e.g. STORM).