Solvent H-bond accepting ability induced conformational change and its influence towards fluorescence enhancement and dual fluorescence of hydroxy meta-GFP chromophore analogue

Abstract

The effect of structural rigidity towards enhancement of fluorescence quantum yield of GFP chromophore analogues has been documented. In the present study, a new way of enhancing the fluorescence quantum yield of two ortho-meta GFP chromophore analogues meta-methoxy-ortho-hydroxy-benzylimidazolidinone (abbreviated as mOMe-HBDI) and meta-diethylamino-ortho-hydroxyl imidazolidinone (abbreviated as MOHIM) has been reported. This enhancement is controlled by the H-bond accepting ability (denoted as β value) of the solvent and happens only in the case of GFP chromophore analogues having ortho (hydroxyl)-meta (electron donating group) and not in the case of analogues having a para electron donating group. The ground state (solid) conformation of mOMe-HBDI has been obtained from single crystal X-ray analysis, exhibiting the existence of strong intramolecular H-bonding. However, in solution phase, as the solvent β value increases, the strength of intramolecular H-bonding decreases. This process has strong influence on the relative conformational orientation of phenyl and imidazolidinone rings. For mOMe-HBDI, fluorescence quantum yield increases with increase in β value of the solvents. However, the effect of solvent polarity cannot be completely ruled out. The lower wavelength emission band (∼480 nm) has been assigned to the normal charge-transferred (CT) species, whereas the highly Stokes shifted emission band (∼660 nm) has been assigned to the proton-transferred (PT) tautomer species for mOMe-HBDI. In solvents of low β value (say hexane) only the PT band and in solvents of high β value (say DMSO) only the CT band is observed. Quite interestingly, in solvents of intermediate β value both CT and PT bands, thus dual emission, are observed. For mOMe-HBDI when fluorescence decay is monitored at the normal CT emission band, it is observed to be biexponential in nature. The short component increases from ∼0.2 ns to 0.6 ns and the long component increases from 1 to 3.6 ns as the β value of the solvent increases. For a particular solvent, when fluorescence decay is monitored at the normal CT band, as the monitoring wavelength increases the amplitude of the long lifetime component increases and that of the short lifetime component decreases. Time-resolved area-normalised emission spectral (TRANES) analysis confirms the possible existence of two conformers having differential stabilisation by solvent polarity. When fluorescence decay is monitored at the PT band an instrument response limited (<60 ps) decay is noted. Strong support in favour of the above-mentioned structural, steady state and time-resolved optical observations and analyses has been obtained from the methoxy derivative mOMe-MBDI and MOMIM.

Innovative Strategy Toward Red Emission: Single-Benzenic, Ultrasmallmeta-Fluorophores

“…Hence, using these novel MBRF s, WLE in solution as well as in solid state (films) could be achieved and our MBRF s have overcome a few major drawbacks shown by other fluorophores. , To the best of our knowledge, this is the first report of such versatile applications with small single-benzenic fluorophores. We believe that in single crystals, we will be able to achieve even more interesting application opportunities such as optoelectronics, waveguides, etc., employing MBRF s. Moreover, green fluorescent protein chromophore analogues, especially the meta -analogues, , need to be explored from the aspect of meta -fluorophores. Whether dual fluorescence can be observed and what could be the ultrafast dynamical reasons for the observations of dual fluorescence in meta -fluorophores need to be investigated in great detail.…”

“…We believe that in single crystals, 31 we will be able to achieve even more interesting application opportunities such as optoelectronics, waveguides, etc., employing MBRFs. Moreover, green fluorescent protein chromophore analogues, especially the meta-analogues, 32,33 need to be explored from the aspect of meta-fluorophores. Whether dual fluorescence 34 can be observed and what could be the ultrafast dynamical reasons 35 for the observations of dual fluorescence in metafluorophores need to be investigated in great detail.…”

Mechanism of Color and Photoacidity Tuning for the Protonated Green Fluorescent Protein Chromophore

“…This is in fact not that easy to test, because HBDI in solutions are fraught with side photoreactions such as photoisomerization and, to a much lesser extent, ESPT . However, for neutral HBDI analogues that suppress these side reactions, solvatofluorochromism ranges as wide as 95 nm (∼3700 cm –1 ) have been observed. − In contrast, absorption and emission maxima for GFP mutants that allow for significant A* emission by disrupting the ESPT chain (e.g., S205V, T203V/S205A, and deGFPs) are all fairly close to 400 and 460 nm, respectively, which could be due to the limited sampling of protein environments and/or the relatively poor ability for protein environments to reorganize as compared to simple solvents.…”

Meta-Fluors—A Unique Way To Create a 200 Da Ultrasmall Fluorophore Emitting in Red with Intense Stokes/Solvatochromic Shift: Imaging Subcellular Nanopolarity in Live Stem Cells

“…In i PrOH, excited-state lifetime of both DEAB and DEAMB are around 1 ns, however, in MeOH, the fluorescence decay falls below the instrument response function signifying the excited-state lifetime is less than 100 ps. Reduction of excited-state lifetime of fluorophores in hydroxylic solvents is quite well known in literature. − …”