Non-trivial ground and excited state photophysics of a substituted phenol.

5-(tert-Butyl)-2-hydroxy-1,3-isophthalaldehyde (5-tBHI) shows solvent dependent single or dual emission. The photophysics of 5-tBHI has been studied in a variety of solvents and the results were compared with that of the methyl derivative of the probe as well as the 5-tBHI anion. It has been found that the intramolecular H-bonded conformer of 5-tBHI predominantly exists in non-polar solvents, and undergoes facile excited state intramolecular proton transfer (ESIPT). On the other hand, a dynamic equilibrium can be found in polar, protic solvents, even in the ground state, except in water. NMR analyses confirm the loss of aromaticity of the probe in the ground state via anion formation, in equilibrium with the solvent mediated intermolecularly H-bonded state, in neat polar protic solvents like methanol. The proton transfer process, either intramolecularly or intermolecularly, was found to be of the order of 1 ps, and even faster than the instrumental resolution in the case of water. The current finding provides important insights on the photophysics of this small, substituted phenol.

Room-Temperature Dual Fluorescence of a Locked Green Fluorescent Protein Chromophore Analogue.

A structurally locked green fluorescent protein (GFP) chromophore with a phenyl group at C(2) of the imidazolone has been synthesized. Rotation around the exocyclic double bond is hindered, resulting in room-temperature fluorescence. The quantum yield in water is 500 times greater than that of unlocked analogues. Unlike the methyl-substituted analogue, the phenyl analogue exhibits a dual emission (cyan and red) that can be used for ultrasensitive ratiometric measurements and fluorescence microscopy. To explain this dual emission, DFT calculations were carried out along with fluorescence upconversion experiments. The Z-isomer was found to be emissive, while the origin of the dual emission was dependent on the phenyl group in the Z-isomer, which stabilizes the Franck-Condon state, resulting in a cyan fluorescence, while the zwitterionic tautomer fluoresces red. These results bring important new insights into the photophysics of the GFP chromophore and provide a new scaffold capable of dual emission with utility in biotechnology.

Unusual absence of FRET in triazole bridged coumarin-hydroxyquinoline, an active sensor for Hg2+ detection.

A triazole-bridged coumarin conjugated quinoline sensor has been 'click'-synthesized by Cu(i) catalyzed Huisgen cycloaddition, and it exhibited high selectivity for toxic Hg2+. Surprisingly, no evidence of energy transfer from the quinoline moiety to coumarin has been found, substantiated by time-resolved fluorescence study. The possible binding mode of this sensor to Hg2+ has been established via NMR study, steady-state and time-resolved fluorescence spectroscopy, which is further supported by TDDFT calculations. The sensor has been found to be cell membrane permeable and non-toxic, and hence is suitable for intracellular Hg2+ detection.

Aggregation-Induced Modulation of Ground and Excited State Photophysics of 5-(tert-Butyl)-2-Hydroxy-1,3-Isophthalaldehyde (5-tBHI).

5-(tert-Butyl)-2-hydroxy-1,3-isophthalaldehyde (5-tBHI) is a photochromic material susceptible to either excited state proton transfer or excited state intramolecular proton transfer, depending upon the solvent. However, it has also been found to aggregate in the presence of sodium dodecyl sulfate. In this current study, based on the steady-state and time-resolved spectroscopy, supported by crystallography, quantum chemical density functional theory calculation, and molecular dynamics (MD) simulation, we report on the aggregation of this potential single benzene-based emitter (SBBE) in neat solvents as well as solid phase to modulate its photophysics. It has been found that 5-tBHI forms mixed aggregates of different orders, owing to the presence of both enolic and tautomeric forms, to yield tunable emission, although the emission intensity is quenched. These findings suggest that the intramolecular hydrogen bonding of 5-tBHI not only limits intermolecular interactions but also promotes nonradiative deactivation pathways. Hence, designing and structural engineering, with a focus to suppressing intramolecular hydrogen bonding as well as increasing through space conjugation by replacing the aldehydic moieties with bulky aliphatic or aromatic ketonic groups, can be a plausible approach to yielding improved probes with tunable emission and higher fluorescence quantum yields.

Ultrafast dynamics of epicocconone, a second generation fluorescent protein stain.

Femtosecond upconversion experiment has been carried out for epicocconone and its butylamine adduct in acetonitrile and tert-butanol. An ultrafast component is found to dominate the decay of fluorescence of epicocconone in acetonitrile solution. Upon reacting with butylamine, a model for the epicocconone-protein adduct, this ultrafast component remains almost unaffected but an additional rise time occurs, indicating the formation of a highly emissive species from the locally excited state. This phenomenon is central to the extraordinary applications of epicocconone in biotechnology. The magnitude of the rise time of the butylamine adduct is similar to that of the longer component of the decay of epicocconone in acetonitrile, suggesting that the dynamics of epicocconone and its butylamine adduct are similar. The ultrafast component is slowed upon increasing the viscosity of the solvent. This results in a marked increase in quantum yield and suggests that it corresponds to rapid bond isomerization, leading to a nonradiative decay. Surprisingly, in water/sucrose mixtures, the ultrafast component remains unaffected but there is still an increase in quantum yield, suggesting that there are at least two nonradiative pathways, one involving bond isomerization and another involving proton transfer. The correct interpretation of these data will allow the design of second generation protein stains based on the epicocconone scaffold with increased quantum yields and photostability.

Excited state dynamics of brightly fluorescent second generation epicocconone analogues.

The natural product epicocconone, owing to its unique fluorescence properties, has been developed into a range of products used in biotechnology, especially proteomics. However, its weak green fluorescence in its native state, while advantageous for proteomics applications, is a disadvantage in other applications that require two-color readouts. Here we report the photophysical characterization of two brightly fluorescent analogues of epicocconone. These analogues, with naphthyl or pyridyl groups replacing the heptatriene chain, resulted in bright fluorescence in both the native state and the long Stokes shifted enamine. Time-resolved fluorescence studies and DFT calculations were carried out to understand the excited state processes involved in fluorescence. Results showed the p-chloro group on the pyridyl is responsible for the high fluorescence of the native fluorophore. The application of one of these compounds for staining electrophoresis gels is exemplified.

The role of different structural motifs in the ultrafast dynamics of second generation protein stains.

Engineering the properties of fluorescent probes through modifications of the fluorophore structure has become a subject of interest in recent times. By doing this, the photophysical and photochemical properties of the modified fluorophore can be understood and this can guide the design and synthesis of better fluorophores for use in biotechnology. In this work, the electronic spectra and fluorescence decay kinetics of four analogues of the fluorescent natural product epicocconone were investigated. Epicocconone is unique in that the native state is weakly green fluorescent, whereas the enamine formed reversibly with proteins is highly emissive in the red. It was found that the ultrafast dynamics of the analogues depends profoundly on the H-bonding effect of solvents and solvent viscosity though solvent polarity also plays a role. Comparing the steady state and time-resolved data, the weak fluorescence of epicocconone in its native state is most likely due to the photoisomerization of the hydrocarbon side chain, while the keto enol moiety also has a role to play in determining the fluorescence quantum yield. This understanding is expected to aid the design of better protein stains from the same family.

Role of spacer in single- or two-step FRET: studies in the presence of two connected cryptands with properly chosen fluorophores.

Two cryptand molecules are connected via p-xyloyl, benzene-1,4-dicarbolyol and 9,10-dimethylene anthracene. Each cryptand is further derivatized with fluorophores such that electronic absorption of one fluorophore overlaps emission of the other. This way, three different systems L(1), L(2) and L(3) have been synthesized to get a better view of the effect on the distance in single- and two-step fluorescence resonance energy transfer (FRET) process. These molecules are probed for FRET in the presence of a metal ion as input. L(1) and L(2) exhibit poor performance in single step FRET while in the case of L(3), a large two-step FRET process is operational with Cu(II) or Hg(II) as input.