Synthesis and photophysical studies of analogues of the GFP chromophore and epicocconone

Thesis by publication.Includes bibliographical references.Chapter 1.Introduction -- Chapter 2. Review on epicocconone -- Chapter 3. Review on green flourescent protein chromophore -- Chapter 4. Materials and methods -- Conclusions.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).Mode of access: World wide web1 online resource (118 pages) illustrations (some colour

An Efficient and concise method to synthesize locked GFP chromophore analogues

A series of GFP analogues, which are fluorescent in the solid state at room temperature, but weakly fluorescent in solution, have been synthesized via an oxazolone formation process that involves a condensation reaction in the presence of a Lewis acid following a Knoevenagel condensation. A ring opened intermediate is formed which cyclizes readily upon heating to produce the imidazolinone. This method is faster, simpler and produces higher yields than alternative methods. A few analogues represent locked GFP derivatives where the exocyclic single bond rotation has been stopped. Weak fluorescence, even after stopping single bond rotation, indicates that restriction of conformation is not effectively controlled and that the double bond rotation is solely responsible for the major non-radiative pathway.4 page(s

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.5 page(s

Ultrafast dynamics of the fluorescent natural product epicocconone

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Excited State Dynamics of Brightly Fluorescent Second Generation Epicocconone Analogues

International audienceThe 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.

International audienceEngineering 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 Dispersive Fluorous Interaction in the Solvation Dynamics of the Perfluoro Group Containing Molecules

Perfluoro group containing molecules possess an important self-aggregation property through the fluorous (F···F) interaction which makes them useful for diverse applications such as medicinal chemistry, separation techniques, polymer technology, and biology. In this article, we have investigated the solvation dynamics of coumarin-153 (C153) and coumarin-6H (C6H) in ethanol (ETH), 2-fluoroethanol (MFE), and 2,2,2-trifluoroethanol (TFE) using the femtosecond upconversion technique and molecular dynamics (MD) simulation to understand the role of fluorous interaction between the solute and solvent molecules in the solvation dynamics of perfluoro group containing molecules. The femtosecond upconversion data show that the time scales of solvation dynamics of C6H in ETH, MFE, and TFE are approximately the same whereas the solvation dynamics of C153 in TFE is slow as compared to that of ETH and MFE. It has also been observed that the time scale of solvation dynamics of C6H in ETH and MFE is higher than that of C153 in the same solvents. MD simulation results show a qualitative agreement with the experimental data in terms of the time scale of the slow components of the solvation for all the systems. The experimental and simulation studies combined lead to the conclusion that the solvation dynamics of C6H in all solvents as well as C153 in ETH and MFE is mostly governed by the charge distribution of ester moieties (CO and O) of dye molecules whereas the solvation of C153 in TFE is predominantly due to the dispersive fluorous interaction (F···F) between the perfluoro groups of the C153 and solvent molecules

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