Synthesis and photochemistry of pH-sensitive GFP chromophore analogs (original) (raw)
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The Journal of Physical Chemistry B, 2016
Dual fluorescence of GFP chromophore analogues has been observed for the first time. OHIM (o-Hydroxy imidazolidinone) shows only Charge transfer (CT) band, CHBDI (p-Cyclicamino o-hydroxy benzimidazolidinone) shows comparable intensity CT and PT (proton transfer) band, and MHBDI (p-methoxy o-hydroxy benzimidazolidinone) shows higher intensity PT band. It could be shown that differential optical behaviour is not due to conformational variation in solid or in solution phase. Rather control of excited state electronic energy level and excited state acidity constant by functional group modification could be shown to be responsible for differential optical behaviour. Chemical modification induced electronic control over relative intensity of charge transfer and proton transfer bands could thus be evidenced. Support from single crystal X-ray structure, NMR, Femtosecond to nanosecond fluorescence decay analysis, TDDFT based calculation provided important information and thus helped us understand the photophysics better.
Chemically Modulating the Photophysics of the GFP Chromophore
The Journal of Physical Chemistry B, 2011
There is growing interest in engineering the properties of fluorescent proteins through modifications to the chromophore structure utilizing mutagenesis with either natural or unnatural amino acids. This entails an understanding of the photophysical and photochemical properties of the modified chromophore. In this work, a range of GFP chromophores with different alkyl substituents are synthesized and their electronic spectra, pH dependence, and ultrafast fluorescence decay kinetics are investigated. The weakly electron donating character of the alkyl substituents leads to dramatic red shifts in the electronic spectra of the anions, which are accompanied by increased fluorescence decay times. This high sensitivity of electronic structure to substitution is also characteristic of some fluorescent proteins. The solvent viscosity dependence of the decay kinetics are investigated, and found to be consistent with a bimodal radiationless relaxation coordinate. Some substituents are shown to distort the planar structure of the chromophore, which results in a blue shift in the electronic spectra and a strong enhancement of the radiationless decay. The significance of these data for the rational design of novel fluorescent proteins is discussed.
Cis-trans photoisomerization properties of GFP chromophore analogs
European Biophysics …, 2011
The photoswitching behaviour of the green fluorescent protein (GFP) chromophore and its analogs opens up exciting horizons for the engineering and development of molecular devices for high sensitivity in vivo studies. In this work we present the synthesis and photophysical study of four GFP chromophore analogs belonging to butenolide and pyrrolinone classes. These chromophores possess an intriguing photoinduced cis– trans isomerization mechanism. Stereochemical structural assignment was unambiguously performed by 1D Nuclear Overhauser Effect NMR measurements. The spectroscopic properties of both cis and trans isomers were studied, and photoconversion quantum yield for cis–trans isomerization was assessed to be in the 0.1–0.4 range. Finally, the 3JC,H coupling constant in the 13C–C=C–H motif was in excellent agreement with theoretical DFT calculations, thus providing a further confirmation of cis–trans photoisomerization of the structurally analog GFP chromophore.
Enhancement for the fluorescent properties of new synthesized GFPs chromophore
The Egyptian Journal of Chemistry, 2021
An alternative method for the synthesis of new GFP chromophore analogous that possess good photo physical properties which can be used as efficient biomarkers. These compounds have low fluorescence most probably due to their Z–E photo isomerization. These chromophores have suitable site for complexation with transitional metals. Forming complexes with metal ions can inhibit their photo isomerization and enhance the fluorescent properties.
Fluorescent Derivatives of the GFP Chromophore Give a New Insight into the GFP Fluorescence Process
Biophysical Journal, 2003
The photophysical properties of synthetic compounds derived from the imidazolidinone chromophore of the green fluorescent protein were determined. Various electron-withdrawing or electron-donating substituents were introduced to mimic the effect of the chromophore surroundings in the protein. The absorption and emission spectra as well as the fluorescence quantum yields in dioxane and glycerol were shown to be highly dependent on the electronic properties of the substituents. We propose a kinetic scheme that takes into account the temperature-dependent twisting of the excited molecule. If the activation energy is low, the molecule most often undergoes an excited-state intramolecular twisting that leads it to the ground state through an avoided crossing between the S 1 and S 0 energy surfaces. For a high activation energy, the torsional motion within the compounds is limited and the ground-state recovery will occur preferentially by fluorescence emission. The excellent correlation between the fluorescence quantum yields and the calculated activation energies to torsion points to the above-mentioned avoided crossing as the main nonradiative deactivation channel in these compounds. Finally, our results are discussed with regard to the chromophore in green fluorescent protein and some of its mutants.
Fluorescence imaging using synthetic GFP chromophores
Current opinion in chemical biology, 2015
Green fluorescent protein and related proteins carry chromophores formed within the protein from their own amino acids. Corresponding synthetic compounds are non-fluorescent in solution due to photoinduced isomerization of the benzylideneimidiazolidinone core. Restriction of this internal rotation by binding to host molecules leads to pronounced, up to three orders of magnitude, increase of fluorescence intensity. This property allows using GFP chromophore analogs as fluorogenic dyes to detect metal ions, proteins, nucleic acids, and other hosts. For example, RNA aptamer named Spinach, which binds to and activates fluorescence of some GFP chromophores, was proved to be a unique label for live-cell imaging of specific RNAs, endogenous metabolites and target proteins. Chemically locked GFP chromophores are brightly fluorescent and represent potentially useful dyes due to their small size and high water solubility.
Dyes and Pigments, 2013
The synthesis, chemical characterization and optical studies of three novel fluorene derivatives is reported. These compounds comprise a DeAeD architecture with fluorene moieties as donor groups and fluorenone or benzothiadiazole derivatives as acceptor groups. Theoretical analysis confirmed the existence and the nature of two principal electronic transitions (p / p* and intramolecular charge transfer). Spectroscopic studies in solution revealed that the intramolecular charge transfer character, and in turn the two-photon activity i.e., the fluorescence induced by two-photon absorption, is strongly affected by solvent polarity. The influence of specific solventesolute interactions over emission properties was also studied through LipperteMataga plot. Evaluation of the two-photon absorption cross-sections, gave a maximum value of 105 GM (1 GM ¼ 10 À50 cm 4 s) in toluene and a minimum value of 23 GM in THF solutions at 750 nm for the fluorenone derivative, a molecule with poor intramolecular charge transfer character and thus weak two-photon absorption; however the benzothiadiazole derivative, with stronger intramolecular charge transfer transition, produced a maximum value of 1000 GM in THF and a minimum value of 236 GM in methanol. Fluorescence quantum efficiency of these compounds was also affected by the medium, with fluorescence quenching in protic solvents such as methanol due to specific solvent interactions (i.e., hydrogen-bonding). Nevertheless, in a more polar medium such as water, nanoaggregates synthesized from the benzothiadiazole derivative exhibited good two-photon activity, i.e., w500 GM and fluorescence quantum efficiency of 0.83. Furthermore, these nanoaggregates exhibited more resistance against photodegradation processes than any of the organic solutions tested.