b. Department of Chemistry, The Pennsylvania State University, University Park, PA 16802
Fluorescent proteins (FP) have been widely used as genetic tags and bio-markers to provide spatial and temporal information of a protein-of-interest in live cells and animals . Fluorescence of these proteins is activated by their chromophore, a chemical moiety that is generated via a series of chemical reactions within a folded FP . For example, the chromophore of green fluorescent protein (GFP) is formed by side chains of three adjacent amino acids (Serine 67, Tyrosine 68, and Glycine 69). During the maturation process, the GFP chromophore forms a 4-hydroxybenzylidene-imidazolinone (HBI) structure via a series of chemical reactions, including cyclization, oxidation, and dehydration. Structural variations of HBI lead to new FPs with diverse photo-physical properties, including excitation/emission spectra, quantum yield, photo stability, and photo switchability . These FPs have enabled a wide range of biological applications in various cell types and organisms.
A new line of application inspired by the FP chromophores is fluorogenic detection (i.e., detection with turn-on fluorescence) of analysts both in test tubes and living cells. This is made possible because most of the FP chromophores as primarily HBI analogues become non-fluorescent when they are synthesized as small molecules and characterized in diluted solvents [4, 5]. In test tubes, the fluorescence is re-activated when synthetic HBI analogues interact with supra-molecular hosts , metal-organic framework , aggregation-induced emission  and protein hosts . In living cells, HBI analogues, represented by represented by 3, 5-difluoro-4-hydroxybenzylidene imid-azolinone (DFHBI), have been used to visualize RNA aptamers , DNA quadruplex , and more recently the detection of protein aggregation .
In particular, GFP analogues recently emerge to enable fluorogenic detection of biomolecules in living cells [11, 12]. Compared to commonly used HBI analogues, RFP analogues harbor an extended moiety at the C2 position of the imidazolinone group (Ib position in left panel, FIG. 1(a)). In the case of the photo-converted Kaede fluorescent protein , an imidazole is connected via a stilbene linker to the C2 of imidazolinone. Derivatives of this scaffold have been employed to enable fluorogenic imaging of DNA quadruplex  and protein aggregates . Given that the Kaede scaffold is increasingly used to develop novel fluorescent probes, we envision that the incorporation of heterocycles to the Ia and Ib positions of this scaffold bears the potential to give rise to novel photochemical properties. In this study, we have synthesized a new molecule wherein indoles were introduced to both the Ia and Ib positions, yielding
All electronic structure calculations were carried out using DFT or the linear response time dependent DFT method at the level of B3LYP/6-31+G(d, p) [14, 15] as implemented in the Gaussian computational chemistry software . Geometries of various stereoisomers of TI3C (FIG. 1(a)) were fully optimized both in the gas phase and in an effective solvent model on the ground- and excited-state. The solvent effect of glycerol was included using the polarizable continuum model (PCM) [17-25] with dielectric constants  (
Commercial grade reagents and anhydrous solvents were used as received unless otherwise stated. Reactions were monitored via thin layer chromatography (TLC) analysis using Silicycle glass sheets precoated with silica gel 60 with detection by UV-absorption (254 nm or 365 nm). Flash column chromatography was performed using Silica Flash F60 silica gel in the indicated solvent mixture.
The synthesis steps for TI3C is shown in FIG. 2.
Condition a: glycine tert-butyl ester hydrochloride (1.1 eq) and NaOH (1.0 eq) were stirred in EtOH for 1 h at room temperature, aldehyde (1.0 eq) was added and stirred overnight. The next day the imidate (1.0 eq) was prepared, added in one portion. The reaction was again stirred overnight, then quenched by water and extracted with DCM. The organic fraction was collected and dried in vacuo. Compounds were further purified by ash chromatography (1:1, ethyl acetate/hexanes) to yield compound 1.
Condition b: aldehyde (2.0 eq), 1 (1.0 eq) were combined in dioxane under Argon. ZnCl
Bridged by CC single/double bonds, the three aromatic rings of TI3C (FIG. 1(a)) constitute a planar
FIG. 4 shows five lowest energy TI3C isomers and their Boltzmann weights at the room temperature,
Two types of electronic transitions, absorption and emission, are considered in this work and shown in FIG. 5, and their characteristics are shown in Table Ⅱ. Vertical absorption spectra were computed using the ground state geometry (S
Spectra of isomer 1 are of a higher oscillator strength than those of 2. As indicated in Table Ⅳ, this is a result of a decrease in transition dipole moment upon isomerization from 1 to 2. For both isomers 1 and 2, the spectral intensity increases significantly in glycerol compared to that in vacuum also due to the increase in transition dipole moment. FIG. 7 shows the electron density difference
Spectral Stokes shifts in vacuum are
FIG. 9 depicts the potential energy surfaces of S
In this study, we presented a theoretical study of the bright TI3C molecule, a Kaede RFP-like chromophore. Thermodynamic isomerization pathways were investigated and statistical distributions of stable isomers were analyzed. The two most stable isomers were selected for photochemical studies. Computed spectral Stokes were in excellent agreement with experiments. An inverted solvatochromic shift of TI3C in glycerol between absorption and emission was observed. Potential energy surface and population analyses suggest that the inverted solvatochromic shift is due to the inhibition of rotational structural reorganization arising from stabilized excited state charge transfer. This work lays the theoretical groundwork for designing RFP-like chromophore with extended conjugation to acquire desired photo-physical properties.Ⅴ. ACKNOWLEDGMENTS
This work was supported by US National Science Foundation (CHE-1565520) to X. Li, Burroughs Wellcome Fund Career Award at the Scientific Interface to X. Zhang, Paul Berg Early Career Professorship to X. Zhang, Lloyd and Dottie Huck Early Career Award to X. Zhang, and the Sloan Research Fellowship to X. Zhang. This work was facilitated through the use of advanced computational, storage, and networking infrastructure provided by the Hyak supercomputer system and funded by the STF at the University of Washington and the National Science Foundation (MRI-1624430).
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b. 美国宾夕法尼亚州立大学化学系, 宾夕法尼亚 16802