Chinese Journal of Chemical Physics  2018, Vol. 31 Issue (4): 599-607

The article information

Hang Hu, Charles H. Wolstenholme, Xin Zhang, Xiaosong Li
胡航, Wolstenholme Charles H., 张鑫, 李晓松
Inverted Solvatochromic Stokes Shift in GFP-like Chromophores with Extended Conjugation
具有延长共轭的绿色发光蛋白生色团的反向溶剂化斯托克斯(Stokes)位移
Chinese Journal of Chemical Physics, 2018, 31(4): 599-607
化学物理学报, 2018, 31(4): 599-607
http://dx.doi.org/10.1063/1674-0068/31/cjcp1806160

Article history

Received on: June 26, 2018
Accepted on: July 7, 2018
Inverted Solvatochromic Stokes Shift in GFP-like Chromophores with Extended Conjugation
Hang Hua, Charles H. Wolstenholmeb, Xin Zhangb, Xiaosong Lia     
Dated: Received on June 26, 2018; Accepted on July 7, 2018
a. Department of Chemistry, University of Washington, Seattle, WA 98195;
b. Department of Chemistry, The Pennsylvania State University, University Park, PA 16802
Author: Xin Zhang (right) is an assistant professor in the Department of Chemistry and Department of Biochemistry and Molecular Biology at the Pennsylvania State University, University Park. He currently holds the Paul Berg Early Career Professorship at the Eberly College of Science at Penn State. Professor Zhang5s research is focused on chemical biology of protein misfolding and aggregation in live cells and organisms. Prior to joining the faculty at Penn State, Professor Zhang was a Helen Hay Whitney Postdoctoral Fellow at the Scripps Research Institute, California. He earned a bachelor's degree at the University of Science and Technology of China in 2001, a master's degree at the Dalian Institute of Chemical Physics in 2004, and a doctoral degree at the California Institute of Technology in 2010. His achievements have been recognized by awards and honors including the Sloan Research Fellowship in 2018, the Lloyd and Dottie Huck Early Career Award in 2015, the Burroughs Wellcome Fund Career Award at the Scientific Interface in 2014, the American Chemical Society Nobel Laureate Signature Award for Graduate Education in Chemistry in 2012, the Helen Hay Whitney Fellowship Award in 2011, and the Herbert Newby McCoy Award in 2010;
Xiaosong Li (left) is the Harry and Catherine Jaynne Boand Endowed Professor of Chemistry at the University of Washington. His research focuses on developing and applying time-dependent relativistic and nonrelativistic electronic structure theories for studying excited state chemical processes that underpin energy conversion, photocatalysis and ultrafast spectroscopies. After completing his undergraduate studies at the University of Science and Technology of China in 1999, Li attended graduate school and received his Ph.D in 2003 from the Wayne State University where he worked with Professor Bernhard Schlegel. He pursued his postdoctoral work at the Yale University with Prof. John C. Tully on nonadiabatic electron-nuclear dynamics. Li joined the University of Washington as Assistant Professor in the fall of 2005 and was promoted to Associate Professor in 2011 and to full Professor in 2015. He has received awards that include the Sloan Research Fellowship, NSF CAREER Award, and ACS Open-Eye Outstanding Junior Faculty Award.
*Author to whom correspondence should be addressed. Xin Zhang, E-mail:xuz31@psu.edu; Xiaosong Li, E-mail:xsli@uw.edu
Part of the special issue for celebration of "the 60th Anniversary of University of Science and Technology of China and the 30th Anniversary of Chinese Journal of Chemical Physics"
Abstract: Chromophore structures inspired by natural green fluorescent protein (GFP) play an important role in the field of bio-imaging. In this work, photochemical properties of a new class of GFP-like chromophores are investigated using computational approaches. Thermodynamically stable isomers are identified in vacuum and in solvent. Spectral Stokes shifts are computed and compared to experiments. An inverted solvatochromic shift between absorption and emission emerging in this new class of GFP-like chromophores is observed, and attributed to the stabilized charge transfer and inhibited rotational structural reorganization in solvent.
Key words: Green fluorescent protein    Solvatochromic shift    Stokes shift    Time-dependent density functional theory    Polarizable continuum model    
Ⅰ. INTRODUCTION

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 [1]. 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 [2]. 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 [3]. 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 [6], metal-organic framework [7], aggregation-induced emission [8] and protein hosts [9]. In living cells, HBI analogues, represented by represented by 3, 5-difluoro-4-hydroxybenzylidene imid-azolinone (DFHBI), have been used to visualize RNA aptamers [10], DNA quadruplex [11], and more recently the detection of protein aggregation [12].

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 [13], 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 [11] and protein aggregates [12]. 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 $t$-butyl (2$E$, 4$Z$)-2-indolevinyl-4-indolidene-imidazolinone-1-carboxylate, (TI3C as shown in the right panel, FIG. 1(a)). Our theoretical results show that TI3C exchanges between five isomers, two of which are energetically favored. Based on the energetically most stable isomer, the calculated Stokes shift matches the experimental data, suggesting that this isomer is the most populated conformation at equilibrium. These results collectively reveal the bright state of fluorophores of the Kaede scaffold, providing guidance to future development of similar fluorophores.

FIG. 1 (a) Molecular structure of TI3C at the global minimum, and its six rotation coordinates. (b) Top- and side-view of TI3C ball-and-stick structure.
Ⅱ. METHODS A. Computational methods

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 [16]. 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 [26] ($\varepsilon_0$=42.82 and $\varepsilon_\infty$=4.0 at $T$=298.15 K). Vibrational frequency calculations confirm that all optimized geometries are true minima. Population analyses, including electron density difference profiles, transition dipole moment, and Mülliken atomic charge change, were also performed.

B. General synthetic and chromatographic methods

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. $^1$H NMR and $^{13}$C NMR spectra were obtained on a Bruker NMR (500/126 MHz) spectrometer in DMSO. The provided $^{13}$C spectra are protondecoupled. Chemical shifts are reported as $\sigma$-values in ppm relative to the DMSO residual solvent peak as an internal standard, all coupling constants are provided in Hz. High resolution mass spectra were recorded using a Waters Q-TOF Premier quadrupole/time-of-light (TOF) mass spectrometer.

C. Synthesis of TI3C

The synthesis steps for TI3C is shown in FIG. 2.

FIG. 2 Steps for synthesis of TI3C

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$_2$ (0.01 eq) was added and refluxed overnight. Solvent was removed and purified by ash chromatography (2:5, ethyl acetate/hexanes) to yield final compound 2.

1: ($Z$)-tert-butyl 2-(4-((1H-indol-3-yl)methylene)-2-methyl-5-oxo-4, 5-dihydro-1H-imidazol-1-yl)acetate. Yellow powder. $^1$H NMR (500 MHz, DMSO-d$_6$) 11.99 (s, 1H), 8.43 (d, $J$=2.8 Hz, 1H), 8.22 (d, $J$=7.7 Hz, 1H), 7.49 (d, $J$=7.9 Hz, 1H), 7.38 (s, 1H), 7.23 7.15 (m, 2H), 4.39 (s, 2H), 2.29 (s, 3H), 1.44 (s, 9H). $^{13}$C NMR (126 MHz, DMSO-d6) 169.21, 167.94, 158.57, 136.90, 133.64, 127.18, 123.06, 121.30, 120.92, 120.18, 112.69, 111.56, 82.48, 42.31, 28.08, 15.54. [M+H]$^+$: calculated 340.1583, observed 340.1873.

2: $tert$-butyl 2-(($Z$)-4-((1H-indol-3-yl)methylene)-2-(($E$)-2-(1H-indol-3-yl)vinyl)-5-oxo-4, 5-dihydro-1H-imidazol-1-yl)acetate. Orange-brown oil. $^1$H NMR (500 MHz, DMSO-d6) 12.02 (s, 1H), 11.84 (s, 1H), 8.60 (d, $J$=2.7 Hz, 1H), 8.27 (d, $J$=15.6 Hz, 1H), 8.20 (d, $J$=6.7 Hz, 1H), 8.08 7.96 (m, 2H), 7.59 7.46 (m, 2H), 7.34 (s, 1H), 7.31 7.16 (m, 4H), 6.76 (d, $J$=15.7 Hz, 1H), 4.65 (s, 2H), 1.42 (s, 9H). $^{13}$C NMR (126 MHz, DMSO-d6) 168.04, 157.09, 137.99, 136.90, 133.32, 127.32, 125.32, 123.21, 123.09, 121.30, 120.69, 119.94, 118.39, 113.89, 112.99, 112.72, 82.43, 42.27, 28.11. [M+H]$^+$: calculated 467.2005, observed 467.2083.

Ⅲ. RESULTS AND DISCUSSION A. Structural and thermodynamic characteristics of chromophore stereo-isomers

Bridged by CC single/double bonds, the three aromatic rings of TI3C (FIG. 1(a)) constitute a planar $\pi$-conjugated framework responsible for its photophysical properties. Rotations of the three rings along the five linkage bonds (dihedral angels $\phi_1$-$\phi_5$ in FIG. 1(a)) give rise to various stereochemical configurations, shown in FIG. 3. 14 configurations were initially considered, while others with strong steric effect were screened out. Among all configurations considered herein, isomer 1 (FIG. 2(a)) in both gas phase and glycerol solvent is thermodynamically most stable. All configurations except 14 are nearly planar both in vacuum and in glycerol, with rotation angles deviating less than 3$^{\circ}$ from the planar structure (see Table Ⅰ).

FIG. 3 Stereo-isomerizations of TI3C chromophore. Note that isomer 1 is at the global energy minimum.
Table Ⅰ Relative Gibbs free energies (in kcal/mol) with respect to that of isomer 1 and rotation angles (in degrees) of TI3C isomers. See FIG. 1(a) for definitions of rotation angles.

FIG. 4 shows five lowest energy TI3C isomers and their Boltzmann weights at the room temperature, $T$=298.15 K. Structural characteristics of all isomers in FIG. 4(a) are very similar, including a $trans$- C6=C7 bond and a $cis$- C4=C10 double bond except for 5, consistent with results from previous work [27-30]. The central framework (central aromatic ring plus three linkage carbon atoms) is relatively fixed in most of the TI3C isomers in glycerol, despite of the strong electron donating character of the extended conjugation (i.e. indolevinyl group). In the following sections, we will only focus on isomers 1 and 2 for spectroscopic and excited state analysis because they are the most abundant species at the room temperature.

FIG. 4 Five lowest energy TI3C isomers: (a) configurations, (b) calculated Gibbs free energies in glycerol, and (c) Boltzmann probability distribution at the room temperature ($T$=298.15 K).
B. Absorption and emission spectra

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$_0$). In order to compute the emission spectra, electronically excited geometries of isomers (1 and 2) were also fully optimized on their first singlet excited state (S$_1$) potential surfaces, respectively. Ground- and excited-state geometric parameters of isomer 1 and 2 are presented in Table Ⅲ. Although the structures of 1 and 2 differ in $\phi_1$ by 180$^{\circ}$, their excited-state electronic characteristics are very similar in both vacuum and glycerol. Orbital analysis using TDDFT calculations suggests that both absorption and emission are dominated by transitions between HOMO and LUMO of the ground state, shown in FIG. 6.

FIG. 5 Absorption and emission spectra of isomers 1 (left panel) and 2 (right panel) in vacuum and glycerol. Spectra are Gaussian-broadened with FWHM=0.3 eV.
Table Ⅱ Electronic characteristics of optical transitions of isomer 1 and 2 in vacuum and glycerol.
Table Ⅲ Geometric parameters of isomer 1 and 2 at S$_0$ and S$_1$ states.
FIG. 6 Top left panel: HOMO (left) and LUMO (right) of isomer 1 in vacuum. Top right panel: HOMO (left) and LUMO (right) of isomer 1 in glycerol. Bottom left panel: HOMO (left) and LUMO (right) of isomer 2 in vacuum. Bottom right panel: HOMO (left) and LUMO (right) of isomer 2 in glycerol.

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 $\delta\rho$=$\rho_{{\textrm{S}_1}}$-$\rho_{{\textrm{S}_0}}$ of 1 and 2 upon photo-excitation S$_0$$\rightarrow$S$_1$ in vacuum and in glycerol. The electron density difference profile in FIG. 7 shows that photo-excitation promotes electron transfer from Ib and Ia to the central frame (see FIG. 1(a) for labeling). Mülliken population analysis presented in Table Ⅴ suggests that the amount of charge transfer upon photo-excitation increases in polar solvent compared to that in vacuum. As a result, spectral oscillator strength also increases.

Table Ⅳ Electric transition dipole moment in a.u.
FIG. 7 Electron density difference, computed as $\delta\rho$=$\rho_{\textrm{S}_1}$-$\rho_{\textrm{S}_0}$, upon photo-excitation S$_0$$\rightarrow$S$_1$. Left panel: isomer 1 in vacuum and in glycerol. Right panel: isomer 2 in vacuum and in glycerol.
Table Ⅴ Mülliken atomic charge changes upon photoabsorption and photoemission.

Spectral Stokes shifts in vacuum are $\sim$77 and $\sim$84 nm for isomers 1 and 2, respectively, corresponding to a reorganization energy of 8.6 and 8.7 kcal/mol. The amounts of Stokes shift decrease in glycerol ($\sim$51 and $\sim$58 nm for isomers 1 and 2, respectively), corresponding to a smaller reorganization energy of 5.5 and 5.8 kcal/mol. Remarkably, the calculated Stokes shift from isomer 1 is almost identical to the experimentally determined value of 48 nm ($E_\textrm{x}$=548 nm, $E_\textrm{m}$=596 nm; FIG. 8), consistent with the observation that isomer 1 is the energetically most stable and populated conformation. The absorption spectra of both isomers exhibit a solvatochromic red-shift of 14-20 nm. However, the emission spectra of both isomers show a solvatochromic blue-shift of $\sim$10 nm. Such an inverted solvatochromic shift between absorption and emission spectra is unusual in photochemical systems.

FIG. 8 Experimental absorption and emission spectra of TI3C in glycerol.

FIG. 9 depicts the potential energy surfaces of S$_1$ and S$_0$ states of isomer 1 in vacuum and in glycerol. In the absence of charge transfer, ground state potential energy surfaces in vacuum and in glycerol have a similar curvature. As a result, solvatochromic shifts are mainly due to the interaction between solvent and solute excited states. In the excited state, charge transfer character is stabilized by the polarity of the solvent compared to that in vacuum, giving rise to the solvatochromic red-shift in the absorption spectra. Such a stabilized charge transfer state is also associated with a smaller reorganization energy. Therefore, the excited state potential energy surface of the charge transfer state in glycerol is flatter than that in vacuum. In addition to the plotted excited state potential energy surface, this observation is verified by excited normal mode analyses and frequency calculations. This also leads to a smaller geometric change (see Table Ⅲ) and smaller reorganization energy. For TI3C isomers, multiple rotation angles allow for modest structural reorganization in the S$_1$ state in vacuum. Rotational structural reorganization is suppressed as a result of stabilized charge transfer in glycerol. As a result, inverted solvatochromic Stokes shift will occur when S$_1$ minimum in vacuum is lower in energy than that in polar solvent, as shown in FIG. 9.

FIG. 9 Potential curve of isomer 1 in vacuum and in glycerol during excitation and emission. Note, S$_0$ minima in vacuum and glycerol are aligned for better comparison of solvatochromic shift.
Ⅳ. CONCLUSION

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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具有延长共轭的绿色发光蛋白生色团的反向溶剂化斯托克斯(Stokes)位移
胡航a, Wolstenholme Charles H.b, 张鑫b, 李晓松a     
a. 美国华盛顿大学化学系, 西雅图 98195;
b. 美国宾夕法尼亚州立大学化学系, 宾夕法尼亚 16802
摘要: 受天然荧光蛋白启发的发色团结构在生物成像领域发挥着重要作用.本文通过计算的方法研究了一类新的绿色荧光蛋白发色团的光化学性质.首先,得到了这一类发色团在真空和溶液中热动力学稳定异构体;然后,计算了它的斯托克斯(Stokes)位移,并与实验数据进行对比.最后,观察到对于这类新的RFP生色团,它们吸收和发射波长发生了反向溶剂化偏移;通过分析,发现了导致反向溶剂化偏移的原因是在溶液中,电荷转移更加稳定,进而常见的旋转结构重组受到抑制.
关键词: 绿色荧光蛋白    溶剂变色偏移    斯托克斯位移    时间密度泛函理论    极化连续模型