Self-Restricted Green Fluorescent Protein Chromophore Analogues

Letter pubs.acs.org/JPCL

Self-Restricted Green Fluorescent Protein Chromophore Analogues: Dramatic Emission Enhancement and Remarkable Solvatofluorochromism Hongping Deng,† Chunyang Yu,*,† Lidong Gong,‡ and Xinyuan Zhu*,† †

School of Chemistry and Chemical Engineering, State Key Laboratory of Metal Matrix Composites, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai 200240, People’s Republic of China ‡ School of Chemistry and Chemical Engineering, Liaoning Normal University, 850 Huanghe Road, Dalian 116029, People’s Republic of China S Supporting Information *

ABSTRACT: The confinement effect of the β-barrel defines the emission profiles of the chromophores of the green fluorescent protein (GFP) family. Here, we describe the design strategy and mimicking of confinement effects via the chromophore itself, termed the self-restricted effect. By systematically tailoring the GFP core, a family of 2,5-dialkoxysubstituted GFP chromophore analogues is found to be highly emissive and show remarkable solvatofluorochromism in fluid solvents. Fluorescence quantum yield (QY) and lifetime measurements, in combination with theoretical calculations, illustrate the mechanism relying on inhibition of torsional rotation around the exocyclic CC bond. Meanwhile, theoretical calculations further reveal that the electrostatic interaction between the solvent and the imidazolinone oxygen can contribute to suppress the radiationless decay channel around the exocyclic CC double bond. Our findings put forward a universal approach toward unlocked highly emissive GFPc analogues, potentially promoting the understanding of the photophysics and biochemical application of GFP chromophore analogues. he green fluorescent protein (GFP) has been extensively used as a genetically encoded fluorescent marker in biology.1−4 The GFP core chromophore, which is formed via autocatalytic dehydration/oxidation of a Ser-Tyr-Gly tripeptide motif and sequestered by a β-barrel, endows GFP with unique photophysical character.5−7 Surprisingly, the fluorescence of either the isolated or synthesized GFP core chromophore diminishes dramatically by 4 orders of magnitude in fluid solvents at room temperature, attributing to an ultrafast internal conversion mainly through two torsional modes along either the exocyclic C−C or the CC bond.8−11 Definitely, the confinement effect of the β-barrel structure makes fluorescence the primary pathway of energy release for the core chromophore.12,13 Inhibition of chromophore nonradiative pathways is a fundamental requirement to enhance the emission response of materials.14−17 Naturally, the confinement effect of the β-barrel has served as a universal mimic to recover the chromophore’s fluorescence over the past years.18 Specifically, encapsulation with supramolecular hosts,10,19 polymers,20,21 porous scaffolds,22 proteins, or ribonucleic acid (RNA),23−26 which generally provides a confined environment, turns out to be innovative and effectively improve the emission response of the corresponding GFP-like chromophores. Meanwhile, suppression of conformationally torsional modes by a chemically locking strategy, which greatly strengthens the molecular rigidity and polarization, results in highly fluorescent GFPc analogues.27−30 However, up until now, it is still a great challenge to obtain unlocked GFPc analogues that are highly emissive in fluid solvents at room temperature. We tried to

T

© XXXX American Chemical Society

learn from previous reports of emission recovery by a confined environment,18,22 which inspires us to investigate the possibility of producing a mimic of the confinement effect by the chromophore itself, termed the self-restricted effect. Prospectively, the successful generation of the self-restricted effect would greatly enhance the emission response of GFPc analogues in bulk solutions. Herein, we attempt to construct the self-restricted effect for GFPc analogues by systematically tailoring the core chromophore without employing the chemical locking strategy. Considering the synthetic convenience and strong electron-donating ability, the phenyl ring of the GFP core is modified by a methoxy substituent with an alkynyl linked to the imidazolinone, favoring further fluorescence labeling;31−33 meanwhile, the corresponding phenolic hydroxyl is removed (Scheme 1). Among 11 synthetic GFPc analogues, we screen out a highly emissive one and further tailor its structure with different substituents, which generates a family of highly fluorescent GFPc analogues. Fluorescence quantum yield (QY) and lifetime measurements, in combination with theoretical calculations, reveal the defined structure that could produce the self-restricted effect and illustrate the mechanism relying on suppression of chromophore rotation around the exocyclic CC bonds. Moreover, the remarkable solvatofluorochromism creates a color palette across blue to yellow and achieves an Received: June 8, 2016 Accepted: July 12, 2016

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DOI: 10.1021/acs.jpclett.6b01251 J. Phys. Chem. Lett. 2016, 7, 2935−2944

Letter

The Journal of Physical Chemistry Letters Scheme 1. Molecular Structures of Synthetic Methoxy-Substituted GFPc Analogues

trimethoxy-substituted GFPc analogues in the absorption spectra (Figure 1C). Notably, whether in the case of the 3 or 5 position, the meta substitution shows a negative effect for the λmax red shift, except for 2,5-MeOBDPI and 2,4,5-MeOBDPI, which could be attributed to the unique influence of 2,5-disubstitution. Compared with 2,5-MeOBDPI, the absorption λmax of 2,4,5MeOBDPI is the most bathochromic one among 11 chromophores. Thus, 2,5-MeOBDPI and 2,4,5-MeOBDPI are most likely to be the desired candidates with the self-restricted effect. The key to the existence of the potential self-restricted effect for GFPc analogues depends on the appearance of bright fluorescence at fluid solvents. After all synthetic GFPc analogues were dissolved in EA at a concentration of 10 μM, the corresponding fluorescence emission spectra were measured at room temperature. Certainly, nearly all chromophores were nonemissive, which is in accord with previous reports.18 However, bright fluorescence was obviously observed for 2,5-MeOBDPI with an intense peak at 475 nm (Figure 1D), demonstrating its potential as a general candidate bearing the self-restricted effect, which inspires us to further modify the GFP core chromophore with different substitutes at the 2,5-position. Encouraged by the above results, we try to further tailor the framework of 2,5-MeOBDPI at the 2,5-position with different substitutes. Considering the electronic and steric effects,37 another six GFPc analogues were successfully prepared (Scheme 2) by the universal 2 + 3 cycloaddition method,34 which followed the same procedure as that for the preparation of methoxy-substituted GFPc analogues. Likewise, the six chromophores were also carefully characterized. Details of synthetic procedures and characterizations are provided in the Supporting Information. To ensure their comparability, the spectroscopic properties of six 2,5-disubstituted GFPc analogues were also tested in EA (Figure 2). Notably, the electron-donating dialkoxyl-substituted analogues all display similar absorption spectra with a relatively longer absorption peak (at around 395 nm), while the electronwithdrawing or weak electron-donating substitution results in a

increased QY and lifetime with enhancing solvent polarity, which is further investigated using theoretical calculations. Therefore, we demonstrate the generation of a family of self-restricted GFPc analogues that show dramatic emission enhancement and remarkable solvatofluorochromism. On the basis of previous reports, methoxy substituent was selected to tailor the core chromophore due to the fact that all of the corresponding mono-, di-, and/or trisubstituted methoxybenzaldehydes are of strong electron-donating ability and also commercially available. Thus, by a universal 2 + 3 cycloaddition method,34 11 GFPc analogues were facilely prepared and linked with alkynyl groups, which will benefit further fluorescence labeling.21 Briefly, all of the methoxybenzaldehydes were stirred with propargylamine in ethanol in a molar ratio of 1:1.1 to get the Schiff bases, which then reacted with 2-(1-ethoxyethylideneamino)acetate (MEEA) to obtain the crude products. All chromophores were purified by silica gel column chromatography and characterized by 1H and 13C NMR, HRMS, and IR spectra. Details of synthetic procedures and characterizations are given in the Supporting Information. The spectroscopic properties of synthetic GFPc analogues were measured in ethyl acetate (EA, a good solvent). Monomethoxysubstituted GFPc analogues characterize similar absorption spectra with variable peaks from 358 to 371 nm due to the influence of different substituent positions on the phenyl ring (Figure 1A). From this result, the electron-donating ability of the methoxy group is ortho > para > meta, which is in accord with previous studies.35,36 Compared to 2-MeOBDPI, the obvious hypochromatic shift to 358 nm for 2,3-MeOBDPI indicates the dominant role of meta substitution, which obeys the same rule in the case of 2,4-MeOBDPI and 3,4-MeOBDPI.36 The absorption λmax of 2,5-MeOBDPI (393 nm) is the greatest among five dimethoxysubstituted GFPc analogues (Figure 1B), which may attribute to the synergistic effect of methoxy 2,5-disubstitution, indicating that 2,5-MeOBDPI may be the target candidate with enhanced emission response due to the formation of the self-restricted effect. Furthermore, a homothetic phenomenon was also observed for 2936


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Figure 1. Normalized absorption spectra of monomethoxy-substituted (A), dimethoxy-substituted (B), and trimethoxy-substituted (C) GFPc analogues in EA. (D) The corresponding fluorescence emission spectra for 11 chromophores excited at absorption λmax in EA; concentration = 10 μM, slits: 2.4/2.4 nm.

Scheme 2. Molecular Structures of Synthetic 2,5-Disubstituted GFPc Analogues with Diverse Steric and Electronic Substitutes

response and molecular structure for the self-restricted GFPc analogues. To compare the chromophores’ emission performance, the fluorescence QY and lifetime were measured in EA using quinine sulfate as the standard (Table 1 and Table S1). As depicted in Table S1, the QY of 2,5-MeOBDPI reaches above 10%, which is comparable to several locked GFP-like chromophores.28,29,35 However, for the other 10 methoxy-substituted GFPc analogues in Scheme 1, the QYs are much lower than that of 2,5-MeOBDPI (almost below 1%), corroborating the general loss of fluorescence for GFP-like chromophores.18 Through the above results, it is found that the substituent number/position has a

blue shift to about 356 nm (Figure 2A), which is also consistent with a previous report.38 Meanwhile, the emission response of these chromophores was also measured with the same concentration in EA. As depicted in Figure 2B, 2,5-MeBDPI is completely nonemissive with undetectable fluorescence. The relatively weak fluorescence is detected in the case of 2,5-FBDPI and 2,5-ClBDPI. Remarkably, four 2,5-dialkoxyl-substituted GFPc analogues all display bright and enhanced emission response under the same conditions. Moreover, with the increase of electrondonating ability or steric hindrance, the emission intensity further increases, and the emission peak red shifts to a longer wavelength, which leads us to investigate the relationship between the emission 2937


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Figure 2. Normalized absorption spectra of different 2,5-disubstituted GFPc analogues in EA (A). The corresponding fluorescence emission spectra excited at absorption λmax in EA (B); concentration = 10 μM; slits: 2.4/2.4 nm.

Moreover, the solvent dependence of fluorescence emission was measured.41 The emission of 2,5-MeOBDPI displays a single intense peak at 440 nm in Hex. Upon going from Hex to MeOH, the fluorescence maximum (λf) exhibits an obvious red shift of about 98 nm (Figure 3C,D), resulting in a remarkable solvatofluorochromism from blue to yellow (Figure 3E).44,45 Although this spectral shift is less prominent than m-DMABDI,43 2,5-MeOBDPI shows enhanced emission response in highly polar solvents. Definitely, the family of self-restricted GFPc analogues shares similar solvent dependence with a spectrum of color across blue to yellow upon going from Hex to MeOH (Figure S2). With the increase of electron-donating ability and steric hindrance, obvious red shifts are generally observed in the same solvent from Hex to MeOH, which demonstrates a universal phenomenon in all examined solvents (Table S4). The dependence of the fluorescence QY and lifetime on solvent polarity was also tested in different solvents. In aprotic solvents, the emission QYs of 2,5-MeOBDPI are relatively high compared to other previous reports.11,35 Contrary to previous phenomenon with the solvent dependence of QYs, which decreases significantly from Hex to ACN,29,35,40,43 the QY of 2,5-MeOBDPI increases gradually from Hex to DMSO (Table 1). Although a similar chromophore was reported to have a QY of 30.62% in ethanol containing 1% DMSO,46 our measurement of the QY for this chromophore in MeOH is much less (below 2%), which can be ascribed to the difference in the testing method. Still, the emission QY of 2,5-MeOBDPI in DMSO reaches 0.18, which is comparatively high for unconstrained and naked GFPc analogues in highly polar solvents. From aprotic to protic solvents, the fluorescence QY displays a great reduction due to the influence of solvent−solute H-bonding in the nonradiative decay pathways.9,43 Nevertheless, it is still notable and relatively higher compared to other unlocked GFPc analogues in protic solvents,29,43,47 which may be attributed to the unique self-restricted effect. The role of hydroxylic solvents in decreasing QYs by H-bonding would be strengthened by including one deuterated solvent. Thus, the emission spectra of 2,5-MeOBDPI in MeOH and MeOH/MeOD mixed solvents were measured. As shown in Figure S1, with the addition of MeOD in MeOH, the emission intensity increases obviously, confirming that solvent−solute H-bonding quenches fluorescence. From Hex to ACN, the QY for the self-restricted GFPc family is uniformly enhanced with increasing solvent polarity and further elevates along with increasing the electron-donating ability for alkoxyl substituents. However, the QY generally decreases nearly 1 order of magnitude in MeOH due to strong solvent−solute H-bonding.43

fundamental influence on the fluorescence performance of the chromophores. The influence of different substituents at the 2,5-position on the fluorescence property was also investigated. As shown in Scheme 2 and Table S2, 2,5-MeBDPI has a QY as low as 0.04%, while 2,5-FBDPI displays increased QY to above 2%. This can be abscribed to the strong electron-withdrawing fluorine substitution compared to that of the weak electron-donating methyl group, which can improve the QY to some extent in low polar solvents, indicating that the strong electronic effect of the substituent plays an important role in enhancing the emission response. Importantly, all four 2,5-dialkoxyl-substituted GFPc analogues display enhanced emission response with fluorescence QY over 10%, implying that only strong electron-donating 2,5-disubstitution results in the generation of the self-restricted effect.39 With increasing steric hindrance of substitution, the corresponding emission QY also enhances whether in the case of 2,5-alkoxyl- or 2,5-haloid-substituted GFPc analogues, confirming that the steric effect also plays an important role in improving the fluorescence emission.40 In the meantime, the fluorescence lifetime of 2,5-dialkoxyl-substituted GFPc analogues was also tested in EA, which generally exceeds 1.5 ns (Table S4). It is also clear that the fluorescent lifetime increases with enhancing electron-donating ability or steric hindrance of 2,5-disubstitutes. Definitely, the substituent number/position, electronic effect, and steric hindrance all contribute to the emission response of GFPc analogues. Subsequently, the common optical properties of four selfrestricted GFPc analogues were studied with 2,5-MeOBDPI as the typical one. The solvent dependence of 2,5-MeOBDPI was readily recorded in both aprotic and protic solvents (Table S3).41 As shown in Figure 3A, the absorption spectra of 2,5-MeOBDPI are characterized with an intense peak at about 392−401 nm, ranging from hexane (Hex) to dimethyl sulfoxide (DMSO), which is of weak dependence on the solvent polarity.42 In protic solvents, the magnitude of the solvatochromic shift from n-butanol (BuOH) to MeOH is 2 nm (Figure 3B). The difference of absorption λmax between aprotic and protic solvents is also small, demonstrating a weak H-bonding effect on λmax.43 Notably, the shoulder peak at around 345 nm strengthens from BuOH to MeOH, indicating a gradually enhanced H-bonding interaction. For the other self-restricted GFPc analogues, the absorption peak red shifts from Hex to MeOH by several nanometers, showing very weak solvent dependence also (Figure S2 and Table S4). It is clear that the shoulder peak for 2,5-IprOBDPI is the most intense in MeOH, suggesting the strongest H-bonding effect for 2,5-IprOBDPI. 2938


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0.17

8.92 9.75

0.25 0.33

11.44 11.85

0.35 0.60

2.70 3.16

0.65 0.60

3.90 4.05

0.55 0.65

5.37

1.00

10.23

Kr

Knr

0.67

Likewise, the fluorescence lifetime of 2,5-MeOBDPI is also solvent-dependent, which increases gradually with solvent polarity in aprotic solvents and reaches a maximum in DMSO as long as 3.03 ns (Figure 4 and Table 1), displaying a comparable lifetime to GFP (3.03 ns) and its mutants (1.88−1.94 ns).22 In protic solvents, the emission lifetime decreases greatly also due to strong solvent−solute H-bonding.40,43 However, a comparatively long fluorescence lifetime is still observed relative to that in Hex, which further increases from BuOH to MeOH. As depicted in Table S4 and Figure S3, the fluorescence lifetime for selfrestricted GFPc analogues generally increases with enhancing solvent polarity from Hex to ACN and further enhances with increasing electron-donating ability or steric hindrance of 2,5-dialkoxyl substituents. However, the emission lifetime decreases from 2,5-MeOBDPI to 2,5-IprMeOBDPI due to increasing solvent−solute H-bonding. Definitely, these self-restricted GFPc analogues show remarkable solvatofluorochromism, exhibiting an enhanced fluorescence QY and lifetime along with increasing solvent polarity or electron-donating ability, which can be greatly reduced by solvent−solute H-bonding. Finally, the general optical properties of 2,5-MeOBDPI were detected. To study the influence of solvent viscosity, the fluorescence QY of 2,5-MeOBDPI was probed with a range of n-alkanes. As shown in Figure S4A, no pronounced change of QY was observed with increasing carbon chain, suggesting that conformational change is not the rate-determining step in nonradiative passways.30 As solvent−solute H-bonding plays a great role in the emission response, the influence on fluorescence quenching was also investigated with MeOH titration of 2,5-MeOBDPI in ACN (Figure S4B). Bright green fluorescence with an emission peak of 508 nm is observed in ACN. With the addition of MeOH, the fluorescence intensity decreases gradually and shows a clear red shift from 508 to 527 nm. However, the fluorescence quenching is less efficient compared to previous results.9,43 Considering the big gap of emission QY between aprotic and protic solvents, we have also explored the possibility of an aggregation-induced emission (AIE) feature for 2,5-MeOBDPI.48 The linear dependence of the fluorescence intensity on the concentration for 2,5-MeOBDPI was measured in DMSO. As shown in Figure S5, good linear dependence was observed with concentrations below 20 μM. After 2,5-MeOBDPI was dissolved in good solvents (DMSO or MeOH), a certain amount of water (a poor solvent) was added before fluorescence measurements. Contrary to the AIE phenomenon, obvious fluorescence quenching accompanied by large red shifts is clearly observed for both systems (Figure S4C,D). In order to disclose the relationship between molecular structure and the fluorescence performance, the optimum geometries and the electron state density distributions of the HOMO and LUMO of the GFPc analogues are shown in Figures S6 and S7. The HOMO and LUMO can be respectively described as an outof-phase and in-phase combination of two localized π bonds.49−52 The bridge region between the aromatic ring and the imidazolinone heterocycle can be explained by two interacting π orbitals.52 We can see from Figures S6 and S7 that the HOMO and LUMO are delocalized over the aromatic ring and the imidazolinone heterocycle. Herein, the position and properties of substituted groups can affect the coplanarity of two rings and the shape of the HOMO and LUMO. For example, the disubstitution at the 2,5-positions can be divided into an electronwithdrawing group and an electron-donating group. The optimized geometries of 2,5-disubstitution indicate that from the S0 state to the S1 state, the location between the aromatic ring and

6.00

1.9

1.10 1.00 0.85 0.82 3.03 2.62 2.22 2.17 1.66 1.50 0.89 τ

538 523

2.5 2.8

521 516

2.9 18.2

510 500

17.1 13.4

508 487

12.0 10.8

476 475

10.1 8.9

440

Φf

λf

MeOH

398 (11 000) 399 (11 800)

EtOH PrOH

400 (10 000) 400 (10 800)

BuOH DMSO

401 (12 700) 399 (15 000)

DMF ACN

395 (11 800) 394 (13 600)

ACT DOX

396 (14 300) 393 (11 500)

EA Hex

392 (2700) λab (ε)

solvent

Table 1. UV−Vis Absorption (λab) and Fluorescence Emission (λf) Maxima (in nm), Extinction Coefficients (ε, M cm)−1, Fluorescence QYs (Φ, %) and Lifetime (τ, ns), and Kr and Knr Values (108 S−1) of 2,5-MeOBDPI in Different Solvents


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Figure 3. Normalized UV−vis absorption (A,B) and fluorescence emission (C,D) spectra of 2,5-MeOBDPI in aprotic (A,C) and protic (B,D) solvents. (E) A spectrum of color from blue to yellow for the fluorescence of 2,5-MeOBDPI in different solvents under a 365 nm lamp.

Figure 4. Time-resolved fluorescence of 2,5-MeOBDPI in different solvents excited at the λmax of absorption and monitored at the λmax of fluorescence emission.

derivatives of F, Cl, and CH3, the other is transferring from the aromatic ring to the imidazolinone heterocycle for 2,5-disubstibution derivatives of alkoxy. The conversion in the atomic charge distribution leads to a significant change of the dipole moment of the S1 state (Table S5). It is well-known that there are remarkably three ways to produce the nonradiative transition for GFPc analogues: (1) rotating around the exocyclic C−C single bond; (2) rotating around the exocyclic

the imidazolelinone heterocycle changes from nearly coplanar to almost perpendicular to each other along with the electronwithdrawing substitution to the electron-donating substitution (Table S5 and Figure S8). Furthermore, the substituent effect can be inspected by the change of the atomic charge distribution. As shown in Figure 6A, the electron transfer upon excitation also has two possible directions: one is transferring from the imidazolinone heterocycle to the aromatic ring for the 2,5-disubstibution 2940


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Figure 5. Computational infrared spectra of GFPc analogues in the S1 state obtained by TDDFT calculations.

the radiationless decay. To explain the enhanced emission of 2,5-HexOBDPI, the dipole moment and Mulliken atomic charge transfer capacity of 2,5-dialkoxy-substituted chromophores with different carbon chain lengths (n = 1−6) upon excitation (S0−S1) are calculated. As shown in Table S7, the dipole of the S1 state of chromophores decreases gradually with increasing carbon chain length, exhibiting a critical transition process. Besides, the charge transfer capacity of chromophores upon excitation (S0−S1) decreases gradually from the aromatic ring to the imidazolinone heterocycle and further reverses transfer direction with the increase of carbon chain length (Table S8), indicating the presence of a critical transition. The critical transition process is caused by the large steric hindrance of the alkoxy group, which explains the reasons for the higher Φf of 2,5-HexOBDPI, confirming that the steric effect also has an important role in enhancing the chromophore’s emission response. Definitely, results of theoretical calculations illustrate the causes for the enhanced emission response of 2,5-dialkoxy-substituted GFPc analogues. Furthermore, the increased fluorescence QY with the increase of solvent polarity in aprotic solvent is also probed using the dipole and Mulliken atomic charges of the ground and excited states. As shown in Table S9, from cyclohexane to DMSO, the dipole in the S1 state is enhanced gradually but decreases heavily in MeOH due to solvent−solute H-bonding. Besides, from cyclohexane to DMSO, the charge translocation direction of 2,5-MeOBDPI is generally from the aromatic ring to the imidazolinone heterocycle (Figure 6B). However, both the negative charge of the imidazolinone heterocycle in the S1 state and the charge transfer capacity from the aromatic ring to the imidazolinone increases with the increase of solvent polarity (Table S10), indicating that the electrostatic interaction between the solvent and the imidazolinone heterocycle oxygen is enhanced

double bond; and (3) simultaneous rotating around both exocyclic bonds.53 First, Figure 5 presents the infrared spectra for methoxy-substituted GFPc analogues at the S1 state in vacuo by TDDFT calculations. From the results, we find out that 2,5-MeOBDPI has a weaker oscillator strength than those of mono-, di-, and trimethoxy-substituted chromophores. The vibrational motions in the S1 state can deplete the excited-state energy of GFPc analogues, which will decrease the emission response of chromophores. Thus, the weaker oscillator strength of 2,5-MeOBDPI in the S1 state results in its better emission response, indicating the formation of rotational inhibition around the CC bond. Second, it has been disclosed by Negri et al.53 that rotating around the exocyclic double bond is as the primary radiationless deactivation channel in solution. Meanwhile, the translocation of the negative charge from the imidazolinone heterocycle to the aromatic ring favors dissipating energy through this channel, but instead, the electrostatic interaction between the solvent and the imidazolinone heterocycle oxygen can contribute to suppress this radiationless decay channel. As shown in Figure 6A and Table S7, the negative charges are respectively located on the aromatic ring and imidazolinone heterocycle for electron-withdrawing and electron-donating substitution, respectively. For 2,5-FBDPI, 2,5-ClBDPI, and 2,5-MeBDPI, the charge translocation direction is from the imidazolinone heterocycle to aromatic ring, favoring rotating around the exocyclic double bond. However, the charge translocation direction for 2,5-MeOBDPI, 2,5-EtOBDPI, and 2,5-IprOBDPI is the opposite, which will inhibit the radiationless decay channel. Thus, the conformational rotation of the exocyclic double bond will be strongly favored for 2,5-FBDPI, 2,5-ClBDPI, and 2,5-MeBDPI but disfavored for 2,5-MeOBDPI, 2,5-EtOBDPI, and 2,5-IprOBDPI. It should be noted that the charge translocation direction of 2,5-HexOBDPI is from the imidazolinone heterocycle to aromatic ring, which is not favorable for decreasing 2941


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Figure 6. (A) Mulliken atomic charge variation upon excitation (S0−S1) of 2,5-substituted GFPc analogues from gas-phase calculations. The atomic charge of the bridge carbon is not included in the sum. (b) Mulliken atomic charges for the ground and excited states of the 2,5-MeOBDPI computed from solvent TDDFT/B3LYP/6-31+G(d) calculations.

raises the possibility toward chemical and biological applications using synthetic GFP-like chromophores.

gradually from cyclohexane to DMSO, which will suppress the radiationless decay channel more efficiently. This helps to explain why the Φf increases with the increase of solvent polarity in aprotic solvents. In MeOH, both the aromatic ring and the imidazolinone acquire negative charge from the S0 to S1 state due to a strong solvent−solute H-bonding effect. However, in the S1 state, the negative charge of the imidazolinone heterocycle will be a benefit for fluorescence, which can explain the enhanced emission response for 2,5-MeOBDPI in MeOH compared with other reported results.9,29,43,47 In conclusion, we have described the overall design strategy of a series of self-restricted GFPc analogues with dramatic emission enhancement and remarkable solvatofluorochromism. By systematically tailoring and screening, a family of highly emissive GFPc analogues is developed. The dramatic emission enhancement of these GFPc analogues is ascribed to the substituent position and electronic/steric effects, which creates the self-restricted effect. Theoretical calculations reveal that the chemical rebuilding of the GFP core affects the chromophore’s rotation dynamics, leading to the suppression of the chromophore radiationless decay channel around the exocyclic CC bonds. Meanwhile, the self-restricted GFPc analogues show remarkable solvatofluorochromism, forming a color palette covering across blue to yellow. More importantly, the emission QY and lifetime increase gradually with enhancing solvent polarity and reach 0.18 and 3.03 ns in DMSO. This unique phenomenon is investigated by theoretical calculations, which can be ascribed to the enhanced interaction between the solvent molecule and the imidazolinone heterocycle oxygen. From aprotic to protic solvent, the fluorescence QY and lifetime decrease heavily due to the influence of solvent−solute H-bonding in the nonradiative decay pathways. Our work provides an approach for a family of unlocked highly emissive GFPc analogues, which

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jpclett.6b01251.

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Synthesis, characterizations, additional data, 1H and 13C NMR spectra, and IR spectra (PDF)

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (C.Y.). *E-mail: [email protected] (X.Z.). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS

We acknowledge financial support from the National Basic Research Program of China (2015CB931801) and the National Natural Science Foundation of China (51473093, 21404070).

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ABBREVIATIONS BDPI:benzylidene-1-propinyl-2-methyl-5-imidazolinone; Hex:n-hexane; EA:ethyl acetate; DOX:1,4-dioxane; ACT:acetone; ACN:acetonitrile; DMF:N,N-dimethylformamide; DMSO:dimethyl sulfoxide; BuOH:n-butyl alcohol; PrOH:n-propyl alcohol; EtOH:ethanol; MeOH:methanol 2942


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Self-Restricted Green Fluorescent Protein Chromophore Analogues

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