Self-restricted oxazolone GFP chromophore for construction of reaction-based fluorescent probe toward dopamine

Materials Today Chemistry 3 (2017) 73e81

Contents lists available at ScienceDirect

Materials Today Chemistry journal homepage: www.journals.elsevier.com/materials-today-chemistry/

Self-restricted oxazolone GFP chromophore for construction of reaction-based fluorescent probe toward dopamine Hongping Deng a, Zhihao Zhang a, Yanjie Zhao a, Chunyang Yu a, *, Lidong Gong b, Deyue Yan a, Xinyuan Zhu a, ** a

School of Chemistry and Chemical Engineering, State Key Laboratory of Metal Matrix Composites, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai 200240, PR China School of Chemistry and Chemical Engineering, Liaoning Normal University, 850 Huanghe Road, Dalian 116029, PR China

b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 14 January 2017 Received in revised form 6 February 2017 Accepted 7 February 2017

The b-barrel provides a confined environment for chromophores of the green fluorescent protein (GFP) family, defining their emission profiles by the chromophore/b-barrel interactions. Here, we describe the generation of self-restricted oxazolone GFP chromophore (GFPc) for construction of reaction-based fluorescent probe toward dopamine by mimicking the confinement effect of the b-barrel. Through standard synthetic method, the first self-restricted GFPc oxazolone analogue (MBDO) and the conventional pyrenyl-based chromophore (PDO) were prepared respectively. Under the same condition, MBDO shows much better emission response with fluorescent quantum yield (QY) over one order of magnitude higher than that of PDO due to the generation of the self-restricted effect. And, the fluorescent QY of MBDO reaches above 30% in dimethyl sulfoxide, which is the largest ever recorded for unlocked GFPc analogues in highly polar solvents. Moreover, theoretical calculations further reveal that the enhanced emission of MBDO is due to the inhibition of conformational motions around the exocyclic CC bonds. Combination the enhanced emission and the reactivity of the lactone, MBDO is applied to construct reaction-based fluorescent probe toward dopamine via a ring-opening reaction of the lactone. Prospectively, the destruction of the oxazolone would break the effective conjugated structure of the chromophore, which can decrease the corresponding fluorescence. This work puts forward a novel approach to generate highly emissive GFPc oxazolone analogue, which can be used to fabricate reactionbased fluorescent probe toward dopamine, potentially promoting the biochemical applications using synthetic GFP chromophore analogues. © 2017 Published by Elsevier Ltd.

Keywords: Green fluorescent protein GFP chromophore Self-restricted effect Fluorescent probe Fluorescent imaging

1. Introduction With unique optical properties, members of the green fluorescent protein family have been widely used as genetically encoded fluorescent markers in biology [1e3]. The GFP chromophore, which is formed in situ via a post-translational autocatalytic cyclization/ oxidation process of a Ser-Tyr-Gly tripeptide motif, exhibits excellent photoluminescent response with intense green fluorescence through excited-state proton transfer (ESPT) derived from the phenolic oxygen to the protein b-barrel matrix [4,5]. Nevertheless, the isolated GFP chromophore losses its fluorescence by four orders

* Corresponding author. ** Corresponding author. E-mail addresses: [email protected] (C. Yu), [email protected] (X. Zhu). http://dx.doi.org/10.1016/j.mtchem.2017.02.002 2468-5194/© 2017 Published by Elsevier Ltd.

of magnitude in fluid solvents at room temperature [6]. Moreover, the chemically synthesized GFPc analogues are also completely non-fluorescent in solutions due to an ultrafast internal conversion along either the exocyclic C-C (4) or C]C (t) bond [7,8]. Obviously, the general loss of fluorescence greatly hinders the potentially biochemical applications using synthetic GFPc analogues [9]. The emission recovery and enhancement of synthetic GFPc analogues have drawn great attention over past decade [9,10]. Physical encapsulation with supramolecular hosts, proteins, ribonucleic acids, polymers or porous scaffolds, which provides a confined/ rigid environment for these chromophores, results in the fluorescence “turn-on” feature of the corresponding chromophores [11e16]. Besides, the structural tunability via chemical modifications has also obtained various “locked” GFPc analogues, which can greatly enhance the emission response [17e19]. Up until now, it is still a great challenge to prepare unlocked highly-emissive GFPc

74

H. Deng et al. / Materials Today Chemistry 3 (2017) 73e81

analogues in fluid solvents. Previously, we developed a unique kind of self-restricted GFPc analogues with dramatic emission enhancement and remarkable solvatofluorochromism, providing another possibility to construct novel fluorescent probes with GFPc analogues [20]. Self-restricted effect may be defined as “restriction of molecular motion via a combinative balance/force of multiple intramolecular electronic effect”. Recently, the development of reaction-based fluorescent probes has become a hot research area due to their excellent functions as biosensors to track the spatial and temporal dynamics of specific targets in cells [21e23]. However, the structural stability of the imidazolinone disfavors the development of reaction-based fluorescent probe under mild conditions, which hinders the fabrication of reaction-based fluorescent probe and only leads to an alternative upon the benzene ring [24]. Thus, by combination of the self-restricted effect and the feasibility of the lactone, the generation of self-restricted GFPc oxazolone hopefully facilitates the construction of novel reactionbased fluorescent probe using GFP-like chromophores. Herein, we report the preparation of a highly emissive GFPc oxazolone for fabrication of a novel reaction-based fluorescent probe toward dopamine. Through the Erlenmeyer azlactone synthesis, the self-restricted GFPc analogue (MBDO), and the compared pyrenyl-based chromophore, PDO, were prepared, respectively. Steady-state absorption and fluorescence measurements, in combination with theoretical calculations, demonstrate that the emission performance of MBDO is much more outstanding than that of PDO in different solutions due to the inhibition of conformational motion around the exocyclic single or double bond. Given the enhanced emission response and the reactivity of the lactone, MBDO has been utilized to develop potential reactionbased fluorescent sensor towards dopamine via a ring-opening reaction, which will destroy the effective conjugated structure and decrease the fluorescence emission intensity of MBDO. Therefore, we demonstrate the generation of the first example of highly emissive GFPc oxazolone, which can be used to construct novel reaction-based fluorescent probe.

2. Materials and methods 2.1. Materials N-Acetylglycine, 2,5-dimethoxybenzaldehyde, acetic anhydride, 1-pyrenecarboxaldehyde and dopamine hydrochloride were purchased and used as received. All other reagents and solvents were purchased commercially and used without further purification.

2.2. Measurements Both 1H and 13C NMR spectra were measured with Varian MERCURY plus-400 spectrometer at 400 MHz and 100 MHz instruments respectively using dimethyl sulfoxide-d6 (DMSO-d6) as the solvents at 298 K. Chemical shifts (d) are quoted in parts per million (ppm) and coupling constant (J) are recorded in Hertz (Hz). The chemical shifts were referenced to the residual peak of deuterated solvent: DMSO-d6 (2.48 ppm). FTIR spectra were tested with a Perkin Elmer Paragon 1000 spectrophotometer between 4000 and 450 cm
1. All sample slices were measured by pestling the solid sample with dry potassium bromide (KBr) and then moulding under high pressure. HRMS was measured on a Waters Micromass Q-TOF Premier Mass Spectrometer with data acquired for each sample from 50 to 1000 Da with a 0.10 s scan time and a 0.01 s interscan delay over a 10 min analysis time.

2.3. Synthetic procedures The synthetic routes for MBDO and PDO employed are given. 2.3.1. Synthesis of 4-[(Z)-2,5-methoxybenzylidene]-2methyloxazol-5(4H)-one (MBDO) A mixture of N-acetylglycine (10 mmol), 2,5dimethoxybenzaldehyde (10 mmol) and sodium acetate (10 mmol) were stirred in 20 mL acetic anhydride at 110  C for 18 h. Upon completion, the reaction was cooled to room temperature and acetic anhydride was removed under reduced pressure. Then, chloroform was added, washed with water and dried over magnesium sulfate. After chloroform was removed by rotary evaporation, ethanol was added and further dropped into water under stirring. The solid was filtered and washed with ethanol three times to get the pure product. Yellow solid, yield 72%. 1H NMR (400 MHz, DMSO-d6, ppm, 298 K): 8.14 (d, J ¼ 2.4 Hz, 1H), 7.34 (s, 1H), 7.05 (m, 2H), 3.81 (s, 3H), 3.72 (s, 3H), 2.35 (s, 3H). 13C NMR (100 MHz, DMSO-d6, ppm, 298 K): 168.12, 167.22, 153.85, 153.56, 132.69, 123.11, 122.35, 119.34, 116.89, 113.22, 56.89, 56.12, 16.09. HRMS: m/z calculated for [C13H14NO4]þ: 248.0845, found: 248.0921. IR (KBr): 3007, 2970, 2926, 2842, 1796, 1773, 1650, 1584, 1499, 1470, 1444, 1422, 1383, 1308, 1296, 1270, 1257, 1229, 1205, 1190, 1165, 1114, 1044, 1021, 1002, 917, 900, 874, 826, 763, 719, 689, 655, 620, 533 cm
1. 2.3.2. Synthesis of 1-pyrenylidene-2-methyloxazol-5(4H)-one (PDO) A mixture of N-acetylglycine (4.2 mmol), 1pyrenecarboxaldehyde (4 mmol) and sodium acetate (6.3 mmol) were stirred in 20 mL acetic anhydride at 110  C for 36 h. Upon completion, the reaction was cooled to room temperature and acetic anhydride was removed under reduced pressure. Then, chloroform was added, washed with water and dried over magnesium sulfate. After chloroform was removed by rotary evaporation, the mixture was purified by silica gel column chromatography using ethyl acetate and petroleum ether as the solvents. Red solid, yield 18%. 1H NMR (400 MHz, DMSO-d6, ppm, 298 K): 9.31 (d, J ¼ 8.4 Hz, 1H), 8.69 (d, J ¼ 9.6 Hz, 1H), 8.40 (t, J ¼ 7.6 Hz, 1H), 8.38 (s, 1H), 8.36 (d, J ¼ 4.4 Hz, 1H), 8.32 (d, J ¼ 8.8 Hz, 1H), 8.20 (m, 2H), 8.13 (t, J ¼ 8.0 Hz, 1H), 2.45 (s, 3H). 13C NMR (100 MHz, DMSO-d6, ppm, 298 K): 169.09, 156.34, 148.66, 140.41, 134.24, 134.11, 132.89, 131.69, 131.41, 130.19, 130.05, 128.63, 128.06, 127.33, 127.14, 125.80, 125.64, 123.71, 123.09, 112.74, 16.18. HRMS: m/z calculated for [C21H14NO2]þ: 312.0946, found: 312.1016. IR (KBr): 3042, 2975, 2926, 1805, 1785, 1759, 1650, 1625, 1604, 1587, 1431, 1419, 1385, 1376, 1362, 1324, 1305, 1257, 1236, 1219, 1198, 1170, 1097, 1047, 904, 889, 852, 843, 829, 774, 759, 713, 632, 606 cm
1. 2.4. Absorption and fluorescence spectroscopy The UVeVis absorption spectra of the sample solutions were tested at room temperature on a Perkin Elmer Lambda 20 UVeVis spectrometer with a scan speed of 480 nm/min. The fluorescence emission spectra were measured with a PTI-QM/TM/IM steadystate & time-resolved fluorescence spectrofluorometer (USA/CAN Photon Technology International Int.). The fluorescence quantum yields were calculated with the method below using the quinine sulfate (F ¼ 0.54 in 0.1 N sulfuric acid) as the standard [25].

Fsample ¼ Fstandard  (Fsample/Fstandard)  (nsample/ fl fl nstandard)2  (Astandard/Asample)

H. Deng et al. / Materials Today Chemistry 3 (2017) 73e81

75

point. We also perform a density functional theory (DFT/B3LYP/631 þ G*) to optimize the first excited state (time-dependent DFT formulation) as well as to calculate vibrational normal modes for these two chromophores [30,31]. 3. Results and discussions 3.1. Synthesis and characterization

Scheme 1. The molecular structures of MBDO, PDO and 2,5-MeOBDPI.

The molecular structures and synthetic routes of MBDO and PDO are given in Scheme 1 and Scheme 2. By the standard synthesis, MBDO was prepared with high yield with just one step [32]. However, the Erlenmeyer synthesis only gets low yield for PDO even with optimized reaction conditions, which might be ascribed to the influence of the large pyrenyl group. Meanwhile, the solubility of PDO is very poor even in even highly polar solvent, such as DMSO, making the signal of its 13C NMR spectra rather weak. However, the structure of PDO can be confirmed via the results of 1 H NMR and HRMS. Details of synthetic procedures and characterizations are given in the Experimental part. 3.2. Optical properties

Scheme 2. The synthetic routes for MBDO and PDO.

where F is the fluorescence quantum yield; F denotes the area under the corrected emission spectra; A is the absorbance at the excitation wavelength; n denotes the refractive index of the solvent. 2.5. Computational details All of the calculations reported here were performed with the Gaussian09 program [26]. Geometry optimization of MBDO and PDO structures was carried out at B3LYP/6-31 þ G* without any symmetry restriction [27e29]. After the geometry optimization was performed, analytical vibration frequencies were calculated at the same level to determine the nature of the located stationary

The photophysical behavior of MBDO and PDO were firstly tested in ethyl acetate (EA). As shown in Fig. 1, the UVeVis absorption spectra of MBDO characterizes two peaks at 323 nm and 396 nm, which can be ascribed to the electron transition from p-p* and n-p*, respectively. This spectrum differs from common reported results with just one broad absorption peak [10], probably due to the strong electron-donating 2,5-disubstitution and the formation of the azalactone. The absorption spectrum of PDO shows triplet peaks similar to that of pyrene, which redshifts compared to that of MBDO and 2,5-MeOBDPI due to the increased conjugation length [20,33e35]. Importantly, the fluorescence spectra of both chromophores display obvious signals with emission peaks at about 490 nm in EA, which reveals the fluorescence enhancement for both chromophores. Notably, the emission peaks show bathochromic-shift compared to that of 2,5-MeOBDPI, which might be attributed to the increased conjugation length or the electron-donating ability of the azalactone [20]. Meanwhile, the fluorescence quantum yields (QY) of MBDO and PDO were also determined, respectively. As given in Table 1 and Table 2, the QY of MBDO reaches 27% in EA, which is nearly 20-fold higher than that of PDO (1.4%), indicating that the self-restricted effect is much more effective than increasing the conjugation length in enhancing the chromophore's emission response.

Fig. 1. The normalized UVeVis absorption (a) and fluorescence emission (b) spectra of MBDO (A) and PDO (B) in EA. Excitation wavelengths are 396 nm and 415 nm for MBDO and PDO, respectively.

76

H. Deng et al. / Materials Today Chemistry 3 (2017) 73e81

Table 1 UVeVis absorption (lab) and fluorescence emission (lf) maxima (in nm), extinction coefficient (ε, M cm)
1, fluorescence quantum yields (F, %) of MBDO in different solvents. Solvent

Hex

EA

ACN

DMSO

MeOH

lab (ε) lf Ff

322 (10 300), 391 (7900) 454 28.7

323 (16 300), 396 (11 600) 489 27.1

322 (13 100), 396 (9000) 520 22.1

327 (16 300), 403 (11 800) 529 30.7

322 (16 100), 397 (10 800) 543 2.4

Table 2 UVeVis absorption (lab) and fluorescence emission (lf) maxima (in nm), extinction coefficient (ε, M cm)
1, fluorescence quantum yields (F, %) of PDO in different solvents. Solvent Hex

EA

ACN

DMSO

MeOH

lab (ε) 396 (3300), 415 (3900), 442 396 (20 500), 417 (21 600), 440 395 (19 900), 416 (20 900), 437 399 (15 900), 423 (18 500), 444 395 (7400), 417 (6300), 438 lf Ff

(4900) 450, 481 2.5

(26 100) 486 1.4

(24 200) 511 0.6

The solvent dependence of MBDO and PDO was further investigated in different solvents. As depicted in Fig. 2A and C, for both chromophores, the absorption peak redshifts several nanometers and reaches the maximum in DMSO with the increasing of solvent polarity from Hex to DMSO. However, bathochromic-shift is still observed for MBDO in MeOH compared to that in DMSO due to the effect of strong solvent-solute H-bonding. The difference of absorption spectra between protic and aprotic solvents is small, demonstrating a weak dependence on the solvent polarity [36,37]. Moreover, the spectra in Hex show the maximum difference compared to that in other solvents, which can be ascribed to the very low polarity of Hex.

(20 000) 520 0.9

(6100) 518 0.4

Subsequently, the solvatofluorochromism feature of MBDO and PDO was also investigated. As shown in Fig. 2B and D, MBDO is highly emissive in both aprotic and protic solvents, which generally shows one intense peak in all examined solvents. Specifically, obvious blue fluorescence with a peak maximum of 454 nm is observed for MBDO in Hex. Upon going from Hex to MeOH, the emission peak shows remarkable bathochromic-shift to 543 nm with a large Stokes shift of 89 nm, which creates a color palette across blue to yellow, resembling to the multicolor of fluorescent proteins [38]. The larger redshift in MeOH is due to the effect of solvent-solute H-bonding [38]. Moreover, the emission half band width rises gradually from 68 nm to 108 nm with increasing solvent

Fig. 2. The normalized UVeVis absorption (A, C) and fluorescence emission (B, D) spectra of MBDO (A, B) and PDO (C, D) in different solvents. The fluorescence emission spectra were excited at 396 nm and 415 nm for MBDO and PDO, respectively.

H. Deng et al. / Materials Today Chemistry 3 (2017) 73e81

77

Fig. 3. (A) The emission spectra of MBDO in MeOH/ACN mixed solvents with a concentration of 10 mM, MeOH contents are given, lex ¼ 396 nm; (B) The emission spectra of MBDO in H2O/DMSO mixed solvents with a concentration of 10 mM, H2O contents are given, lex ¼ 401 nm.

polarity. However, the solvatofluorochromism feature of PDO is less prominent compared to that of MBDO. Upon going from Hex to MeOH, the emission peak redshifts gradually with the maximum shift of 70 nm, which is 19 nm smaller than that of MBDO, indicating the greater influence of solvent effect on the emission of the self-restricted chromophore. Notably, the emission peak of PDO in MeOH is only at 518 nm, which means that the increased conjugation length can decrease the influence of solvent-solute hydrogen bonding. Moreover, the fluorescence QYs of MBDO and PDO in different solvents were also measured and listed in Tables 1 and 2. In aprotic solvents, the QYs of MBDO are more than 10-fold higher than that of PDO, indicating that the self-restricted effect is much more efficient in enhancing the emission performance. The QYs of MBDO are enhanced greatly and all exceed 20% in aprotic solvents, which are comparable to some locked GFPc analogues [17e19]. From Hex to ACN, the QY of MBDO decreases slightly, which is in contrary to that of 2,5-MeOBDPI. Instead, the QY further increases and reaches 30% in DMSO, which is the largest ever recorded for unlocked GFPc analogues in highly polar solvents [9,10]. Compared to aprotic solvents, a tremendous reduction is observed in MeOH (2%), which

is due to the strong influence of solvent-solute H-bonding effect [37]. In short, an unlocked highly emissive GFPc analogue with a lactone has been developed, which is much more outstanding than conventional GFPc dyes. Considering the fact that the emission response of MBDO is much more effective than PDO, only the optical property of MBDO is further explored. It is widely reported that solvent-solute Hbonding has a great influence on the emission response of GFP-like chromophores [36,37]. Thus, the emission spectra of MBDO with different MeOH in ACN were recorded. As shown in Fig. 3A, the emission intensity decreases heavily and obvious redshift is observed with the increasing of MeOH content, attributing to the effect of strong solvent-solute H-bonding interaction [20,36]. However, the fluorescence quenching is less significant compared to reported results [37]. The aggregation-induced emission (AIE) feature of some GFPc analogue has been reported [39e41], which inspires us to further explore the possibility of AIE feature for MBDO. As shown in Fig. 3B, the emission intensity decreases greatly and the emission peak redshifts significantly with the increasing of water content, displaying typical aggregation-caused quenching (ACQ) characteristic. 3.3. Theoretical calculations

Fig. 4. Molecular orbital diagrams for the (top) LUMOs and (bottom) HOMOs of MBDO and PDO obtained from DFT calculations.

In order to investigate the impact of the molecular structure on the fluorescence performance, the optimum geometries and the electron density distributions of the highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO) orbitals of MBDO and PDO are shown in Fig. 4. Herein, the HOMO and LUMO of MBDO and PDO are the out-of-phase and in-phase combination of two localized p bonds [42e45], and all of them are delocalized over the aromatic/pyrene ring and the azalactone heterocycle. Moreover, Fig. 5 presents the infrared spectra for MBDO and PDO at S1 state in vacuo by TDDFT calculations. From the results, we find out that PDO has a much stronger oscillator strength than that of MBDO in the range of 1000e1500 cm
1. After careful inspection of the vibration mode, we find that this range is mainly corresponding to the in-plane torsional movement. The vibrational motions in the S1 state can deplete the excited-state energy of PDO to some extent, which may decrease the emission response of the chromophore. Thus, the better emission response of MBDO molecule might be related to the weaker oscillator strength of MBDO in the S1 state. In addition, the previous study have shown that motion around the exocyclic double bond is the primary radiationless deactivation channel in solution [46]. Meanwhile, the translocation of the

78

H. Deng et al. / Materials Today Chemistry 3 (2017) 73e81

GFPc analogue shows obvious fluorescence among all eleven methoxy substituted D-A structured GFPc chromophores [20]. Furthermore, the energy profiles of S1 state of MBDO and PDO were plotted to display the rotation barriers around the exocyclic CC single and double bonds (Fig. 7). The energy profiles were conducted by 10 increments of the dihedral angle a and b (1-2-34/2-3-4-5). We can observe that the first excited-state energy increases with the deviation from the co-planar structure. Moreover, two conclusions can be drawn from these energy profiles. The first is the rotation barriers around the exocyclic CC double bond is slight larger than that around the CC single bond in both of these molecules. The second is both rotation barriers in MBDO molecule are higher than the corresponding ones in PDO molecule when the rotation angle is less than 60 . The relatively high rotation barriers will contribute to reduce the probability of the energy dissipation through rotating around the exocyclic CC bonds. Consequently, the MBDO molecule manifests as a molecule with the highly fluorescence quantum yield. Fig. 5. The computational infrared spectra of MBDO and PDO in the S1 state obtained by TDDFT calculations.

3.4. Construction of reaction-based fluorescent probe negative charge from the aromatic ring to the azalactone heterocycle favors to inhibit this radiation channel. As shown in Fig. 6 and Table 3, the negative charges are mainly located on aromatic ring for MBDO molecule at S0 state, and then it has an obviously charge translocation from aromatic ring to azalactone heterocycle upon excitation, which will be favored to inhibit the radiationless decay channel around the exocyclic double bond. On the contrary, the strong conjugation of pyrene molecule makes the charge distribution more uniform, which disfavors to the charge translocation. Although, the electronic structure of MBDPO is a conventional and typical D-A structure, its emission enhancement can't be ascribed to the excited-state charge transfer effect [47,48] from methoxyphenyl to the oxzaolone, which was proved in our previous publication [17]. The evidence is that only 2,5-dimethoxy substituted

Previously, the methods for dopamine detection usually depend on the reactivity or oxidation/reduction properties of the two phenol groups [49e51]. Given the enhanced emission and the reactivity of the lactone, MBDO is further used to fabricate reactionbased fluorescent sensor for potential dopamine detection, which utilizes the amino group of dopamine. As shown in Fig. 8, the absorption peaks of MBDO in DMSO decreased with the addition of different amount of dopamine, which is more obvious for the long wavelength peak. And, the absorption peak decreased about 35% with a dosage of 2.5 equivalent after 24 h. At the same time, the emission peak of MBDO at 529 nm diminished gradually without obvious shifts. Similarly, the maximum reduction degree is about 31% with the dosage of 2.5 equivalent. From the absorption and emission spectra, the reaction efficiency between MBDO and dopamine is a bit low.

Fig. 6. The Mulliken atomic charge variation upon excitation (S0-S1) of MBDO and PDO from gas phase calculations. The atomic charge of the bridge carbon is not included in the sum.

H. Deng et al. / Materials Today Chemistry 3 (2017) 73e81

79

Table 3 The Mulliken atomic charge and charge transfer (D ¼ S1-S0) upon excitation (S0-S1) for the aromatic ring and the azalactone heterocyclic ring of MBDO. Aromatic/Pyrene ring (a) Charge

S0 S1

D

Heterocycle (b) Charge

S0 S1

D

MBDO

PDO


0.3691
0.1788 0.1903

0.1346 0.1404 0.0058

MBDO

PDO

0.0003
0.0868
0.0871

0.0815 0.0882 0.0067

Fig. 7. The Energy profile of the S1 state as a function of the dihedral angle b (from
90 to 90 ). The zero value corresponds to the energy of the molecule with b ¼ 0.0 .

Based on the above results, the time-dependent absorption and emission spectra of MBDO with 2.5 equivalent dopamine were recorded within 3 h. As depicted in Fig. 9, the absorption peaks and emission intensity both decrease gradually with the addition of dopamine. The attenuation amplitudes obtained through absorption and emission spectra are 18% and 16%, respectively, which demonstrates the low reactivity between the lactone and dopamine. To accelerate the reactivity, the reaction temperature was increased to 65  C. As shown in Fig. 10, the reduction for absorption and emission increase to 27% and 25% respectively in the same condition, which indicates that the reactivity enhanced with the increasing of temperature. Actually, the low reactivity between MBDO and dopamine hinders the fabrication of biological sensors, which needs to be improved via molecular design of novel selfrestricted GFPc oxazolone analogues. However, as a

demonstration, the detection of dopamine using MBDO gives light to the potential construction of novel reaction-based fluorescent sensor with GFPc oxazolone analogues. It has been widely reported that primary amine can react with the oxazolone of GFPc analogue to open the lactone [52,53]. Based on these reports, we put forward the reaction mechanism between MBDO and dopamine. As shown in Fig. 11, the amino group of dopamine can attack the lactone of MBDO and open the lactone, destroying the conjugated structure of MBDO and further decreasing the corresponding fluorescence intensity. So, it can be predicted that the selectivity of MBDO is based upon the reactivity of the amino group, which surely has no specificity for dopamine. However, with proper molecular design and modification, this kind of self-restricted GFPc oxazolone analogues can hopefully be applied to generate novel fluorescent sensors toward dopamine.

Fig. 8. The absorption (A) and emission (B) spectra of MBDO (20 mM) with different mole ratio of dopamine in DMSO at 37  C, lex ¼ 401 nm.

80

H. Deng et al. / Materials Today Chemistry 3 (2017) 73e81

Fig. 9. The absorption (A) and emission (B) spectra of MBDO (20 mM) with 2.5 equivalent dopamine in DMSO within 3 h at 37  C, lex ¼ 401 nm.

Fig. 10. The absorption (A) and emission (B) spectra of MBDO (20 mM) with 2.5 equivalent dopamine in DMSO within 3 h at 65  C, lex ¼ 401 nm.

Notes The authors declare no competing financial interests. Acknowledgment

Fig. 11. The proposed mechanism of reaction-based fluorescent sensor for potential dopamine detection.

We acknowledge the financial support from the National Basic Research Program of China (2015CB931801) and National Natural Science Foundation of China (51690151, 21404070, 51473093). Appendix A. Supplementary data

4. Conclusion In conclusion, we have developed a self-restricted GFPc oxazolone with dramatic emission enhancement and obvious solvatofluorochromism, which has the potential to be used to fabricate novel reaction-based fluorescent sensors. The self-restricted strategy is much more effective than conventional strategy of increasing conjugation length in enhancing the emission responses, which is further explained via theoretical calculations. Combination the enhanced emission and the reactivity of lactone, the self-restricted GFPc analogue has been further utilized to construct reactionbased fluorescent probe for dopamine, which shows potential for the fabrication of novel reaction-based fluorescent probe using GFP-like chromophores.

NMR and FTIR characterization data. This material is available free of charge via the Internet at http://dx.doi.org/10.1016/j. mtchem.2017.02.002. References [1] R.Y. Tsien, The green fluorescent protein, Annu. Rev. Biochem. 67 (1998) 509e544. [2] M. Zimmer, GFP: from jellyfish to the Nobel prize and beyond, Chem. Soc. Rev. 38 (2009) 2823e2832. [3] R.H. Newman, M.D. Fosbrink, J. Zhang, Genetically encodable fluorescent biosensors for tracking signaling dynamics in living cells, Chem. Rev. 111 (2011) 3614e3666. [4] G.N. Phillips, Structure and dynamics of green fluorescent protein, Curr. Opin. Struct. Biol. 7 (1997) 821e827. [5] T.D. Craggs, Green fluorescent protein: structure, folding and chromophore maturation, Chem. Soc. Rev. 38 (2009) 2865e2875.

H. Deng et al. / Materials Today Chemistry 3 (2017) 73e81 [6] E.A. Dolgopolova, T.M. Moore, W.B. Fellows, M.D. Smith, N.B. Shustova, Photophysics of GFP-related chromophores imposed by a scaffold design, Dalton Trans. 45 (2016) 9884e9891. [7] N.Y.A. Baffour-Awuah, M. Zimmer, Hula-twisting in green fluorescent protein, Chem. Phys. 303 (2004) 7e11. [8] Y.O. Jung, J.H. Lee, J. Kim, M. Schmidt, K. Moffat, V. Srajer, H. Ihee, Volumeconserving trans-cis isomerization pathways in photoactive yellow protein visualized by picosecond X-ray crystallography, Nat. Chem. 5 (2013) 212e220. [9] H.P. Deng, X.Y. Zhu, Emission enhancement and application of synthetic green fluorescent protein chromophore analogs, Mater. Chem. Front. (2017), http:// dx.doi.org/10.1039/c6qm00148c. [10] L.M. Tolbert, A. Baldridge, J. Kowalik, K.M. Solntsev, Collapse and recovery of green fluorescent protein chromophore emission through topological effects, Acc. Chem. Res. 45 (2012) 171e181. [11] A. Baldridge, S.R. Samanta, N. Jayaraj, V. Ramamurthy, L.M. Tolbert, Activation of fluorescent protein chromophores by encapsulation, J. Am. Chem. Soc. 132 (2010) 1498e1499. [12] A. Baldridge, S.H. Feng, Y.T. Chang, L.M. Tolbert, Recapture of GFP chromophore fluorescence in a protein host, ACS Comb. Sci. 13 (2011) 214e217. [13] J.S. Paige, K.Y. Wu, S.R. Jaffrey, RNA mimics of green fluorescent protein, Science 333 (2011) 642e646. [14] H.P. Deng, Q. Zhu, D.L. Wang, C.L. Tu, B.S. Zhu, X.Y. Zhu, GFP-inspired fluorescent polymer, Polym. Chem. 3 (2012) 1975e1977. [15] D.E. Williams, E.A. Dolgopolova, P.J. Pellechia, A. Palukoshka, T.J. Wilson, R. Tan, J.M. Maier, A.B. Greytak, M.D. Smith, J.A. Krause, N.B. Shustova, Mimic of the green fluorescent protein b-barrel: photophysics and dynamics of confined chromophores defined by a rigid porous scaffold, J. Am. Chem. Soc. 137 (2015) 2223e2226. [16] H.P. Deng, Y. Su, M.X. Hu, X. Jin, L. He, Y. Pang, R.J. Dong, X.Y. Zhu, Multicolor fluorescent polymers inspired from green fluorescent protein, Macromolecules 48 (2015) 5969e5979. ̌ [17] L.X. Wu, K. Burgess, Syntheses of highly fluorescent GFP-chromophore analogues, J. Am. Chem. Soc. 130 (2008) 4089e4096. [18] M.S. Baranov, K.A. Lukyanov, A.O. Borissova, J. Shamir, D. Kossenkov, L.V. Slipchenko, L.M. Tolbert, I.V. Yampolsky, K.M. Solntsev, Conformationally locked chromophores as models of excited-state proton transfer in fluorescent proteins, J. Am. Chem. Soc. 134 (2012) 6025e6032. [19] Y.H. Hsu, Y.A. Chen, H.W. Tseng, Z.Y. Zhang, J.Y. Shen, W.T. Chuang, T.C. Lin, C.S. Lee, W.Y. Hung, B.C. Hong, S.H. Liu, P.T. Chou, Locked Ortho-and para-core chromophores of green fluorescent protein; dramatic emission enhancement via structural constraint, J. Am. Chem. Soc. 136 (2014) 11805e11812. [20] H.P. Deng, C.Y. Yu, L.D. Gong, X.Y. Zhu, Self-restricted green fluorescent protein chromophore analogues: dramatic emission enhancement and remarkable solvatofluorochromism, J. Phys. Chem. Lett. 7 (2016) 2935e2944. [21] H. Kobayashi, M. Ogawa, R. Alford, P.L. Choyke, Y. Urano, New strategies for fluorescent probe design in medical diagnostic, Chem. Rev. 110 (2010) 2620e2640. [22] J. Chan, S.C. Dodani, C.J. Chang, Reaction-based small-molecule fluorescent probes for chemoselective bioimaging, Natur. Chem. 4 (2012) 973e984. [23] L. Yuan, W.Y. Lin, K.B. Zheng, L.W. He, W.M. Huang, Rar-red to near infrared analyte-responsive fluorescent probes based on organic fluorophore platforms for fluorescence imaging, Chem. Soc. Rev. 42 (2013) 622e661. [24] X.Y. Liu, L. Shi, Z.F. Ding, Y.T. Long, New insight into the application of GFP chromophore inspired derivatives: a F
fluorescent chemodosimeter, RSC Adv. 4 (2014) 53557e53560. [25] H. Sunahara, Y. Urano, H. Kojima, T. Nagano, Design and synthesis of a library of BODIPY-based environmental polarity sensors utilizing photoinduced electron-transfer-controlled fluorescence ON/OFF switching, J. Am. Chem. Soc. 129 (2007) 5597e5604. [26] M.J. Frisch, G.W. Trucks, H.B. Schlegel, G.E. Scuseria, M.A. Robb, J.R. Cheeseman, G. Scalmani, V. Barone, B. Mennucci, G.A. Petersson, H. Nakatsuji, M. Caricato, X. Li, H.P. Hratchian, A.F. Izmaylov, J. Bloino, G. Zheng, J.L. Sonnenberg, M. Hada, M. Ehara, K. Toyota, R. Fukuda, J. Hasegawa, M. Ishida, T. Nakajima, Y. Honda, O. Kitao, H. Nakai, T. Vreven, J.A. Montgomery, J.E. Peralta, F. Ogliaro, M. Bearpark, J.J. Heyd, E. Brothers, K.N. Kudin, V.N. Staroverov, T. Keith, R. Kobayashi, J. Normand, K. Raghavachari, A. Rendell, J.C. Burant, S.S. Iyengar, J. Tomasi, M. Cossi, N. Rega, J.M. Millam, M. Klene, J.E. Knox, J.B. Cross, V. Bakken, C. Adamo, J. Jaramillo, R. Gomperts, R.E. Stratmann, O. Yazyev, A.J. Austin, R. Cammi, C. Pomelli, J.W. Ochterski, R.L. Martin, K. Morokuma, V.G. Zakrzewski, G.A. Voth, P. Salvador, J.J. Dannenberg, S. Dapprich, A.D. Daniels, O. Farkas, J.B. Foresman, J.V. Ortiz, J. Cioslowski, D.J. Fox, Gaussian 09, Inc., Wallingford CT, 2013. [27] S. Vosko, L. Wilk, M. Nusair, Accurate spin-dependent electron liquid correlation energies for local spin density calculations: a critical analysis, Can. J. Phys. 58 (1980) 1200e1211.

81

[28] A.D. Becke, Density-functional thermochemistry. V. Systematic optimization of exchange-correlation functionals, J. Chem. Phys. 107 (1997) 8554e8560. [29] W. Kohn, A.D. Becke, R.G. Parr, Density functional theory of electronic structure, J. Phys. Chem. 100 (1996) 12974e12980. [30] O. Treutler, R. Ahlrichs, Efficient molecular numerical integration schemes, J. Chem. Phys. 102 (1995) 346e354. [31] F. Furche, R. Ahlrichs, Adiabatic time-dependent density functional methods for excited state properties, J. Chem. Phys. 117 (2002) 7433e7447. [32] I.V. Yampolsky, S.J. Remington, V.I. Martynov, V.K. Potapov, S. Lukyanov, K.A. Lukyanov, Synthesis and properties of the chromophore of the asFP595 chromoprotein from Anemonia Sulcate, Biochemistry 44 (2005) 5788e5793. [33] X. He, A.F. Bell, P.J. Tonge, Synthesis and spectroscopic studies of model red fluorescent protein chromophores, Org. Lett. 4 (2002) 1523e1526. [34] T.M. Figueira-Duarte, K. Müllen, Pyrene-based materials for organic electronics, Chem. Rev. 111 (2011) 7260e7314. [35] M. Shellaiah, Y.H. Wu, A. Singh, M.V. Ramakrishnam Raju, H.C. Liu, Novel pyrene-and anthracene-based schiff base derivatives as Cu2þ and Fe3þ fluorescence turn-on sensors and for aggregation induced emissions, J. Mater. Chem. A 1 (2013) 1310e1318. [36] C.W. Cheng, G.J. Huang, H.Y. Hsu, C. Prabhakar, Y.P. Lee, E.W.G. Diau, J.S. Yang, Effects of hydrogen bonding on internal conversion of GFP-like chromophores. II. The meta-amino systems, J. Phys. Chem. B 117 (2013) 2705e2716. [37] G.-J. Huang, J.H. Ho, C. Prabhakar, Y.-H. Liu, S.-M. Peng, J.-S. Yang, Site-selective hydrogen-bonding-induced fluorescence quenching of highly solvatofluorochromic GFP-like chromophores, Org. Lett. 14 (2012) 5034e5037. [38] D.M. Chudakov, S. Lukyanov, K.A. Lukyanov, Fluorescent proteins as a toolkit for in vivo imaging, Trends Biotechnol. 23 (2005) 605e613. [39] S.L. Tou, G.J. Huang, P.C. Chen, H.T. Chang, J.Y. Tsai, J.S. Yang, Aggregationinduced emission of GFP-like chromophores via exclusion of solvent-solute hydrogen bonding, Chem. Commun. 50 (2014) 620e622. [40] J. Mei, N.L.C. Leung, R.T.K. Kwok, J.W.Y. Lam, B.Z. Tang, Aggregation-induced emission: together we shine, united we soar, Chem. Rev. 115 (2015) 11718e11940. [41] Y. Hong, J.W.Y. Lam, B.Z. Tang, Aggregation-induced emission, Chem. Soc. Rev. 40 (2011) 5361e5388. [42] J.B. Greenwood, J. Miles, S.D. Camillis, P. Mulholland, L. Zhang, M.A. Parkes, H.C. Hailes, H.H. Fielding, Resonantly enhanced multiphoton ionization spectrum of the neutral green fluorescent protein chromophore, J. Phys. Chem. Lett. 5 (2014) 3588e3592. [43] K.B. Bravaya, B.L. Grigorenko, A.V. Nemukhin, A.I. Krylov, Quantum chemistry behind bioimaging: insights from ab initio studies of fluorescent proteins and their chromophores, Acc. Chem. Res. 45 (2012) 265e275. [44] I. Polyakov, E. Epifanovsky, B. Grigorenko, A.I. Krylov, A. Nemukhin, Quantum chemical benchmark studies of the electronic properties of the green fluorescent protein chromophore: 2. Cis-trans isomerization in water, J. Chem. Theory Comput. 5 (2009) 1907e1914. [45] E. Epifanovsky, I. Polyakov, B. Grigorenko, A. Nemukhin, A.I. Krylov, Quantum chemical benchmark studies of the electronic properties of the green fluorescent protein chromophore. 1. Electronically excited and ionized states of the anionic chromophore in the gas phase, J. Chem. Theory Comput. 5 (2009) 1895e1906. [46] P. Altoe, F. Bernardi, M. Garavelli, G. Orlandi, F. Negri, Solvent effects on the vibrational activity and photodynamics of the green fluorescent protein chromophore: a quantum-chemical study, J. Am. Chem. Soc. 127 (2005) 3952e3963. [47] A.P. Demchenko, K.C. Tang, P.T. Chou, Excited-state proton coupled charge transfer modulated by molecular structure and media polarization, Chem. Soc. Rev. 42 (2013) 1379e1408. [48] D.M. Stoltzfus, J.E. Donaghey, A. Armin, P.E. Shaw, P.L. Burn, P. Meredith, Charge generation pathways in organic solar cells: assessing the contribution from the electron acceptor, Chem. Rev. (2016), http://dx.doi.org/10.1021/ acs.chemrev.6b00126. [49] Y. Teng, X.F. Jia, J. Li, E.K. Wang, Ratiometric fluorescence detection of tyrosinase activity and dopamine using thiolate-protected gold nanoclusters, Anal. Chem. 87 (2015) 4897e4902. [50] L. Zhang, Y. Cheng, J.P. Lei, Y.T. Liu, Q. Hao, H.X. Ju, Stepwise chemical reaction strategy for highly sensitive electrochemiluminescent eetection of dopamine, Anal. Chem. 85 (2013) 8001e8007. [51] X. Ji, G. Palui, T. Avellini, H.B. Na, C.Y. Yi, K.L. Knappenberger, H. Mattoussi, On the pH-dependent quenching of quantum dot photoluminescence by redox active dopamine, J. Am. Chem. Soc. 134 (2012) 6006e6017. [52] I.V. Yampolsky, T.A. Balashova, K.A. Lukyanov, Synthesis and spectral and chemical properties of the yellow fluorescent protein zFP538 chromophore, Biochemistry 48 (2009) 8077e8082. [53] M.Å. Petersen, P. Riber, L.H. Andersen, M.B. Nielsen, Synthesis and characterization of model compounds for the neutral green fluorescent protein chromophore, Synthesis 23 (2007) 3635e3638.