Benzothiazole-Based AIEgen with Tunable Excited-State

Apr 18, 2018 - High-sensitive detecting methods for physiological pH are essential for ... (6−8) As a noninvasive and highly sensitive technique, fl...
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Benzothiazole-based AIEgen with tunable excited-state intramolecular proton transfer (ESIPT) and restricted intramolecular rotation (RIR) processes for highly sensitive physiological pH sensing Kai Li, Qi Feng, Guangle Niu, Weijie Zhang, Yuanyuan Li, Miaomiao Kang, Kui Xu, Juan He, Hongwei Hou, and Ben Zhong Tang ACS Sens., Just Accepted Manuscript • DOI: 10.1021/acssensors.7b00820 • Publication Date (Web): 18 Apr 2018 Downloaded from http://pubs.acs.org on April 19, 2018

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ACS Sensors

Benzothiazole-based AIEgen with tunable excited-state intramolecular proton transfer (ESIPT) and restricted intramolecular rotation (RIR) processes for highly sensitive physiological pH sensing Kai Li,*,†,§,▼ Qi Feng,†,▼ Guangle Niu,§,▼ Weijie Zhang,§ Yuanyuan Li,‡ Miaomiao Kang,§ Kui Xu,† Juan He,‡ Hongwei Hou*,† and Ben Zhong Tang*,§ †

College of Chemistry and Molecular Engineering, Zhengzhou University, Zhengzhou 450001, P. R. China College of Chemistry, Chemical and Environmental Engineering, Henan University of Technology, Henan 450001, P. R. China § Department of Chemistry and Hong Kong Branch of Chinese National Engineering Research Center for Tissue Restoration and Reconstruction, The Hong Kong University of Science & Technology, Clear Water Bay, Kowloon, Hong Kong, China ‡

ABSTRACT: In this work, a benzothiazole-based aggregation-induced emission luminogen (AIEgen) of 2-(5-(4-carboxyphenyl)-2-hydroxyphenyl)benzothiazole (3) was designed and synthesized, which exhibited multi-fluorescence emissions in different dispersed or aggregated state based on tunable excited-state intramolecular proton transfer (ESIPT) and restricted intramolecular rotation (RIR) processes. 3 was successfully used as a ratiometric fluorescent chemosensor for the detection of pH, which exhibited reversible acid/base-switched yellow/cyan emission transition. More importantly, the pH jump of 3 was very precipitous from 7.0 to 8.0 with a midpoint of 7.5, which was well matched with the physiological pH. This feature makes 3 very suitable for the highly sensitive detection of pH fluctuation in biosamples and neutral water samples. 3 was also successfully used as a ratiometric fluorescence chemosensor for the detection of acidic and basic organic vapors in test papers. KEYWORDS: aggregation-induced emission, excited-state intramolecular proton transfer, restricted intramolecular rotation, physiological pH, ratiometric fluorescent chemosensor

As a key parameter in biological samples, pH plays an essential role in many physiological processes such as cell proliferation and apoptosis, ion transport, enzyme activity, protein degradation, etc.1-3 In healthy individuals, the pH of blood and intracellular fluid is in the range of 7.35-7.45.4, 5 This weakly alkaline pH range is often referred as the “physiological pH”. High-sensitive detecting methods for physiological pH are essential for medical examination, environmental monitoring and food security.6-8 As a noninvasive and high sensitive technique, fluorescent chemosensors have attracted considerable interest in pH sensing, especially in the detection of local pH in live cells due to its real-time monitoring property. 9, 10 However, high-sensitive physiological pH sensing is still challenging because of the lack of effective chemosensors. On one hand, the “physiological pH” means that both of the concentrations of hydrated proton and hydroxide ion are very low (10-6 ~ 10-7 mol/L) in this pH range, which raises higher requirement to the detection limit. On the other hand, most of the reported chemosensors for pH sensing exhibit only one emission channel, which makes the detection easily influenced by environmental interferences such as variation of chemosensor concentration, background fluorescence, and photobleaching.11-15 In contrast, ratiometric fluorescent chemosensors with two emission channels provides a built-in correction for the environmental interferences, which can

effectively increase the sensitivity, enlarge the dynamic range and improve the accuracy of measurements.16-21 The main challenge for the design of fluorescent chemosensors is the notorious aggregation caused quenching (ACQ) effect in most of the conventional luminogens, which usually show high emission in dispersed state but weak or even no emission in aggregated state.22 The ACQ effect is unfavourable for the fluorescence detections in aqueous solution, because most of the organic fluorescent chemosensors are hydrophobic and tend to aggregate in aqueous media, resulting in a fluorescence quenching. Unforturnately, physiological processes usually take placed in enviroments with high water fration, which makes the pH sensing in physiological samples difficult when using luminogens with ACQ effect. In contrast to the luminogens with ACQ effect, aggregation-induced emission luminogens (AIEgens) exhibited stronger emission in aggregated state than that in dispersed state, which is benificial to the design of fluorescent chemosensor used in systems with high water fraction.23-28 The unique aggregation-induced emission (AIE) effect is usually originated from the mechanism of restricted intramolecular rotation (RIR): The rotor-containing AIEgens undergo dynamic intramolecular rotations in dilute solutions, which will cause the "excited state non-radiative transition" and result in fluorescence quenching. While in the aggregated 1

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state (or solid state), the rotations are greatly restricted, which makes the "radiative transition" be the primary pathway for the excited state electrons back to ground state.29-31 The RIR mechanism is applicable to most of the propeller-like AIEgens including tetraphenylethene (TPE), hexaphenylsilole (HPS), tetraphenylpyrazine (TPP), 2,5-bis(4-alkoxycarbonylphenyl)-1,4-diaryl-1,4-dihydropyrrol o[3,2-b]pyrrole (AAPP), etc.32-35 For some other AIEgens with intramolecular H-bond such as 2-(2-hydroxyphenyl)benzothiazole (HBT), salicylaldehyde azine (SAA), 4-N,N-dimethylaminoaniline salicylaldehyde hydrazone (DAS), 2-(2-hydroxyphenyl)quinazolin-4(3H)-one (HPQ), however, the excited-state intramolecular proton transfer (ESIPT) process rather than the RIR process should be the primary cause for their AIE effect.36-41 For example, 2-(2-hydroxyphenyl)benzothiazole (HBT) is a classical ESIPT molecular with an intramolecular H-bond between its proton donor (-OH ) and proton acceptor (-N=) groups (Figure 1).42, 43 In the electronic ground state, HBT exists as an enol form (HBT-Enol), which is stabilized by the intramolecular H-bond. Upon photo excitation, an exited enol form of HBT-Enol* generated. Then a fast proton transfer reaction accompanied with a tautomeric transformation from HBT-Enol* to the excited keto form (HBT-Keto*) occurred due to the redistribution of electronic charge. The HBT-Keto* can decay radiatively to its ground state (HBT-Keto). After that, HBT-Keto will turn back to HBT-Enol by proton transfer. This fast four-level cycle in HBT usually results in two different types of emission bands, which are dependent on solvent environments.44, 45 One belongs to the decay from HBT-Enol* to HBT-Enol, which is called Enol fluorescence emission (Enol FL). This emission band was predominantly observed in protic solvents because the intramolecular hydrogen bond in HBT can be replaced by intermolecular hydrogen bond between HBT and protic solvent molecules, which hinders the proton transfer in HBT. The other emission band is called ESIPT fluorescence emission (Keto FL), which exhibited much larger Stokes’ shift and weaker intensity than that of Enol FL. Keto FL was mainly observed in aprotic solvents or aggregated state where the intramolecular H-bond is impregnable by solvent molecules. From this point of view, the AIE fluorescence caused by the aggregation of intramolecular H-bond containing AIEgens is exactly the Keto FL.46-48

highly dependent on solvent environments owing to their alterable H-bond types. Similarly, acids or bases in the solvents can also change their fluroescence: Acidic environment is benificial to the stablility of intramolecular H-bond while alkaline environment will break the intramolecular H-bond by deprotonation.49-51 This character suggests that the intramolecular H-bond containing AIEgens may be an ideal strategy for the development of fluorescent chemosensors for pH sensing. In this work, a new AIEgen of 2-(5-(4-carboxyphenyl)-2-hydroxyphenyl)benzothiazole (3) was designed and synthesized, which exhibited multi-fluorescence emissions based on tunable ESIPT and RIR processes. As shown in Scheme 1, 3 was designed by connecting HBT with rotatable conjugated planes through a single bond. The ESIPT and RIR processes endow 3 a typical AIE property with intense fluorescence emission in aggregated state. Meanwhile, the ESIPT process makes 3 very sensitive to the solvent environments, especially to solvents with different pH. In alkaline conditions, the fluorescence emission of 3 is around 484 nm and a cyan fluorescence can be observed by naked eyes under UV light irradiation. In acidic conditions, the cyan fluorescence quenches while a yellow fluorescence appears with a fluorescence emission around 551 nm. More importantly, the pH jump range is very precipitous form 7.0 to 8.0 and its midpoint is 7.5, which is very close to the “physiological pH”. This feature makes 3 very suitable for the pH sensing in biosamples as well as neutral water samples. The pH-switched emission transition of 3 is reversible and the fatigue resistance is excellent. As a ratiometric fluorescence chemosensor, 3 was also successfully used for the detection of acidic and basic organic vapors in test papers.

Experimental Section Materials. All of the reagents and solvents were commercially available and used as received. 2-aminothiophenol, 5-bromosalicylaldehyde, 5-bromo-2-methoxybenzaldehyde, 4-methoxycarbonylphenylboronic acid, tetrakis(triphenylphosphine)palladium, triethylamine (TEA) and phosphate buffered saline (PBS) were purchased from J&K Chemical Co., Beijing, China. Minimum essential media (MEM), fetal bovine serum (FBS), 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT), MitoTracker Deep-Red FM, sodium dodecyl sulfate (SDS), penicillin and streptomycin were purchased from Invitrogen. Nigericin and monensin was purchased from Sigma-Aldrich. All the other materials were purchased from Sinopharm Chemical Reagent Beijing Co., Beijing, China. Milli-Q water was used in the whole experiment which was supplied by Milli-Q Plus System (Millipore Corporation, United States). Instruments. The nuclear magnetic resonance (1H NMR and 13C NMR) spectra measurements were performed on a Bruker 400 Avance NMR spectrometer operated at 400 MHz. High resolution mass spectra (HRMS) were recorded on Agilent Technologies 6420 triple quadrupole LC/MS without using the LC part. Fluorescence spectra were recorded on a JASCO-FP-8300 fluorescence spectrophotometer (JASCO International Co., Ltd, Japan) with 1 cm quartz cell at 25 oC.

Figure 1. Four-level energy diagram of ESIPT process of HBT.

According to the mechanism of ESIPT, the luminescence properties of intramolecular H-bond containing AIEgens are 2

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ACS Sensors Absorption spectra were recorded on a JASCO-750 UV-vis spectrophotometer (JASCO International Co., Ltd, Japan) with 1 cm quartz cell at 25 oC. The temperatures in fluorescence and absorption measurements were controlled by ETC-815 peltier thermostatted single cell holder (water cooled) and ETCS-761 peltier thermostatted single cell holder (water cooled), respectively, which offered temperature control accuracy of ±0.1 oC. Fluorescence lifetimes were recorded on an Edinburgh FLS-980 fluorescence spectrometer (Edinburgh instruments Ltd, UK) with a light source of EPL-405 picosecond pulsed diode laser. Dynamic light scattering (DLS) measurements were performed on a NanoPlus-3 DLS particle size/zeta potential analyzer (Micromeritics Instrument Corporation, CA, USA). The pH was determined by a PHS-3C pH meter (Shanghai INESA Scientific Instrument Co., LTD, China). Single-crystal X-ray diffraction intensity data were recorded using a Rigaku Saturn 724 CCD diffractometer (Rigaku Corporation, Japan) with Mo Ka radiation (λ = 0.71073 Å) at room temperature. Fluorescence images were taken using a LSM7 DUO confocal laser scanning microscope (Carl Zeiss Co., Ltd.) with an objective lens (×63). Perkin-Elmer Victor 3 plate reader with excitation at 440±10 nm (Perkin-Elmer, Wellesley, MA, USA) was used in cell viability test. The photos were taken by a Nikon D5500 camera.

absorbance (570 nm) of the wells were recorded by plate reader. Synthesis and Characterizations. The synthetic routes of the compounds mentioned in this work were shown in Scheme 1 and Scheme S1. The new compounds were characterized by nuclear magnetic resonance (1H and 13C NMR) spectroscopy and high-resolution mass spectroscopy (HRMS). Detailed synthetic procedures and characterization can be found in Electronic Supplementary Information. Scheme 1. Synthesis of compound 3

Results and discussion Fluorescence characteristics of 3. The fluorescence characteristics of 3 in mixed solvents of water/ethanol (water fraction (fw) from 0% to 99%, v/v) were investigated first. It is very interesting that there were three fluorescence peaks (398 nm, 484 nm and 551nm) in the fluorescence spectra in solvents with different fw (Figure 2). As shown in Figure 2B, a strong fluorescence peak around 398 nm could be observed in pure ethanol (fw = 0). This emission decreased gradually along with the increase of fw while a new emission around 484 nm appeared and reached its maximum when fw was 70%. Once fw was beyond 70%, the fluorescence emission of 484 nm decreased rapidly and a yellow emission around 551 nm emerged. These three fluorescence emissions suggested that there might be three different luminescence processes in 3 which existed in different forms depending on the solvents.

Cell imaging. HeLa cells were seeded on a 35 mm (diameter) petri dish. The cells were incubated overnight in MEM supplemented with 10% FBS, penicillin (100 units/mL), and streptomycin (100 mg/mL) at 37 oC under a humidified atmosphere containing 5% CO2. For co-staining experiments, after incubation with 50 µmol/L 3 for 3 h, the cells were washed with PBS and further incubated with MitoTracker Deep-Red FM (100 nM) for another 20 min. For pH calibration experiments, after removing the medium, the cells were incubated with a solution containing 50 µmol/L 3 for 3 h under the same condition. The pH calibration buffers with different pH values of 6.86, 7.38, 7.63 and 8.07 were obtained by adding very small amount of NaOH solution, which contain 125 mmol/L KCl, 25 mmol/L NaCl, 0.5 mmol/L CaCl2, 0.5 mM MgCl2, 5 µmol/L nigericin, 5 µmol/L monensin, 10 mmol/L Na2HPO4 and 10 mmol/L citric acid. Before cell imaging, the medium was removed and the cells were washed with the pH calibration buffer (without nigericin and monensin) to wipe off the surplus material. Then the 3-labeled cells were treated with the pH calibration buffer for 20 min at 25 oC and washed with corresponding buffer (without nigericin and monensin) three times. The cells were imaged with Zeiss laser scanning confocal microscope with two channels. For channel I, the mission signals from 450 to 520 nm were collected. For channel II, the mission signals from 520 to 600 nm were collected. The excitation wavelength was 405 nm. The images were analyzed with Image-Pro Plus software. Cell viability test. HeLa cells were seeded in 96-well plates at density of 5000 cells/well and cultured overnight. Then the medium in the wells were replaced by new medium containing compound 3 with different concentrations. After 24 h’s treatment, 10 µL of 5 mg/mL MTT solution was added to the wells and the systems was incubated for another 4 h at 37 oC. Afterward, 0.1 mL SDS-HCl solutions which contain 10% SDS and 0.01 mol/L HCl were added to the wells. After 6 h’s incubation, the

Figure 2. A) Fluorescence photograph and B) fluorescence emission spectra of 3 in water/ethanol mixtures with different fw. C) The fluorescence emissions at 398 nm, 484 nm and 551 nm as functions of fw. Conditions: The concentration of 3 was 50 µmol/L. Water was buffered by PBS at pH 6.0. The photograph was taken under an irradiation of 365 nm UV light.

3

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As a compound with intramolecular H-bond, the multi-fluorescence emissions of 3 might be originated from the prototropic equilibria in different solvents. A possible mechanism was proposed in Figure 3, which shows the excited state structures giving different emission wavelengths. In pure ethanol, the intsramolecular H-bond of 3 was mostly replaced by the intermolecular H-bond between 3 and ethanol, which hindered the ESIPT process. Therefore, normal decay from the excited form (Enol*) to ground-state form (Enol) took place with strong fluorescence emission at 398 nm (Enol FL). In the mixed solevent with increased fw, 3 formed anions in the excited state by deprotonation due to the increase of solvent polarity, resulting in a new emission band at 484 nm (Deprotonated FL). When fw was higher than 70%, aggregated state of 3 was mostly preferred because of its insolubility in water, in which the intramolecular H-bond of 3 was stable. Thus, an ESIPT process occurred, resuting in a fluorescence emission at 551 nm (Keto FL), i.e., an AIE emission.

intermolecular H-bond was formed. It was noticed that in some aprotic solvents with low polarity such as toluene and chloroform, a fluorescence emission at 540 nm could be clearly observed. Accoding to the ESIPT theory mentioned above, a built-in intramolecular H-bond is stable in aprotic solvents and is essential for the efficient ESIPT process. Therefore, the emission at 540 nm belongs the Keto FL of 3, which is intense in aprotic solvents but weak in protic solvents.

Figure 4. Fluorescence spectra of 3 in different solvents. Conditions: The concentration of 3 was 50 µmol/L. Figure 3. Proposed excited state structures for the multi-fluorescence emissions of 3 in solvents with different fw.

Here is the question: both of the emissions of 3 in aggregated state and aprotic solvents are Keto FL, why the wavelengths are different (551 nm and 540 nm, respectively)? In order to clarify this question, the structures of 3 in different environments were first investigated. Crystal characterization was performed by X-ray crystallography to demonstrate the structure of 3 in aggregated state, while density functional theory (DFT) caculation was used to optimize the structure of 3 in aprotic solvents. As can be seen in Figure 5, the dihedral angle between benzoic acid rotor and HBT plane was 31.87o in aggregated state, while it was 36.52o in aprotic solvents. The smaller dihedral angle in aggregated state was attribute to the packing effect, which enables 3 a more planar structure with better conjugation. It was reported that a better conjugation in the luminogens usually exhibits a red-shifted emission.54, 55 Therefore, the fluorescence wavelength of 3 in aggregated state was longer than that in aprotic solvents. Furthermore, a control compound of 2-(2-hydroxy-5-carboxyphenyl)benzothiazole (3a) was designed and synthesized (Figure 6). Compared with 3, there was no biphenyl moiety in 3a, which endow it analogous degree of conjugation in aggregated state and in aprotic solvents. As expected, the emission wavelength of 3a in aprotic solvent (toluene) was very similar to that in aggregated state (Figure 6). This result demonstrated that the rotation of the conjugated plane (biphenyl moiety) in different state had a significant impact on the Keto FL wavelength of 3, which also explained that why there were two different wavelengths of 551 nm and 540 nm in aggregated state and aprotic solvents, respectively.

To verify the proposed mechanism, a series of experiments were carried out. Firstly, the AIE fluorescence at 551 nm was investigated. As shown in Figure S1, the fluorescence spectra and fluorescence lifetime of 3 in aqueous solution (fw = 99%) were very similar to that in solid state , which supported that the fluorescence emission at 551 nm was originated from the aggregation. Another evidence for the aggregation of 3 in aqueous solution was obtained from absorption spectra. As can be seen in Figure S2, 3 displayed a structured absorption spectrum in ethanol, indicating its good solubility. In contrast, the fine structures of the absorption spectrum of 3 disappeared in aqueous solution and a level-off tail could be clearly seen, which was due to the light scattering of aggregated suspensions.52, 53 Moreover, dynamic light scattering (DLS) measurement (Figure S2, inset) showed that there were particles around 1000 nm in aqueous solution but no particles in ethanol. Because of the aggregation in aqueous solution, the intramolecular H-bond in 3 was stablized, which was benifical to the ESIPT process. Thus, a fast four-level cycle occured after photo excitation, resulting in an Keto FL at 551 nm. Meanwhile, the minimum distance of the adjacent conjugate planes of 3 was 3.98 Å (Figure S3), which was larger than 3.8 Å and not conducive for π-π stacking interaction in the crystal.36 This structural feature was favorable for AIE fluorecence. The above evidences about the aggregation of 3 fully demonstrated that the Keto FL was an AIE fluorescence. As had mentioned, the ESIPT process are very sensitive to the solvent environments.44, 45 Thus, the fluorescence emissions of 3 in solvents with different structure and structure were investigated. As shown in Figure 4 and Figure S4, 3 exhibited strong deep blue fluorescence emission around 398 nm in polar protic solvents such as methanol and ethanol. This emission was believed to be originated from the Enol form of 3, which was stabilized by the intermolecular H-bond between 3 and solvent molecules. In contrast, the Enol FL of 3 in polar aprotic solvents was extremely weak since no 4

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ACS Sensors solvents. As show in Figure S7, all of the emission wavelengths were around 484 nm and the correspondinsg lifetimes were about 3 ns, which were analogous to that of 3 in the mixture of water/ethanol (fw = 70%). These results indicated that the cyan fluorescence at 484 nm was originated from the deprotonated form of 3. Ratiometric pH sensing using 3. According to the above proposed mechanism, the proton/deproton processes can effectively influence the luminescence properties of compound 3, which suggested that it might be a potential candidate for pH sensing. Thus, the fluorescence of 3 in aqueous solutions with different pH were investigated. As shown in Figure 7A, 3 shows yellow fluorescence at pH 2.0-6.0 while cyan fluorescence at pH 8.0-12.0. Fluorescence titration experiment of 3 was carried out in aqueous solution and ratiometric fluorescence patterns were observed (Figure 7B). The emission peaks in acidic and alkaline condition were 551 nm and 484 nm, respectively. Meanwhile, a unique narrow jump range at pH 6.9-8.0 was obtained (Figure 7B inset), which fitted well with the physiological pH range. It was noticed that in this pH range, a satisfying linear relationship between the emission ratio and pH can be observed (Figure S8, which is a partial enlargement of the data shown in Figure 7B inset), whose R value was as high as 0.98. More importantly, the midpoint of the jump range was 7.5, which was very close to the pH of healthy tissue. These properties suggest that 3 could be used for physiological pH sensing with high sensitivity. The fatigue resistance of 3 was further investigated to evaluate its performance as a reversible fluorescent chemosensor (Figure 7C). The pH value of the solution was switched back and forth between 2.0 and 12.0 by adding concentrated hydrochloric acid and aqueous sodium hydroxide for 10 times. The fluorescence intensity ratio of 3 (I484/I551) stayed almost constant without apparent degradation, indicating a good fatigue resistance. The reversible, ratiometric and highly sensitive fluorescence response as well as the good fatigue resistance of 3 indicate that it can be served as promising chemosensor for physiological pH sensing.

Figure 5. The dihedral angle between benzoic acid rotor and HBT plane of 3. A) Crystal structure of 3. B) Optimized configuration of 3 in Toluene obtained by DFT calculation (Frontier molecular orbitals and energy levels of 3 are shown in Figure S5).

Figure 6. Fluorescence spectra of 3a in solid state and in toluene. Conditions: The concentration of 3a in toluene was 50 µmol/L.

As well as the emission wavelength, the fluorescence intensity is also influenced by the rotatable conjugated planes in 3. As we mentioned above, RIR process is a primary cause for the AIE fluorescence in most of the rotor-containing AIEgens. To verify that the RIR process also contributed to the AIE intensity of 3, the fluorescence emissions of 3 in solvent with high viscosity (ethanol/glycerol) were investigated. As shown in Figure S6, 3 exhibited strong fluorescence emission at 398 nm in all of the mixtures because both ethanol and glycerol are protic solvents and beneficial to Enol FL. For the Keto FL at 540 nm, gradual enhancements were found with the increase of glycerol fraction (fg). The most likely cause should be that the intramolecular rotations were restricted by the high viscosity of solvent and the radiative transition was enhanced, which resulted in intense fluorescense. Therefore, the rotatable conjugated planes caused RIR process also makes a great contribution to the AIE intensity of 3. The above experiments demonstrated that the fluorescence emissions of 3 at 398 nm and 551 nm (or 540 nm) are originated from its Enol FL and Keto FL, respectively. Beside these two emissions, it can be seen in Figure 2 that there was another cyan fluorescence at 484 nm. This fluorescence might be stemed from the deprotonated 3, whose active hydrogen could be removed in excited state (Figure 3, the middle one).42 To confirm this assumption, the fluorescence spectra and lifetimes of 3 in methanol, acetonitrile and water with excessive alkali were investigated. The excessive alkali insured that 3 existed in deprotonated form in all of these

Figure 7. A) Fluorescence photograph and B) fluorescence emission spectra of 3 in water/ethanol (fw = 99%, v/v) with different pH. C) Reversible fluorescence changes of 3 by adjusting its solution to acidic and basic environments repeatedly. The inset of B) shows the fluorescence intensity ratio of I484/I551 as a function of pH. Conditions: The photograph was taken under an irradiation of 365 nm UV light. The concentration of 3 was 50 µmol/L. 5

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To understand the pH response of 3, a possible mechanism is proposed: There are two typical proton-responsive moieties of carboxyl and phenolic hydroxyl groups in 3 (Figure 8 inset). In acidic condition, 3 is in its neutral state with the carboxyl and phenolic hydroxyl being protonated, which results in low solubility in aqueous solution and an aggregation with yellow fluorescence. Upon the addition of alkali, the carboxyl and phenolic hydroxyl are deprotonated successively with the formation of 3-H+ and 3-2H+, which exhibited cyan fluorescence. To confirm this assumption, a series of experiments were carried out. Absorption spectra titration of compound 3 in buffer solution from pH 2.0 to 12.0 was investigated firstly (Figure S9). The spectra at pH 6.0, 8.3 and 10.0 were specially picked out for comparision (Figure 8). As shown, 3 exhibited an absorption peak around 282 nm at pH 6.0. Along with the addition of alkali, this absorbance increased gradually and reached to its maximum at pH 8.3. This increasement suggests the formation of new compound, which should be 3-H+. The first acid dissociation constant (pKa1) was obtained by nonlinear fitting of the titration curve, which is 7.3 (R2 = 0.995, Figure S9A). When the pH of the solution was higher than 8.3, the absorbance at 282 nm decreased and a new absorption band at 301 nm emerged simultaneously (Figure 8). It was also noticed that a new absorption peak around 390 nm could be observed when the pH of the solution was higher than 7.4 (Figure S9B), which indicated the formation of 3-2H+. Analogous to pKa1, the second acid dissociation constant pKa2 was calculated to be 8.6 by nonlinear fitting of the titration curve (R2 = 0.992). It showed that pKa1 is very close to pKa2 , which results in a narrow pH jump range and is conductive to the highly sensitive pH sensing. As shown in Figure 8, level-off tails in visible region can be clearly observed for 3 and 3-H+, suggesting their poor solubility in aqueous solution.52, 53 For 3-2H+, the tailing in UV-Vis spectra is almost disappeared, which may be attributed to the improved solubility by the high charge.

changed to 8.0 (Figure S10C). These results demostrate that the carboxyl proton is more reactive than the phenolic hydroxyl proton. Unsurprisingly, when the carboxyl proton and phenolic hydroxyl proton of 3 were both repalced by methoxyl (compound 6), the compound was insensitive to pH due to the lack of active proton (Figure S10D). It was noticed that there was no intramolecular hydrogen bond in 5 and 6 between their HBT moiety, so they exhibited no fluorescence. As shown in Figure S11, the fluorescence change of 2 from acid to basic condition was analogous to that of 3, which was form yellow to cyan. The pKa of 2 was 11.2, which means that 2 could be used in the detection of pH change in basic conditions. A more direct evidences for the deprotonate reaction of 3 was observed from the 1H-NMR spectra. Figure S12 shows the 1 H-NMR spectra (aromatic proton peaks) of 3 in DMSO-d6 before and after the addition of triethylamine (TEA), which can adjust the solvent enviroment from neutral to alkaline condition. Before the addition of TEA, the carboxyl proton and phenolic hydroxyl proton resonance peaks (denoted by blue letters of a and b) could be well assigned since 3 existed in its original form. After addition of TEA into the mixture, these two peaks disappeared while other protons signals keep almost no change, which demonstrated the deprotonation of 3 visually. Miscellaneous applications. Not only for detecting pH in solutions, 3 can also be used for distinguishing acidic and alkaline volatile vapors. As shown in Figure 9, yellow and cyan fluorescence were observed in the dye-loaded test papers upon treating with hydrogen chloride and ammonia vapour, respectively. Meanwhile, the fatigue resistance of dye-loaded test papers was investigated by switching between the yellow and cyan emissive states repeatedly. The fluorescence intensity ratio remained almost constant without apparent degradation after 10 times cycle, indicating a good fatigue resistance.

Figure 8. Absorption spectra of 3 in different pH. Inset: Sensing mechanism of 3 to pH. The concentration of 3 was 50 µmol/L.

Figure 9. Reversible fluorescence ratio changes of 3 by fuming with HCl and NH3 vapors repeatedly. Inset: Fluorescence photograph of 3 on a test paper after fuming with HCl and NH3 vapors.

To gain further insight into the deprotonation process of 3 upon the addition of alkali, three control compounds, whose carboxyl proton and phenolic hydroxyl proton are part or all replaced by methoxyl, were prepared (Figure S10A). When the carboxyl proton of 3 was replaced by methoxyl (compound 2), an acid dissociation constant of 11.2 could be obtained from the absorption spectra titration experiment (Figure S10B). In contrast, when the phenolic hydroxyl proton of 3 was replaced (compound 5), the acid dissociation constant

Furthermore, fluorescence imaging of HeLa cells after incubation with 3 were carried out. As shown in Figure 10, 3 stained the linear reticulum-like structures in HeLa cells with high contrast. The fluorescence of 3 was perfectly overlapped with the fluorescence of a commercial mitochondrial probe of MitoTracker Deep-Red FM, and Pearson correlation 6

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ACS Sensors images and ratiometric fluorescence images. The scale bar is 20 µm.

coefficient was 0.92. This result revealed the high mitochondrial staining of 3. Then the intracellular pH was adjusted by a standard approach using nigericin and monensin.57-60 As shown in Figure 11, the fluorescence brightness of channel I (450-520 nm) was enhanced while that of channel II (520-600 nm) decreased when the pH increased from 6.86 to 8.07. The plot of the ratiometric fluorescence intensity of 3 in HeLa cells as a function of pH indicated a satisfactory linearity of R = 0.93 (Figure S13). These two opposite changes indicated that 3 exhibits distinct ratiometric fluorescence response upon the varied pH, which demonstrated that 3 can served as a good candidate for monitoring pH fluctuations in live cells. Moreover, MTT assay was performed to assess the cytotoxicity of 3 to the cells. As can been seen in Figure S14, the cell viability was over 90% after treating with 50 µmol/L of 3, which indicated that 3 exhibited low cytotoxicity to cells at the test concentration.

Conclusions In conclusion, a new HBT-based AIEgen of 3 was designed and synthesized, which exihibited uniuqe multi-fluorescence emissions in different solvents or solid state. Mechanism study showed that the multi-fluorescence emissions of 3 were originated from its tunable ESIPT and RIR processes. The yellow fluorescence (in nonpolar solvents or solid state) and blue fluorescence (in protic solvents) were determined to be originated from its Enol form and Keto form in ESIPT process, while the cyan fluorescence (in solvents with high water fraction or alkaline environment) was attributed to the deprotonated excited state. Sensitive and ratiometric response of 3 to the change of pH were achieved by the proton/deproton processes, which can effectively influence the luminescence properties. More importantly, the pH jump range was very precipitous form 7.0 to 8.0 and its midpoint was 7.5, which was well matched with the physiological pH. The pH-switched emission transition of 3 was reversible and the fatigue resistance was excellent. 3 was successfully used for monitoring pH fluctuations in neutral water sample and live cells. Meanwhile, it was also successfully applied for the detection of acidic and basic organic vapors in test papers.

ASSOCIATED CONTENT Supporting Information Experimental details and additional experimental data referred in the paper. This material is available free of charge via the Internet at http://pubs.acs.org.

Figure 10. A) Bright-field and fluorescent images of HeLa cells co-stained with B) 3 (50 umol/L) and C) MitoTracker Deep-Red FM (100 nM) in culture medium for 20 min. D) Merged image of B) and C). Excitation wavelength: 405 nm for 3 and 635 nm for MitoTracker Deep-Red FM. For 3, the mission signals from 480 to 580 nm were collected. For MitoTracker Deep-Red FM, the mission signals from 650 to 750 nm were collected. The scale bar is 20 µm.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]. *E-mail: [email protected]. *E-mail: [email protected].

Author Contributions ▼

K. Li, Q. Feng and G. Niu contributed equally to this work and should be considered co-first authors.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This research was supported by the National Natural Science Foundation of China (No. 21501150, 51502079, 21371155, 21671174, 21575034), the Funding of Henan Key Laboratory of Cereal Resource Transformation and Utilization (PL2017006), the Fundamental Research Funds for the Henan Provincial Colleges and Universities (2015QNJH09), Science Foundation of Henan University of Technology (2015BS004), the Research Grants Council of Hong Kong (16301614, 16305015, N_HKUST604/14) and Innovation and Technology Commission (ITC-CNERC14SC01).

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Figure 11. Confocal fluorescence images of HeLa cells incubated with 3 (50 µmol/L) and different pH buffers. From left to right: bright-field images, channel I (emission collected from 450 to 520 nm) images, channel II (emission collected from 520 to 600 nm) 7

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SYNOPSIS TOC: Benzothiazole-based AIEgen exhibited multi-fluorescence emissions with tunable excited-state intramolecular proton transfer (ESIPT) and restricted intramolecular rotation (RIR) processes for highly sensitive physiological pH sensing.

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