Turn-On Fluorescent pH Probes for Monitoring Alkaline pHs Using Bis[2-(2′-hydroxyphenyl)benzazole] Derivatives

For surveilling human health, industries, and the environment, pH monitoring is important. Numerous studies on fluorescent probes have been conducted to monitor various pH ranges. However, fluorescent probes that are capable of sensing alkaline regions are rare. In this study, we propose turn-on-type fluorescent probes for detecting alkaline pHs using bis[2-(2′-hydroxyphenyl)benzazole] (bis(HBX)) derivatives. These probes have high pKa values (from 9.7 to 10.8) and exhibit strong fluorescence intensity and color changes at alkaline pHs. Probes derived from bis(HBX) exhibit good photostability, reversibility, and anti-interference toward pH variations, which can be identified as a certain fluorescence change toward a basic pH. Therefore, compounds would be advantageous to use fluorescent probes for monitoring alkaline pH changes.


Introduction
Accurate monitoring of pH is valuable for human health, diseases, industry, and the environment [1]. Therefore, various methods have been developed to monitor pH, such as those utilizing microelectrodes, nuclear magnetic resonance (NMR), colorimetry, and fluorescence [2][3][4][5][6]. Compared to other pH measurement methods, fluorescence technology is beneficial because of its easy visualization and operation, nondestructive character, high sensitivity and selectivity, and the ease of availability of a wide range of dyes as pH indicators [7][8][9][10].
Although the developed fluorescent pH probes have been successful in monitoring acidic to near-neutral pH conditions [17][18][19][20], probes capable of monitoring alkaline regions are still rare. pH detection in the alkaline range is valuable in several fields, such as leather processing, wastewater treatment, the paper industry, nuclear fuel reprocessing, agriculture, detection of corrosion of steel-reinforced concrete structures, and metal mining and finishing [21,22]. Therefore, several fluorescent pH probes for detecting alkaline pHs have been developed, such as perylene tetra-(alkoxycarbonyl) derivative-based [21], BODIPY-based [22], and coumarin-based [23] alkaline pH fluorescent probes. However, most fluorescent probes reported to monitor pHs above 10 are turn-off type, in which fluorescence intensity decreases in the alkaline pH [4,11,21]. Fluorescent probes with turnon signals are generally considered more efficient. Compared with "turn-off" probes, the main advantage of "turn-on" probes is that they can easily measure low-concentration contrasts against a background, which increases sensitivity [24]. Therefore, it is particularly meaningful to develop new turn-on-type fluorescent probes that can detect an extremely alkaline pH.
In this study, we synthesized bis [2-(2 -hydroxyphenyl)benzazole] (bis(HBX)) derivatives that can be used in monitoring alkaline pHs. HBX-based probes exhibit a significant Stokes shift because of their intramolecular hydrogen-bonding properties, which exhibit excited-state intramolecular proton transfer (ESIPT) [25,26]. Owing to this property, HBX derivatives have been developed as fluorescent pH probes. However, the reported pH probes using mono HBX monitored a limited range of pHs (from acidic to near neutral) [1,27,28]. Herein, we envisioned that this limitation could be solved by combining the two HBX moieties into one molecule. According to studies, molecules that combine two benzazole groups have extra proton-binding sites and more effective ESIPT than mono HBX [25]. The intramolecular hydrogen bonding energies were found to be around 10 kcal/mol [29,30]. Unlike the previous mono HBX, bis(HBX) derivatives provide two intramolecular hydrogen-bonding acceptors, as shown in Scheme 1. The hydrogen bond can increase the deprotonation energy of the OH group in bis(HBX) derivatives [31,32]. Because of these characteristics, it is also expected that bis(HBX) derivatives should exhibit higher pK a values than mono HBX and interesting fluorescence patterns under extreme basic conditions. In addition, we speculate that the properties of the synthesized molecules could be tuned by changing the benzazole and functional groups bound to the central phenol. Consequently, nine bis(HBX) derivatives with two benzazole groups and various functional groups attached to the central phenol were synthesized, and the fluorescence change was measured according to the pH. Among the synthesized nine compounds, three compounds (A1, A2, and C1) with better properties were selected as alkaline pH probes. Probes exhibited the characteristics of "turn-on" and color change in the probe solution from colorless to yellow when the pH value increased from acidic to basic. In addition, unlike other probes that have a sigmoid-type pH titration plot, Al, A2, and C1 show a pH titration plot in the form of a step function with a relatively narrow transition range, which has the advantage of clearly checking the pH before and after pK a . on signals are generally considered more efficient. Compared with "turn-off" probes, main advantage of "turn-on" probes is that they can easily measure low-concentrat contrasts against a background, which increases sensitivity [24]. Therefore, it is parti larly meaningful to develop new turn-on-type fluorescent probes that can detect an tremely alkaline pH.
In this study, we synthesized bis[2-(2′-hydroxyphenyl)benzazole] (bis(HBX)) deri tives that can be used in monitoring alkaline pHs. HBX-based probes exhibit a signific Stokes shift because of their intramolecular hydrogen-bonding properties, which exhi excited-state intramolecular proton transfer (ESIPT) [25,26]. Owing to this property, H derivatives have been developed as fluorescent pH probes. However, the reported probes using mono HBX monitored a limited range of pHs (from acidic to near neutr [1,27,28]. Herein, we envisioned that this limitation could be solved by combining the t HBX moieties into one molecule. According to studies, molecules that combine two b zazole groups have extra proton-binding sites and more effective ESIPT than mono H [25]. The intramolecular hydrogen bonding energies were found to be around 10 kcal/m [29,30]. Unlike the previous mono HBX, bis(HBX) derivatives provide two intramolecu hydrogen-bonding acceptors, as shown in Scheme 1. The hydrogen bond can increase deprotonation energy of the OH group in bis(HBX) derivatives [31,32]. Because of th characteristics, it is also expected that bis(HBX) derivatives should exhibit higher pKa v ues than mono HBX and interesting fluorescence patterns under extreme basic conditio In addition, we speculate that the properties of the synthesized molecules could be tun by changing the benzazole and functional groups bound to the central phenol. Con quently, nine bis(HBX) derivatives with two benzazole groups and various functio groups attached to the central phenol were synthesized, and the fluorescence change w measured according to the pH. Among the synthesized nine compounds, three co pounds (A1, A2, and C1) with better properties were selected as alkaline pH prob Probes exhibited the characteristics of "turn-on" and color change in the probe solut from colorless to yellow when the pH value increased from acidic to basic. In additi unlike other probes that have a sigmoid-type pH titration plot, Al, A2, and C1 show a titration plot in the form of a step function with a relatively narrow transition range, wh has the advantage of clearly checking the pH before and after pKa.

Calculation of the pK a and Quantum Yield (Φ)
The pK a value of each bis(HBX) derivative was obtained by fitting the Boltzmann function to the titration data of its fluorescence response to pH. To calculate the quantum yield, coumarin 153 (C-153, Φ st = 0.53) in EtOH was used as the standard dye. The slope of each compound was calculated by plotting the absorbance on the x-axis and the integration of the fluorescence intensity on the y-axis. The photoluminescence quantum yield (Φ) was calculated using Equation (1) [35].
compound solvent re f ractive index 2 standard solvent re f ractive index 2 (1)
To The fluorescence reversibility of the bis(HBX) derivatives was analyzed using solutions comprising each bis(HBX) derivative (A1, A2, and C1: 25 µM; 30% DMSO) and B-R buffers (pH 12.0 for A1, A2 and pH 13.0 for C1; 10 mM). The fluorescence spectrum of each solution was measured before the pH was fitted through the dropwise addition of HCl and NaOH aqueous solutions. The fluorescence spectrum of the fitted solution was then measured again. The pH decreased and subsequently increased during the four cycles.
The data were collected using an Agilent fluorescence spectrophotometer at 25 • C. Each experiment was repeated thrice. For all measurements, the PMT was 400 V, and the slit width was 5/5 nm.

Spectroscopic Properties of the Bis(HBX) Derivatives
The UV-vis and fluorescence responses of the nine bis(HBX) derivatives according to pH changes were investigated under a wide range of pH conditions (pH 1.0-13.9). The UV-vis spectra of the nine compounds exhibited similar patterns, depending on the type of bound benzazole ( Figure S1). The fluorescence spectra according to the pH are listed in Figure 1. As we expected, among the synthesized nine bis(HBX) derivatives, the benzothiazole (A1-3) and benzoxazole (C1-3) conjugated compounds exhibited strong turn-on fluorescence responses under basic pH conditions in contrast to their HBX mono forms, which exhibited fluorescence changes in acidic to neutral pH ranges [1,27,28]. In which exhibited fluorescence changes in acidic to neutral pH ranges [1,27,28]. In contrast compounds bearing benzimidazole (B1-3) exhibited unique spectra, showing ratiometric fluorescence responses from pH 1.0 to 13.9. To figure out the compound showing the best performance as a pH fluorescence probe among the nine bis(HBX) derivatives, we compared these in terms of photostability and solubility. First, photostability was studied by measuring the fluorescence intensity for 30 min in the pH regions, including the transition ranges ( Figure S2). While fluores cence changes for compounds A1-3, C1, and C3 were negligible, compounds B1-3 and C2 exhibited unstable properties where the emission wavelengths or fluorescence intensity changed over time. The photochemical instability of a fluorescent dye is well correlated with the excited-state lifetime of the fluorescent dye because the fluorescent dye in an excited state can react with its surrounding molecules [39]. The excited state lifetimes o mono HBX were determined to be 2-(2′-hydroxyphenyl)benzothiazole (HBT) (14 ps in ACN) [40], 2-(2′-hydroxyphenyl)benzoxazole (HBO) (1080 ps in DMSO) [41], and 2-(2′ hydroxyphenyl)benzimidazole (HBI) (1.5 ns in EtOH) [42], respectively. Therefore, the differences in the photochemical instabilities of the bis(HBX) derivatives may come from the length of their excited-state lifetimes. Further, the solubility of the compounds was confirmed in a solvent, and A3 exhibited an extremely low solubility compared with those of the other compounds ( Figure S3).
For the four compounds (A1, A2, C1, and C3), the pKa and quantum yield calcula tions were performed (Table 1). Various pKa values of 9.8 (A2), 10.4 (A1, C3), and 10.8 (C1 indicated that the synthesized probes were capable of sensing a wide range of alkaline pHs. Based on the data, A1, A2, and C1 with various pKa values between 9.7 and 10.8 were selected as fluorescent probes for monitoring alkaline pH ranges, and further studies were conducted (C3 exhibited a pKa value similar to that of A1 but a smaller quantum yield than A1; thus, A1 was selected).  To figure out the compound showing the best performance as a pH fluorescence probe among the nine bis(HBX) derivatives, we compared these in terms of photostability and solubility. First, photostability was studied by measuring the fluorescence intensity for 30 min in the pH regions, including the transition ranges ( Figure S2). While fluorescence changes for compounds A1-3, C1, and C3 were negligible, compounds B1-3 and C2 exhibited unstable properties where the emission wavelengths or fluorescence intensity changed over time. The photochemical instability of a fluorescent dye is well correlated with the excited-state lifetime of the fluorescent dye because the fluorescent dye in an excited state can react with its surrounding molecules [39]. The excited state lifetimes of mono HBX were determined to be 2-(2 -hydroxyphenyl)benzothiazole (HBT) (14 ps in ACN) [40], 2-(2 -hydroxyphenyl)benzoxazole (HBO) (1080 ps in DMSO) [41], and 2-(2hydroxyphenyl)benzimidazole (HBI) (1.5 ns in EtOH) [42], respectively. Therefore, the differences in the photochemical instabilities of the bis(HBX) derivatives may come from the length of their excited-state lifetimes. Further, the solubility of the compounds was confirmed in a solvent, and A3 exhibited an extremely low solubility compared with those of the other compounds ( Figure S3).
For the four compounds (A1, A2, C1, and C3), the pK a and quantum yield calculations were performed (Table 1). Various pK a values of 9.8 (A2), 10.4 (A1, C3), and 10.8 (C1) indicated that the synthesized probes were capable of sensing a wide range of alkaline pHs. Based on the data, A1, A2, and C1 with various pK a values between 9.7 and 10.8 were selected as fluorescent probes for monitoring alkaline pH ranges, and further studies were conducted (C3 exhibited a pK a value similar to that of A1 but a smaller quantum yield than A1; thus, A1 was selected).  A1, A2, and C1) to pH Figure 2 shows the pH titration plots of the selected probes (A1, A2, and C1). The fluorescence intensity of A1, having a pK a value of 10.4, was extremely low from pH 1.0 to 9.0, but the fluorescence intensity increased approximately 16-fold for pH 9.7 to 10.7 and saturated above pH 10.7. Similar to the fluorescence response of A1, A2, having a pK a value of 9.8, exhibited an extremely low fluorescence intensity from pH 1.0 to 8.0, and the intensity dramatically increased approximately 12-fold for pH 9.0 to 10.7 and saturated over pH 10.7. The fluorescence intensity of C1, with a pK a value of 10.8, was extremely low from pH 1.0 to 10.0, exhibiting a fluorescence change that increased approximately 24-fold from pH 10.3 to 13.9.

C3
10.408 507.0 0.34 (A1, A2, and C1) to pH Figure 2 shows the pH titration plots of the selected probes (A1, A2, and C1 fluorescence intensity of A1, having a pKa value of 10.4, was extremely low from p to 9.0, but the fluorescence intensity increased approximately 16-fold for pH 9.7 t and saturated above pH 10.7. Similar to the fluorescence response of A1, A2, having value of 9.8, exhibited an extremely low fluorescence intensity from pH 1.0 to 8.0, a intensity dramatically increased approximately 12-fold for pH 9.0 to 10.7 and satu over pH 10.7. The fluorescence intensity of C1, with a pKa value of 10.8, was extr low from pH 1.0 to 10.0, exhibiting a fluorescence change that increased approxim 24-fold from pH 10.3 to 13.9.

Fluorescence Response of the Selected Probes
The probes exhibited an extremely sharp increase in fluorescence intensity w narrow pH transition range. By having the pH titration plots in the form of a step fun the probes could clearly monitor the pH before and after pKa. Moreover, the solutio the three compounds changed from colorless to yellow as the pH increased to the al range, showing that the pH change for the alkaline range could be checked with the eye using the probes. The compounds exhibited high pKa values and strong turn-on rescence signals at alkaline pH values, indicating that these compounds are suitab monitoring alkaline pHs.

Photostability, Reversibility, Ionic Interference Study, and the Applicability of the Sele Probes
For the three compounds (A1, A2, and C1) selected as fluorescent probes, instudies were conducted, such as photostability, reversibility, and ionic interference ies, for evaluating the performance of fluorescent probes. To evaluate the photost of the probes, changes in the fluorescence intensity of the three probes were measur 30 min at 5 min intervals at each pH that the fluorescence signal turned on and o shown in Figure 3, the fluorescence intensity of A1 remained stable at pH 6.0 and and the other probes also exhibited good photostability under acidic and alkaline tions ( Figure S4). The probes exhibited an extremely sharp increase in fluorescence intensity with a narrow pH transition range. By having the pH titration plots in the form of a step function, the probes could clearly monitor the pH before and after pK a . Moreover, the solutions for the three compounds changed from colorless to yellow as the pH increased to the alkaline range, showing that the pH change for the alkaline range could be checked with the naked eye using the probes. The compounds exhibited high pK a values and strong turn-on fluorescence signals at alkaline pH values, indicating that these compounds are suitable for monitoring alkaline pHs.

Photostability, Reversibility, Ionic Interference Study, and the Applicability of the Selected Probes
For the three compounds (A1, A2, and C1) selected as fluorescent probes, in-depth studies were conducted, such as photostability, reversibility, and ionic interference studies, for evaluating the performance of fluorescent probes. To evaluate the photostability of the probes, changes in the fluorescence intensity of the three probes were measured for 30 min at 5 min intervals at each pH that the fluorescence signal turned on and off. As shown in Figure 3, the fluorescence intensity of A1 remained stable at pH 6.0 and 12.0, and the other probes also exhibited good photostability under acidic and alkaline conditions ( Figure S4).  The reversibility of the fluorescence response was investigated by changing using aqueous HCl and NaOH solutions. As shown in Figure 4, the reversibility tes started from pH 12.0 to 8.0, for four cycles. During the cycles, the enhanced fluore signal at pH 12.0 was quenched immediately at pH 8.0 by adding HCl solution, exh good reversibility. Simultaneously, the solution changed from yellow to colorles 12.0 and 8.0 under natural light. Similar reversible results were obtained for A2 ( Figure S5). These results show that the fluorescence responses of the probes po clear on-off switch with pH changes, and the probes have good reversibility.  Figure 5, the fluorescence intensity of A1 was unaffected by th ence of various ionic species at pH 12.0 and 6.0, and similar results were obtained and C1 ( Figure S6). The interference test showed that A1, A2, and C1 could be r used to monitor alkaline pHs without interference in the presence of various ion human serum level. The reversibility of the fluorescence response was investigated by changing the pH using aqueous HCl and NaOH solutions. As shown in Figure 4, the reversibility test of A1 started from pH 12.0 to 8.0, for four cycles. During the cycles, the enhanced fluorescence signal at pH 12.0 was quenched immediately at pH 8.0 by adding HCl solution, exhibiting good reversibility. Simultaneously, the solution changed from yellow to colorless at pH 12.0 and 8.0 under natural light. Similar reversible results were obtained for A2 and C1 ( Figure S5). These results show that the fluorescence responses of the probes possess a clear on-off switch with pH changes, and the probes have good reversibility. The reversibility of the fluorescence response was investigated by changing using aqueous HCl and NaOH solutions. As shown in Figure 4, the reversibility tes started from pH 12.0 to 8.0, for four cycles. During the cycles, the enhanced fluore signal at pH 12.0 was quenched immediately at pH 8.0 by adding HCl solution, exh good reversibility. Simultaneously, the solution changed from yellow to colorless 12.0 and 8.0 under natural light. Similar reversible results were obtained for A2 a ( Figure S5). These results show that the fluorescence responses of the probes po clear on-off switch with pH changes, and the probes have good reversibility.  Figure 5, the fluorescence intensity of A1 was unaffected by th ence of various ionic species at pH 12.0 and 6.0, and similar results were obtained and C1 ( Figure S6). The interference test showed that A1, A2, and C1 could be r used to monitor alkaline pHs without interference in the presence of various ions human serum level. Further, we measured the fluorescence responses of the probes to confirm the interference of various ionic species, including metal cations (Na + , K + , Ca 2+ , Mg 2+ , Fe 2+ , Cu 2+ , and Zn 2+ ) and anions (F − , Cl − , HSO 4 − , H 2 PO 4 − , NO 3 − , and SCN − ), under different pH conditions. As shown in Figure 5, the fluorescence intensity of A1 was unaffected by the presence of various ionic species at pH 12.0 and 6.0, and similar results were obtained for A2 and C1 ( Figure S6). The interference test showed that A1, A2, and C1 could be reliably used to monitor alkaline pHs without interference in the presence of various ions at the human serum level. To show the applicability of the pH probes to practical samples, we conduc measurements using chlorine bleach, a basic solution that can be easily seen in r When the fluorescence intensity of the chlorine bleach solution with probes w trasted with the pH titration plots of A1, A2, and C1, the pH of the chlorine bleach s measured using fluorescence probes coincided with the pH values measured with electrode ( Figure S7). In addition, when the chlorine bleach solution was mixed w solutions of probes A1, A2, and C1, the approximate pH range of the chlorine ble lution was immediately confirmed from the color change in each mixed solution results show that the three probes (A1, A2, and C1) as pH indicators can be app practical examples with high reliability.

Conclusions
We synthesized nine bis(HBX) derivatives and selected three compounds as f cent pH probes. The three probes (A1, A2, and C1) exhibited high pKa values betw to 10.8 and a narrow pH transition range, and they effectively detected alkaline pH showed the properties of multifunctional alkaline pH probes with both color c from colorless to yellow and fluorescence turn-on responses at alkaline pHs. The exhibited excellent photostability in the extreme alkaline pH range (pH > 10), pote competing ionic species, with good reversibility. Moreover, it showed that the pro measure the pH of practical examples with high accuracy. Thus, we envision that and C1 can be applied in various fields as effective fluorescent probes for monito kaline pHs.
Supplementary Materials: The following supporting information can be downloa www.mdpi.com/xxx/s1, Scheme S1: Synthesis route of 2,6-diformyl-phenol derivative Scheme S2: Synthesis route of bis(HBX) derivatives (A1-C3); Table S1: Two-step synt bis(HBX) derivatives; Figure S1: UV-vis spectra of nine bis(HBX) derivatives; Figure S2: P bility by fluorescence intensity of A1-3 (pH 7.0-10.0), B1-3, C1, and C3 (pH 8.0-11.0) for ([bis(HBX)] = 50 μM in 10 mM B-R buffers; 10% DMF); Figure S3: Solubility of A1-3, C1, an mM in DMSO); Figure S4: Plots of the fluorescence intensity for 30 min; Figure S5: The pH To show the applicability of the pH probes to practical samples, we conducted pH measurements using chlorine bleach, a basic solution that can be easily seen in real life. When the fluorescence intensity of the chlorine bleach solution with probes was contrasted with the pH titration plots of A1, A2, and C1, the pH of the chlorine bleach solution measured using fluorescence probes coincided with the pH values measured with the pH electrode ( Figure S7). In addition, when the chlorine bleach solution was mixed with the solutions of probes A1, A2, and C1, the approximate pH range of the chlorine bleach solution was immediately confirmed from the color change in each mixed solution. These results show that the three probes (A1, A2, and C1) as pH indicators can be applied to practical examples with high reliability.

Conclusions
We synthesized nine bis(HBX) derivatives and selected three compounds as fluorescent pH probes. The three probes (A1, A2, and C1) exhibited high pK a values between 9.7 to 10.8 and a narrow pH transition range, and they effectively detected alkaline pHs. They showed the properties of multifunctional alkaline pH probes with both color changes from colorless to yellow and fluorescence turn-on responses at alkaline pHs. The probes exhibited excellent photostability in the extreme alkaline pH range (pH > 10), potentially competing ionic species, with good reversibility. Moreover, it showed that the probes can measure the pH of practical examples with high accuracy. Thus, we envision that A1, A2, and C1 can be applied in various fields as effective fluorescent probes for monitoring alkaline pHs.