- Email: [email protected]
Novel benzimidazole-based ratiometric fluorescent probes for acidic pH
Accepted Manuscript Novel benzimidazole-based ratiometric fluorescent probes for acidic pH Yan-Cheng Wu, Jia-Yi You, Kai Jiang, Han-Qing Wu, Jin-Feng Xiong, Zhao-Yang Wang PII:
S0143-7208(17)31504-8
DOI:
10.1016/j.dyepig.2017.09.043
Reference:
DYPI 6268
To appear in:
Dyes and Pigments
Received Date: 9 July 2017 Revised Date:
2 September 2017
Accepted Date: 17 September 2017
Please cite this article as: Wu Y-C, You J-Y, Jiang K, Wu H-Q, Xiong J-F, Wang Z-Y, Novel benzimidazole-based ratiometric fluorescent probes for acidic pH, Dyes and Pigments (2017), doi: 10.1016/j.dyepig.2017.09.043. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT
Novel benzimidazole-based ratiometric fluorescent probes for acidic pH
Yan-Cheng Wuab, Jia-Yi Youa, Kai Jianga, Han-Qing Wua, Jin-Feng Xiong*c and Zhao-Yang
a
RI PT
Wang*a
School of Chemistry and Environment, South China Normal University; Key Laboratory of Theoretical Chemistry of
SC
Environment, Ministry of Education, Guangzhou 510006, China
College of Materials Science and Engineering, South China University of Technology, Guangzhou 510640, China
c
HEC Pharm R&D Center, Dongguan 523871, China
* Corresponding Author
TE D
M AN U
b
E-mail: [email protected]. Tel: 8620-39310258.
AC C
EP
E-mail: [email protected].
1
ACCEPTED MANUSCRIPT
ABSTRACT: 2,6-Bis(1-alkyl-1H-benzo[d]imidazol-2-yl)naphthalene (2a-2c) as three new benzimidazole molecules have been designed, synthesized and applied as ratiometric fluorescent pH probes in
RI PT
the acidic pH region with high-efficiency and fast response. For probe 2a, upon decreasing the pH from 7.5 to 2.5, the emission spectra exhibit a large red shift from 402 to 472 nm, and the emission ratio (I472/I402) changes dramatically from 0.10 to 3.42 with a pKa value of 2.92. No
SC
significant interference on emission behavior is observed in the presence of various metal ions.
M AN U
Similar results are obtained for probes 2b and 2c. 1H NMR and DFT calculations studies demonstrate that the ratiometric response of the probes to acidic pH is due to H+ binding with one N atom in C=N of benzimidazole ring and the subsequent enhancement of the intramolecular charger transfer (ICT) process. In addition, these probes can be used in real-time and reversible
TE D
pH sensing. Finally, the probes have been successfully applied to the visual detection of pH not
Keywords:
EP
only in solution, but also in test paper.
AC C
Benzimidazole; Fluorescent probe; pH sensing, Ratiometric response; ICT
2
ACCEPTED MANUSCRIPT
1. Introduction Since pH is a key parameter in a wide range of fields, such as chemical process control, biological systems and environmental monitoring, the measurement of pH is of great significance
RI PT
to the protection of the environment and life [1-6]. Owing to many advantages of the fluorescent detection technique, such as high selectivity and sensitivity, operational simplicity, low cost and real-time monitoring, the design and synthesis of fluorescent pH probes with high-efficiency and
SC
fast response have become a highly popular topic of scientists' interests [7-12].
M AN U
Recently, a number of fluorescent probes have been used to the measurement of pH [13-23]. Amongst them, most fluorescent probes only have an intensity change in single-emission window with the response to the variation of pH [18-23]. However, fluorescence signals are easily influenced by environmental factors, such as temperature, solvent polarity and excitation
TE D
wavelength [24,25]. It is well-known that, the ratiometric fluorescent probe can encompass a built-in correction for the above-mentioned environmental effects to improve the selectivity and sensitivity since the ratiometric measurements are based on the change in the ratio of the
EP
emission intensity at two wavelengths [26-30]. Therefore, there are some reports about
AC C
ratiometric fluorescent pH probes in recent year, and the typical examples include polymers [31-33], carbon nanodots [34-36], metal-organic frameworks [37,38], and organic small molecules [39-43]. In these efforts, organic small molecules-based fluorescent ratiometric probes are easier to be designed and prepared. However, some deficiencies still exist in organic small molecule-based ratiometric fluorescent pH probes. For instance, more steps are necessary in the synthesis [39-41]. Utilizing that the N-H unit and the N atom in C=Ν of benzimidazole ring can interact with
3
ACCEPTED MANUSCRIPT hydroxyl and hydrogen ions respectively, benzimidazole fluorophore has been widely used to the design of fluorescent pH probes [25,44-49]. Unfortunately, only a little research has been reported on the development of ratiometric pH probes based on benzimidazole compounds
RI PT
[25,45,49]. In addition, only a few benzimidazole-based probes can be applicable for strong acidic conditions with pH below 4 [45,48,49]. Therefore, it is important to develop benzimidazole-based ratiometric pH probes for extreme acidity with high-efficiency and fast
SC
response.
M AN U
Herein, for a continuation of our ongoing program aimed at developing novel fluorescent probes for different analytes [50-53], we have synthesized and characterized three benzimidazole-based fluorescent probes as new ratiometric fluorescent pH probes (Scheme 1). These probes display excellent performance and potential application as anticipated. Their
TE D
sensing mechanism via the enhanced intramolecular charger transfer (ICT) effect has been
AC C
EP
established with the help of 1H NMR and DFT calculations studies.
Scheme 1.
Synthesis of probes 2.
2. Experimental 2.1. Materials and instruments 4
ACCEPTED MANUSCRIPT Melting point was performed on an X-5 digital melting point apparatus without correcting. 1
H and 13C NMR spectra were collected on a BRUKER DRX-400 spectrometer in CDCl3 using
TMS as an internal standard. Mass spectra (MS) were recorded on a Thermo LCQ DECA XP
UV-vis
spectra
were
measured
by using
a
Shimazu
RI PT
MAX mass spectrometer. Elemental analysis was obtained with Perkin Elmer Series II 2400. UV-2550
ultraviolet
visible
spectrophotometer at room temperature. The fluorescence spectra were recorded with a Hitachi
SC
F-2700 spectrophotometer at room temperature; and the slit width was 5 nm for both excitation
M AN U
and emission. pH values were recorded on a LIDA Model PHS-25C Bench pH/MV meter. All reagents were purchased from commercial suppliers and used without purification. 2,6-Bis(1H-benzo[d]imidazol-2-yl)naphthalene 1 was prepared according to our literature [52].
TE D
2.2. Synthesis
According to the literature [50], a 100 mL round-bottom flask was charged with compound 1 (3 mmol, 1.080 g), the selected bromoalkane (6.2 mmol) and solid NaOH (12 mmol, 0.480 g)
EP
in CH3CN (30 mL). The reaction mixture was stirred at 80 °C for 2-4 h under the monitoring of
AC C
TLC. After the solvent was removed, the residue was dissolved in dichloromethane. The organic layers were washed with water three times and then dried over anhydrous MgSO4. The concentration under vacuum gave a crude product, which was purified by column chromatography on silica gel with gradient eluents of petroleum ether and ethyl acetate to afford pure compound 2. 2,6-Bis(1-decyl-1H-benzo[d]imidazol-2-yl)naphthalene (2a). White solid, 1.702 g, yield 88.5%, m.p. 97.2-98.5
; UV-vis (THF) λmax: 327.0 nm; 1H NMR (CDCl3-TMS, 400 MHz): δ =
5
ACCEPTED MANUSCRIPT 0.86 (t, J = 8.0 Hz, 6H), 1.18-1.31 (m, 28H), 1.85-1.97 (m, 4H), 4.35 (t, J = 8.0 Hz, 4H), 7.29-7.41 (m, 4H), 7.44-7.51 (m, 2H), 7.82-7.90 (m, 2H), 7.93 (d, J = 8.0 Hz, 2H), 8.09 (d, J = 8.0 Hz, 2H), 8.32 (s, 2H); 13C NMR (CDCl3-TMS, 100 MHz): δ = 14.1, 22.6, 26.7, 29.0, 29.2, 29.4, 29.7, 29.9,
RI PT
31.8, 45.0, 110.2, 120.1, 122.5, 122.9, 127.1, 129.0, 129.1, 129.4, 133.2, 135.8, 143.3, 153.2; IR (film), ν, cm-1: 3055, 2955, 2922, 2855, 1616, 1489, 1454, 1329, 893, 824, 746; ESI-MS, m/z (%):
C 82.45, H 8.81, N 8.74, Found: C 82.58, H 9.01, N 8.72.
SC
Calcd for C44H57N4+ ([M+H]+): 641.46 (100%), Found: 641.50 (100%); Anal. Calcd for C44H56N4:
83.7%, m.p. 93.3-94.2
M AN U
2,6-Bis(1-dodecyl-1H-benzo[d]imidazol-2-yl)naphthalene (2b). White solid, 1.750 g, yield ; UV-vis (THF) λmax: 327.0 nm; 1H NMR (CDCl3-TMS, 400 MHz): δ =
0.85 (t, J = 8.0 Hz, 6H), 1.06-1.41 (m, 36H), 1.85-1.95 (m, 4H), 4.34 (t, J = 8.0 Hz, 4H), 7.31-7.39 (m, 4H), 7.44-7.50 (m, 2H), 7.84-7.90 (m, 2H), 7.92 (d, J = 8.0 Hz, 2H), 8.09 (d, J = 8.0 Hz, 2H),
TE D
8.31 (s, 2H); 13C NMR (CDCl3-TMS, 100 MHz): δ = 14.1, 22.7, 26.8, 29.1, 29.3, 29.4, 29.5, 29.6, 29.9, 31.9, 45.0, 110.2, 120.1, 122.5, 122.9, 127.2, 129.1, 129.4, 133.2, 135.8, 143.3, 153.2; IR
EP
(film), ν, cm-1: 3063, 2930, 2855, 1616, 1489, 1458, 1329, 893, 819, 746; ESI-MS, m/z (%): Calcd for C48H65N4+ ([M+H]+): 697.52 (100%), Found: 697.47 (100%); Anal. Calcd for C48H64N4: C
AC C
82.71, H 9.25, N 8.04, Found: C 82.78, H 9.34, N 7.96.
2,6-Bis(1-tetradecyl-1H-benzo[d]imidazol-2-yl)naphthalene (2c). White solid, 1.625 g, yield 71.9%, m.p. 88.6-89.3
; UV-vis (THF) λmax: 328.0 nm; 1H NMR (CDCl3-TMS, 400 MHz):
δ = 0.86 (t, J = 8.0 Hz, 6H), 1.07-1.39 (m, 44H), 1.85-1.95 (m, 4H), 4.35 (t, J = 8.0 Hz, 4H), 7.30-7.39 (m, 4H), 7.44- 7.50 (m, 2H), 7.84-7.90 (m, 2H), 7.92 (d, J = 8.0 Hz, 2H), 8.09 (d, J = 8.0 Hz, 2H), 8.31 (s, 2H);
13
C NMR (CDCl3-TMS, 100 MHz): δ = 14.1, 22.7, 26.7, 29.4, 29.5,
6
ACCEPTED MANUSCRIPT 29.6, 29.7, 29.9, 31.9, 45.0, 110.2, 120.1, 122.5, 122.9, 127.2, 129.0, 129.1, 129.4, 133.2, 135.8, 143.3, 153.2; IR (film), ν, cm-1: 3069, 2930, 2862, 1616, 1493, 1458, 1329, 893, 819, 743; ESI-MS, m/z (%): Calcd for C52H73N4+ ([M+H]+): 753.58 (100%), Found: 753.69 (100%); Anal.
RI PT
Calcd for C52H72N4: C 82.93, H 9.64, N 7.44, Found: C 82.88, H 9.75, N 7.52.
SC
2.3. Computational methods
According to the literature [24,54], all of the calculations were obtained using density
3. Results and discussion
M AN U
functional theory (DFT) with the B3LYP/6-31G (d) level of the Gaussian 09 program.
TE D
3.1. Synthesis and Photophysical Properties of 2
As shown in Scheme 1, the target molecules 2a-2c were synthesized via a simple and efficient N-alkylation reaction of compound 1 and the selected 1-bromoalkane with the yields of 13
C NMR, ESI-MS [the corresponding
EP
71.9-88.5%. Their structures were characterized by 1H,
AC C
spectra (Figs. S1-S9) can be seen in Supporting Information (SI)], IR, and elemental analysis. In an effort to gain insight into the photophysical process of the target molecules, we firstly investigated the absorption and emission behaviors of compounds 2a-2c in different non-polar or polar solvents. They show similar absorption spectra, and there is a slight blueshift with the increase of the solvent polarity (Figs. S10a-S12a). The emission spectra of compounds 2a-2c show two shoulder peaks in less polar solvents, but merging into a single peak in polar solvents (Figs. S10b-S12b). It's worth noting that the fluorescence intensity of 2 is relatively high in THF,
7
ACCEPTED MANUSCRIPT and it can be completely miscible with water. Therefore, we selected THF as the solvent for the
Fig. 1.
M AN U
SC
RI PT
studies on the fluorescent properties of compounds 2a-2c.
The fluorescence spectra (λex = 325 nm) of compound 2c (10 µM) in H2O/THF system
EP
TE D
with different water fractions.
The emission spectra of compound 2c in THF-H2O mixed solvents had also been
AC C
investigated. As shown in Fig. 1, compound 2c exhibits a strong emission at 400 nm with a shoulder peak at 389 nm in THF solution. When the water fraction (fw) is less than 60%, there is a slight change in fluorescence intensity. Further increasing fw may lead to a dramatic decline in the emission intensity, indicating that the maximum water content is 60%. Perhaps, the probes may aggregate in the presence of a large amount of water, resulting in fluorescence quenching (aggregation-caused quenching effect). However, all the aqueous mixtures are homogenous without precipitates, suggesting that the aggregates are of nano-dimensions. This is proved by the 8
ACCEPTED MANUSCRIPT level-off tail observed in the UV spectrum (Fig. S13) in the longer wave-length region when the fw values exceeded 60% [53,55]. These results show that compounds 2 has a high tolerance to water. Thus, we chose the binary system with an fw of 60% (H2O/THF, v/v, 3/2) as the solvent for
RI PT
studying the pH responsive properties of the probes.
3.2. Optical Response to pH
SC
To study the optical responses of compounds 2 to pH, standard pH titrations of absorption
M AN U
and emission spectra were performed. The spectroscopic properties of probe 2a under different pH conditions in H2O/THF (v/v, 3/2) solution were first selected to be examined (Fig. 2). As the pH is decreased from 7.5 to 2.5, the absorbance at 282 and 325 nm is gradually decreased accompanying by the formation of a new peak at 245 nm and a shoulder peak at around 370 nm
AC C
EP
TE D
(Fig. 2a). Further decrease in pH does not induce any change in the absorption spectrum.
(a) Fig. 2.
(b)
(a) UV-vis absorption spectra and (b) emission spectra of probe 2a (10 µM in H2O/THF, v/v, 3/2) with the different pH (Inset: plot of ratio of I472/I402 as a function of pH).
9
ACCEPTED MANUSCRIPT As shown in Fig. 2b, upon decreasing the pH from 7.5 to 2.5, the emission spectra of probe 2a exhibits a shift from 402 to 472 nm with an obvious red shift of 70 nm. The large red shift of the signals may be attributed to the enhanced ICT effect because of the H+ binding-induced
RI PT
enhancement of the electron-withdrawing ability of benzimidazole ring. A well-defined isoemission point is found at 460 nm, indicating that only one benzimidazole ring could bind with H+ [24]. The large red shift makes probe 2a suitable for ratiometric pH sensing in the acidic
SC
region. When the pH is decreased from 7.5 to 2.5, the emission intensity ratio (I472/I402) changes
as a function of pH (Fig. S14).
M AN U
from 0.10 to 3.42 (Inset Fig. 2b). The pKa value of 2.92 can be calculated from the emission ratio
Similar behaviors were also observed for both 2b (Fig. S15) and 2c (Fig. S16). As the pH is decreased, the emission spectra of both 2b (Fig. S15b) and 2c (Fig. S16b) exhibit an apparent
TE D
red shift from 402 to 472 nm, with the emission ratio (I472/I402) changing from 0.11 and 0.10 to 3.13 and 3.53 respectively (Inset Figs. S15b and S16b). Similarly, the pKa value of 2b and 2c can
EP
be calculated to be 2.90 and 2.51 respectively (Fig. S17).
AC C
3.3. Possible Sensing Mechanism
In order to get insight into the sensing mechanism of probes 2 for acidic pH, 1H NMR experiments were carried out according to the literature [24,56]. As shown in Fig. 3, with the addition of incremental amounts of CF3COOD (1-10 equiv.) to the solution of probe 2a (20 mM in CDCl3), a downfield shift is observed for the chemical shift values of the benzimidazole protons (Hd, He, Hf) and naphthalene protons (Ha, Hb). When 20 equiv. of CF3COOD is added to above solution, the chemical shift values of these protons remain almost constant. The downfield
10
ACCEPTED MANUSCRIPT chemical shift of these protons is obviously due to the H+ binding with the N atom in C=Ν of benzimidazole ring, which results in the decrease of electron density around these protons [24,56]. In addition, the chemical shift values of the benzimidazole protons (Hg) and naphthalene
RI PT
protons (Hc) are almost unchanged, indicating that the alkylated N atom in benzimidazole ring does not bind with H+ [24]. Therefore, it is clear that the H+ binding with the N atom in C=Ν of
AC C
EP
TE D
M AN U
SC
benzimidazole ring in probes 2 results in the significant optical response to acidic pH.
Fig. 3.
Partial 1H NMR spectra of probe 2a (20 mM in CDCl3) and probe 2a with the different equiv. CF3COOD.
11
Fig. 4.
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
Calculated (DFT B3LYP/6-31G (d) level) HOMO-LUMO energy levels and the
TE D
interfacial plots of the orbitals for probe 2a, the 2a-H+ complex and the 2a-2H+ complex.
To better understand the optical responses of probes 2 upon binding with H+, we further
EP
carried out DFT calculations using probe 2a as an example (Fig. 4). The DFT calculations study shows a HOMO→LUMO charge transfer in both 2a and 2a-H+. For probe 2a, the LUMO is
AC C
mainly localized on the central naphthalene core and partly distributed on both benzimidazole rings, whereas the HOMO is evenly distributed on the central naphthalene core and both benzimidazole rings. For 2a-H+ complex, the LUMO is mainly localized on the central naphthalene core and benzimidazolium moiety, whereas the HOMO is mainly distributed on another benzimidazole ring without the appendage of H+. These results indicate that the ICT process in 2a-H+ is enhanced comparing with that in 2a [24]. In addition, the energy gap (△E) between the HOMO and LUMO of the 2a-H+ complex (2.65583 eV) is smaller than that of probe 12
ACCEPTED MANUSCRIPT 2a (3.84089 eV), indicating that the 2a-H+ complex is more stable than probe 2a. The result is in good agreement with the red shift in emission spectra of probe 2a upon binding with H+. For bis-protonation species (2a-2H+), the LUMO is mainly localized on the central
RI PT
naphthalene core and partly distributed on both benzimidazole rings, whereas the HOMO is mainly distributed on one N-alkyl chain. It is worth noting that the ∆E of 2a-2H+ is increased (3.20387 eV) comparing with that of 2a-H+. These results further confirm that the
SC
mono-protonation is difficult to further formation of bis-protonation [54]. What is more, the
M AN U
results of DFT calculations for probe 2b and its protonated species (Fig. S18) are basically similar. Thus, the above conclusions are further confirmed.
Based on the above research results, the sensing mechanism for pH can be concluded in the following (Scheme 2): When proton is added into the solution of probes 2, H+ can bind with one
TE D
N atom in C=Ν of benzimidazole ring [57], and H+ binding induces an enhancement of the electron-withdrawing ability of benzimidazole. Therefore, the fluorescence changes via
AC C
EP
enhancing the ICT process in 2-H+ complex [45,56].
Scheme 2.
Proposed mechanism of fluorescence change for probes 2 upon addition of H+.
13
ACCEPTED MANUSCRIPT 3.4. Selectivity Studies The selectivity response of probes 2 to H+ over various metal ions at pH 7.0 and 3.0 were investigated (Fig. 5 and Fig. S19). At pH = 7.0, there is no observed effect on the emission ratio
RI PT
of the probes 2 within the additives except for Fe3+ causing a slight increase of I472/I402. Meanwhile, the addition of different metal ions has no obvious influence on the emission ratio of the probes 2 at pH = 3.0. These results demonstrate that probes 2 can be considered as selective
AC C
EP
TE D
M AN U
SC
fluorescent probes for acidic pH sensing.
Fig. 5.
The ratio of fluorescence intensity of probe 2a (10 µM) at I472/I402 containing different
metal ions at pH 7.0 (black bar) and 3.0 (blue bar). All the concentration of the metal ions is 500 µM (λex = 325 nm).
14
ACCEPTED MANUSCRIPT 3.5. Real-time Response and Reversibility Study For practical application, the real-time determination is very important [24,30]. Thus, using probe 2a as a representative, the reaction-time profile of probes 2 in the solution of different pH
RI PT
values was measured. As shown in Fig. 6a, the probe 2a can respond to the variation of pH rapidly, the emission ratios change markedly within 5 seconds and then reach a steady state.
Fig. 6.
EP
TE D
M AN U
is very much shorter than the reported before [30,58].
SC
Therefore, the probes 2 can be used to monitor the pH variation in real time. Their response time
(a) Reaction-time profile of probe 2a (10 µM in H2O/THF, v/v, 3/2) at different pH
AC C
values; (b) Reversibility of the emission ratio (I472/I407) of probe 2a (10 µM in H2O/THF, v/v, 3/2) between pH 7.4 and 2.5.
If the fluorescence change of pH probe is reversible, the potential application in real-time pH monitoring will be increased greatly [59]. To examine the reversibility of these pH-dependent emission ratio changes, the pH value of the solution was adjusted back and forth between 7.4 and
15
ACCEPTED MANUSCRIPT 2.5 by using trifluoroacetic acid and aqueous sodium hydroxide [30,47]. As shown in Fig. 6b, the results clearly reveal that these processes are fully reversible. This indicates that there is a reversibility between the protonated and deprotonated forms of the probe. Therefore, the cycle of
RI PT
pH-mediated fluorescence conversion and the real-time pH monitoring by the probes is feasible.
3.6. Visual Detection of pH
SC
In order to further investigate the application of probes 2, we studied the visualization of
M AN U
fluorescent color change by using probe 2a at H2O/THF solution (v/v, 3/2, 10 µM) under different pH conditions (Fig. 7a) [11,22]. Under irradiation with a 365 nm UV light, the color of the solution is changed from blue-violet to cyan with the decrease of pH value, which is consistent with the change of fluorescence spectra. Therefore, probe 2a can be used as a
AC C
EP
naked-eye.
TE D
convenient probe to distinguish different pH value through fluorescent color change by
Fig. 7.
Visual photographs of (a) probe 2a (10 µM in H2O/THF, v/v, 3/2) and (b) change in
color of probe 2a test strips with different pH solutions under 365 nm UV light.
Visual detection of pH by the method of test paper was also carried out according to the 16
ACCEPTED MANUSCRIPT literature [16,60]. After the test paper were dipped in neutral (pH = 7.0) and acidic (pH = 2.5) solutions containing probe 2a (10 µM in H2O/THF, v/v, 3/2) for 1 min and then dried in air, the observed colour changes of the test strips were recorded as shown in Fig. 7b. Under irradiation
RI PT
with 365 nm UV light, the neutral (pH = 7.0) test strip is blue-violet, and the acidic (pH = 2.5) test strip is cyan. Therefore, the test strip experiment demonstrates that probe 2a can be
SC
conveniently and efficiently used as a pH probe.
M AN U
4. Conclusions
In summary, we have designed and synthesized three novel benzimidazole molecules as ratiometric fluorescent pH probes. The emission spectra of the probes exhibit marked red shifts, resulting in large changes in the emission ratios. The ratiometric response of the probes to acidic
TE D
pH is due to the H+ binding with one N atom in C=Ν of benzimidazole ring and the induced enhancement of the ICT process, which is confirmed by 1H NMR experiments and DFT calculations. In addition, there is no significant interference caused by common metal ions to
EP
these pH probes. Finally, they display potential applications to monitor the pH variation in real
AC C
time and by naked-eyes.
Acknowledgements
We are grateful to Guangzhou Science and Technology Project Scientific Special (General Items, No. 201607010251), Guangdong Provincial Science and Technology Project (No. 2017A010103016) and the NSF of Guangdong Province (No. 2014A030313429) for financial
17
ACCEPTED MANUSCRIPT support.
Appendix A. Supplementary data
RI PT
Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/
Han JY, Burgess K. Fluorescent indicators for intracellular pH. Chem Rev 2010; 110:
M AN U
[1]
SC
References
2709-28. [2]
Hamilton GRC, Sahoo SK, Kamila S, Singh N, Kaur N, Hyland BW, et al. Optical probes for the detection of protons, and alkali and alkaline earth metal cations. Chem Soc Rev
[3]
TE D
2015; 44: 4415-32.
Yue YK, Huo FJ, Lee SY, Yin CX, Yoon J. A review: the trend of progress about pH probes in cell application in recent years. Analyst 2017; 142: 30-41. Fei X, Lu T, Ma J, Wang W-L, Zhu S-M, Zhang D. Bioinspired polymeric photonic
EP
[4]
AC C
crystals for high cycling pH-sensing performance. ACS Appl Mater Interfaces 2016; 8: 27091-8.
[5]
Weidman JL, Mulvenna RA, Boudouris BW, Phillip WA. Unusually stable hysteresis in
the pH-response of poly(acrylic acid) brushes confined within nanoporous block polymer
thin films. J Am Chem Soc 2016; 138: 7030-9. [6]
Kishimoto M, Kondo K, Akita M, Yoshizawa M. A pH-responsive molecular capsule with an acridine shell: catch and release of large hydrophobic compounds. Chem Commun 2017;
18
ACCEPTED MANUSCRIPT 53: 1425-8. [7]
Jokic T, Borisov SM, Saf R, Nielsen D, Kühl M, Klimant I. Highly photostable near-infrared
fluorescent
pH
indicators
and
sensors
based
on
BF2-chelated
[8]
RI PT
tetraarylazadipyrromethene dyes. Anal Chem 2012; 84: 6723-30. Berbasova T, Nosrati M, Vasileiou C, Wang W, Lee KSS, Yapici I, et al. Rational design of a colorimetric pH sensor from a soluble retinoic acid chaperone. J Am Chem Soc 2013;
Asanuma D, Takaoka Y, Namiki S, Takikawa K, Kamiya M, Nagano T, et al.
M AN U
[9]
SC
135: 16111-9.
Acidic-pH-activatable fluorescence probes for visualizing exocytosis dynamics. Angew Chem Int Ed 2014; 53: 6085-9. [10]
Hariharan PS, Mothi EM, Moon D, Anthony SP. Halochromic isoquinoline with
TE D
mechanochromic triphenylamine: smart fluorescent material for rewritable and self-erasable fluorescent platform. ACS Appl Mater Interfaces 2016; 8: 33034-42. [11]
Niu G-L, Zhang P-P, Liu W-M, Wang M-Q, Zhang H-Y, Wu J-S, et al. Near-infrared
EP
probe based on rhodamine derivative for highly sensitive and selective lysosomal pH
[12]
AC C
tracking. Anal Chem 2017; 89: 1922-9. Vukotic VN, Zhu KL, Baggi G, Loeb SJ. Optical distinction between “slow” and “fast”
translational motion in degenerate molecular shuttles. Angew Chem Int Ed 2017; 56: 6136-41.
[13]
Singh P, Baheti A, Thomas KRJ. Synthesis and optical properties of acidochromic amine-substituted benzo[a]phenazines. J Org Chem 2011; 76: 6134-45.
[14]
Zhou J, Liu H-Y, Jin B, Liu X-J, Fu H-B, Shangguan D-H. A guanidine derivative of
19
ACCEPTED MANUSCRIPT naphthalimide with excitedstate deprotonation coupled intramolecular charge transfer properties and its application. J Mater Chem C 2013; 1: 4427-36. [15] Reddy GU, Anila AH, Ali F, Taye N, Chattopadhyay S, Das A. FRET-based probe for
[16]
RI PT
monitoring pH changes in lipid-dense region of Hct116 cells. Org Lett 2015; 17: 5532-5. Li X-Q, Yue Y-K, Wen Y, Yin C-X. Huo F-J. Hemicyanine based fluorimetric and colorimetric pH probe and its application in bioimaging. Dyes Pigments 2016; 134: 291-6. Matsui M, Yamamoto T, Kakitani K, Biradar S, Kubota Y, Funabiki K. UV-vis absorption
SC
[17]
M AN U
and fluorescence spectra, solvatochromism, and application to pH sensors of novel xanthene dyes having thienyl and thieno[3,2-b]thienyl rings as auxochrome. Dyes Pigments 2017; 139: 533-40. [18]
Liu W, Sun R, Ge J-F, Xu Y-J, Xu Y, Liu J-M, et al. Reversible near-infrared pH probes
[19]
TE D
based on benzo[a]phenoxazine. Anal Chem 2013; 85: 7419-25. Zhu H, Fan J-L, Xu Q-L, Li H-L, Wang J-Y, Gao P, et al. Imaging of lysosomal pH changes with a fluorescent sensor containing a novel lysosome-locating group. Chem
EP
Commun 2012; 48: 11766-8.
AC C
[20] Shen S-L, Chen X-P, Zhang X-F, Miao J-Y, Zhao B-X. A rhodamine B-based lysosomal pH probe. J Mater Chem B 2015; 3: 919-25.
[21]
Takano H, Narumi T, Nomura W, Tamamura H. Microwave-assisted synthesis of
azacoumarin fluorophores and the fluorescence characterization. J Org Chem 2017; 82: 2739-44.
[22]
Hu J-L, Wu F, Feng S, Xu J-H, Xu Z-H, Chen Y-Q, et al. A convenient ratiomeric pH probe and its application for monitoring pH change in living cells. Sens Actuators B 2015;
20
ACCEPTED MANUSCRIPT 196: 194-202. [23]
Sun R, Liu X-D, Xun Z, Lu J-M, Xu Y-J, Ge, J-F. A rosamine-based red-emitting fluorescent sensor for detecting intracellular pH in live cells. Sens Actuators B 2014; 201:
[24]
RI PT
426-32. Long L-L, Li X-F, Zhang D-D, Meng S-C, Zhang J-F, Sun X-L, et al. Amino-coumarin based fluorescence ratiometric sensors for acidic pH and their application for living cells
You QH, Fan L, Chan WH, Lee AWM, Shuang SM. Ratiometric spiropyran-based
M AN U
[25]
SC
imaging. RSC Adv 2013; 3: 12204-9.
fluorescent pH probe. RSC Adv 2013; 3: 15762-8.
[26] Park HJ, Lim CS, Kim ES, Han JH, Lee TH, Chun HJ, et al. Measurement of pH values in human tissues by two-photon microscopy. Angew Chem Int Ed 2012; 51: 2673-6. Zhou XF, Su FY, Lu HG, Senechal-Willis P, Tian YQ, Johnson RH, et al. An FRET-based
TE D
[27]
ratiometric chemosensor for in vitro cellular fluorescence analyses of pH. Biomaterials 2012; 33: 171-80.
Chan Y-P, Fan L, You QH, Chan W-H, Lee AWM, Shuang SM. Ratiometric pH
EP
[28]
[29]
AC C
responsive fluorescent probes operative on ESIPT. Tetrahedron 2013; 69: 5874-9. Mukherjee S, Paul AK, Rajak KK, Stoeckli-Evans H. ICT based ratiometric fluorescent
pH sensing using quinoline hydrazones. Sens Actuators B 2014; 203: 150-6.
[30]
Zhang X-F, Zhang T, Shen S-L, Miao J-Y, Zhao B-X. A ratiometric lysosomal pH probe
based on the naphthalimide-rhodamine system. J Mater Chem B 2015; 3: 3260-6. [31]
Li ZY, Zhang PS, Wei Lu, Peng L, Zhao Y, Chen GJ. Ratiometric fluorescent pH probes based on glycopolymers. Macromol Rapid Commun 2016; 37: 1513-9.
21
ACCEPTED MANUSCRIPT [32] Chen J, Tang Y, Wang H, Zhang PS, Li Y, Jiang JH. Design and fabrication of fluorescence resonance energy transfer-mediated fluorescent polymer nanoparticles for ratiometric sensing of lysosomal pH. J Colloid Interface Sci 2016; 484: 298-307. Zhang LQ, Su FY, Kong XX, Lee F, Day K, Gao WM, et al. Ratiometric fluorescent
RI PT
[33]
pH-sensitive polymers for high-throughput monitoring of extracellular pH. RSC Adv 2016; 6: 46134-42.
[35]
M AN U
pH sensing. ChemistrySelect 2017; 2: 572-8.
SC
[34] Chandra A, Singh N. Biocompatible fluorescent carbon dots for ratiometric intracellular
Liu WJ, Li C, Sun XB, Pan W, Wang JP. Carbon-dot-based ratiometric fluorescent pH sensor for the detections of very weak acids assisted by auxiliary reagents that contribute to the release of protons. Sens Actuators B 2017; 244: 441-9. Shangguan J-F, He D-G, He X-X, Wang K-M, Xu F-Z, Liu J-Q, et al. Label-free
TE D
[36]
carbon-dots-based ratiometric fluorescence pH nanoprobes for intracellular pH sensing. Anal Chem 2016; 88: 7837-43.
Lu Y, Yan B. A ratiometric fluorescent pH sensor based on nanoscale metal-organic
EP
[37]
AC C
frameworks (MOFs) modified by europium(III) complexes. Chem Commun 2014; 50: 13323-6.
[38]
Xia TF, Zhu FL, Jiang K, Cui YJ, Yang Y, Qian GD. A luminescent ratiometric pH sensor
based on a nanoscale and biocompatible Eu/Tb-mixed MOF. Dalton Trans 2017; 46: 7549-55.
[39]
Luo WF, Jiang H, Tang XL, Liu WS. A reversible ratiometric two-photon lysosometargeted probe for real-time monitoring of pH changes in living cells. J Mater Chem B
22
ACCEPTED MANUSCRIPT 2017; 5: 4768-73. [40]
Liu XJ, Su YA, Tian HH, Yang L, Zhang HY, Song XZ, et al. Ratiometric fluorescent probe for lysosomal pH measurement and imaging in living cells using single-wavelength
RI PT
excitation. Anal. Chem. 2017; 89: 7038-45. [41] Shi BJ, Gao YL, Liu CX, Feng W, Li ZX, Wei LH, et al. A ratiometric fluorescent sensor for pH fluctuation and its application in living cells with low dark toxicity. Dyes Pigments
SC
2017; 136: 522-8.
M AN U
[42] Chen SY, Zhao M, Su J, Zhang Q, Tian XH, Li SL, et al. Two novel two-photon excited fluorescent pH probes based on the A-π-D-π-A system for intracellular pH mapping. Dyes Pigments 2017; 136: 807-16. [43]
Zhu Q, Li Z, Mu L, Zeng X, Redshaw C, Wei G. A quinoline-based fluorometric and
TE D
colorimetric dual-modal pH probe and its application in bioimaging. Spectrochim Acta A Part A, 2018; 188: 230-6. [44]
Lirag RC, Le HTM, Miljanic OS. L-shaped benzimidazole fluorophores: synthesis,
[45]
AC C
4304-6.
EP
characterization and optical response to bases, acids and anions. Chem Commun 2013; 49:
Kim HJ, Heo CH, Kim HM. Benzimidazole-based ratiometric two-photon fluorescent
probes for acidic pH in live cells and tissues. J Am Chem Soc 2013; 135: 17969-77.
[46]
Zhu X, Lin Q, Zhang Y-M, Wei T-B. Nitrophenylfuran-benzimidazole-based reversible
alkaline fluorescence switch accurately controlled by pH. Sens Actuators B 2015; 219: 8-42. [47]
Zhu X, Lin Q, Chen P, Fu Y-P, Zhang Y-M, Wei T-B. A novel pH sensor which could
23
ACCEPTED MANUSCRIPT respond to multi-scale pH changes via different fluorescence emissions. New J Chem 2016; 40: 4562-5. [48]
Li Z-S, Li L-J, Sun T-T, Liu L-M, Xie Z-G. Benzimidazole-BODIPY as optical and
[49]
RI PT
fluorometric pH sensor. Dyes Pigments 2016; 128:165-9. Horak E, Hranjec M, Vianello R, Steinberg IM. Reversible pH switchable aggregation-
solution. Dyes Pigments 2017; 142: 108-15.
Xiong J-F, Li J-X, Mo G-Z, Huo J-P, Liu J-Y, Chen X-Y, et al. Benzimidazole derivatives:
M AN U
[50]
SC
induced emission of self-assembled benzimidazole-based acrylonitrile dye in aqueous
Selective fluorescent chemosensors for the picogram detection of picric acid. J Org Chem 2014; 79: 11619-30.
[51] Wu Y-C, Huo J-P, Cao L, Ding S, Wang L-Y, Cao DR, et al. Design and application of
TE D
tri-benzimidazolyl star-shape molecules as fluorescent chemosensors for the fast-response detection of fluoride ion. Sens Actuators B 2016; 237: 865-75. [52] Wu Y-C, You J-Y, Jiang K, Xie J-C, Li S-L, Cao DR, et al. Colorimetric and ratiometric
EP
fluorescent sensor for F- based on benzimidazole-naphthalene conjugate: Reversible and
AC C
reusable study & design of logic gate function. Dyes Pigments 2017; 140: 47-55. [53] Wu Y-C, Luo S-H, Cao L, Jiang K, Wang L-Y, Xie J-C, et al. Self-assembled structures of N-alkylated bisbenzimidazolyl naphthalene in aqueous media for highly sensitive detection of picric acid. Anal Chim Acta 2017; 976: 74-83.
[54]
Beneto AJ, Thiagarajan V, Siva A. A tunable ratiometric pH sensor based on phenanthro[9,10-d]imidazole covalently linked with vinylpyridine. RSC Adv 2015; 5: 67849-52.
24
ACCEPTED MANUSCRIPT [55] Wang LY, Yang LL, Cao DR. Probes based on diketopyrrolopyrrole and anthracenone conjugates with aggregation-induced emission characteristics for pH and BSA sensing. Sens Actuators B 2015; 221: 155-66. Niu W-F, Fan L, Nan M, Li Z-B, Lu D-T, Wong M-S, et al. Ratiometric emission
RI PT
[56]
fluorescent pH probe for imaging of living cells in extreme acidity. Anal Chem 2015; 87: 2788-93.
Niu W-F, Fan L, Nan M, Wong M-S, Shuang S-M, Dong C. A novel fluorescent probe for
SC
[57]
[58]
M AN U
sensing and imaging extreme acidity. Sens Actuators B 2016; 234: 534-40. Zhao X-X, Chen X-P, Shen S-L, Li D-P, Zhou S, Zhou Z-Q, et al. A novel pH probe based on a rhodamine-rhodamine platform. RSC Adv 2014; 4: 50318-24. [59]
Hua JL, Wu F, Feng S, Xu JH, Xu ZH, Chen YQ, et al. A convenient ratiomeric pH probe
194-202.
TE D
and its application for monitoring pH change in living cells. Sens Actuators B 2014; 196:
[60] Cheng JH, Gou F, Zhang XH, Shen GY, Zhou XG, Xiang HF. A class of multiresponsive
EP
colorimetric and fluorescent pH probes via three different reaction mechanisms of salen
AC C
complexes: A selective and accurate pH measurement. Inorg Chem 2016; 55: 9221-9.
25
ACCEPTED MANUSCRIPT
1. Three novel ratiometric fluorescent probes for acidic pH are developed. 2. The enhanced ICT mechanism is confirmed by 1H NMR and DFT calculations studies.
RI PT
3. There is no significant interference caused by common metal ions to these pH probes.
4. These probes can be used in real-time and reversible pH sensing.
SC
5. There is a potential application of monitoring the pH variation by
AC C
EP
TE D
M AN U
naked-eyes.
S0143-7208(17)31504-8
DOI:
10.1016/j.dyepig.2017.09.043
Reference:
DYPI 6268
To appear in:
Dyes and Pigments
Received Date: 9 July 2017 Revised Date:
2 September 2017
Accepted Date: 17 September 2017
Please cite this article as: Wu Y-C, You J-Y, Jiang K, Wu H-Q, Xiong J-F, Wang Z-Y, Novel benzimidazole-based ratiometric fluorescent probes for acidic pH, Dyes and Pigments (2017), doi: 10.1016/j.dyepig.2017.09.043. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT
Novel benzimidazole-based ratiometric fluorescent probes for acidic pH
Yan-Cheng Wuab, Jia-Yi Youa, Kai Jianga, Han-Qing Wua, Jin-Feng Xiong*c and Zhao-Yang
a
RI PT
Wang*a
School of Chemistry and Environment, South China Normal University; Key Laboratory of Theoretical Chemistry of
SC
Environment, Ministry of Education, Guangzhou 510006, China
College of Materials Science and Engineering, South China University of Technology, Guangzhou 510640, China
c
HEC Pharm R&D Center, Dongguan 523871, China
* Corresponding Author
TE D
M AN U
b
E-mail: [email protected]. Tel: 8620-39310258.
AC C
EP
E-mail: [email protected].
1
ACCEPTED MANUSCRIPT
ABSTRACT: 2,6-Bis(1-alkyl-1H-benzo[d]imidazol-2-yl)naphthalene (2a-2c) as three new benzimidazole molecules have been designed, synthesized and applied as ratiometric fluorescent pH probes in
RI PT
the acidic pH region with high-efficiency and fast response. For probe 2a, upon decreasing the pH from 7.5 to 2.5, the emission spectra exhibit a large red shift from 402 to 472 nm, and the emission ratio (I472/I402) changes dramatically from 0.10 to 3.42 with a pKa value of 2.92. No
SC
significant interference on emission behavior is observed in the presence of various metal ions.
M AN U
Similar results are obtained for probes 2b and 2c. 1H NMR and DFT calculations studies demonstrate that the ratiometric response of the probes to acidic pH is due to H+ binding with one N atom in C=N of benzimidazole ring and the subsequent enhancement of the intramolecular charger transfer (ICT) process. In addition, these probes can be used in real-time and reversible
TE D
pH sensing. Finally, the probes have been successfully applied to the visual detection of pH not
Keywords:
EP
only in solution, but also in test paper.
AC C
Benzimidazole; Fluorescent probe; pH sensing, Ratiometric response; ICT
2
ACCEPTED MANUSCRIPT
1. Introduction Since pH is a key parameter in a wide range of fields, such as chemical process control, biological systems and environmental monitoring, the measurement of pH is of great significance
RI PT
to the protection of the environment and life [1-6]. Owing to many advantages of the fluorescent detection technique, such as high selectivity and sensitivity, operational simplicity, low cost and real-time monitoring, the design and synthesis of fluorescent pH probes with high-efficiency and
SC
fast response have become a highly popular topic of scientists' interests [7-12].
M AN U
Recently, a number of fluorescent probes have been used to the measurement of pH [13-23]. Amongst them, most fluorescent probes only have an intensity change in single-emission window with the response to the variation of pH [18-23]. However, fluorescence signals are easily influenced by environmental factors, such as temperature, solvent polarity and excitation
TE D
wavelength [24,25]. It is well-known that, the ratiometric fluorescent probe can encompass a built-in correction for the above-mentioned environmental effects to improve the selectivity and sensitivity since the ratiometric measurements are based on the change in the ratio of the
EP
emission intensity at two wavelengths [26-30]. Therefore, there are some reports about
AC C
ratiometric fluorescent pH probes in recent year, and the typical examples include polymers [31-33], carbon nanodots [34-36], metal-organic frameworks [37,38], and organic small molecules [39-43]. In these efforts, organic small molecules-based fluorescent ratiometric probes are easier to be designed and prepared. However, some deficiencies still exist in organic small molecule-based ratiometric fluorescent pH probes. For instance, more steps are necessary in the synthesis [39-41]. Utilizing that the N-H unit and the N atom in C=Ν of benzimidazole ring can interact with
3
ACCEPTED MANUSCRIPT hydroxyl and hydrogen ions respectively, benzimidazole fluorophore has been widely used to the design of fluorescent pH probes [25,44-49]. Unfortunately, only a little research has been reported on the development of ratiometric pH probes based on benzimidazole compounds
RI PT
[25,45,49]. In addition, only a few benzimidazole-based probes can be applicable for strong acidic conditions with pH below 4 [45,48,49]. Therefore, it is important to develop benzimidazole-based ratiometric pH probes for extreme acidity with high-efficiency and fast
SC
response.
M AN U
Herein, for a continuation of our ongoing program aimed at developing novel fluorescent probes for different analytes [50-53], we have synthesized and characterized three benzimidazole-based fluorescent probes as new ratiometric fluorescent pH probes (Scheme 1). These probes display excellent performance and potential application as anticipated. Their
TE D
sensing mechanism via the enhanced intramolecular charger transfer (ICT) effect has been
AC C
EP
established with the help of 1H NMR and DFT calculations studies.
Scheme 1.
Synthesis of probes 2.
2. Experimental 2.1. Materials and instruments 4
ACCEPTED MANUSCRIPT Melting point was performed on an X-5 digital melting point apparatus without correcting. 1
H and 13C NMR spectra were collected on a BRUKER DRX-400 spectrometer in CDCl3 using
TMS as an internal standard. Mass spectra (MS) were recorded on a Thermo LCQ DECA XP
UV-vis
spectra
were
measured
by using
a
Shimazu
RI PT
MAX mass spectrometer. Elemental analysis was obtained with Perkin Elmer Series II 2400. UV-2550
ultraviolet
visible
spectrophotometer at room temperature. The fluorescence spectra were recorded with a Hitachi
SC
F-2700 spectrophotometer at room temperature; and the slit width was 5 nm for both excitation
M AN U
and emission. pH values were recorded on a LIDA Model PHS-25C Bench pH/MV meter. All reagents were purchased from commercial suppliers and used without purification. 2,6-Bis(1H-benzo[d]imidazol-2-yl)naphthalene 1 was prepared according to our literature [52].
TE D
2.2. Synthesis
According to the literature [50], a 100 mL round-bottom flask was charged with compound 1 (3 mmol, 1.080 g), the selected bromoalkane (6.2 mmol) and solid NaOH (12 mmol, 0.480 g)
EP
in CH3CN (30 mL). The reaction mixture was stirred at 80 °C for 2-4 h under the monitoring of
AC C
TLC. After the solvent was removed, the residue was dissolved in dichloromethane. The organic layers were washed with water three times and then dried over anhydrous MgSO4. The concentration under vacuum gave a crude product, which was purified by column chromatography on silica gel with gradient eluents of petroleum ether and ethyl acetate to afford pure compound 2. 2,6-Bis(1-decyl-1H-benzo[d]imidazol-2-yl)naphthalene (2a). White solid, 1.702 g, yield 88.5%, m.p. 97.2-98.5
; UV-vis (THF) λmax: 327.0 nm; 1H NMR (CDCl3-TMS, 400 MHz): δ =
5
ACCEPTED MANUSCRIPT 0.86 (t, J = 8.0 Hz, 6H), 1.18-1.31 (m, 28H), 1.85-1.97 (m, 4H), 4.35 (t, J = 8.0 Hz, 4H), 7.29-7.41 (m, 4H), 7.44-7.51 (m, 2H), 7.82-7.90 (m, 2H), 7.93 (d, J = 8.0 Hz, 2H), 8.09 (d, J = 8.0 Hz, 2H), 8.32 (s, 2H); 13C NMR (CDCl3-TMS, 100 MHz): δ = 14.1, 22.6, 26.7, 29.0, 29.2, 29.4, 29.7, 29.9,
RI PT
31.8, 45.0, 110.2, 120.1, 122.5, 122.9, 127.1, 129.0, 129.1, 129.4, 133.2, 135.8, 143.3, 153.2; IR (film), ν, cm-1: 3055, 2955, 2922, 2855, 1616, 1489, 1454, 1329, 893, 824, 746; ESI-MS, m/z (%):
C 82.45, H 8.81, N 8.74, Found: C 82.58, H 9.01, N 8.72.
SC
Calcd for C44H57N4+ ([M+H]+): 641.46 (100%), Found: 641.50 (100%); Anal. Calcd for C44H56N4:
83.7%, m.p. 93.3-94.2
M AN U
2,6-Bis(1-dodecyl-1H-benzo[d]imidazol-2-yl)naphthalene (2b). White solid, 1.750 g, yield ; UV-vis (THF) λmax: 327.0 nm; 1H NMR (CDCl3-TMS, 400 MHz): δ =
0.85 (t, J = 8.0 Hz, 6H), 1.06-1.41 (m, 36H), 1.85-1.95 (m, 4H), 4.34 (t, J = 8.0 Hz, 4H), 7.31-7.39 (m, 4H), 7.44-7.50 (m, 2H), 7.84-7.90 (m, 2H), 7.92 (d, J = 8.0 Hz, 2H), 8.09 (d, J = 8.0 Hz, 2H),
TE D
8.31 (s, 2H); 13C NMR (CDCl3-TMS, 100 MHz): δ = 14.1, 22.7, 26.8, 29.1, 29.3, 29.4, 29.5, 29.6, 29.9, 31.9, 45.0, 110.2, 120.1, 122.5, 122.9, 127.2, 129.1, 129.4, 133.2, 135.8, 143.3, 153.2; IR
EP
(film), ν, cm-1: 3063, 2930, 2855, 1616, 1489, 1458, 1329, 893, 819, 746; ESI-MS, m/z (%): Calcd for C48H65N4+ ([M+H]+): 697.52 (100%), Found: 697.47 (100%); Anal. Calcd for C48H64N4: C
AC C
82.71, H 9.25, N 8.04, Found: C 82.78, H 9.34, N 7.96.
2,6-Bis(1-tetradecyl-1H-benzo[d]imidazol-2-yl)naphthalene (2c). White solid, 1.625 g, yield 71.9%, m.p. 88.6-89.3
; UV-vis (THF) λmax: 328.0 nm; 1H NMR (CDCl3-TMS, 400 MHz):
δ = 0.86 (t, J = 8.0 Hz, 6H), 1.07-1.39 (m, 44H), 1.85-1.95 (m, 4H), 4.35 (t, J = 8.0 Hz, 4H), 7.30-7.39 (m, 4H), 7.44- 7.50 (m, 2H), 7.84-7.90 (m, 2H), 7.92 (d, J = 8.0 Hz, 2H), 8.09 (d, J = 8.0 Hz, 2H), 8.31 (s, 2H);
13
C NMR (CDCl3-TMS, 100 MHz): δ = 14.1, 22.7, 26.7, 29.4, 29.5,
6
ACCEPTED MANUSCRIPT 29.6, 29.7, 29.9, 31.9, 45.0, 110.2, 120.1, 122.5, 122.9, 127.2, 129.0, 129.1, 129.4, 133.2, 135.8, 143.3, 153.2; IR (film), ν, cm-1: 3069, 2930, 2862, 1616, 1493, 1458, 1329, 893, 819, 743; ESI-MS, m/z (%): Calcd for C52H73N4+ ([M+H]+): 753.58 (100%), Found: 753.69 (100%); Anal.
RI PT
Calcd for C52H72N4: C 82.93, H 9.64, N 7.44, Found: C 82.88, H 9.75, N 7.52.
SC
2.3. Computational methods
According to the literature [24,54], all of the calculations were obtained using density
3. Results and discussion
M AN U
functional theory (DFT) with the B3LYP/6-31G (d) level of the Gaussian 09 program.
TE D
3.1. Synthesis and Photophysical Properties of 2
As shown in Scheme 1, the target molecules 2a-2c were synthesized via a simple and efficient N-alkylation reaction of compound 1 and the selected 1-bromoalkane with the yields of 13
C NMR, ESI-MS [the corresponding
EP
71.9-88.5%. Their structures were characterized by 1H,
AC C
spectra (Figs. S1-S9) can be seen in Supporting Information (SI)], IR, and elemental analysis. In an effort to gain insight into the photophysical process of the target molecules, we firstly investigated the absorption and emission behaviors of compounds 2a-2c in different non-polar or polar solvents. They show similar absorption spectra, and there is a slight blueshift with the increase of the solvent polarity (Figs. S10a-S12a). The emission spectra of compounds 2a-2c show two shoulder peaks in less polar solvents, but merging into a single peak in polar solvents (Figs. S10b-S12b). It's worth noting that the fluorescence intensity of 2 is relatively high in THF,
7
ACCEPTED MANUSCRIPT and it can be completely miscible with water. Therefore, we selected THF as the solvent for the
Fig. 1.
M AN U
SC
RI PT
studies on the fluorescent properties of compounds 2a-2c.
The fluorescence spectra (λex = 325 nm) of compound 2c (10 µM) in H2O/THF system
EP
TE D
with different water fractions.
The emission spectra of compound 2c in THF-H2O mixed solvents had also been
AC C
investigated. As shown in Fig. 1, compound 2c exhibits a strong emission at 400 nm with a shoulder peak at 389 nm in THF solution. When the water fraction (fw) is less than 60%, there is a slight change in fluorescence intensity. Further increasing fw may lead to a dramatic decline in the emission intensity, indicating that the maximum water content is 60%. Perhaps, the probes may aggregate in the presence of a large amount of water, resulting in fluorescence quenching (aggregation-caused quenching effect). However, all the aqueous mixtures are homogenous without precipitates, suggesting that the aggregates are of nano-dimensions. This is proved by the 8
ACCEPTED MANUSCRIPT level-off tail observed in the UV spectrum (Fig. S13) in the longer wave-length region when the fw values exceeded 60% [53,55]. These results show that compounds 2 has a high tolerance to water. Thus, we chose the binary system with an fw of 60% (H2O/THF, v/v, 3/2) as the solvent for
RI PT
studying the pH responsive properties of the probes.
3.2. Optical Response to pH
SC
To study the optical responses of compounds 2 to pH, standard pH titrations of absorption
M AN U
and emission spectra were performed. The spectroscopic properties of probe 2a under different pH conditions in H2O/THF (v/v, 3/2) solution were first selected to be examined (Fig. 2). As the pH is decreased from 7.5 to 2.5, the absorbance at 282 and 325 nm is gradually decreased accompanying by the formation of a new peak at 245 nm and a shoulder peak at around 370 nm
AC C
EP
TE D
(Fig. 2a). Further decrease in pH does not induce any change in the absorption spectrum.
(a) Fig. 2.
(b)
(a) UV-vis absorption spectra and (b) emission spectra of probe 2a (10 µM in H2O/THF, v/v, 3/2) with the different pH (Inset: plot of ratio of I472/I402 as a function of pH).
9
ACCEPTED MANUSCRIPT As shown in Fig. 2b, upon decreasing the pH from 7.5 to 2.5, the emission spectra of probe 2a exhibits a shift from 402 to 472 nm with an obvious red shift of 70 nm. The large red shift of the signals may be attributed to the enhanced ICT effect because of the H+ binding-induced
RI PT
enhancement of the electron-withdrawing ability of benzimidazole ring. A well-defined isoemission point is found at 460 nm, indicating that only one benzimidazole ring could bind with H+ [24]. The large red shift makes probe 2a suitable for ratiometric pH sensing in the acidic
SC
region. When the pH is decreased from 7.5 to 2.5, the emission intensity ratio (I472/I402) changes
as a function of pH (Fig. S14).
M AN U
from 0.10 to 3.42 (Inset Fig. 2b). The pKa value of 2.92 can be calculated from the emission ratio
Similar behaviors were also observed for both 2b (Fig. S15) and 2c (Fig. S16). As the pH is decreased, the emission spectra of both 2b (Fig. S15b) and 2c (Fig. S16b) exhibit an apparent
TE D
red shift from 402 to 472 nm, with the emission ratio (I472/I402) changing from 0.11 and 0.10 to 3.13 and 3.53 respectively (Inset Figs. S15b and S16b). Similarly, the pKa value of 2b and 2c can
EP
be calculated to be 2.90 and 2.51 respectively (Fig. S17).
AC C
3.3. Possible Sensing Mechanism
In order to get insight into the sensing mechanism of probes 2 for acidic pH, 1H NMR experiments were carried out according to the literature [24,56]. As shown in Fig. 3, with the addition of incremental amounts of CF3COOD (1-10 equiv.) to the solution of probe 2a (20 mM in CDCl3), a downfield shift is observed for the chemical shift values of the benzimidazole protons (Hd, He, Hf) and naphthalene protons (Ha, Hb). When 20 equiv. of CF3COOD is added to above solution, the chemical shift values of these protons remain almost constant. The downfield
10
ACCEPTED MANUSCRIPT chemical shift of these protons is obviously due to the H+ binding with the N atom in C=Ν of benzimidazole ring, which results in the decrease of electron density around these protons [24,56]. In addition, the chemical shift values of the benzimidazole protons (Hg) and naphthalene
RI PT
protons (Hc) are almost unchanged, indicating that the alkylated N atom in benzimidazole ring does not bind with H+ [24]. Therefore, it is clear that the H+ binding with the N atom in C=Ν of
AC C
EP
TE D
M AN U
SC
benzimidazole ring in probes 2 results in the significant optical response to acidic pH.
Fig. 3.
Partial 1H NMR spectra of probe 2a (20 mM in CDCl3) and probe 2a with the different equiv. CF3COOD.
11
Fig. 4.
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
Calculated (DFT B3LYP/6-31G (d) level) HOMO-LUMO energy levels and the
TE D
interfacial plots of the orbitals for probe 2a, the 2a-H+ complex and the 2a-2H+ complex.
To better understand the optical responses of probes 2 upon binding with H+, we further
EP
carried out DFT calculations using probe 2a as an example (Fig. 4). The DFT calculations study shows a HOMO→LUMO charge transfer in both 2a and 2a-H+. For probe 2a, the LUMO is
AC C
mainly localized on the central naphthalene core and partly distributed on both benzimidazole rings, whereas the HOMO is evenly distributed on the central naphthalene core and both benzimidazole rings. For 2a-H+ complex, the LUMO is mainly localized on the central naphthalene core and benzimidazolium moiety, whereas the HOMO is mainly distributed on another benzimidazole ring without the appendage of H+. These results indicate that the ICT process in 2a-H+ is enhanced comparing with that in 2a [24]. In addition, the energy gap (△E) between the HOMO and LUMO of the 2a-H+ complex (2.65583 eV) is smaller than that of probe 12
ACCEPTED MANUSCRIPT 2a (3.84089 eV), indicating that the 2a-H+ complex is more stable than probe 2a. The result is in good agreement with the red shift in emission spectra of probe 2a upon binding with H+. For bis-protonation species (2a-2H+), the LUMO is mainly localized on the central
RI PT
naphthalene core and partly distributed on both benzimidazole rings, whereas the HOMO is mainly distributed on one N-alkyl chain. It is worth noting that the ∆E of 2a-2H+ is increased (3.20387 eV) comparing with that of 2a-H+. These results further confirm that the
SC
mono-protonation is difficult to further formation of bis-protonation [54]. What is more, the
M AN U
results of DFT calculations for probe 2b and its protonated species (Fig. S18) are basically similar. Thus, the above conclusions are further confirmed.
Based on the above research results, the sensing mechanism for pH can be concluded in the following (Scheme 2): When proton is added into the solution of probes 2, H+ can bind with one
TE D
N atom in C=Ν of benzimidazole ring [57], and H+ binding induces an enhancement of the electron-withdrawing ability of benzimidazole. Therefore, the fluorescence changes via
AC C
EP
enhancing the ICT process in 2-H+ complex [45,56].
Scheme 2.
Proposed mechanism of fluorescence change for probes 2 upon addition of H+.
13
ACCEPTED MANUSCRIPT 3.4. Selectivity Studies The selectivity response of probes 2 to H+ over various metal ions at pH 7.0 and 3.0 were investigated (Fig. 5 and Fig. S19). At pH = 7.0, there is no observed effect on the emission ratio
RI PT
of the probes 2 within the additives except for Fe3+ causing a slight increase of I472/I402. Meanwhile, the addition of different metal ions has no obvious influence on the emission ratio of the probes 2 at pH = 3.0. These results demonstrate that probes 2 can be considered as selective
AC C
EP
TE D
M AN U
SC
fluorescent probes for acidic pH sensing.
Fig. 5.
The ratio of fluorescence intensity of probe 2a (10 µM) at I472/I402 containing different
metal ions at pH 7.0 (black bar) and 3.0 (blue bar). All the concentration of the metal ions is 500 µM (λex = 325 nm).
14
ACCEPTED MANUSCRIPT 3.5. Real-time Response and Reversibility Study For practical application, the real-time determination is very important [24,30]. Thus, using probe 2a as a representative, the reaction-time profile of probes 2 in the solution of different pH
RI PT
values was measured. As shown in Fig. 6a, the probe 2a can respond to the variation of pH rapidly, the emission ratios change markedly within 5 seconds and then reach a steady state.
Fig. 6.
EP
TE D
M AN U
is very much shorter than the reported before [30,58].
SC
Therefore, the probes 2 can be used to monitor the pH variation in real time. Their response time
(a) Reaction-time profile of probe 2a (10 µM in H2O/THF, v/v, 3/2) at different pH
AC C
values; (b) Reversibility of the emission ratio (I472/I407) of probe 2a (10 µM in H2O/THF, v/v, 3/2) between pH 7.4 and 2.5.
If the fluorescence change of pH probe is reversible, the potential application in real-time pH monitoring will be increased greatly [59]. To examine the reversibility of these pH-dependent emission ratio changes, the pH value of the solution was adjusted back and forth between 7.4 and
15
ACCEPTED MANUSCRIPT 2.5 by using trifluoroacetic acid and aqueous sodium hydroxide [30,47]. As shown in Fig. 6b, the results clearly reveal that these processes are fully reversible. This indicates that there is a reversibility between the protonated and deprotonated forms of the probe. Therefore, the cycle of
RI PT
pH-mediated fluorescence conversion and the real-time pH monitoring by the probes is feasible.
3.6. Visual Detection of pH
SC
In order to further investigate the application of probes 2, we studied the visualization of
M AN U
fluorescent color change by using probe 2a at H2O/THF solution (v/v, 3/2, 10 µM) under different pH conditions (Fig. 7a) [11,22]. Under irradiation with a 365 nm UV light, the color of the solution is changed from blue-violet to cyan with the decrease of pH value, which is consistent with the change of fluorescence spectra. Therefore, probe 2a can be used as a
AC C
EP
naked-eye.
TE D
convenient probe to distinguish different pH value through fluorescent color change by
Fig. 7.
Visual photographs of (a) probe 2a (10 µM in H2O/THF, v/v, 3/2) and (b) change in
color of probe 2a test strips with different pH solutions under 365 nm UV light.
Visual detection of pH by the method of test paper was also carried out according to the 16
ACCEPTED MANUSCRIPT literature [16,60]. After the test paper were dipped in neutral (pH = 7.0) and acidic (pH = 2.5) solutions containing probe 2a (10 µM in H2O/THF, v/v, 3/2) for 1 min and then dried in air, the observed colour changes of the test strips were recorded as shown in Fig. 7b. Under irradiation
RI PT
with 365 nm UV light, the neutral (pH = 7.0) test strip is blue-violet, and the acidic (pH = 2.5) test strip is cyan. Therefore, the test strip experiment demonstrates that probe 2a can be
SC
conveniently and efficiently used as a pH probe.
M AN U
4. Conclusions
In summary, we have designed and synthesized three novel benzimidazole molecules as ratiometric fluorescent pH probes. The emission spectra of the probes exhibit marked red shifts, resulting in large changes in the emission ratios. The ratiometric response of the probes to acidic
TE D
pH is due to the H+ binding with one N atom in C=Ν of benzimidazole ring and the induced enhancement of the ICT process, which is confirmed by 1H NMR experiments and DFT calculations. In addition, there is no significant interference caused by common metal ions to
EP
these pH probes. Finally, they display potential applications to monitor the pH variation in real
AC C
time and by naked-eyes.
Acknowledgements
We are grateful to Guangzhou Science and Technology Project Scientific Special (General Items, No. 201607010251), Guangdong Provincial Science and Technology Project (No. 2017A010103016) and the NSF of Guangdong Province (No. 2014A030313429) for financial
17
ACCEPTED MANUSCRIPT support.
Appendix A. Supplementary data
RI PT
Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/
Han JY, Burgess K. Fluorescent indicators for intracellular pH. Chem Rev 2010; 110:
M AN U
[1]
SC
References
2709-28. [2]
Hamilton GRC, Sahoo SK, Kamila S, Singh N, Kaur N, Hyland BW, et al. Optical probes for the detection of protons, and alkali and alkaline earth metal cations. Chem Soc Rev
[3]
TE D
2015; 44: 4415-32.
Yue YK, Huo FJ, Lee SY, Yin CX, Yoon J. A review: the trend of progress about pH probes in cell application in recent years. Analyst 2017; 142: 30-41. Fei X, Lu T, Ma J, Wang W-L, Zhu S-M, Zhang D. Bioinspired polymeric photonic
EP
[4]
AC C
crystals for high cycling pH-sensing performance. ACS Appl Mater Interfaces 2016; 8: 27091-8.
[5]
Weidman JL, Mulvenna RA, Boudouris BW, Phillip WA. Unusually stable hysteresis in
the pH-response of poly(acrylic acid) brushes confined within nanoporous block polymer
thin films. J Am Chem Soc 2016; 138: 7030-9. [6]
Kishimoto M, Kondo K, Akita M, Yoshizawa M. A pH-responsive molecular capsule with an acridine shell: catch and release of large hydrophobic compounds. Chem Commun 2017;
18
ACCEPTED MANUSCRIPT 53: 1425-8. [7]
Jokic T, Borisov SM, Saf R, Nielsen D, Kühl M, Klimant I. Highly photostable near-infrared
fluorescent
pH
indicators
and
sensors
based
on
BF2-chelated
[8]
RI PT
tetraarylazadipyrromethene dyes. Anal Chem 2012; 84: 6723-30. Berbasova T, Nosrati M, Vasileiou C, Wang W, Lee KSS, Yapici I, et al. Rational design of a colorimetric pH sensor from a soluble retinoic acid chaperone. J Am Chem Soc 2013;
Asanuma D, Takaoka Y, Namiki S, Takikawa K, Kamiya M, Nagano T, et al.
M AN U
[9]
SC
135: 16111-9.
Acidic-pH-activatable fluorescence probes for visualizing exocytosis dynamics. Angew Chem Int Ed 2014; 53: 6085-9. [10]
Hariharan PS, Mothi EM, Moon D, Anthony SP. Halochromic isoquinoline with
TE D
mechanochromic triphenylamine: smart fluorescent material for rewritable and self-erasable fluorescent platform. ACS Appl Mater Interfaces 2016; 8: 33034-42. [11]
Niu G-L, Zhang P-P, Liu W-M, Wang M-Q, Zhang H-Y, Wu J-S, et al. Near-infrared
EP
probe based on rhodamine derivative for highly sensitive and selective lysosomal pH
[12]
AC C
tracking. Anal Chem 2017; 89: 1922-9. Vukotic VN, Zhu KL, Baggi G, Loeb SJ. Optical distinction between “slow” and “fast”
translational motion in degenerate molecular shuttles. Angew Chem Int Ed 2017; 56: 6136-41.
[13]
Singh P, Baheti A, Thomas KRJ. Synthesis and optical properties of acidochromic amine-substituted benzo[a]phenazines. J Org Chem 2011; 76: 6134-45.
[14]
Zhou J, Liu H-Y, Jin B, Liu X-J, Fu H-B, Shangguan D-H. A guanidine derivative of
19
ACCEPTED MANUSCRIPT naphthalimide with excitedstate deprotonation coupled intramolecular charge transfer properties and its application. J Mater Chem C 2013; 1: 4427-36. [15] Reddy GU, Anila AH, Ali F, Taye N, Chattopadhyay S, Das A. FRET-based probe for
[16]
RI PT
monitoring pH changes in lipid-dense region of Hct116 cells. Org Lett 2015; 17: 5532-5. Li X-Q, Yue Y-K, Wen Y, Yin C-X. Huo F-J. Hemicyanine based fluorimetric and colorimetric pH probe and its application in bioimaging. Dyes Pigments 2016; 134: 291-6. Matsui M, Yamamoto T, Kakitani K, Biradar S, Kubota Y, Funabiki K. UV-vis absorption
SC
[17]
M AN U
and fluorescence spectra, solvatochromism, and application to pH sensors of novel xanthene dyes having thienyl and thieno[3,2-b]thienyl rings as auxochrome. Dyes Pigments 2017; 139: 533-40. [18]
Liu W, Sun R, Ge J-F, Xu Y-J, Xu Y, Liu J-M, et al. Reversible near-infrared pH probes
[19]
TE D
based on benzo[a]phenoxazine. Anal Chem 2013; 85: 7419-25. Zhu H, Fan J-L, Xu Q-L, Li H-L, Wang J-Y, Gao P, et al. Imaging of lysosomal pH changes with a fluorescent sensor containing a novel lysosome-locating group. Chem
EP
Commun 2012; 48: 11766-8.
AC C
[20] Shen S-L, Chen X-P, Zhang X-F, Miao J-Y, Zhao B-X. A rhodamine B-based lysosomal pH probe. J Mater Chem B 2015; 3: 919-25.
[21]
Takano H, Narumi T, Nomura W, Tamamura H. Microwave-assisted synthesis of
azacoumarin fluorophores and the fluorescence characterization. J Org Chem 2017; 82: 2739-44.
[22]
Hu J-L, Wu F, Feng S, Xu J-H, Xu Z-H, Chen Y-Q, et al. A convenient ratiomeric pH probe and its application for monitoring pH change in living cells. Sens Actuators B 2015;
20
ACCEPTED MANUSCRIPT 196: 194-202. [23]
Sun R, Liu X-D, Xun Z, Lu J-M, Xu Y-J, Ge, J-F. A rosamine-based red-emitting fluorescent sensor for detecting intracellular pH in live cells. Sens Actuators B 2014; 201:
[24]
RI PT
426-32. Long L-L, Li X-F, Zhang D-D, Meng S-C, Zhang J-F, Sun X-L, et al. Amino-coumarin based fluorescence ratiometric sensors for acidic pH and their application for living cells
You QH, Fan L, Chan WH, Lee AWM, Shuang SM. Ratiometric spiropyran-based
M AN U
[25]
SC
imaging. RSC Adv 2013; 3: 12204-9.
fluorescent pH probe. RSC Adv 2013; 3: 15762-8.
[26] Park HJ, Lim CS, Kim ES, Han JH, Lee TH, Chun HJ, et al. Measurement of pH values in human tissues by two-photon microscopy. Angew Chem Int Ed 2012; 51: 2673-6. Zhou XF, Su FY, Lu HG, Senechal-Willis P, Tian YQ, Johnson RH, et al. An FRET-based
TE D
[27]
ratiometric chemosensor for in vitro cellular fluorescence analyses of pH. Biomaterials 2012; 33: 171-80.
Chan Y-P, Fan L, You QH, Chan W-H, Lee AWM, Shuang SM. Ratiometric pH
EP
[28]
[29]
AC C
responsive fluorescent probes operative on ESIPT. Tetrahedron 2013; 69: 5874-9. Mukherjee S, Paul AK, Rajak KK, Stoeckli-Evans H. ICT based ratiometric fluorescent
pH sensing using quinoline hydrazones. Sens Actuators B 2014; 203: 150-6.
[30]
Zhang X-F, Zhang T, Shen S-L, Miao J-Y, Zhao B-X. A ratiometric lysosomal pH probe
based on the naphthalimide-rhodamine system. J Mater Chem B 2015; 3: 3260-6. [31]
Li ZY, Zhang PS, Wei Lu, Peng L, Zhao Y, Chen GJ. Ratiometric fluorescent pH probes based on glycopolymers. Macromol Rapid Commun 2016; 37: 1513-9.
21
ACCEPTED MANUSCRIPT [32] Chen J, Tang Y, Wang H, Zhang PS, Li Y, Jiang JH. Design and fabrication of fluorescence resonance energy transfer-mediated fluorescent polymer nanoparticles for ratiometric sensing of lysosomal pH. J Colloid Interface Sci 2016; 484: 298-307. Zhang LQ, Su FY, Kong XX, Lee F, Day K, Gao WM, et al. Ratiometric fluorescent
RI PT
[33]
pH-sensitive polymers for high-throughput monitoring of extracellular pH. RSC Adv 2016; 6: 46134-42.
[35]
M AN U
pH sensing. ChemistrySelect 2017; 2: 572-8.
SC
[34] Chandra A, Singh N. Biocompatible fluorescent carbon dots for ratiometric intracellular
Liu WJ, Li C, Sun XB, Pan W, Wang JP. Carbon-dot-based ratiometric fluorescent pH sensor for the detections of very weak acids assisted by auxiliary reagents that contribute to the release of protons. Sens Actuators B 2017; 244: 441-9. Shangguan J-F, He D-G, He X-X, Wang K-M, Xu F-Z, Liu J-Q, et al. Label-free
TE D
[36]
carbon-dots-based ratiometric fluorescence pH nanoprobes for intracellular pH sensing. Anal Chem 2016; 88: 7837-43.
Lu Y, Yan B. A ratiometric fluorescent pH sensor based on nanoscale metal-organic
EP
[37]
AC C
frameworks (MOFs) modified by europium(III) complexes. Chem Commun 2014; 50: 13323-6.
[38]
Xia TF, Zhu FL, Jiang K, Cui YJ, Yang Y, Qian GD. A luminescent ratiometric pH sensor
based on a nanoscale and biocompatible Eu/Tb-mixed MOF. Dalton Trans 2017; 46: 7549-55.
[39]
Luo WF, Jiang H, Tang XL, Liu WS. A reversible ratiometric two-photon lysosometargeted probe for real-time monitoring of pH changes in living cells. J Mater Chem B
22
ACCEPTED MANUSCRIPT 2017; 5: 4768-73. [40]
Liu XJ, Su YA, Tian HH, Yang L, Zhang HY, Song XZ, et al. Ratiometric fluorescent probe for lysosomal pH measurement and imaging in living cells using single-wavelength
RI PT
excitation. Anal. Chem. 2017; 89: 7038-45. [41] Shi BJ, Gao YL, Liu CX, Feng W, Li ZX, Wei LH, et al. A ratiometric fluorescent sensor for pH fluctuation and its application in living cells with low dark toxicity. Dyes Pigments
SC
2017; 136: 522-8.
M AN U
[42] Chen SY, Zhao M, Su J, Zhang Q, Tian XH, Li SL, et al. Two novel two-photon excited fluorescent pH probes based on the A-π-D-π-A system for intracellular pH mapping. Dyes Pigments 2017; 136: 807-16. [43]
Zhu Q, Li Z, Mu L, Zeng X, Redshaw C, Wei G. A quinoline-based fluorometric and
TE D
colorimetric dual-modal pH probe and its application in bioimaging. Spectrochim Acta A Part A, 2018; 188: 230-6. [44]
Lirag RC, Le HTM, Miljanic OS. L-shaped benzimidazole fluorophores: synthesis,
[45]
AC C
4304-6.
EP
characterization and optical response to bases, acids and anions. Chem Commun 2013; 49:
Kim HJ, Heo CH, Kim HM. Benzimidazole-based ratiometric two-photon fluorescent
probes for acidic pH in live cells and tissues. J Am Chem Soc 2013; 135: 17969-77.
[46]
Zhu X, Lin Q, Zhang Y-M, Wei T-B. Nitrophenylfuran-benzimidazole-based reversible
alkaline fluorescence switch accurately controlled by pH. Sens Actuators B 2015; 219: 8-42. [47]
Zhu X, Lin Q, Chen P, Fu Y-P, Zhang Y-M, Wei T-B. A novel pH sensor which could
23
ACCEPTED MANUSCRIPT respond to multi-scale pH changes via different fluorescence emissions. New J Chem 2016; 40: 4562-5. [48]
Li Z-S, Li L-J, Sun T-T, Liu L-M, Xie Z-G. Benzimidazole-BODIPY as optical and
[49]
RI PT
fluorometric pH sensor. Dyes Pigments 2016; 128:165-9. Horak E, Hranjec M, Vianello R, Steinberg IM. Reversible pH switchable aggregation-
solution. Dyes Pigments 2017; 142: 108-15.
Xiong J-F, Li J-X, Mo G-Z, Huo J-P, Liu J-Y, Chen X-Y, et al. Benzimidazole derivatives:
M AN U
[50]
SC
induced emission of self-assembled benzimidazole-based acrylonitrile dye in aqueous
Selective fluorescent chemosensors for the picogram detection of picric acid. J Org Chem 2014; 79: 11619-30.
[51] Wu Y-C, Huo J-P, Cao L, Ding S, Wang L-Y, Cao DR, et al. Design and application of
TE D
tri-benzimidazolyl star-shape molecules as fluorescent chemosensors for the fast-response detection of fluoride ion. Sens Actuators B 2016; 237: 865-75. [52] Wu Y-C, You J-Y, Jiang K, Xie J-C, Li S-L, Cao DR, et al. Colorimetric and ratiometric
EP
fluorescent sensor for F- based on benzimidazole-naphthalene conjugate: Reversible and
AC C
reusable study & design of logic gate function. Dyes Pigments 2017; 140: 47-55. [53] Wu Y-C, Luo S-H, Cao L, Jiang K, Wang L-Y, Xie J-C, et al. Self-assembled structures of N-alkylated bisbenzimidazolyl naphthalene in aqueous media for highly sensitive detection of picric acid. Anal Chim Acta 2017; 976: 74-83.
[54]
Beneto AJ, Thiagarajan V, Siva A. A tunable ratiometric pH sensor based on phenanthro[9,10-d]imidazole covalently linked with vinylpyridine. RSC Adv 2015; 5: 67849-52.
24
ACCEPTED MANUSCRIPT [55] Wang LY, Yang LL, Cao DR. Probes based on diketopyrrolopyrrole and anthracenone conjugates with aggregation-induced emission characteristics for pH and BSA sensing. Sens Actuators B 2015; 221: 155-66. Niu W-F, Fan L, Nan M, Li Z-B, Lu D-T, Wong M-S, et al. Ratiometric emission
RI PT
[56]
fluorescent pH probe for imaging of living cells in extreme acidity. Anal Chem 2015; 87: 2788-93.
Niu W-F, Fan L, Nan M, Wong M-S, Shuang S-M, Dong C. A novel fluorescent probe for
SC
[57]
[58]
M AN U
sensing and imaging extreme acidity. Sens Actuators B 2016; 234: 534-40. Zhao X-X, Chen X-P, Shen S-L, Li D-P, Zhou S, Zhou Z-Q, et al. A novel pH probe based on a rhodamine-rhodamine platform. RSC Adv 2014; 4: 50318-24. [59]
Hua JL, Wu F, Feng S, Xu JH, Xu ZH, Chen YQ, et al. A convenient ratiomeric pH probe
194-202.
TE D
and its application for monitoring pH change in living cells. Sens Actuators B 2014; 196:
[60] Cheng JH, Gou F, Zhang XH, Shen GY, Zhou XG, Xiang HF. A class of multiresponsive
EP
colorimetric and fluorescent pH probes via three different reaction mechanisms of salen
AC C
complexes: A selective and accurate pH measurement. Inorg Chem 2016; 55: 9221-9.
25
ACCEPTED MANUSCRIPT
1. Three novel ratiometric fluorescent probes for acidic pH are developed. 2. The enhanced ICT mechanism is confirmed by 1H NMR and DFT calculations studies.
RI PT
3. There is no significant interference caused by common metal ions to these pH probes.
4. These probes can be used in real-time and reversible pH sensing.
SC
5. There is a potential application of monitoring the pH variation by
AC C
EP
TE D
M AN U
naked-eyes.