An acidic pH fluorescent probe based on Tröger's base

A novel pH fluorescent probe 2,8-(6H,12H-5,11-methanodibenzo[b,f]diazocineylene)-di(p-ethenylpyridine) (TBPP) incorporating an electron-donating amine moiety and electron-accepting pyridine group through Tröger's base linker was designed and synthesized. TBPP exhibits an intramolecular charge transfer effect caused by the donor–acceptor interaction between its amine and pyridine units. Its emission can be reversibly switched between blue and dark states by protonation and deprotonation. Such behavior enables it to work as a turn-off fluorescent pH sensor in solution state. H NMR spectroscopy analysis suggests that the change in electron affinity of the pyridinyl unit upon protonation and deprotonation is responsible for such sensing processes.


Introduction
pH is a key parameter in a wide range of elds such as environmental analysis, chemical process control, food production, medical diagnosis and life science. [1][2][3][4][5][6] Many important physiological processes of cells and organelles are also related to pH values. 7,8 Consequently, the measurement of pH is of great importance in the eld of environmental, chemical, medical and life sciences. Many techniques have been used for measuring pH value including UV-vis absorbance spectroscopy, 9,10 uorescence spectroscopy, 11,12 nuclear magnetic resonance 13,14 and electrochemistry. 15,16 Among these methods, uorescent probes for pH detection have attracted much more attention due to their convenient operation, particularly high sensitivity, non-invasiveness and real-time detection. 17 Towards this end, many kinds of pH uorescent probes have been explored including uoresceins, 18,19 coumarins 20,21 and rhodamines 22,23 etc. However, most reported uorescence pH probes are practical for near-neutral pH range while there are limited probes for monitoring pH changes in acidic organelles. And some pH probes still suffer from the severe excitation interference caused by a shorter Stokes shi. There is a great pressing need to develop uorescent probes toward high sensitivity and large Stokes shi for monitoring acid physiological pH uctuations.
Tröger's base (TB), 24 rst synthesized in 1887, has gained steady interest in recent years because of its C2 symmetry, chirality, and rigid concave shape. 25,26 Our recent work indicated pyridinium salts based on TB display a special aggregationinduced emission (AIE) property that it is non-emissive in solution but exhibit strong uorescence in solid state. 27,28 In addition, N-heterocyclic derivatives are ideal substances for use as hydronium ion indicators owing to the high sensitivity and the exceptionally rapid rates of proto transfer occurring in acidbase equilibrium process. 11,29,30 With this in mind, we designed and synthesized a new compound TBPP based on TB by introducing the pyridine electron-withdrawing group into TB framework. We expect that optical change could be achieved through the transformation between the neutral TBPP and non-uorescence ionic pyridinium salt by protonation. Proton titration experiments indicated that TBPP can be used as a turnoff pH uorescent probe for acidic pH detection with high sensitivity.

Materials and instrumentation
All commercially available chemicals were purchased from Adamas, Aldrich and TCI, and used as received without further purication except N-methyl pyrrolidone was dried over 3Å molecular sieves. 2,8-Dibromo-6H,12H-5,11-methanodibenzo [b,f] [1,5]diazocine was prepared according to literature procedures. 31 1 H and 13 C NMR spectra were recorded on a Bruker AVANCE 400 spectrometer. The chemical shis are reported in d ppm with reference to residual protons and carbons of CDCl 3 (7.26 ppm in 1 H NMR and 77.16 ppm in 13 C NMR) and DMSO-d 6 (2.50 ppm in 1 H NMR and 39.52 ppm in 13 C NMR). Coupling constants J are given in hertz. Mass spectra was measured with a Bruker Daltonics micrOTOF-focus using the APCI-TOF method in the positive-ion mode in toluene. The UV-visible absorption spectra were measured on a TU-1800 spectrophotometer using a quartz cuvette having 1 cm path length. The photoluminescence spectra were collected on a Hitachi F-4500 uorescence spectrophotometer with a 150 W Xe lamp. Fluorescence quantum yields (F) were determined using quinine sulfate in 0.1 N sulfuric acid (F ¼ 0.577) as standard. Solvents were puried and dried according to standard procedures. Thin layer chromatography (TLC) was performed on glass plates coated with 0.20 mm thickness of silica gel. Column chromatography was performed using neutral silica gel PSQ100B.

General procedure for spectroscopic measurements
Stock solution of probe with a concentration of 1.0 mM was prepared in DMSO and the solution for spectroscopic determination was obtained by diluting the stock solution to 10.0 mM in DMSO. The metal ions were provided by NaCl, KCl, CaCl 2 , In the pH titrations experiments, the slight pH variations of the solutions were achieved by adding the minimum volumes of HCl (1.0 mM). Spectral data were recorded aer each addition. The excitation wavelength was 340 nm. The resulting solution was shaken well and kept at room temperature for 30 min before taking its absorption and uorescence spectra.
To a stirring of 2,8-dibromo-6H,12H-5,11-methanodibenzo[b,f] [1,5]diazocine (5.0 mmol, 1.9 g), 4-vinyl pyridine (20.0 mmol, 2.2 mL) and K 2 CO 3 (20.0 mmol, 2.8 g) in N-methyl pyrrolidone (10 mL) was added tris(2-methylphenyl)phosphine (0.001 mmol, 0.003 g) and palladium acetate (0.001 mmol, 0.0023 g) as catalysts. The mixture was stirred at 130 C for 10 h under N 2 atmosphere and monitored by TLC. Aer the reaction was nished, the reaction mixture was cooled to room temperature and extracted by CH 2 Cl 2 for three times. Combined organic phase was washed with water for three times and dried over anhydrous MgSO 4 . Aer evaporation of the solvent, the mixture was puried by a silica gel column chromatography (eluent: CH 2 Cl 2 /ethanol ¼ 40/1 in the ratio of volume) to afford TBPP (

Synthesis
As outlined in Scheme 1, compound TBPP was synthesized in moderate yield directly by Heck coupling reaction of 4-vinyl [1,5]diazocine in the presence of a base and palladium catalyst.
The product was characterized by 1 H NMR, 13 C NMR and HRMS methods. It is readily soluble in normal solvents such as benzene, CH 2 Cl 2 , CHCl 3 , THF, methanol, ethanol, CH 3 CN, DMF, DMSO, but insoluble in hexane and water.

Photophysical properties of probe TBPP
In our experiment, we found that both of solutions and solids of TBPP show strong blue emissions (the photos were shown in the inset of Fig. 1(a)). The normalized absorption and uorescence Scheme 1 Synthesis of probe TBPP. emission spectra of TBPP in various solvents with different polarity were shown in Fig. 1. The absorption peak wavelength, uorescence peak wavelength, Stokes' shi and uorescence quantum yields were summarized in Table 1. Increase the polarity of solvent (benzene, CH 2 Cl 2 , THF, ethanol, acetonitrile, DMF, DMSO), no obvious spectral shi was observed in the absorption spectra with the absorption peaks locate at the range from 335 to 340 nm while uorescence spectra shown a large red-shi that the uorescence peaks changed from 409 to 489 nm. The Stokes' shi is changed from 74 nm to 149 nm. This can be explained by the fact that the excited state of the compound TBPP may possess higher polarity than that of the ground state. For the solvatochromism is associated with the energy level lowering. Increased dipole-dipole interaction between the solute and solvent leads to lowering the energy level greatly. 32,33 The amine on TB bridge and pyridine group act as electron donor and acceptor, respectively. Each wing of compound TBPP is a D-p-A charge transfer structure. Therefore, the intense uorescence of TBPP in solution and solid state should contributed from the effective intramolecular charge transfer (ICT) through strong push-pull interaction between the electron-donor amine moiety on TB bridge and the electron-acceptor pyridine group.

pH titrations of probe TBPP
To study the optical responses of probe TBPP to pH, pH titrations of absorption spectra and uorescence emission spectra were performed. As shown in Fig. 2(a), along with the H + addition, the original absorption band at 340 nm decreased and a new band around 390 nm increased gradually. A well-dened isosbestic point at 356 nm was observed. A notable color change from colorless to yellow (the inset in Fig. 2(a)) was observed upon increasing the acidity. Therefore, this probe can serve as a "naked-eye" colorimetric indicator for acidic pH.
The uorescence property of TBPP as a function of pH were displayed in Fig. 2(b) and (c). Before the addition of acid, DMSO solution of TBPP exhibited an intense emission band around 490 nm with a large Stokes shi of 149 nm. 29,30,34,35 The large Stokes shi should help to reduce the excitation interference. Upon increasing the amount of H + , the uorescence intensity reduced gradually until quenched completely. The uorescence change was well demonstrated in the uorescence photos which were shown in the inset of Fig. 2(b). It is should be noted that there was no spectral shi was observed during the titration experiments. This means the new species resulted from the protonation of N atoms in pyridine group is non-emissive in solution state. This property of TBPP-H + is similar to our previous pyridinium salts. 27,28 The uorescence color change  was due to the protonation of N atoms in pyridine group. Under strongly acid condition, the protonation of N atoms in pyridine group enhanced their electron withdrawing ability, which resulted in the of ICT effect from pyridine group to amine group. The structures of TBPP, TBPP-H + and the sensing process were depicted in Fig. 3. The UV-vis absorption and corresponding uorescence emission properties during the pH titrations enable probe TBPP to serve as a highly sensitive probe for precisely pH measuring in acidic regions through colorimetric and uorimetric changes.

Proposed mechanism
To study the proton-binding behavior, the 1 H NMR spectroscopy of probe TBPP in DMSO-d 6 with different equivalent hydrochloric acid has been acquired. As illustrated in Fig. 3, with the addition of incremental amounts of HCl to TBPP solution in DMSO-d 6 , the pyridine proton signals (H1 and H2) were gradually shied down-eld because of the transformation of the pyridine ring in TBPP to an electron-decient pyridinium unit in TBPP-H + . The resonances of phenyl and vinyl protons (H3 and H4) also move to lower elds for the same reason. However, the chemical shis of amine protons had almost no change (H8, H9 and H10), which indicated that the protonation occurred at the pyridine N rather than amine N. The downeld chemical shi of these protons was obviously due to H + binding with N atom of pyridine, which resulted in the decrease of electron density around these protons. The presence of acid-base equilibrium between the two forms of TBPP was given in Scheme 2.

Selectivity of probe TBPP
The selectivity response of probe to H + over various metal ions were examined at pH 5.83 and 4.10, respectively. It is noteworthy that TBPP shows high selectivity toward H + . As shown in Fig. 4

Photostability of probe TBPP
The stability of the probe was tested by measuring the uorescent response during 2 h with pH 5.83, 5.12, and 4.10, respectively (Fig. 5). The uorescence intensity was continuously monitored and recorded at set time intervals at 490 nm. The results indicated that the probe can instantly respond to the change of H + concentration, and the probe solution is stable. Thus, the probe can be used to monitor the pH variation in real time.

Conclusion
In summary, we have designed and synthesized a new pH uorescent probe TBPP based on TB by introducing the pyridine electron-withdrawing group into TB framework. The UV-vis absorption and uorescence titrations indicated that TBPP can serve as a highly sensitive probe for pH measuring in acidic regions through colorimetric and uorimetric changes. Furthermore, the detection mechanism has been veried by 1 H NMR spectroscopy. The large Stokes shi (149 nm), high sensitivity, good selectivity, and extremely short response time under extreme acidic condition of the probe makes it an extremely effective pH sensor. We believe it will be great bene-cial to study chemical and biological system.

Conflicts of interest
There are no conicts to declare.