A novel benzothiazole-based enaminone as a fluorescent probe for highly selective and sensitive detection of CN⁻

Abstract

A novel benzothiazole derived enaminone BTP as a fluorescent probe for CN⁻ recognition has been developed. In DMSO/H₂O (95/5, v/v, HEPES 10 mM, pH = 7.4) solution, BTP exhibits high selectivity toward CN⁻ among various anions through colorimetric and fluorescence dual channel changes. On treatment with CN⁻, BTP displays a blue shifted fluorescence enhancement and a naked eye observable color change from orange to yellow green. Moreover, the CN⁻ sensing event possesses an excellent anti-interference ability over other anions. Sensing mechanism investigations prove that the CN⁻ recognition process undergoes a typical nucleophilic addition of CN⁻ to the enaminone formed BTP. Simple test paper experiments evidenced the practicability of BTP for CN⁻ detection.

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1. Introduction

Considerable efforts have been devoted to the development of selective optical signaling systems for anionic species due to the crucial roles played by anions in many areas including biology, medicine, catalysis, and environment.^1–6 Among the various important anions, cyanide (CN⁻) is particularly important and has received substantial attention because of its extreme high toxicity to human and aquatic life. For instance, CN⁻ can bind heme cofactors and leads to suppression of the cellular respiration process in mammals.⁷ Therefore, intensive investigations have been devoted to the detection of CN⁻ with the aid of highly selective, sensitive and easy to operate chemical probes, since the response of the probes can be monitored through changes in color and/or fluorescence intensity (or emission wavelength). A number of cyanide sensing strategies based on various mechanisms have been developed, such as hydrogen bonding interactions,^8–11 generation of cyanide complexes,^12–15 nucleophilic addition of CN⁻ to an activated double bond^16–25 or a boron center,^26–28 demetalation of pre-assembled ‘probe-metal ion’ complexes,^29–33 and interaction with quantum dots.^34,35 Among the methods documented, reaction-based receptors have the advantage of high specificity and thus can effectively avoid the interference of other anions including F⁻ and AcO⁻.³⁶

2-(2′-Hydroxyphenyl)benzothiazole (HBT) is a well known fluorophore with the characteristic of excited-state intramolecular proton transfer (ESIPT),³⁷ and has been widely used as a key building block for construction of novel fluorescent probes.^38–41 With these considerations in mind, we designed and synthesized a HBT-derived fluorescent probe BTP (Scheme 1), which is hoped to act as an anion probe since it contains multiple hydrogen bonding donating sites as well as the activated double bond. The results show that probe BTP displayed a high fluorescence selectivity toward CN⁻ in DMSO/H₂O (95/5, v/v, HEPES 10 mM, pH = 7.4) with a significant enhancement of the blue shifted emission. The observed CN⁻ recognition could accomplish within a very short time. Sensing mechanism investigations reveal that CN⁻ undergoes a nucleophilic addition to the probe.

Scheme 1 Synthesis of probe BTP.

2. Experimental

2.1 Materials and instruments

Unless otherwise stated, solvents and reagents were of analytical grade and were used as received. 3-(Benzo[d]thiazol-2-yl)-2-hydroxy-5-methylbenzaldehyde (1) was synthesized following the previous reported method.^{42 1}H NMR and ¹³C NMR spectra were checked on an Agilent 400 MR spectrometer, and the chemical shifts (δ) were expressed in ppm and coupling constants (J) in Hertz. High-resolution mass spectroscopy (HRMS) was carried out on a Bruker micrOTOF-Q mass spectrometer (Bruker Daltonik, Bremen, Germany). Fluorescence measurements were conducted on a Sanco 970-CRT spectrofluorometer (Shanghai, China). The pH measurements were conducted with a Model PHS-25B meter (Shanghai, China). UV-vis absorption spectra were measured on a SP-1900 spectrophotometer (Shanghai Spectrum instruments Co., Ltd., China).

2.2 Synthesis of probe BTP

A mixture of compound 1 (269.32 mg, 1.0 mmol) and 2-aminophenol (2) (109.13 mg, 1.0 mmol) in absolute ethanol (30 mL) was heated to refluxed for 4 h. After cooled to room temperature, the precipitate formed was separated by filtration, which was washed several times with cold ethanol to give receptor BTP as red solid (246 mg, 65%). Mp 170–171 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 16.26 (d, J = 4.8 Hz, 1H), 10.37 (s, 1H), 9.18 (d, J = 4.8 Hz, 1H), 8.44 (s, 1H), 8.12 (d, J = 7.6 Hz, 1H), 8.02 (d, J = 8.4 Hz, 1H), 7.65 (d, J = 8.0 Hz, 1H), 7.54–7.50 (m, 2H), 7.41 (t, J = 7.6 Hz, 1H), 7.21 (t, J = 7.6 Hz, 1H), 7.05 (d, J = 7.6 Hz, 1H), 6.97 (t, J = 7.6 Hz, 1H), 2.37 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ 165.91, 162.58, 159.60, 151.85, 150.55, 136.38, 136.01, 134.79, 130.12, 129.15, 126.48, 125.02, 124.79, 122.48, 122.36, 122.32, 120.30, 119.04, 118.38, 116.95, 20.41. HRMS (ESI⁺) m/z calcd for C₂₁H₁₇N₂O₂S [M + H]⁺ 361.1011; found 361.0998.

3. Results and discussion

Probe BTP was readily prepared by simple condensation of compounds 1 and 2 in absolute ethanol as depicted in Scheme 1, and the structure of BTP was fully characterized by ¹H NMR, ¹³C NMR and HRMS analysis (see ESI†). Interestingly, ¹H NMR spectrum data of BTP manifest that it exists exclusively in the enaminone form in DMSO-d₆, owing to the characteristic splitting of NH proton signal and the absence of singlet imine proton signal. The proton signal appeared at 16.26 ppm displays doublet peak with a coupling constant of 4.8 Hz can be ascribed to the NH group, such a downfield chemical shift indicates that the NH proton is involved in intramolecular hydrogen bonding and the enaminone exists in cis-form. Be closely related with the NH signal, the olefinic proton appeared at 9.18 ppm with a coupling constant as 4.8 Hz. The appearance of these characteristic splitting protons and the absence of the singlet imine proton (ca. 8.5–9.5 ppm) strongly support that BTP exists in enaminone form, which may attributed to the combined effects of solvent polarity and the bifurcated intramolecular hydrogen bond formation capability of the NH moiety.^43,44

With compound BTP in hand, we firstly explored the fluorescence response of BTP toward different anions (Fig. 1). Due to the poor solubility of BTP, we selected DMSO/H₂O (95/5, v/v, HEPES 10 mM, pH = 7.4) co-solvent as the working moiety. Upon excitation at 382 nm, solely BTP solution (20 μM) displays a very weak emission band centered at 602 nm, which may attributed to the photo-induced electron transfer (PET) from aminophenol to benzothiazole skeleton, and may also partly due to the ESIPT process (from NH to C=O) at the excited state, these effects combined together suppressed the emission. On addition of CN⁻ (60 equiv.) to BTP solution, a dramatic fluorescence enhancement at 487 nm was observed, and the fluorescence color changed from non-emissive to cyanine blue (Fig. 1, inset). However, upon individual addition of other anions including AcO⁻, NO₂⁻, SO₃²⁻, SCN⁻, HS⁻, F⁻, CO₃²⁻, SO₄²⁻, PO₄³⁻, ClO₄⁻, Br⁻, HPO₄²⁻, H₂PO₄⁻, HSO₃⁻, HCO₃⁻, S₂O₃²⁻, they promoted no significant fluorescence variations. The anion induced UV-vis absorption spectrum variations of BTP was also examined. The intense absorption of BTP at 480 nm was greatly decreased on addition of CN⁻, meanwhile, a new absorption band centered at 426 nm was observed, and the CN⁻ caused color change from orange to yellow green is naked eye observable (Fig. S1, ESI†). This obvious color change suggests that the CN⁻ may involve in a nucleophilic addition to BTP,^45,46 which perturbed the conjugation system of BTP. Whereas, other added anions do not promote such a significant absorption changes. Thus, probe BTP exhibited a high selectivity toward CN⁻ over other anions.

Fig. 1 Fluorescence spectrum changes of BTP (20 μM) in DMSO/H₂O (95/5, v/v, HEPES, 10 mM, pH = 7.4) solution upon respective addition 60.0 equiv. of different anions (λ_ex = 382 nm). Inset: fluorescence color changes of BTP solution with and without CN⁻ under illumination with a portable UV lamp at 365 nm.

Subsequently, the fluorescence titration experiment was conducted to evaluate the sensing behavior of BTP to CN⁻. As depicted in Fig. 2, upon gradual increase in added CN⁻ amount (0 to 60 equiv.) to BTP solution, the emission intensity at 487 nm enhanced gradually and reached a plateau when 60 equiv. of CN⁻ was added. The fluorescence intensity displayed a good linear dependence against CN⁻ concentration (Fig. 2, inset). Job's plot analysis show that the emission intensity reaches a maximum when the mole ratio of CN⁻ appeared at 0.5, advocating a 1 : 1 interaction ratio of BTP with CN⁻ (Fig. S2, ESI†). This interaction stoichiometry was further confirmed by ESI-mass spectrometry analysis (negative mode) (Fig. S3, ESI†). The observed peak at m/z = 418.1226 could be assigned to the species of [BTP + CN⁻ + CH₃OH]⁻ (calcd m/z = 418.1231), suggesting the formation of BTP–CN⁻ product. The detection limit of BTP for CN⁻ was then calculated based on LOD = 3σ/ρ (σ is the standard deviation of blank measurement, ρ is the slop between the fluorescence intensity versus CN⁻ concentration)⁴⁷ and was estimated to be 3.94 × 10⁻⁷ M.

Fig. 2 Fluorescence spectrum changes of BTP (20 μM) in DMSO/H₂O (95/5, v/v, HEPES 10 mM, pH = 7.4) solution on incremental addition of CN⁻ (0 to 60.0 equiv.). Inset: linear dependence of fluorescence intensity (λ_em = 487 nm) of BTP against CN⁻ concentration. Fluorescence titration experiments disclosed that utilization of 60 equiv. of CN⁻ is sufficient for the rapid sensing event. Based on this fact, the kinetic of this reaction was subsequently explored with the pseudo-first-order reaction equation I_t = I_max + A × exp(−k × t).^48–50 In which, I_t represents the fluorescence intensity (487 nm) of the test solution at time t, I_max represents the maximum fluorescence intensity of the test solution. Time-dependent fluorescence changes of BTP (20 μM) in the presence of 60 equiv. of CN⁻ was monitored and the results were shown in Fig. 3. The results indicate that the CN⁻ sensing event is very quick and can accomplish within 10 seconds. The pseudo-first-order rate constant k was calculated to be 1.296 s⁻¹, indicating the remarkable high reactivity of BTP with CN⁻.

Fig. 3 Fluorescence intensity changes (λ_em = 487 nm) of BTP (20 μM) in the presence of CN⁻ (60 equiv.) as a function of time.

To further confirm the high selectivity and the potential applicability of probe BTP for CN⁻ detection, the competition experiments were then conducted (Fig. 4). When other anion containing BTP solution was further treated with CN⁻, similar fluorescence enhancements at 487 nm were observed, demonstrating that the potential competitive anions elicit no significant interference on CN⁻ detection. These results indicate that BTP can act as a good probe for CN⁻ over other competing anions in the given solution system.

Fig. 4 Changes in fluorescence intensity (at 487 nm) of BTP (20 μM) in DMSO/H₂O (95/5, v/v, HEPES, 10 mM, pH = 7.4) in the individual presence of various anions (60.0 equiv.) (red bar) and on subsequent addition of the same amount of CN⁻ (gray bar).

To further verify the practicability of BTP for CN⁻ detection, pH effect on fluorescence changes of BTP (at 487 nm) with and without CN⁻ was then explored (Fig. 5). Solely BTP solution exhibits weak fluorescence emission within pH range from 4 to 12. In the presence of 60 equiv. of CN⁻, the solution displays strong fluorescence emission from pH 6 to 10 (Caution! pH effects evaluation experiments in the presence of CN⁻ should be carried out in the hood due to the formation of HCN at low pH value), suggesting that probe BTP is suitable for CN⁻ detection in a wide pH range, especially at near neutral pH conditions. Moreover, the stabilities of BTP and its CN⁻ adduct in the working solvent were also examined (Fig. S4†). The results demonstrate that BTP and its CN⁻ adduct are stable enough in DMSO/H₂O (95/5, v/v, HEPES, 10 mM, pH = 7.4) solution due to the almost negligible variation of emission intensity within 30 min, which further advocates the potential applicability of BTP for CN⁻ detection.

Fig. 5 The fluorescence intensity changes (at 487 nm) of BTP and BTP–CN⁻ solution at various pH conditions. λ_ex = 382 nm.

Previous UV-vis studies showed that addition of CN⁻ to BTP solution resulted in a distinct color change from orange to yellow green (Fig. S1†), indicating that the conjugation system of BTP might be interrupted by the nucleophilic addition reaction of CN⁻ to BTP.^8,46,51 Thus, a plausible CN⁻ sensing mechanism was proposed and illustrated in Scheme 2. To further verify this hypothesis, ¹H NMR spectrum of BTP in the absence and presence of CN⁻ was compared (Fig. 6). On addition of CN⁻ to BTP in DMSO-d₆, the typical peaks of BTP at 16.26 (Ha), 10.37 (Hc) and 9.18 ppm (Hb) (Fig. 6a) were all disappeared (Fig. 6b), suggesting that the addition reaction of CN⁻ to BTP take place at the β-olefinic carbon atom^52,53 as shown in Scheme 2. Concomitantly, CN⁻ addition also led to all the aromatic protons shifted to up-field, indicating that the nucleophilic addition resulted negative charge might be delocalized through the whole product molecule. Meanwhile, two doublet peaks with identical coupling constant (10.4 Hz) were observed at 6.07 and 5.50 ppm, respectively. The doublet peak at 6.07 ppm disappeared and the doublet peak at 5.50 ppm changed to a singlet peak after addition of a few drops of D₂O (Fig. 6c), revealing that the former is the NH (Ha′) signal and the latter is the CN–CH (Hb′) signal.^18,54 These results as well as the HRMS analysis could unambiguously prove the supposed nucleophilic addition of CN⁻ to enaminone BTP as depicted in Scheme 2. To the best of our knowledge, exploit nucleophilic addition of enaminone as the fluorescent probe sensing mechanism has not been documented.

Scheme 2 Proposed sensing mechanism of probe BTP for CN⁻.

Fig. 6 Partial ¹H NMR spectrum of BTP (a), BTP + CN⁻ (60 equiv.) (b) in DMSO-d₆, and BTP + CN⁻ (60 equiv.) (c) in DMSO-d₆/D₂O (95 : 5, v/v).

To prove the potential applicability of BTP for CN⁻ detection, test paper experiments were then conducted. The filter papers were immersed into the CH₃CN solution of BTP (1 mM) and then dried in air. When the filter papers coated with BTP were immersed into the aqueous solutions of CN⁻ with different concentrations, the obvious emission color changes from dark-pink to blue-green was observed under illumination at 365 nm with a portable UV lamp (Fig. 7). These results manifest that BTP is practicable for simple CN⁻ detection.

Fig. 7 Photographs of test strips of BTP to various concentration of CN⁻ (×10⁻⁶ M): (A) 0, (B) 1, (C) 5, (D) 10, (E) 30, (F) 50.

4. Conclusions

In conclusion, we have developed a novel benzothiazole derived enaminone-based fluorescent probe BTP for CN⁻ detection based on nucleophilic addition mechanism. BTP exhibits high selectivity and sensitivity to CN⁻ over other anions via colorimetric and ‘off–on’ fluorescence dual channel variations. The CN⁻ detection mechanism was proved to undergo a nucleophilic addition of CN⁻ to the β-olefinic carbon atom of enaminone BTP. In addition, the CN⁻ detection event also has some advantages including rapid response, excellent anti-interference ability, and potential applicability.

Acknowledgements

The project was supported by the National Natural Science Foundation of China (No. 21476029, 21176029), Liaoning BaiQianWan Talents Program (No. 2012921057), and the Program for Liaoning Excellent Talents in University (No. LR2015001).

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Footnote

† Electronic supplementary information (ESI) available: ¹H NMR, ¹³C NMR and HRMS of probe BTP. See DOI: 10.1039/c6ra07909a

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