Fluorescent chemodosimeter based on spirobenzopyran for organophosphorus nerve agent mimics (DCP)

Abstract

A new chromogenic as well as fluorogenic protocol based on the spirobenzopyran system for the selective detection of nerve agent mimics (diethyl chlorophosphate or DCP vapour) within a few seconds (∼30 s) is designed, synthesized and characterized in this study. The nucleophilic attack from the oxygen atom of the spiro ring on the electrophilic phosphonyl group of DCP (diethyl chlorophosphate) causes the opening of the spiro (SP) framework and ultimately gives rise to the meta stable merocyanine (MC) form to give a fluorescent species, which gives a signal in the red region (∼675 nm). The ‘turn-on’ red fluorescence and a colorimetric change from colourless to yellow was observed upon the addition of DCP, which evokes almost 124 and 84 fold enhancement in the absorbance and emission intensity, respectively, compare to the probe itself. To the best of our knowledge, such a DCP sensor based on the spirobenzopyran network has not been reported to date. Moreover, the detection limit of this probe was found to be in 10⁻⁸ M level in the solution phase. We also developed it as a portable chemosensor kit for DCP and demonstrated its practical application in real-time monitoring.

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Introduction

From an international point of view, the present increase of criminal terrorist attacks via chemical warfare (CW) agents has caused an increased risk of exposure to dangerous chemicals. Among all these CW species, nerve agents are extremely hazardous due to their extremely high poisonous effects and their simplicity of fabrication. This emphasizes the need to recognize these lethal chemicals through rapid and consistent procedures.

The high simplicity of production combined with the dangerous poisonous effects of these organophosphorus (OP)-containing nerve agents, including sarin, soman, tabun and cyclosarin, which are inhibitors of serine proteases, especially acetylcholinesterase, underscores the need to detect these odorless and colorless chemicals. These highly toxic compounds bind with the hydroxyl groups and can inhibit acetylcholinesterase (AChE), a critical central-nervous enzyme,^1–4 thus making it non-functional. Many detection methods for nerve agents have been developed based on potentiometry,⁵ surface acoustic wave spectroscopy,⁶ gas chromatography/mass spectrometry⁷ and interferometry.⁸ However, these methods suffer from limitations, such as slow response, lack of specificity, limited selectivity, low sensitivity, operational complexity, non-portability, difficulties in real-time monitoring, or false positive readings.⁹ Therefore, the discovery of simple and efficient methods for the detection of nerve agents remain of high interest.¹⁰ In addition, spirobenzopyran moieties are widely used as molecular optical switches as well as memories.^11,12 They have been classified as a group of photochromes because under UV irradiation they are converted into their merocyanine form and revert back to their original spiro-network in day-light.¹³ These spirobenzopyran rings exhibit a variety of advantages, including fast response, good quantum yield and naked eye detection, with respect to their use as a promising fluorescent sensor. The main importance of such a platform is to exploit the reversibility of (merocyanine) MC and spiro platforms, which ultimately change the color of the solution as well as turn-on fluorescence. In recent years, a number of sensors that take advantage of the abovementioned properties and technologies that employ spirobenzopyran as a photo-switchable bright sensor have been reported; however, these sensors are mostly based on sensing metal ions.¹⁴ Despite these elegant studies, there is significant room for improvement. Therefore, it is necessary to develop a fluorescence probe that can be used for the detection of nerve agent mimics, preferably with ‘turn on’ emission located in the red region. Herein, an ICT based strategy has been implemented with the combination of a spirobenzopyran and a naphthalene system to detect a toxic chemical agent (DCP) even in the presence of nontoxic compounds. The sensing of an organophosphorus compound (OP) with a spirobenzopyran moiety has not been reported to date. We envisioned here that the oxygen atom of the spiro ring could be readily used as a good nucleophile and can undergo a nucleophilic attack with OP nerve agents. The handle can react with electrophilic OP nerve agents within a few seconds (within 30 s). This actually opens up the spiro framework and gives rise to a signal in the red region (∼675 nm). Furthermore, the probe exhibits a noticeable color change under UV light and is even observable by the naked eye. Finally, it can simultaneously detect both liquid and gas nerve agents (DCP) (Fig. 1).

Fig. 1 Structure of chemical warfare nerve agents and simulants.

Results and discussion

The synthetic route of SBN is shown in Scheme 1. The compound R1 was prepared using the procedure given in literature.¹⁵ Compounds R1 and R2 were treated together in dry methanol to afford the desired probe (SBN). The structure of SBN was confirmed by ¹H NMR, ¹³C NMR and HRMS spectroscopy (ESI, Fig. S6–S10†).

Scheme 1 Synthesis of the probe (SBN).

Photo-physical studies

First, the reactivity of SBN was tested with an organophosphorus compound, diethyl chlorophosphate (DCP), and it responded promptly. The objective of the preparation of such a type of an ICT induced benzopyran–naphthalene based probe was to get a beautiful spectrum both in emission and absorbance, which was due to the change in the photophysical properties upon interaction with DCP. These organophosphates have been widely used as simulants because they mimic the reactivity of the well-known nerve agents (such as tabun, sarin and soman), but have much less toxicity. The chemodosimetric approach of SBN towards DCP was observed in both the liquid and vapour states. The charge-transfer was due to the presence of an electron donor oxygen atom, which attacks the electron acceptor phosphonyl group. The opening of the spiro ring changes the color of the solution from colorless to yellow, which is noticeable by the naked eye. It was observed that our probe, SBN, exhibited high selectivity towards only DCP among other tested analytes (including ^tBuOOH, CH₃COCl, H₂O₂, NaOCl and some interfering metal ions such as Na⁺, K⁺, Ca²⁺, Cr³⁺, Ni²⁺, Cu²⁺, Zn²⁺, Cd²⁺, Hg²⁺, Pb²⁺ and nerve agent mimics like DCNP) both colorimetrically as well as fluorometrically.

UV-vis study

A solution of SBN (1 × 10⁻⁵ M, 25 °C in CH₃CN/H₂O, 1/1) and different analytes (1 × 10⁻⁴ M, 25 °C) were prepared in CH₃CN and added separately to the prepared solution of SBN to better understand the change in absorbance. Only DCP was found to perturb the spectral behavior of SBN, which showed an enhancement of absorption.

In contrast, no other analytes of interest showed any significant effect on the absorption profile of SBN (ESI, Fig. S2†). SBN itself showed two λ_max at 295 nm and 355 nm and no peak at 440 nm, which indicates that the probe exists predominantly in its spiro form and is stable in the CH₃CN–H₂O solution. Upon the addition of DCP, SBN showed a huge change in its spectroscopic behavior; moreover, a new peak was generated at 440 nm and it gradually increased with increasing concentration of DCP, as shown in Fig. 2. Upon the incremental addition of DCP, the absorbance intensity of SBN at 440 nm increased almost 124 times; consequently, the color of the solution changed from colorless to yellow. This observation indicated the opening of the spiro network and formation of the merocyanine (MC) system (Scheme 2). During the addition of DCP, the absorbance intensity exhibited a linear curve of fitness relationship with DCP concentration (0 to 1.12 × 10⁻⁵ M) with a good R² value of 0.9964 (ESI, Fig. S4†).

Fig. 2 Change of absorption spectra of SBN (10 μM) upon the gradual addition of DCP (0 to 3 equivalents). Inset: an image of SBN (10 μM), showing a visible color in the absence and presence of 2 equivalents of DCP.

Scheme 2 Probable sensing scheme of SBN to DCP.

The identity of the reaction product generated in situ in the reaction assay was confirmed through a ¹H NMR titration experiment, wherein free –OH gives a peak nearly at δ 13 ppm (ESI, Fig. S10 and 11†). This observation indicates that the SBN–DCP complex may hydrolyze in assay conditions to give the hydrolysis product. The formation of the hydrolysed product was further confirmed through HRMS, wherein the peak at 488.2339 strongly indicates the formation of the hydrolysed product (Fig. S13 and 14, ESI†). The chemodosimetric approach is depicted in Scheme 3.

Scheme 3 Chemodosimetric approach of SBN towards DCP.

Competition study

Selectivity and interference are two very important parameters that are used to evaluate the performance of a receptor. To utilize SBN (1 × 10⁻⁵ M, 25 °C in CH₃CN/H₂O, 1/1) as a selective sensor for the DCP, a competing experiment was also performed by adding DCP (2.0 equiv.) in the presence of 5.0 equivalents of different analytes. The studies revealed that DCP can be detected in the presence of almost all the interfering analytes (Fig. 3).

Fig. 3 A comparative study of emission intensity after the addition of different analytes (5 equivalents) in the solution of SBN in the presence of DCP (2 equivalents).

Emission study

A clear readout was obtained when the sensing experiment was studied by fluorescence titration in an acetonitrile–H₂O solution. The fluorescence spectrum of SBN in aqueous acetonitrile was recorded upon excitation at 460 nm. SBN (1 × 10⁻⁵ M, 25 °C in CH₃CN/H₂O, 1/1) itself showed a weak emission peak at 675 nm. As expected, after the addition of DCP (0–1.2 × 10⁻⁵ M) there was an enormous change in the emission profile. A large enhancement of the emission intensity at 675 nm was noticed, which was actually accompanied by the opening of the spiro network and formation of the merocyanine form (Fig. 4). This observation indicates that after the addition of DCP, the nucleophilic attack from the oxygen atom towards the phosphonyl group of DCP concomitantly opens the spiro ring (Scheme 3). This mechanism may play a part in the opening of the spirobenzopyran ring of SBN to achieve the formation of the meta-stable merocyanine form, which is accompanied by intramolecular charge transfer (ICT).

Fig. 4 Change of emission spectra of SBN (10 μM) upon the gradual addition of DCP. Inset: an image of SBN (6 μM), showing a visible color in the absence and presence of 2 equivalents of DCP.

The fluorogenic response of SBN was also studied using the abovementioned guest analytes. Almost all the guest analytes are mute towards SBN. Only DCP was found to perturb the fluorescence profile remarkably. The addition of other examined analytes even in excess amounts leads to no significant changes in the emission spectrum of the receptor. This experiment indicates that this probe is exclusive in detecting DCP.

The detection limit of SBN for DCP was determined from the emission spectral data, using the equation DL = K × S_b1/S, where K = 3, S_b1 is the standard deviation of the blank solution and S is the slope of the calibration curve.¹⁶ From the graph, the detection limit was found to be 2.1 × 10⁻⁸ M, indicating that SBN has a very good efficiency to detect DCP (ESI, Fig. S3†).

The fluorescence quantum yield of the probe was calculated. The quantum yields of SBN and hydrolyzed SBN–DCP were found to be 0.015 and 0.36, respectively, using rhodamine-B as reference (Φ = 0.66 in ethanol).

Reaction kinetics study

In the case of chemodosimetric reactions, a reaction kinetics study is very important. In this case also we have calculated the rate constant of the reaction. The fluorescence spectra of SBN after addition of 2 equivalents of DCP were recorded, and are depicted below. It was observed that after 30 seconds, the increment almost reached its highest peak. Though the total experiment was recorded for up to 2 minutes, the intensity (675 nm) vs. time plot shows a plateau at almost 30 s. This indicates that SBN is a promising receptor for DCP, which not only detects it but also responds to it very promptly (Fig. 5).

Fig. 5 Change of emission spectra of SBN (10 μM) after the addition of DCP (2 equivalents) with time interval.

Pseudo first order rate constant was calculated from the changes of emission curve of SBN (10 μM) at different time intervals by the addition of DCP (20 μM). From the time vs. emission plot (ESI, Fig. S1†) at a fixed wavelength of 675 nm using first order rate equation, we get the rate constant: k = slope × 2.303 = 0.55 × 10⁻² s⁻¹.

pH studies

In order to investigate the sensitivity of the probe towards change in pH, a pH titration experiment is extremely important. It has been observed that an acid–base titration experiment with the probe does not undergo any significant fluorescence enhancement within the pH 6–14 range; moreover, this experiment suggests that the sensor predominantly exists in the spirobenzopyran form in this pH range. In strong acidic conditions (pH < 4), the probe exhibits a different nature; however, with decreasing pH, protonation causes the opening of the spiro form and conversion to the merocyanine form, which can be observed by the naked eye as well as strong red fluorescence was observed. From the abovementioned experiment, it can be concluded that SBN can be employed for the detection of DCP in near-neutral pH range (pH = 7.2) (Fig. 6).

Fig. 6 Fluorescence response of SBN at 675 nm (10 μM, λ_ex = 460 nm) as a function of pH in CH₃CN/H₂O (1 : 1, v/v), pH is adjusted using aqueous solutions of 1 M HCl or 1 M NaOH.

Test kit

Motivated by the favourable in situ and rapid sensing response of SBN towards DCP, we took a step towards the potential application by using it as a portable kit for sensing DCP. In order to perform this application in an innovative way, it is preferential to detect the toxic DCP in the gas phase. It is a very simple but very important experiment because it gives instant qualitative information without resorting to instrumental analysis. In order to perform this experiment, we prepared TLC plates, which were immersed in the solution of SBN (50 μM) in acetonitrile, and then evaporated the solvent to dryness. Herein, the SBN probe exhibits a visible as well as a fluorometric color change in the presence of DCP in its gaseous form; this can be achieved by bubbling a larger volume of contaminated air into the dried TLC plate. This color change is depicted in Fig. 7. From this experiment it can be concluded that the probe offers a good nerve agent assay kit.

Fig. 7 Images of TLC plates after immersion in a SBN–acetonitrile solution (a & c) and after exposure to DCP vapour for 2 minutes (b & d) taken in ambient light (left) and under hand-held UV light (right).

Conclusions

In summary, we have reported for the first time, the fluorescence sensing of DCP (a nerve agent mimic) based on a spirobenzopyran–naphthalene system. The probe is a highly selective, rapid and sensitive chemodosimeter for DCP. It can detect DCP within 30 seconds. In our approach, we take advantage of the nucleophilic attack from the oxygen atom to the electrophilic phosphonyl group that leads to the opening of the spirobenzopyran ring thereby displaying a yellow color. Other guest analytes, such as metal cations and peroxides, do not interfere in the DCP detection. The detection limit was found to be of the 10⁻⁸ M level, which indicates that our probe, SBN, is a highly efficient sensor of DCP in acetonitrile. To demonstrate the possibility of its practical applications, a dip-stick method was developed, which can detect DCP in a closed vessel in its gaseous phase.

Experimental

General

Unless otherwise mentioned, materials were obtained from commercial suppliers and were used without further purification. Thin layer chromatography (TLC) was carried out using the Merck 60 F₂₅₄ plates with a thickness of 0.25 mm. Melting points were determined on a hot-plate melting point apparatus in an open mouth capillary and are uncorrected. ¹H and ¹³C NMR spectra of SBN were recorded on a JEOL 400 the MHz and 125 MHz instruments, respectively. For NMR spectra, CDCl₃ was used as a solvent and TMS as an internal standard. Chemical shifts are expressed in δ units and ¹H–¹H and ¹H–C coupling constants in Hz. Fluorescence spectra were recorded on a Perkin Elmer LS 55 spectrophotometer and UV-vis titration experiments were performed on a JASCO V-630 spectrophotometer.

General method of UV-vis and fluorescence titration

UV-vis method. For UV-vis titration we used the host solution in the order of 1 × 10⁻⁵ M. The solution was prepared in CH₃CN/H₂O (1/1, v/v). The guest analyte solutions in the order of 1 × 10⁻⁴ M were prepared in acetonitrile. Different concentrations of the host solution and increasing concentrations of analytes were prepared separately. The spectra of these solutions were recorded by means of the UV-vis method.

Fluorescence method. The receptor solution for the fluorescence titration was prepared (1 × 10⁻⁵ M) in CH₃CN/H₂O (1/1, v/v). The guest analyte solutions in the order of 1 × 10⁻⁴ M were prepared in CH₃CN. Herein, also various concentrations of the guest analyte and increasing concentration of the host solution were prepared and the spectra were recorded by means of a fluorescence method.

Synthesis of the probe (SBN)

A mixture of R1 (0.5 g, 1.56 mmol) and R2 (0.45 g, 1.57 mmol) was dissolved in 20 mL of dry ethanol. The resulting solution was stirred for 12 h at room temperature. The solvent was evaporated under reduced pressure to get the crude product, which was purified through silica gel (100–200, mesh size) column chromatography using 10–15% ethyl acetate in petroleum ether as eluent. The final product (SBN) was obtained as an off-white solid. The solid obtained was dissolved in a minimum volume of dichloromethane solution and re-precipitated using pentane. The precipitate was filtered and dried under vacuum, which afforded a better purified white colored compound (yield = 62%).

M_p = 276–278 °C

¹H NMR (400 MHz, CDCl₃). δ 1.12 (s, 3H), 1.24 (s, 3H), 2.26 (s, 3H), 2.68 (s, 3H), 5.66 (d, J = 8 Hz, 1H), 6.34 (d, J = 8 Hz, 1H), 6.67 (t, J = 6 Hz, 1H), 6.80 (d, J = 8 Hz, 1H), 6.93 (t, J = 8 Hz, 2H), 7.01 (t, J = 8 Hz, 1H), 7.40 (t, J = 6 Hz, 1H), 7.48 (m, 2H), 7.62 (d, J = 8 Hz, 1H), 7.83 (m, 3H), 8.08 (s, 1H), 8.31 (t, J = 8 Hz, 1H), 9.25 (s, 1H).

¹³C NMR (125 MHz, CDCl₃). δ 20.4, 20.6, 26.2, 28.9, 52.0, 104.9, 106.7, 118.9, 119.1, 119.2, 121.7, 124.6, 125.7, 126.6, 127.2, 127.4, 127.6, 128.3, 129.4, 129.8, 130.0, 130.6, 131.3, 132.2, 133.8, 136.8, 142.5, 148.1, 151.2, 165.5.

HRMS (ESI, positive). Calcd for C₃₂H₂₉N₃O₂Na [M + Na]⁺ (m/z): 510.2157; found: 510.1941.

Acknowledgements

The authors thank CSIR and DST, Govt. of India, for the financial support. S.D. and K.A. acknowledge CSIR for providing their fellowship.

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Footnote

† Electronic supplementary information (ESI) available: NMR, HRMS of the probe and reaction product. See DOI: 10.1039/c5ra01216c

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