Get 20M+ Full-Text Papers For Less Than $1.50/day. Start a 7-Day Trial for You or Your Team.

Learn More →

Chromo-Fluorogenic Detection of Soman and Its Simulant by Thiourea-Based Rhodamine Probe

Chromo-Fluorogenic Detection of Soman and Its Simulant by Thiourea-Based Rhodamine Probe molecules Article Chromo-Fluorogenic Detection of Soman and Its Simulant by Thiourea-Based Rhodamine Probe 1 , † 1 , 2 , , † 3 4 1 , 2 Shengsong Li , Yongchao Zheng * , Weiqiang Chen , Meiling Zheng , He Zheng , 1 1 , 2 1 , 2 , 1 , 2 , Zhe Zhang , Yan Cui , Jinyi Zhong * and Chonglin Zhao * Research Institute of Chemical Defense, Beijing 102205, China; [email protected] (S.L.); [email protected] (H.Z.); [email protected] (Z.Z.); [email protected] (Y.C.) State Key Laboratory of NBC Protection for Civilian, Beijing 102205, China Institute of Modern Physics, Chinese Academy of Sciences, Lanzhou 730000, China; [email protected] Laboratory of Organic NanoPhotonics and CAS Key Laboratory of Bio-Inspired Materials and Interfacial Science, Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, Beijing 100190, China; [email protected] * Correspondence: [email protected] (Y.Z.); [email protected] (J.Z.); [email protected] (C.Z.); Tel.: +86-10-66758321(C.Z.) † These authors have contributed equally to this work. Academic Editor: Simone Morais Received: 3 February 2019; Accepted: 18 February 2019; Published: 26 February 2019 Abstract: Here, we introduced a novel thiourea-based rhodamine compound as a chromo-fluorogenic indicator of nerve agent Soman and its simulant diethyl chlorophosphate (DCP). The synthesized probe N-(rhodamine B)-lactam-2-(4-cyanophenyl) thiourea (RB-CT), which has a rhodamine core linked by a cyanophenyl thiosemicarbazide group, enabled a rapidly and highly sensitive response to DCP with clear fluorescence and color changes. The detection limit was as low as 2  10 M. The sensing mechanism showed that opening of the spirolactam ring following the phosphorylation of thiosemicarbazides group formed a seven-membered heterocycle adduct, according to MS analysis and TD-DFT calculations. RB-CT exhibited high detecting selectivity for DCP, among other organophosphorus compounds. Moreover, two test kits were employed and successfully used to detect real nerve agent Soman in liquid and gas phase. Keywords: Nerve agents; Soman; Rhodamine B; Thiosemicarbazide; Chromo-fluorogenic probe; Detection 1. Introduction Nerve agents are a class of highly toxic organophosphates, mainly including Tabun (GA), Sarin (GB), Soman (GD), and VX [1] (Scheme 1). These compounds are extremely dangerous because they are able to enter human body through respiration or penetration and inhibit the activity of the acetylcholinesterase, then the accumulation of acetylcholine in the synapse causes neuromuscular paralysis and eventually death [2]. Due to the easy production, high toxicity, being colorless and odorless, and the possible use in terrorist attacks, the development of reliable and rapid detection systems for nerve agents is highly desirable [3]. In addition, rapidly discriminating the distribution of nerve agents in the contaminated areas can also provide effective guidance for subsequent decontamination operations and further safety confirmation. Molecules 2019, 24, 827; doi:10.3390/molecules24050827 www.mdpi.com/journal/molecules Molecules 2019, 24, x 2 of 12 imprinted polymers[12], nanoparticles[13], and chromo-fluorogenic probes[14-22]. Above all, chromo-fluorogenic probes have recently gained increasing interest due to their cost-effectiveness, simplicity, and “naked-eye” detection [23,24]. A typical chromo-fluorogenic probe is usually formed by two moieties: (1) a chromo-fluorogenic reporter group, which translates the binding event into the change of color and fluorescence, mainly containing rhodamine[22], fluorescein[15], boron dipyrromethene (BODIPY) [16,17], azo[14,20], and cyanine dye[18]; (2) a selective reactive group, which provides a reactive binding site for nucleophilic attack, mainly containing hydroxyl[15,25], oxime[19], and amino groups[26]. Recently, thiourea has been proved to be capable of reacting with nerve agents through hydrogen-bond interaction between N-H protons of thiourea and phosphonate oxygen or hydrolyzed products[27,28]. It has been also proven that the reaction of thiosemicarbazide group can induce opening of the spirolactam ring in rhodamine B accompanied with color change and enhanced fluorescence[29]. Thus, the thiosemicarbazide group is expected to become a reactive group bonding with rhodamine B core for chromo-fluorogenic detection of nerve agents and simulants. Herein, a rhodamine B based probe N-(rhodamine B)-lactam-2-(4-cyanophenyl) thiourea (RB- CT) was designed and synthesized, and as shown in Scheme 2, the electron-withdrawing cyanophenyl group in the molecule could promote the intramolecular charge transfer (ICT) process to enhance activity of ring-opening reaction and strengthen the change of color. Spectra analysis and mechanism study confirmed the probe was capable of the rapid detection of diethyl chlorophosphate (DCP, a simulant of the Soman) with remarkable change of color and fluorescence through the Molecules 2019, 24, 827 2 of 12 phosphorylation of the thiosemicarbazide group. Finally, the probe was also successfully used in the detection of GD in both liquid and gas phase. Scheme 1. Chemical structures of nerve-agents Sarin, Soman, Tabun, and VX, their simulant DCP, and Scheme 1. Chemical structures of nerve-agents Sarin, Soman, Tabun, and VX, their simulant DCP, some organophosphorus compounds as potential interferences. and some organophosphorus compounds as potential interferences Several methodologies have been employed for the detection of nerve agents, including enzymatic assays [4,5], interferometry [6], ion mobility spectroscopy [7], electrochemistry [8], micro-cantilevers [9,10], and photonic crystals [11]. Nevertheless, these protocols usually have some drawbacks, such as operation complexity, non-portability, difficulties in real-time monitoring, etc. As alternatives to these procedures, some new approaches have been explored involving molecularly imprinted polymers [12], nanoparticles [13], and chromo-fluorogenic probes [14–22]. Above all, chromo-fluorogenic probes have recently gained increasing interest due to their cost-effectiveness, simplicity, and “naked-eye” detection [23,24]. A typical chromo-fluorogenic probe is usually formed by two moieties: (1) a chromo-fluorogenic reporter group, which translates the binding event into the change of color and fluorescence, mainly containing rhodamine [22], fluorescein [15], boron dipyrromethene (BODIPY) [16,17], azo [14,20], and cyanine dye [18]; (2) a selective reactive group, which provides a reactive binding site for nucleophilic attack, mainly containing hydroxyl [15,25], oxime [19], and amino groups [26]. Recently, thiourea has been proved to be capable of reacting with nerve agents through hydrogen-bond interaction between N-H protons of thiourea and phosphonate oxygen or hydrolyzed products [27,28]. It has been also proven that the reaction of thiosemicarbazide group can induce opening of the spirolactam ring in rhodamine B accompanied with color change and enhanced fluorescence [29]. Thus, the thiosemicarbazide group is expected to become a reactive group bonding with rhodamine B core for chromo-fluorogenic detection of nerve agents and simulants. Herein, a rhodamine B based probe N-(rhodamine B)-lactam-2-(4-cyanophenyl) thiourea (RB-CT) was designed and synthesized, and as shown in Scheme 2, the electron-withdrawing cyanophenyl group in the molecule could promote the intramolecular charge transfer (ICT) process to enhance Molecules 2019, 24, x 5 of 12 TD-DFT calculations were used to confirm the structure of the adduct through optimizing the geometries formed by nucleophilic attack with different nitrogen sites in the thiosemicarbazide group. The proposed mechanism was presented in Scheme 2; a seven-membered heterocycle was proved to be a favorable adduct structure owing to its relatively lower energy, which also agreed Molecules 2019, 24, 827 3 of 12 well with the mass spectrometry result (Figure S5). The optimized structures of RB-CT and RB- CT/DCP adducts were shown in Figure 3. The HOMO–LUMO energy gap (2.651 eV) of the adduct is less than that of RB-CT (3.885 eV) and the electron densities in LUMO are more concentrated in the activity of ring-opening reaction and strengthen the change of color. Spectra analysis and mechanism thiourea group after the reaction. The calculated main contributing electronic transitions for S0→S1 study confirmed the probe was capable of the rapid detection of diethyl chlorophosphate (DCP, a energy state are HOMO→LUMO (2.14 eV/578 nm) and HOMO-1→LUMO+1 (2.34 eV/530 nm), which simulant of the Soman) with remarkable change of color and fluorescence through the phosphorylation is consistent with the absorption band at 560 nm obtained experimentally. The considerable of the thiosemicarbazide group. Finally, the probe was also successfully used in the detection of GD in difference in the energy and electronic transition can shed light on the changes of absorption spectra both liquid and gas phase. and color. CN CN EtO O OEt N NH NH Cl OEt N O N N O N Colorless Pink Fluorescence 'off' Fluorescence 'on' Scheme 2. Proposed mechanism of RB-CT with DCP. Scheme 2. Proposed mechanism of RB-CT with DCP. 2. Results and Discussion 2.1. Spectroscopic Properties To demonstrate the response of RB-CT to nerve agents, absorption and fluorescence spectra were first studied. DCP was used as low-toxic simulant because of its similar chemical structure and reactivity with nerve agents (Tabun, Sarin and Soman). Et N (3 Vt%) was added in the CH CN 3 3 solution of RB-CT to avoid the interference of proton. As shown in Figure 1a, an obvious absorption band at 560 nm was observed after the addition of DCP in the RB-CT solution. The change in color from colorless to pink implied the spirolactam ring-opening reaction of RB-CT caused by DCP (Figure 1b). The thiosemicarbazide group of RB-CT is likely to react with DCP, resulting in a remarkable enhancement of absorption intensity. The result highlights that RB-CT is of high potential as a naked-eye probe for the detection of nerve agents. Figure 1c presented the fluorescence spectra of RB-CT with increasing concentration of DCP; a new fluorescent band at 583 nm appears and shows remarkable enhancement. The spectral changes provided further evidence for the opening of the spirolactam ring, according to the changes in absorption spectra. Moreover, the fluorescence intensity of RB-CT showed a good linear relationship 3 3 at the concentration of DCP in the range of 0.1  10 –1.9  10 M (Figure 1d). The limit of detection (LOD) was determined from the fluorescence spectral data, using the equation K  S /S, where K = 3, S is the standard deviation of blank measurements and S is b1 b1 the slope of the calibration curve. The limit of detection was found to be 2  10 M, indicating high sensitivity to detect DCP by RB-CT. The sensing ability for the naked eye was evaluated by immobilizing RB-CT on silica plates. After the plates were dipped in the CH CN solution of DCP (2–2000 ppm), clear enhancement of the fluorescence was observed by the naked eye in the range of 20 and 2000 ppm (Figure S1), which demonstrates the LOD for the naked eye is as low as the value calculated. Molecules 2019, 24, x 3 of 12 2. Results and Discussion 2.1. Spectroscopic Properties To demonstrate the response of RB-CT to nerve agents, absorption and fluorescence spectra were first studied. DCP was used as low-toxic simulant because of its similar chemical structure and reactivity with nerve agents (Tabun, Sarin and Soman). Et3N (3 Vt%) was added in the CH3CN solution of RB-CT to avoid the interference of proton. As shown in Figure 1a, an obvious absorption band at 560 nm was observed after the addition of DCP in the RB-CT solution. The change in color from colorless to pink implied the spirolactam ring-opening reaction of RB-CT caused by DCP (Figure 1b). The thiosemicarbazide group of RB-CT is likely to react with DCP, resulting in a remarkable enhancement of absorption intensity. The result highlights that RB-CT is of high potential as a naked- eye probe for the detection of nerve agents. Figure 1c presented the fluorescence spectra of RB-CT with increasing concentration of DCP; a new fluorescent band at 583 nm appears and shows remarkable enhancement. The spectral changes provided further evidence for the opening of the spirolactam ring, according to the changes in absorption spectra. Moreover, the fluorescence intensity of RB-CT showed a good linear relationship −3 −3 at the concentration of DCP in the range of 0.1 × 10 –1.9 × 10 M (Figure 1d). The limit of detection (LOD) was determined from the fluorescence spectral data, using the equation K × Sb1/S, where K = 3, Sb1 is the standard deviation of blank measurements and S is the slope −6 of the calibration curve. The limit of detection was found to be 2 × 10 M, indicating high sensitivity to detect DCP by RB-CT. The sensing ability for the naked eye was evaluated by immobilizing RB- CT on silica plates. After the plates were dipped in the CH3CN solution of DCP (2–2000 ppm), clear Molecules 2019, 24, 827 4 of 12 enhancement of the fluorescence was observed by the naked eye in the range of 20 and 2000 ppm (Figure S1), which demonstrates the LOD for the naked eye is as low as the value calculated. 5 4 Figure 1. (a) Absorption spectra of RB-CT (1.0  10 M) upon the addition of DCP (0.5  10 –8.0 10 M) in CH CN (3% Et N). (b) The images of the RB-CT solution before and after the addition of 3 3 DCP under sun light (left) and UV light (right). (c) Fluorescence spectra of RB-CT (1.0  10 M) at 3 3 = 540 nm, upon the addition of DCP (0.1  10 –1.9  10 M) in CH CN (3% Et N). (d) Plot of ex 3 3 emission intensity of RB-CT at 583 nm. 2.2. Reaction Kinetics Study To investigate the reaction kinetics of RB-CT and DCP, time-dependent fluorescence change was performed under different DCP concentrations at room temperature. As shown in Figure 2a, the reaction was almost complete and the fluorescence intensity was saturated within 1200 s in all cases. The kinetic constants were further examined following pseudo-first-order kinetic rate law as the clear linear relationships between the change of fluorescence intensity and reaction time (Figure S2). The observed rate constants k , half-life time t , and the constant rates (k) were summarized in Table 1. obs 1/2 The average half-life time was about 280 s, less than 5 min. The short half-life times are the basis for the rapid detection of nerve agents. Molecules 2019, 24, x 4 of 12 −5 −4 −4 Figure 1. (a) Absorption spectra of RB-CT (1.0 × 10 M) upon the addition of DCP (0.5 × 10 –8.0 × 10 M) in CH3CN (3% Et3N). (b) The images of the RB-CT solution before and after the addition of DCP −6 under sun light (left) and UV light (right). (c) Fluorescence spectra of RB-CT (1.0 × 10 M) at λex = 540 −3 −3 nm, upon the addition of DCP (0.1 × 10 –1.9 × 10 M) in CH3CN (3% Et3N). (d) Plot of emission intensity of RB-CT at 583 nm. 2.2. Reaction Kinetics Study To investigate the reaction kinetics of RB-CT and DCP, time-dependent fluorescence change was performed under different DCP concentrations at room temperature. As shown in Figure 2a, the reaction was almost complete and the fluorescence intensity was saturated within 1200 s in all cases. The kinetic constants were further examined following pseudo-first-order kinetic rate law as the clear linear relationships between the change of fluorescence intensity and reaction time (Figure S2). The observed rate constants kobs, half-life time t1/2, and the constant rates (k) were summarized in Table 1. Molecules 2019, 24, 827 5 of 12 The average half-life time was about 280 s, less than 5 min. The short half-life times are the basis for the rapid detection of nerve agents. −6 Figure 2. (a) Kinetic profiles of the fluorescence intensity at 583 nm of RB-CT ((1.0 × 10 M, 6 CH3CN, Figure 2. (a) Kinetic profiles of the fluorescence intensity at 583 nm of RB-CT ((1.0  10 M, CH CN, −4 −4 −4 −3 3% Et3N) after the addition of 4.0 × 10 M, 5.0 × 10 M, 6.0 × 10 M, and 1.2 × 10 M DCP. (b)The 4 4 4 3 3% Et N) after the addition of 4.0  10 M, 5.0  10 M, 6.0  10 M, and 1.2  10 M DCP. correlation between kobs and the concentration of DCP. (b)The correlation between k and the concentration of DCP. obs Table 1. Observed reaction rates (kobs), half-life time (t1/2), and the constant rates (k) for the reaction of Table 1. Observed reaction rates (k ), half-life time (t ), and the constant rates (k) for the reaction of obs 1/2 6−6 RB-CT RB-CT ((1.0 ((1.0  × 1 10 0 M, M, CH CH 3C CN, N, 3% 3%Et Et 3N) and N) and DCP with different concentrations. DCP with different concentrations. 3 3 The Concentrations The 4 4 4 3 4  10 5  10 6  10 1.2  10 of DCP −4 −4 −4 −3 Concentrations 4 × 10 5 × 10 6 × 10 1.2 × 10 of DCP 0.00244 0.00245 0.00246 0.00251 K (s ) obs t (s) 284 283 282 276 1/2 2 1 −1 6 Kobs (s ) 0.00244 0.00245 0.00246 0.00251 k (M S ) 8.645  10 t1/2 (s) 284 283 282 276 2.3. Mechanism Study −2 −1 −6 k (M S ) 8.645 × 10 The absorption and fluorescence changes can be attributed to the opening of spirolactam ring via nucleophilic reaction between RB-CT and DCP. An ESI analysis was employed to investigate the 2.3. Mechanism Study possible products and a major peak at 708.3223 was observed, which indicated the phosphorylation of RB-CT The ab (Figur sorp e t S3). ion and Theflnew uorescence ch singlet observed anges can b ( e0.32) attrib in uted to the openi the P NMR spectr ng of spi um rola ofcDCP tam ring after addition via nuc of leophilic RB-CT re gives action be further tween RB evidence -CT for and DC the occurr P. An ESI an ence of aly phosphorylation sis was employed reaction to investig (Figur ate e the S4). possible products and a major peak at 708.3223 was observed, which indicated the phosphorylation As the formation of a heterocycle in RB-CT/DCP adduct is a reasonable mechanism [30,31], DFT of RB-CT (Figure S3). The new singlet observed (δ 0.32) in the P NMR spectrum of DCP after and TD-DFT calculations were used to confirm the structure of the adduct through optimizing the addition of RB-CT gives further evidence for the occurrence of phosphorylation reaction (Figure S4). geometries formed by nucleophilic attack with different nitrogen sites in the thiosemicarbazide group. As the formation of a heterocycle in RB-CT/DCP adduct is a reasonable mechanism [30,31], DFT and The proposed mechanism was presented in Scheme 2; a seven-membered heterocycle was proved to be a favorable adduct structure owing to its relatively lower energy, which also agreed well with the mass spectrometry result (Figure S5). The optimized structures of RB-CT and RB-CT/DCP adducts were shown in Figure 3. The HOMO–LUMO energy gap (2.651 eV) of the adduct is less than that of RB-CT (3.885 eV) and the electron densities in LUMO are more concentrated in the thiourea group after the reaction. The calculated main contributing electronic transitions for S !S energy state are 0 1 HOMO!LUMO (2.14 eV/578 nm) and HOMO-1!LUMO+1 (2.34 eV/530 nm), which is consistent with the absorption band at 560 nm obtained experimentally. The considerable difference in the energy and electronic transition can shed light on the changes of absorption spectra and color. Molecules 2019, 24, x 5 of 12 TD-DFT calculations were used to confirm the structure of the adduct through optimizing the geometries formed by nucleophilic attack with different nitrogen sites in the thiosemicarbazide group. The proposed mechanism was presented in Scheme 2; a seven-membered heterocycle was proved to be a favorable adduct structure owing to its relatively lower energy, which also agreed well with the mass spectrometry result (Figure S5). The optimized structures of RB-CT and RB- CT/DCP adducts were shown in Figure 3. The HOMO–LUMO energy gap (2.651 eV) of the adduct is less than that of RB-CT (3.885 eV) and the electron densities in LUMO are more concentrated in the thiourea group after the reaction. The calculated main contributing electronic transitions for S0→S1 energy state are HOMO→LUMO (2.14 eV/578 nm) and HOMO-1→LUMO+1 (2.34 eV/530 nm), which is consistent with the absorption band at 560 nm obtained experimentally. The considerable difference in the energy and electronic transition can shed light on the changes of absorption spectra and color. CN CN EtO O H OEt N NH NH Cl OEt N O N N O N Colorless Pink Fluorescence 'off' Fluorescence 'on' Molecules 2019, 24, 827 6 of 12 Scheme 2. Proposed mechanism of RB-CT with DCP. Figure 3. Energy optimized structures, HOMO-LUMO energy levels, and interfacial plots of the orbitals of RB-CT and RB-CT/DCP adduct. 2.4. Interferents As organophosphorus pesticides often act as interferences to render false positives during the detection of nerve agents, several conventional organophosphorus compounds were chosen as target species to study the selectivity of RB-CT. As shown in Figure 4, the interferences did not cause any obvious color and fluorescence changes in DCP, which indicated the selective detection of DCP can be achieved by RB-CT, among other conventional organophosphorus compounds. It is noted that although proton can induce the spirolactam ring opening of the rhodamine molecule [32], RB-CT exhibited little changes of color and fluorescence intensity in the presence of hydrochloric acid. Thus, the false-positive caused by proton can be effectively avoided under the detection condition with the addition of 3% Et N, since the suitable detection pH range of the probe is 7.0 to 10.0 (Figure S6). 3 Molecules 2019, 24, x 6 of 12 Molecules 2019, 24, x 6 of 12 Figure 3. Energy optimized structures, HOMO-LUMO energy levels, and interfacial plots of the Figure 3. Energy optimized structures, HOMO-LUMO energy levels, and interfacial plots of the orbitals of RB-CT and RB-CT/DCP adduct. orbitals of RB-CT and RB-CT/DCP adduct. 2.4. Interferents 2.4. Interferents As organophosphorus pesticides often act as interferences to render false positives during the As organophosphorus pesticides often act as interferences to render false positives during the detection of nerve agents, several conventional organophosphorus compounds were chosen as target detection of nerve agents, several conventional organophosphorus compounds were chosen as target species to study the selectivity of RB-CT. As shown in Figure 4, the interferences did not cause any species to study the selectivity of RB-CT. As shown in Figure 4, the interferences did not cause any obvious color and fluorescence changes in DCP, which indicated the selective detection of DCP can obvious color and fluorescence changes in DCP, which indicated the selective detection of DCP can be achieved by RB-CT, among other conventional organophosphorus compounds. It is noted that be achieved by RB-CT, among other conventional organophosphorus compounds. It is noted that although proton can induce the spirolactam ring opening of the rhodamine molecule [32], RB-CT although proton can induce the spirolactam ring opening of the rhodamine molecule [32], RB-CT exhibited little changes of color and fluorescence intensity in the presence of hydrochloric acid. Thus, exhibited little changes of color and fluorescence intensity in the presence of hydrochloric acid. Thus, the false-positive caused by proton can be effectively avoided under the detection condition with the Molecules 2019, 24, 827 7 of 12 the false-positive caused by proton can be effectively avoided under the detection condition with the addition of 3% Et3N, since the suitable detection pH range of the probe is 7.0 to 10.0 (Figure S6). addition of 3% Et3N, since the suitable detection pH range of the probe is 7.0 to 10.0 (Figure S6). −5 5 Figure 4. (a) Relative fluorescence intensity of RB-CT (1 × 10 M, CH3CN, 3% Et3N) at 583 nm after Figure 4. (a) Relative fluorescence intensity of RB-CT (1  10 M, CH CN, 3% Et N) at 583 nm 3 3 −5 −3 Figure 4. (a) Relative fluorescence intensity of RB-CT (1 × 10 M, CH3CN, 3% Et 3 3N) at 583 nm after the addition of different organophosphorus compounds (1.0 × 10 M). Blank is the CH3CN solution after the addition of different organophosphorus compounds (1.0  10 M). Blank is the CH CN −3 the addition of different organophosphorus compounds (1.0 × 10 M). Blank is the CH3CN solution of RB-CT without Et3N. (b) Relative color change of RB-CT after the addition of different solution of RB-CT without Et N. (b) Relative color change of RB-CT after the addition of different of RB-CT without Et3N. (b) Relative color change of RB-CT after the addition of different organophosphorus compounds under sunlight and UV light. organophosphorus compounds under sunlight and UV light. organophosphorus compounds under sunlight and UV light. 2.5. 2.Practical 5. Practical App Application lication toward toward Real Real Nerve Nerve Agent Agent 2.5. Practical Application toward Real Nerve Agent To confirm in situ and rapid sensing ability of RB-CT toward real nerve agents, we have To confirm in situ and rapid sensing ability of RB-CT toward real nerve agents, we have To confirm in situ and rapid sensing ability of RB-CT toward real nerve agents, we have investigated the response characteristics of the probe to GD in both liquid and gas phase. Clear color investigated the response characteristics of the probe to GD in both liquid and gas phase. Clear investigated the response characteristics of the probe to GD in both liquid and gas phase. Clear color changes under sunlight and fluorescence enhancement irradiated by UV lamp were all observed by color changes under sunlight and fluorescence enhancement irradiated by UV lamp were all observed changes under sunlight and fluorescence enhancement irradiated by UV lamp were all observed by the naked eye immediately in liquid and within 10 min in gas phase (40 ppm GD introduced in a by the naked eye immediately in liquid and within 10 min in gas phase (40 ppm GD introduced in a the naked eye immediately in liquid and within 10 min in gas phase (40 ppm GD introduced in a flask as an aerosol). The remarkable visual differences did not fade or quench after 24 h, which flask as an aerosol) (as shown in Figure 5). The remarkable visual differences did not fade or quench flask as an aerosol). The remarkable visual differences did not fade or quench after 24 h, which indicated the irreversible cyclization had occurred[33]. The above results illustrate the potential after 24 h, which indicated the irreversible cyclization had occurred [33]. The above results illustrate indicated the irreversible cyclization had occurred[33]. The above results illustrate the potential application of RB-CT in the rapid and facile naked-eye detection of nerve agents in both liquid and the potential application of RB-CT in the rapid and facile naked-eye detection of nerve agents in both application of RB-CT in the rapid and facile naked-eye detection of nerve agents in both liquid and vapor phases. liquid and vapor phases. vapor phases. Figure 5. (a) Color change of filter paper treated with RB-CT solution (1.0  10 M in CH CN, 3% Et N) and GD in liquid phase under sunlight (left) and UV light (right). (b) Color change of filter paper treated with RB-CT solution (1.0 10 M in CH CN, 3% Et N) and exposed to GD in gas phase under 3 3 sunlight (left) and UV light (right). 3. Materials and Methods 3.1. Materials All of the solvents were obtained from Beijing Chemical Reagent Company and used without further purification. Rhodamine B, 4-cyanophenyl isothiocyanate and anhydrous magnesium sulfate (MgSO ) were purchased from Aladdin In. Co. (Los Angeles, CA, USA). Diethyl chlorophosphate (DCP) was purchased from Sigma Co. (Louis, MO, USA). Nerve agent GD (the purity is 80 %) was provided by the Research Institute of Chemical Defense of China. Molecules 2019, 24, x 7 of 12 −4 Figure 5. (a) Color change of filter paper treated with RB-CT solution (1.0 × 10 M in CH3CN, 3% Et3N) and GD in liquid phase under sunlight (left) and UV light (right). (b) Color change of filter −4 paper treated with RB-CT solution (1.0 × 10 M in CH3CN, 3% Et3N) and exposed to GD in gas phase under sunlight (left) and UV light (right). 3. Materials and Methods 3.1. Materials All of the solvents were obtained from Beijing Chemical Reagent Company and used without Molecules 2019, 24, 827 8 of 12 further purification. Rhodamine B, 4-cyanophenyl isothiocyanate and anhydrous magnesium sulfate (MgSO4) were purchased from Aladdin In. Co. (Los Angeles, CA, USA). Diethyl chlorophosphate (DCP) was purchased from Sigma Co. (Louis, MO, USA). Nerve agent GD (the purity is 80 %) was 3.2. Measurements provided by the Research Institute of Chemical Defense of China. The ESI mass analysis was performed using an Q Exactive HF-X Hybrid Quadrupole-Orbitrap Mass Spectrometer (Thermo Scientific, Waltham, MA, USA). The H NMR spectra were recorded 3.2. Measurements on AV 400 spectrometer (Bruker, Karlsruhe, Germany). Chemical shifts were expressed in parts per The ESI mass analysis was performed using an Q Exactive HF-X Hybrid Quadrupole-Orbitrap millions () downfield from the internal standard tetramethyl silane and were reported as s (singlet), Mass Spectrometer (Thermo Scientific, Waltham, MA, USA). The H NMR spectra were recorded on d (doublet), bs (broad singlet), t (triplet), and m (multiplet). Absorbance and fluorescence spectra AV 400 spectrometer (Bruker, Karlsruhe, Germany). Chemical shifts were expressed in parts per were recorded at room temperature with a Hitachi U-3900 UV-Visible spectrophotometer and F-4500 millions (δ) downfield from the internal standard tetramethyl silane and were reported as s (singlet), fluorescence spectrophotometer, respectively, using a fluorescence cell of 10 mm path. The excitation d (doublet), bs (broad singlet), t (triplet), and m (multiplet). Absorbance and fluorescence spectra wavelength was set to 540 nm (slit width 5 nm), and emission was monitored from 560–700 nm (slit were recorded at room temperature with a Hitachi U-3900 UV-Visible spectrophotometer and F-4500 width 5 nm). Column chromatography was conducted over silica gel (mesh 100–200). fluorescence spectrophotometer, respectively, using a fluorescence cell of 10 mm path. The excitation wavelength was set to 540 nm (slit width 5 nm), and emission was monitored from 560–700 nm (slit 3.3. Synthetic Procedures width 5 nm). Column chromatography was conducted over silica gel (mesh 100–200). As shown in Scheme 3, compound 1 was synthesized using the reported procedure [34]. Compound 2 was synthesized in a facile way. 3.3. Synthetic Procedures Scheme 3. Synthesis route of RB-TU. Reagents and conditions: (a) CH OH, hydrazine hydrate, 80 C, Scheme 3. Synthesis route of RB-TU. Reagents and conditions: (a) CH3OH, hydrazine hydrate, 80 °C, 6 h; (b) DMF, 4-cyanophenyl isothiocyanate, r.t, 12 h. 6 h; (b) DMF, 4-cyanophenyl isothiocyanate, r.t, 12 h. Synthesis of compound 1: In a 100 mL flask, 1 mL hydrazine hydrate was drop-wise added to a As shown in Scheme 3, compound 1 was synthesized using the reported procedure [34]. solution of rhodamine B (1.67 mmol, 800 mg) in anhydrous CH OH (30 mL), the mixed solution was Compound 2 was synthesized in a facile way. then stirred and heated to 80 °C, and refluxed for 6 h. Pure water (30 mL) was added and the solvent Synthesis of compound 1: In a 100 mL flask, 1 mL hydrazine hydrate was drop-wise added to was extracted with EtOAc (3  60 mL), after the organic phase was dried with anhydrous MgSO a solution of rhodamine B (1.67 mmol, 800 mg) in anhydrous CH3OH (30 mL), the mixed solution and evaporated, orange solid compound 1 (356 mg, 48.8% yield) was obtained, and used without was then stirred and heated to 80 ℃, and refluxed for 6 h. Pure water (30 mL) was added and the further purification. solvent was extracted with EtOAc (3 × 60 mL), after the organic phase was dried with anhydrous The H NMR (400 MHz, DMSO-d6)  7.82 (d, J = 8.3 Hz, 1H), 7.58-7.48 (m, 2H), 7.04 (d, J = 8.0 Hz, MgSO4 and evaporated, orange solid compound 1 (356 mg, 48.8% yield) was obtained, and used 1H), 6.41 (d, J = 17.1 Hz, 6H), 4.32 (s, 2H), 3.36 (d, J = 7.0 Hz, 8H), 1.14 (t, J = 6.9 Hz, 12H) (Figure S7). without further purification. Synthesis of compound 2(RB-TU probe): To a solution of compound 1 (0.22 mmol, 100 mg) in The H NMR (400 MHz, DMSO-d6) δ 7.82 (d, J = 8.3 Hz, 1H), 7.58-7.48 (m, 2H), 7.04 (d, J = 8.0 1.5 mL DMF, a solution of 4-cyanophenyl isothiocyanate (0.31 mmol,50 mg) in 1.5 mL DMF was added, Hz, 1H), 6.41 (d, J = 17.1 Hz, 6H), 4.32 (s, 2H), 3.36 (d, J = 7.0 Hz, 8H), 1.14 (t, J = 6.9 Hz, 12H) (Figure the reaction mixture was stirred for 12 h at room temperature. After the solution evaporated, the S7). residue was purified by flash chromatography (EtOAc: hexane = 1:10) to afford RB-TU probe (36 mg, Synthesis of compound 2(RB-TU probe): To a solution of compound 1 (0.22 mmol, 100 mg) in 26.6% yield). 1.5 mL DMF, a solution of 4-cyanophenyl isothiocyanate (0.31 mmol,50 mg) in 1.5 mL DMF was The H NMR (400 MHz, Chloroform-d)  8.01 (d, J = 7.6 Hz, 1H), 7.72 - 7.57 (m, 3H), 7.44 (d, added, the reaction mixture was stirred for 12 h at room temperature. After the solution evaporated, J = 8.4 Hz, 2H), 7.34 (d, J = 8.5 Hz, 2H), 7.29 (d, J = 7.6 Hz, 1H), 6.97 (s, 1H), 6.47 (d, J = 8.8 Hz, 2H), 6.42 (d, J = 2.5 Hz, 2H), 6.29 (d, J = 2.6 Hz, 1H), 6.27 (d, J = 2.6 Hz, 1H), 3.32 (qd, J = 7.2, 2.5 Hz, 8H), 1.15 (t, J = 7.0 Hz, 12H) (Figure S8). HR-MS (C H N O S): Calcd.: 616.2620; Found: 617.2681(9.9 ppm) (Figure S9). 36 36 6 2 3.4. Reaction Kinetics Study The fluorescence spectra of RB-CT (1.0  10 M) were recorded after the addition of DCP in 4 4 4 3 the concentrations of 4.0  10 M, 5.0  10 M, 6.0  10 M, and 1.2  10 M over a 0–1200 s Molecules 2019, 24, 827 9 of 12 incubation period at room temperature. The observed rate constants k and corresponding half-life obs time t were determined according to Equations (1) and (2). 1/2 ln[(F -F )/F ] = k t (1) max t max obs t = ln2/k (2) 1/2 obs where F and F are maximum fluorescence intensity obtained after the reaction was completed and max t the fluorescence intensities at 583 nm at reaction time t. 3.5. Computational Methods Quantum chemical calculations were carried out by Gaussian 09 package [35]. Geometry was optimized using density functional theory (DFT) functional B3LYP [36–38] and 6–31G (d) basis set [39,40]. Time-dependent density functional theory (TD-DFT) calculation [41] was also performed at the same level of theory. Considering the effects of the environment on the calculated energies, solvation effects were considered throughout geometry optimizations via the self-consistent reaction field (SCRF) and conductor-like polarizable continuum model (CPCM) method [42]. 3.6. Interferents Selectivity and specificity tests were performed with different organophosphorus compounds (OPs), i.e., dimethyl methylphosphonate (DMMP), triphenyl phosphate (TPP), trimethyl phosphate (TMP), acephate, isocarbophos, and dimethoate (Scheme 1). To 2 mL reagent solution of the probe 5 1 molecule (1.0  10 M) in CH CN containing 3% Et N, 20 L stock solution of OPs (1.0  10 M) 3 3 was added. The solutions were incubated at room temperature for 5 min and the fluorescence spectra was recorded. 3.7. The Preparation of Test Kits of Real Nerve Agents in Liquid and Gas Phase The test kits toward GD in liquid and gas phase were produced by the following steps. For liquid phase, a waterman filter paper was soaked in the CH CN solution of RB-CT (1.0  10 M, 3% Et N), 3 3 after the solvent was air-dried, a “GD” logo was patterned by GD. For gas phase, a solution of RB-CT in CH CN (1.0  10 M, 3% Et N) was sprayed onto a filter paper to spell out “GD” first, after the 3 3 solvent was air-dried, the paper was exposed to GD vapor in a flask (40 ppm introduced as an aerosol) for 10 mins. The LCt and LD of Soman are estimated to be 100 mg min/m and 300 mg/individual, 50 50 respectively [43]. Safety Note: Only highly qualified and experienced personnel should work with CWAs employed here, as described in the experimental part below. Due to the high volatility and toxicity of the CWAs, all of the experiments in this part were carried out inside a safety fume hood and operated under efficient ventilation systems. To avoid any risk of CWA inhalation, respiratory protection of involved personnel was provided by protective breathing masks equipped with combined NBC filters. 4. Conclusions In conclusion, we have designed and synthesized a novel probe N-(rhodamine B)-lactam-2-(4-cyanophenyl) thiourea for chromogenic and fluorogenic detection of nerve agents. The probe undergoes an irreversible opening-ring reaction following the form of a seven-membered heterocycle adduct in the presence of DCP, accompanied by the obvious color change from colorless to pink, which has been confirmed by MS analysis and TD-DFT calculations. The response is that instantaneously half-life time is about 280 s at room temperature and the detection limit was found to be 2  10 M. Furthermore, we have demonstrated that the probe is applicable for rapid and facile in situ detection of GD with the naked eye in both liquid and vapor phase. Molecules 2019, 24, 827 10 of 12 Supplementary Materials: The following are available online. Figure S1: The limit of detection for naked eye. Figure S2: Calibration plots by using fluorescence intensity of RB-CT (1 M) as a function of reaction time t in different DCP concentrations. Figure S3: ESI-MS spectrum of RB-CT/DCP adduct. Figure S4: P NMR spectra of DCP and RB-CT with DCP in CD CN. Figure S5: HOMO-LUMO energy levels of TD-DFT optimized geometries of probe and predicted RB-CT/DCP adducts. Figure S6: The plot of pH versus fluorescence intensity of free 1 1 RB-CT and RB-CT+DCP. Figure S7: H NMR Spectrum of Compound 1. Figures S8 and S9: H NMR and ESI-MS Spectra of RB-CT. Author Contributions: Y.Z. and W.C. conceived the idea and designed the experiments; S.L. and Y.Z. performed the synthesis, characterization, and co-wrote the manuscript draft; S.L., H.Z., Y.C., and Z.Z. discussed the data and the mechanisms; M.Z. and C.Z. contributed to spectra analysis; C.Z., J.Z., M.Z., and W.C. edited and revised the manuscript; Y.Z. and M.Z. assisted in DFT and TD-DFT calculation; Authors S.L. and Y.Z. contributed equally. All authors discussed the results and commented on the manuscript. Funding: This research was funded by the National Key Research and Development Program of China (Grant No. 2017YFC0108500). Acknowledgments: The authors thank the financial support of the National Key Research and Development Program of China (Grant No. Grant No. 2017YFC0108500). The authors would like to thank Prof. Dr. Shi-Lu Chen for valuable guidance and discussion about the quantum chemistry calculation, and Dr. Xiang-Xi Meng for discussions in the synthesis and purification process of RB-CT. Conflicts of Interest: The authors declare no conflict of interest. References 1. Szinicz, L. History of chemical and biological warfare agents. Toxicology 2005, 214, 167–181. [CrossRef] [PubMed] 2. Gunderson, C.H.; Lehmann, C.R.; Sidell, F.R.; Jabbari, B. Nerve agents: A review. Neurology 1992, 42, 946–950. [CrossRef] [PubMed] 3. Lei, Z.; Yang, Y. A concise colorimetric and fluorimetric probe for sarin related threats designed via the "covalent-assembly" approach. J. Am. Chem. Soc. 2014, 136, 6594–6597. [CrossRef] [PubMed] 4. Russell, R.J.; Pishko, M.V.; Simonian, A.L.; Wild, J.R. Poly(ethylene glycol) hydrogel-encapsulated fluorophore-enzyme conjugates for direct detection of organophosphorus neurotoxins. Anal. Chem. 1999, 71, 4909–4912. [CrossRef] [PubMed] 5. Russell, A.J.; Berberich, J.A.; Drevon, G.F.; Koepsel, R.R. Biomaterials for mediation of chemical and biological warfare agents. Annu. Rev. Biomed. Eng. 2003, 5, 1–27. [CrossRef] [PubMed] 6. Kim, S.; Cho, B.; Sohn, H. Detection of nerve agent stimulants based on photoluminescent porous silicon interferometer. Nanoscale Res. Lett. 2012, 7, 527. [CrossRef] [PubMed] 7. Sorribes-Soriano, A.; de la Guardia, M.; Esteve-Turrillas, F.A.; Armenta, S. Trace analysis by ion mobility spectrometry: From conventional to smart sample preconcentration methods. A review. Anal. Chim. Acta 2018, 1026, 37–50. [CrossRef] [PubMed] 8. Maresova, E.; Tomecek, D.; Fitl, P.; Vlcek, J.; Novotny, M.; Fiser, L.; Havlova, S.; Hozak, P.; Tudor, A.; Glennon, T.; et al. Textile chemiresistors with sensitive layers based on polymer ionic liquids: Applicability for detection of toxic gases and chemical warfare agents. Sens. Actuator B-Chem. 2018, 266, 830–840. [CrossRef] 9. Walker, J.P.; Kimble, K.W.; Asher, S.A. Photonic crystal sensor for organophosphate nerve agents utilizing the organophosphorus hydrolase enzyme. Anal. Bioanal Chem. 2007, 389, 2115–2124. [CrossRef] [PubMed] 10. He, W.; Liu, Z.; Du, X.; Jiang, Y.; Xiao, D. Analytical application of poly{methyl[3-(2-hydroxy-3,4-difluoro)phenyl]propyl siloxane} as a QCM coating for DMMP detection. Talanta 2008, 76, 698–702. [CrossRef] [PubMed] 11. Kittle, J.D.; Fisher, B.P.; Esparza, A.J.; Morey, A.M.; Iacono, S.T. Sensing Chemical Warfare Agent Simulants via Photonic Crystals of the Morpho didius Butterfly. ACS Omega 2017, 2, 8301–8307. [CrossRef] [PubMed] 12. Roy, K.S.; Mazumder, A.; Goud, D.R.; Dubey, D.K. A simplistic designing of molecularly imprinted polymers for derivative of nerve agents marker using P-31{H-1}NMR. Eur. Polym. J. 2018, 98, 105–115. [CrossRef] 13. Nanda Kumar, D.; Satija, J.; Chandrasekaran, N.; Mukherjee, A. Acetylcholinesterase-based inhibition screening through in situ synthesis of gold nanoparticles: Application for detection of nerve agent simulant. J. Mol. Liq. 2018, 249, 623–628. [CrossRef] Molecules 2019, 24, 827 11 of 12 14. Costero, A.M.; Parra, M.; Gil, S.; Gotor, R.; Mancini, P.M.E.; Martinez-Manez, R.; Sancenon, F.; Royo, S. Chromo-fluorogenic detection of nerve-agent mimics using triggered cyclization reactions in push-pull dyes. Chem. Asian J. 2010, 5, 1573–1585. [CrossRef] [PubMed] 15. Goud, D.R.; Pardasani, D.; Tak, V.; Dubey, D.K. A highly selective visual detection of tabun mimic diethyl cyanophosphate (DCNP): effective discrimination of DCNP from other nerve agent mimics. RSC Adv. 2014, 4, 24645. [CrossRef] 16. Barba-Bon, A.; Costero, A.M.; Gil, S.; Martinez-Manez, R.; Sancenon, F. Selective chromo-fluorogenic detection of DFP (a Sarin and Soman mimic) and DCNP (a Tabun mimic) with a unique probe based on a boron dipyrromethene (BODIPY) dye. Org. Biomol. Chem. 2014, 12, 8745–8751. [CrossRef] [PubMed] 17. Kim, Y.; Jang, Y.J.; Lee, D.; Kim, B.-S.; Churchill, D.G. Real nerve agent study assessing pyridyl reactivity: Selective fluorogenic and colorimetric detection of Soman and simulant. Sens. Actuator B-Chem. 2017, 238, 145–149. [CrossRef] 18. Ali, S.S.; Gangopadhyay, A.; Maiti, K.; Mondal, S.; Pramanik, A.K.; Guria, U.N.; Uddin, M.R.; Mandal, S.; Mandal, D.; Mahapatra, A.K. A chromogenic and ratiometric fluorogenic probe for rapid detection of a nerve agent simulant DCP based on a hybrid hydroxynaphthalene-hemicyanine dye. Org. Biomol. Chem. 2017, 15, 5959–5967. [CrossRef] [PubMed] 19. Cai, Y.-C.; Li, C.; Song, Q.-H. Selective and visual detection of a nerve agent mimic by phosphorylation and protonation of quinolin oximes. J. Mater. Chem. C 2017, 5, 7337–7343. [CrossRef] 20. Royo, S.; Costero, A.M.; Parra, M.; Gil, S.; Martinez-Manez, R.; Sancenon, F. Chromogenic, specific detection of the nerve-agent mimic DCNP (a tabun mimic). Chemistry 2011, 17, 6931–6934. [CrossRef] [PubMed] 21. Pascual, L.; Sayed, S.E.; Martínez-Máñez, R.; Costero, A.M.; Gil, S.; Gaviña, P.; Sancenón, F. Acetylcholinesterase-Capped Mesoporous Silica Nanoparticles That Open in the Presence of Diisopropylfluorophosphate (a Sarin or Soman Simulant). Org. Lett. 2016, 18, 5548–5551. [CrossRef] [PubMed] 22. Sarkar, H.S.; Ghosh, A.; Das, S.; Maiti, P.K.; Maitra, S.; Mandal, S.; Sahoo, P. Visualisation of DCP, a nerve agent mimic, in Catfish brain by a simple chemosensor. Sci. Rep. 2018, 8, 3402. [CrossRef] [PubMed] 23. Chen, L.; Wu, D.; Yoon, J. Recent Advances in the Development of Chromophore-Based Chemosensors for Nerve Agents and Phosgene. ACS Sens. 2018, 3, 27–43. [CrossRef] [PubMed] 24. Royo, S.; Martínez-Máñez, R.; Sancenón, F.; Costero, A.M.; Parra, M.; Gil, S. Chromogenic and fluorogenic reagents for chemical warfare nerve agents’ detection. Chem. Commun. 2007, 4839–4847. [CrossRef] 25. El Sayed, S.; Pascual, L.; Agostini, A.; Martinez-Manez, R.; Sancenon, F.; Costero, A.M.; Parra, M.; Gil, S. A Chromogenic Probe for the Selective Recognition of Sarin and Soman Mimic DFP. ChemistryOpen 2014, 3, 142–145. [CrossRef] [PubMed] 26. Khan, M.S.J.; Wang, Y.-W.; Senge, M.O.; Peng, Y. Sensitive fluorescence on-off probes for the fast detection of a chemical warfare agent mimic. J. Hazard Mater. 2018, 342, 10–19. [CrossRef] [PubMed] 27. Ha, S.; Lee, M.; Seo, H.O.; Song, S.G.; Kim, K.S.; Park, C.H.; Kim, I.H.; Kim, Y.D.; Song, C. Structural Effect of Thioureas on the Detection of Chemical Warfare Agent Simulants. ACS Sens. 2017, 2, 1146–1151. [CrossRef] [PubMed] 28. Kumar, V.; Rana, H.; Raviraju, G.; Garg, P.; Baghel, A.; Gupta, A.K. Chromogenic and fluorogenic multianalyte detection with a tuned receptor: refining selectivity for toxic anions and nerve agents. RSC Adv. 2016, 6, 59648–59656. [CrossRef] 29. Yang, Y.K.; Keunjeong Yook, A.; Tae, J. A Rhodamine-Based Fluorescent and Colorimetric Chemodosimeter 2+ for the Rapid Detection of Hg Ions in Aqueous Media. J. Am. Chem. Soc. 2005, 127, 16760–16761. [CrossRef] [PubMed] 30. Goswami, S.; Manna, A.; Paul, S. Rapid ‘naked eye’ response of DCP, a nerve agent simulant: from molecules to low-cost devices for both liquid and vapour phase detection. RSC Adv. 2014, 4, 21984–21988. [CrossRef] 31. So, H.-S.; Angupillai, S.; Son, Y.-A. Prompt liquid-phase visual detection and low-cost vapor-phase detection of DCP, a chemical warfare agent mimic. Sens. Actuators B: Chem. 2016, 235, 447–456. [CrossRef] 32. Ramette, R.W.; Sandell, E.B. Rhodamine B Equilibria. J. Am. Chem. Soc. 1956, 78, 4872–4878. [CrossRef] 33. Gotor, R.; Gaviña, P.; Ochando, L.E.; Chulvi, K.; Lorente, A.; Martínez-Máñez, R.; Costero, A.M. BODIPY dyes functionalized with 2-(2-dimethylaminophenyl)ethanol moieties as selective OFF–ON fluorescent chemodosimeters for the nerve agent mimics DCNP and DFP. RSC Adv. 2014, 4, 15975–15982. [CrossRef] Molecules 2019, 24, 827 12 of 12 34. Yang, X.-F.; Guo, X.-Q.; Zhao, Y.-B. Development of a novel rhodamine-type fluorescent probe to determine peroxynitrite. Talanta 2002, 57, 883–890. [CrossRef] 35. Frisch, M.J.; Trucks, G.W.; Schlegel, H.B.; Scuseria, G.E.; Robb, M.A.; Cheeseman, J.R.; Scalmani, G.; Barone, V.; Mennucci, B.; Petersson, G.A.; et al. Gaussian 09, Revision D. 01; Gaussian. Inc.: Wallingford, CT, USA, 2009. 36. Lee, C.; Yang, W.; Parr, R.G. Development of the Colle-Salvetti correlation-energy formula into a functional of the electron density. Phys. Rev. B 1988, 37, 785–789. [CrossRef] 37. Becke, A.D. A new mixing of Hartree–Fock and local density-functional theories. J. Chem. Phys. 1993, 98, 1372–1377. [CrossRef] 38. Becke, A.D. Density-functional thermochemistry. III. The role of exact exchange. J. Chem. Phys. 1993, 98, 5648–5652. [CrossRef] 39. Hehre, W.J.; Ditchfield, R.; Pople, J.A. Self—Consistent Molecular Orbital Methods. XII. Further Extensions of Gaussian—Type Basis Sets for Use in Molecular Orbital Studies of Organic Molecules. J. Chem. Phys. 1972, 56, 2257–2261. [CrossRef] 40. Francl, M.M.; Pietro, W.J.; Hehre, W.J.; Binkley, J.S.; Gordon, M.S.; DeFrees, D.J.; Pople, J.A. Self-consistent molecular orbital methods. XXIII. A polarization-type basis set for second-row elements. J. Chem. Phys. 1982, 77, 3654–3665. [CrossRef] 41. Bauernschmitt, R.; Ahlrichs, R. Treatment of electronic excitations within the adiabatic approximation of time dependent density functional theory. Chem. Phys. Lett. 1996, 256, 454–464. [CrossRef] 42. Ming, W.W.; Wiberg, K.B.; Frisch, M.J. Solvent effects. 3. Tautomeric Equilibria of Formamide and 2-pyridone in the Gas Phase and Solution: an ab Initio SCRF Study. J. Am. Chem. Soc. 1992, 114, 1645–1652. [CrossRef] 43. Sadik, O.A.; Land, W.H., Jr.; Wang, J. Targeting Chemical and Biological Warfare Agents at the Molecular Level. Electroanalysis 2003, 15, 1149–1159. [CrossRef] Sample Availability: Samples of compounds 1–2 are available from the authors. © 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Molecules Multidisciplinary Digital Publishing Institute

Chromo-Fluorogenic Detection of Soman and Its Simulant by Thiourea-Based Rhodamine Probe

Download PDF
12 pages

Loading next page...
 
/lp/multidisciplinary-digital-publishing-institute/chromo-fluorogenic-detection-of-soman-and-its-simulant-by-thiourea-XWk5JQBOH4

References

References for this paper are not available at this time. We will be adding them shortly, thank you for your patience.

Publisher
Multidisciplinary Digital Publishing Institute
Copyright
© 1996-2019 MDPI (Basel, Switzerland) unless otherwise stated
ISSN
1420-3049
DOI
10.3390/molecules24050827
Publisher site
See Article on Publisher Site

Abstract

molecules Article Chromo-Fluorogenic Detection of Soman and Its Simulant by Thiourea-Based Rhodamine Probe 1 , † 1 , 2 , , † 3 4 1 , 2 Shengsong Li , Yongchao Zheng * , Weiqiang Chen , Meiling Zheng , He Zheng , 1 1 , 2 1 , 2 , 1 , 2 , Zhe Zhang , Yan Cui , Jinyi Zhong * and Chonglin Zhao * Research Institute of Chemical Defense, Beijing 102205, China; [email protected] (S.L.); [email protected] (H.Z.); [email protected] (Z.Z.); [email protected] (Y.C.) State Key Laboratory of NBC Protection for Civilian, Beijing 102205, China Institute of Modern Physics, Chinese Academy of Sciences, Lanzhou 730000, China; [email protected] Laboratory of Organic NanoPhotonics and CAS Key Laboratory of Bio-Inspired Materials and Interfacial Science, Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, Beijing 100190, China; [email protected] * Correspondence: [email protected] (Y.Z.); [email protected] (J.Z.); [email protected] (C.Z.); Tel.: +86-10-66758321(C.Z.) † These authors have contributed equally to this work. Academic Editor: Simone Morais Received: 3 February 2019; Accepted: 18 February 2019; Published: 26 February 2019 Abstract: Here, we introduced a novel thiourea-based rhodamine compound as a chromo-fluorogenic indicator of nerve agent Soman and its simulant diethyl chlorophosphate (DCP). The synthesized probe N-(rhodamine B)-lactam-2-(4-cyanophenyl) thiourea (RB-CT), which has a rhodamine core linked by a cyanophenyl thiosemicarbazide group, enabled a rapidly and highly sensitive response to DCP with clear fluorescence and color changes. The detection limit was as low as 2  10 M. The sensing mechanism showed that opening of the spirolactam ring following the phosphorylation of thiosemicarbazides group formed a seven-membered heterocycle adduct, according to MS analysis and TD-DFT calculations. RB-CT exhibited high detecting selectivity for DCP, among other organophosphorus compounds. Moreover, two test kits were employed and successfully used to detect real nerve agent Soman in liquid and gas phase. Keywords: Nerve agents; Soman; Rhodamine B; Thiosemicarbazide; Chromo-fluorogenic probe; Detection 1. Introduction Nerve agents are a class of highly toxic organophosphates, mainly including Tabun (GA), Sarin (GB), Soman (GD), and VX [1] (Scheme 1). These compounds are extremely dangerous because they are able to enter human body through respiration or penetration and inhibit the activity of the acetylcholinesterase, then the accumulation of acetylcholine in the synapse causes neuromuscular paralysis and eventually death [2]. Due to the easy production, high toxicity, being colorless and odorless, and the possible use in terrorist attacks, the development of reliable and rapid detection systems for nerve agents is highly desirable [3]. In addition, rapidly discriminating the distribution of nerve agents in the contaminated areas can also provide effective guidance for subsequent decontamination operations and further safety confirmation. Molecules 2019, 24, 827; doi:10.3390/molecules24050827 www.mdpi.com/journal/molecules Molecules 2019, 24, x 2 of 12 imprinted polymers[12], nanoparticles[13], and chromo-fluorogenic probes[14-22]. Above all, chromo-fluorogenic probes have recently gained increasing interest due to their cost-effectiveness, simplicity, and “naked-eye” detection [23,24]. A typical chromo-fluorogenic probe is usually formed by two moieties: (1) a chromo-fluorogenic reporter group, which translates the binding event into the change of color and fluorescence, mainly containing rhodamine[22], fluorescein[15], boron dipyrromethene (BODIPY) [16,17], azo[14,20], and cyanine dye[18]; (2) a selective reactive group, which provides a reactive binding site for nucleophilic attack, mainly containing hydroxyl[15,25], oxime[19], and amino groups[26]. Recently, thiourea has been proved to be capable of reacting with nerve agents through hydrogen-bond interaction between N-H protons of thiourea and phosphonate oxygen or hydrolyzed products[27,28]. It has been also proven that the reaction of thiosemicarbazide group can induce opening of the spirolactam ring in rhodamine B accompanied with color change and enhanced fluorescence[29]. Thus, the thiosemicarbazide group is expected to become a reactive group bonding with rhodamine B core for chromo-fluorogenic detection of nerve agents and simulants. Herein, a rhodamine B based probe N-(rhodamine B)-lactam-2-(4-cyanophenyl) thiourea (RB- CT) was designed and synthesized, and as shown in Scheme 2, the electron-withdrawing cyanophenyl group in the molecule could promote the intramolecular charge transfer (ICT) process to enhance activity of ring-opening reaction and strengthen the change of color. Spectra analysis and mechanism study confirmed the probe was capable of the rapid detection of diethyl chlorophosphate (DCP, a simulant of the Soman) with remarkable change of color and fluorescence through the Molecules 2019, 24, 827 2 of 12 phosphorylation of the thiosemicarbazide group. Finally, the probe was also successfully used in the detection of GD in both liquid and gas phase. Scheme 1. Chemical structures of nerve-agents Sarin, Soman, Tabun, and VX, their simulant DCP, and Scheme 1. Chemical structures of nerve-agents Sarin, Soman, Tabun, and VX, their simulant DCP, some organophosphorus compounds as potential interferences. and some organophosphorus compounds as potential interferences Several methodologies have been employed for the detection of nerve agents, including enzymatic assays [4,5], interferometry [6], ion mobility spectroscopy [7], electrochemistry [8], micro-cantilevers [9,10], and photonic crystals [11]. Nevertheless, these protocols usually have some drawbacks, such as operation complexity, non-portability, difficulties in real-time monitoring, etc. As alternatives to these procedures, some new approaches have been explored involving molecularly imprinted polymers [12], nanoparticles [13], and chromo-fluorogenic probes [14–22]. Above all, chromo-fluorogenic probes have recently gained increasing interest due to their cost-effectiveness, simplicity, and “naked-eye” detection [23,24]. A typical chromo-fluorogenic probe is usually formed by two moieties: (1) a chromo-fluorogenic reporter group, which translates the binding event into the change of color and fluorescence, mainly containing rhodamine [22], fluorescein [15], boron dipyrromethene (BODIPY) [16,17], azo [14,20], and cyanine dye [18]; (2) a selective reactive group, which provides a reactive binding site for nucleophilic attack, mainly containing hydroxyl [15,25], oxime [19], and amino groups [26]. Recently, thiourea has been proved to be capable of reacting with nerve agents through hydrogen-bond interaction between N-H protons of thiourea and phosphonate oxygen or hydrolyzed products [27,28]. It has been also proven that the reaction of thiosemicarbazide group can induce opening of the spirolactam ring in rhodamine B accompanied with color change and enhanced fluorescence [29]. Thus, the thiosemicarbazide group is expected to become a reactive group bonding with rhodamine B core for chromo-fluorogenic detection of nerve agents and simulants. Herein, a rhodamine B based probe N-(rhodamine B)-lactam-2-(4-cyanophenyl) thiourea (RB-CT) was designed and synthesized, and as shown in Scheme 2, the electron-withdrawing cyanophenyl group in the molecule could promote the intramolecular charge transfer (ICT) process to enhance Molecules 2019, 24, x 5 of 12 TD-DFT calculations were used to confirm the structure of the adduct through optimizing the geometries formed by nucleophilic attack with different nitrogen sites in the thiosemicarbazide group. The proposed mechanism was presented in Scheme 2; a seven-membered heterocycle was proved to be a favorable adduct structure owing to its relatively lower energy, which also agreed Molecules 2019, 24, 827 3 of 12 well with the mass spectrometry result (Figure S5). The optimized structures of RB-CT and RB- CT/DCP adducts were shown in Figure 3. The HOMO–LUMO energy gap (2.651 eV) of the adduct is less than that of RB-CT (3.885 eV) and the electron densities in LUMO are more concentrated in the activity of ring-opening reaction and strengthen the change of color. Spectra analysis and mechanism thiourea group after the reaction. The calculated main contributing electronic transitions for S0→S1 study confirmed the probe was capable of the rapid detection of diethyl chlorophosphate (DCP, a energy state are HOMO→LUMO (2.14 eV/578 nm) and HOMO-1→LUMO+1 (2.34 eV/530 nm), which simulant of the Soman) with remarkable change of color and fluorescence through the phosphorylation is consistent with the absorption band at 560 nm obtained experimentally. The considerable of the thiosemicarbazide group. Finally, the probe was also successfully used in the detection of GD in difference in the energy and electronic transition can shed light on the changes of absorption spectra both liquid and gas phase. and color. CN CN EtO O OEt N NH NH Cl OEt N O N N O N Colorless Pink Fluorescence 'off' Fluorescence 'on' Scheme 2. Proposed mechanism of RB-CT with DCP. Scheme 2. Proposed mechanism of RB-CT with DCP. 2. Results and Discussion 2.1. Spectroscopic Properties To demonstrate the response of RB-CT to nerve agents, absorption and fluorescence spectra were first studied. DCP was used as low-toxic simulant because of its similar chemical structure and reactivity with nerve agents (Tabun, Sarin and Soman). Et N (3 Vt%) was added in the CH CN 3 3 solution of RB-CT to avoid the interference of proton. As shown in Figure 1a, an obvious absorption band at 560 nm was observed after the addition of DCP in the RB-CT solution. The change in color from colorless to pink implied the spirolactam ring-opening reaction of RB-CT caused by DCP (Figure 1b). The thiosemicarbazide group of RB-CT is likely to react with DCP, resulting in a remarkable enhancement of absorption intensity. The result highlights that RB-CT is of high potential as a naked-eye probe for the detection of nerve agents. Figure 1c presented the fluorescence spectra of RB-CT with increasing concentration of DCP; a new fluorescent band at 583 nm appears and shows remarkable enhancement. The spectral changes provided further evidence for the opening of the spirolactam ring, according to the changes in absorption spectra. Moreover, the fluorescence intensity of RB-CT showed a good linear relationship 3 3 at the concentration of DCP in the range of 0.1  10 –1.9  10 M (Figure 1d). The limit of detection (LOD) was determined from the fluorescence spectral data, using the equation K  S /S, where K = 3, S is the standard deviation of blank measurements and S is b1 b1 the slope of the calibration curve. The limit of detection was found to be 2  10 M, indicating high sensitivity to detect DCP by RB-CT. The sensing ability for the naked eye was evaluated by immobilizing RB-CT on silica plates. After the plates were dipped in the CH CN solution of DCP (2–2000 ppm), clear enhancement of the fluorescence was observed by the naked eye in the range of 20 and 2000 ppm (Figure S1), which demonstrates the LOD for the naked eye is as low as the value calculated. Molecules 2019, 24, x 3 of 12 2. Results and Discussion 2.1. Spectroscopic Properties To demonstrate the response of RB-CT to nerve agents, absorption and fluorescence spectra were first studied. DCP was used as low-toxic simulant because of its similar chemical structure and reactivity with nerve agents (Tabun, Sarin and Soman). Et3N (3 Vt%) was added in the CH3CN solution of RB-CT to avoid the interference of proton. As shown in Figure 1a, an obvious absorption band at 560 nm was observed after the addition of DCP in the RB-CT solution. The change in color from colorless to pink implied the spirolactam ring-opening reaction of RB-CT caused by DCP (Figure 1b). The thiosemicarbazide group of RB-CT is likely to react with DCP, resulting in a remarkable enhancement of absorption intensity. The result highlights that RB-CT is of high potential as a naked- eye probe for the detection of nerve agents. Figure 1c presented the fluorescence spectra of RB-CT with increasing concentration of DCP; a new fluorescent band at 583 nm appears and shows remarkable enhancement. The spectral changes provided further evidence for the opening of the spirolactam ring, according to the changes in absorption spectra. Moreover, the fluorescence intensity of RB-CT showed a good linear relationship −3 −3 at the concentration of DCP in the range of 0.1 × 10 –1.9 × 10 M (Figure 1d). The limit of detection (LOD) was determined from the fluorescence spectral data, using the equation K × Sb1/S, where K = 3, Sb1 is the standard deviation of blank measurements and S is the slope −6 of the calibration curve. The limit of detection was found to be 2 × 10 M, indicating high sensitivity to detect DCP by RB-CT. The sensing ability for the naked eye was evaluated by immobilizing RB- CT on silica plates. After the plates were dipped in the CH3CN solution of DCP (2–2000 ppm), clear Molecules 2019, 24, 827 4 of 12 enhancement of the fluorescence was observed by the naked eye in the range of 20 and 2000 ppm (Figure S1), which demonstrates the LOD for the naked eye is as low as the value calculated. 5 4 Figure 1. (a) Absorption spectra of RB-CT (1.0  10 M) upon the addition of DCP (0.5  10 –8.0 10 M) in CH CN (3% Et N). (b) The images of the RB-CT solution before and after the addition of 3 3 DCP under sun light (left) and UV light (right). (c) Fluorescence spectra of RB-CT (1.0  10 M) at 3 3 = 540 nm, upon the addition of DCP (0.1  10 –1.9  10 M) in CH CN (3% Et N). (d) Plot of ex 3 3 emission intensity of RB-CT at 583 nm. 2.2. Reaction Kinetics Study To investigate the reaction kinetics of RB-CT and DCP, time-dependent fluorescence change was performed under different DCP concentrations at room temperature. As shown in Figure 2a, the reaction was almost complete and the fluorescence intensity was saturated within 1200 s in all cases. The kinetic constants were further examined following pseudo-first-order kinetic rate law as the clear linear relationships between the change of fluorescence intensity and reaction time (Figure S2). The observed rate constants k , half-life time t , and the constant rates (k) were summarized in Table 1. obs 1/2 The average half-life time was about 280 s, less than 5 min. The short half-life times are the basis for the rapid detection of nerve agents. Molecules 2019, 24, x 4 of 12 −5 −4 −4 Figure 1. (a) Absorption spectra of RB-CT (1.0 × 10 M) upon the addition of DCP (0.5 × 10 –8.0 × 10 M) in CH3CN (3% Et3N). (b) The images of the RB-CT solution before and after the addition of DCP −6 under sun light (left) and UV light (right). (c) Fluorescence spectra of RB-CT (1.0 × 10 M) at λex = 540 −3 −3 nm, upon the addition of DCP (0.1 × 10 –1.9 × 10 M) in CH3CN (3% Et3N). (d) Plot of emission intensity of RB-CT at 583 nm. 2.2. Reaction Kinetics Study To investigate the reaction kinetics of RB-CT and DCP, time-dependent fluorescence change was performed under different DCP concentrations at room temperature. As shown in Figure 2a, the reaction was almost complete and the fluorescence intensity was saturated within 1200 s in all cases. The kinetic constants were further examined following pseudo-first-order kinetic rate law as the clear linear relationships between the change of fluorescence intensity and reaction time (Figure S2). The observed rate constants kobs, half-life time t1/2, and the constant rates (k) were summarized in Table 1. Molecules 2019, 24, 827 5 of 12 The average half-life time was about 280 s, less than 5 min. The short half-life times are the basis for the rapid detection of nerve agents. −6 Figure 2. (a) Kinetic profiles of the fluorescence intensity at 583 nm of RB-CT ((1.0 × 10 M, 6 CH3CN, Figure 2. (a) Kinetic profiles of the fluorescence intensity at 583 nm of RB-CT ((1.0  10 M, CH CN, −4 −4 −4 −3 3% Et3N) after the addition of 4.0 × 10 M, 5.0 × 10 M, 6.0 × 10 M, and 1.2 × 10 M DCP. (b)The 4 4 4 3 3% Et N) after the addition of 4.0  10 M, 5.0  10 M, 6.0  10 M, and 1.2  10 M DCP. correlation between kobs and the concentration of DCP. (b)The correlation between k and the concentration of DCP. obs Table 1. Observed reaction rates (kobs), half-life time (t1/2), and the constant rates (k) for the reaction of Table 1. Observed reaction rates (k ), half-life time (t ), and the constant rates (k) for the reaction of obs 1/2 6−6 RB-CT RB-CT ((1.0 ((1.0  × 1 10 0 M, M, CH CH 3C CN, N, 3% 3%Et Et 3N) and N) and DCP with different concentrations. DCP with different concentrations. 3 3 The Concentrations The 4 4 4 3 4  10 5  10 6  10 1.2  10 of DCP −4 −4 −4 −3 Concentrations 4 × 10 5 × 10 6 × 10 1.2 × 10 of DCP 0.00244 0.00245 0.00246 0.00251 K (s ) obs t (s) 284 283 282 276 1/2 2 1 −1 6 Kobs (s ) 0.00244 0.00245 0.00246 0.00251 k (M S ) 8.645  10 t1/2 (s) 284 283 282 276 2.3. Mechanism Study −2 −1 −6 k (M S ) 8.645 × 10 The absorption and fluorescence changes can be attributed to the opening of spirolactam ring via nucleophilic reaction between RB-CT and DCP. An ESI analysis was employed to investigate the 2.3. Mechanism Study possible products and a major peak at 708.3223 was observed, which indicated the phosphorylation of RB-CT The ab (Figur sorp e t S3). ion and Theflnew uorescence ch singlet observed anges can b ( e0.32) attrib in uted to the openi the P NMR spectr ng of spi um rola ofcDCP tam ring after addition via nuc of leophilic RB-CT re gives action be further tween RB evidence -CT for and DC the occurr P. An ESI an ence of aly phosphorylation sis was employed reaction to investig (Figur ate e the S4). possible products and a major peak at 708.3223 was observed, which indicated the phosphorylation As the formation of a heterocycle in RB-CT/DCP adduct is a reasonable mechanism [30,31], DFT of RB-CT (Figure S3). The new singlet observed (δ 0.32) in the P NMR spectrum of DCP after and TD-DFT calculations were used to confirm the structure of the adduct through optimizing the addition of RB-CT gives further evidence for the occurrence of phosphorylation reaction (Figure S4). geometries formed by nucleophilic attack with different nitrogen sites in the thiosemicarbazide group. As the formation of a heterocycle in RB-CT/DCP adduct is a reasonable mechanism [30,31], DFT and The proposed mechanism was presented in Scheme 2; a seven-membered heterocycle was proved to be a favorable adduct structure owing to its relatively lower energy, which also agreed well with the mass spectrometry result (Figure S5). The optimized structures of RB-CT and RB-CT/DCP adducts were shown in Figure 3. The HOMO–LUMO energy gap (2.651 eV) of the adduct is less than that of RB-CT (3.885 eV) and the electron densities in LUMO are more concentrated in the thiourea group after the reaction. The calculated main contributing electronic transitions for S !S energy state are 0 1 HOMO!LUMO (2.14 eV/578 nm) and HOMO-1!LUMO+1 (2.34 eV/530 nm), which is consistent with the absorption band at 560 nm obtained experimentally. The considerable difference in the energy and electronic transition can shed light on the changes of absorption spectra and color. Molecules 2019, 24, x 5 of 12 TD-DFT calculations were used to confirm the structure of the adduct through optimizing the geometries formed by nucleophilic attack with different nitrogen sites in the thiosemicarbazide group. The proposed mechanism was presented in Scheme 2; a seven-membered heterocycle was proved to be a favorable adduct structure owing to its relatively lower energy, which also agreed well with the mass spectrometry result (Figure S5). The optimized structures of RB-CT and RB- CT/DCP adducts were shown in Figure 3. The HOMO–LUMO energy gap (2.651 eV) of the adduct is less than that of RB-CT (3.885 eV) and the electron densities in LUMO are more concentrated in the thiourea group after the reaction. The calculated main contributing electronic transitions for S0→S1 energy state are HOMO→LUMO (2.14 eV/578 nm) and HOMO-1→LUMO+1 (2.34 eV/530 nm), which is consistent with the absorption band at 560 nm obtained experimentally. The considerable difference in the energy and electronic transition can shed light on the changes of absorption spectra and color. CN CN EtO O H OEt N NH NH Cl OEt N O N N O N Colorless Pink Fluorescence 'off' Fluorescence 'on' Molecules 2019, 24, 827 6 of 12 Scheme 2. Proposed mechanism of RB-CT with DCP. Figure 3. Energy optimized structures, HOMO-LUMO energy levels, and interfacial plots of the orbitals of RB-CT and RB-CT/DCP adduct. 2.4. Interferents As organophosphorus pesticides often act as interferences to render false positives during the detection of nerve agents, several conventional organophosphorus compounds were chosen as target species to study the selectivity of RB-CT. As shown in Figure 4, the interferences did not cause any obvious color and fluorescence changes in DCP, which indicated the selective detection of DCP can be achieved by RB-CT, among other conventional organophosphorus compounds. It is noted that although proton can induce the spirolactam ring opening of the rhodamine molecule [32], RB-CT exhibited little changes of color and fluorescence intensity in the presence of hydrochloric acid. Thus, the false-positive caused by proton can be effectively avoided under the detection condition with the addition of 3% Et N, since the suitable detection pH range of the probe is 7.0 to 10.0 (Figure S6). 3 Molecules 2019, 24, x 6 of 12 Molecules 2019, 24, x 6 of 12 Figure 3. Energy optimized structures, HOMO-LUMO energy levels, and interfacial plots of the Figure 3. Energy optimized structures, HOMO-LUMO energy levels, and interfacial plots of the orbitals of RB-CT and RB-CT/DCP adduct. orbitals of RB-CT and RB-CT/DCP adduct. 2.4. Interferents 2.4. Interferents As organophosphorus pesticides often act as interferences to render false positives during the As organophosphorus pesticides often act as interferences to render false positives during the detection of nerve agents, several conventional organophosphorus compounds were chosen as target detection of nerve agents, several conventional organophosphorus compounds were chosen as target species to study the selectivity of RB-CT. As shown in Figure 4, the interferences did not cause any species to study the selectivity of RB-CT. As shown in Figure 4, the interferences did not cause any obvious color and fluorescence changes in DCP, which indicated the selective detection of DCP can obvious color and fluorescence changes in DCP, which indicated the selective detection of DCP can be achieved by RB-CT, among other conventional organophosphorus compounds. It is noted that be achieved by RB-CT, among other conventional organophosphorus compounds. It is noted that although proton can induce the spirolactam ring opening of the rhodamine molecule [32], RB-CT although proton can induce the spirolactam ring opening of the rhodamine molecule [32], RB-CT exhibited little changes of color and fluorescence intensity in the presence of hydrochloric acid. Thus, exhibited little changes of color and fluorescence intensity in the presence of hydrochloric acid. Thus, the false-positive caused by proton can be effectively avoided under the detection condition with the Molecules 2019, 24, 827 7 of 12 the false-positive caused by proton can be effectively avoided under the detection condition with the addition of 3% Et3N, since the suitable detection pH range of the probe is 7.0 to 10.0 (Figure S6). addition of 3% Et3N, since the suitable detection pH range of the probe is 7.0 to 10.0 (Figure S6). −5 5 Figure 4. (a) Relative fluorescence intensity of RB-CT (1 × 10 M, CH3CN, 3% Et3N) at 583 nm after Figure 4. (a) Relative fluorescence intensity of RB-CT (1  10 M, CH CN, 3% Et N) at 583 nm 3 3 −5 −3 Figure 4. (a) Relative fluorescence intensity of RB-CT (1 × 10 M, CH3CN, 3% Et 3 3N) at 583 nm after the addition of different organophosphorus compounds (1.0 × 10 M). Blank is the CH3CN solution after the addition of different organophosphorus compounds (1.0  10 M). Blank is the CH CN −3 the addition of different organophosphorus compounds (1.0 × 10 M). Blank is the CH3CN solution of RB-CT without Et3N. (b) Relative color change of RB-CT after the addition of different solution of RB-CT without Et N. (b) Relative color change of RB-CT after the addition of different of RB-CT without Et3N. (b) Relative color change of RB-CT after the addition of different organophosphorus compounds under sunlight and UV light. organophosphorus compounds under sunlight and UV light. organophosphorus compounds under sunlight and UV light. 2.5. 2.Practical 5. Practical App Application lication toward toward Real Real Nerve Nerve Agent Agent 2.5. Practical Application toward Real Nerve Agent To confirm in situ and rapid sensing ability of RB-CT toward real nerve agents, we have To confirm in situ and rapid sensing ability of RB-CT toward real nerve agents, we have To confirm in situ and rapid sensing ability of RB-CT toward real nerve agents, we have investigated the response characteristics of the probe to GD in both liquid and gas phase. Clear color investigated the response characteristics of the probe to GD in both liquid and gas phase. Clear investigated the response characteristics of the probe to GD in both liquid and gas phase. Clear color changes under sunlight and fluorescence enhancement irradiated by UV lamp were all observed by color changes under sunlight and fluorescence enhancement irradiated by UV lamp were all observed changes under sunlight and fluorescence enhancement irradiated by UV lamp were all observed by the naked eye immediately in liquid and within 10 min in gas phase (40 ppm GD introduced in a by the naked eye immediately in liquid and within 10 min in gas phase (40 ppm GD introduced in a the naked eye immediately in liquid and within 10 min in gas phase (40 ppm GD introduced in a flask as an aerosol). The remarkable visual differences did not fade or quench after 24 h, which flask as an aerosol) (as shown in Figure 5). The remarkable visual differences did not fade or quench flask as an aerosol). The remarkable visual differences did not fade or quench after 24 h, which indicated the irreversible cyclization had occurred[33]. The above results illustrate the potential after 24 h, which indicated the irreversible cyclization had occurred [33]. The above results illustrate indicated the irreversible cyclization had occurred[33]. The above results illustrate the potential application of RB-CT in the rapid and facile naked-eye detection of nerve agents in both liquid and the potential application of RB-CT in the rapid and facile naked-eye detection of nerve agents in both application of RB-CT in the rapid and facile naked-eye detection of nerve agents in both liquid and vapor phases. liquid and vapor phases. vapor phases. Figure 5. (a) Color change of filter paper treated with RB-CT solution (1.0  10 M in CH CN, 3% Et N) and GD in liquid phase under sunlight (left) and UV light (right). (b) Color change of filter paper treated with RB-CT solution (1.0 10 M in CH CN, 3% Et N) and exposed to GD in gas phase under 3 3 sunlight (left) and UV light (right). 3. Materials and Methods 3.1. Materials All of the solvents were obtained from Beijing Chemical Reagent Company and used without further purification. Rhodamine B, 4-cyanophenyl isothiocyanate and anhydrous magnesium sulfate (MgSO ) were purchased from Aladdin In. Co. (Los Angeles, CA, USA). Diethyl chlorophosphate (DCP) was purchased from Sigma Co. (Louis, MO, USA). Nerve agent GD (the purity is 80 %) was provided by the Research Institute of Chemical Defense of China. Molecules 2019, 24, x 7 of 12 −4 Figure 5. (a) Color change of filter paper treated with RB-CT solution (1.0 × 10 M in CH3CN, 3% Et3N) and GD in liquid phase under sunlight (left) and UV light (right). (b) Color change of filter −4 paper treated with RB-CT solution (1.0 × 10 M in CH3CN, 3% Et3N) and exposed to GD in gas phase under sunlight (left) and UV light (right). 3. Materials and Methods 3.1. Materials All of the solvents were obtained from Beijing Chemical Reagent Company and used without Molecules 2019, 24, 827 8 of 12 further purification. Rhodamine B, 4-cyanophenyl isothiocyanate and anhydrous magnesium sulfate (MgSO4) were purchased from Aladdin In. Co. (Los Angeles, CA, USA). Diethyl chlorophosphate (DCP) was purchased from Sigma Co. (Louis, MO, USA). Nerve agent GD (the purity is 80 %) was 3.2. Measurements provided by the Research Institute of Chemical Defense of China. The ESI mass analysis was performed using an Q Exactive HF-X Hybrid Quadrupole-Orbitrap Mass Spectrometer (Thermo Scientific, Waltham, MA, USA). The H NMR spectra were recorded 3.2. Measurements on AV 400 spectrometer (Bruker, Karlsruhe, Germany). Chemical shifts were expressed in parts per The ESI mass analysis was performed using an Q Exactive HF-X Hybrid Quadrupole-Orbitrap millions () downfield from the internal standard tetramethyl silane and were reported as s (singlet), Mass Spectrometer (Thermo Scientific, Waltham, MA, USA). The H NMR spectra were recorded on d (doublet), bs (broad singlet), t (triplet), and m (multiplet). Absorbance and fluorescence spectra AV 400 spectrometer (Bruker, Karlsruhe, Germany). Chemical shifts were expressed in parts per were recorded at room temperature with a Hitachi U-3900 UV-Visible spectrophotometer and F-4500 millions (δ) downfield from the internal standard tetramethyl silane and were reported as s (singlet), fluorescence spectrophotometer, respectively, using a fluorescence cell of 10 mm path. The excitation d (doublet), bs (broad singlet), t (triplet), and m (multiplet). Absorbance and fluorescence spectra wavelength was set to 540 nm (slit width 5 nm), and emission was monitored from 560–700 nm (slit were recorded at room temperature with a Hitachi U-3900 UV-Visible spectrophotometer and F-4500 width 5 nm). Column chromatography was conducted over silica gel (mesh 100–200). fluorescence spectrophotometer, respectively, using a fluorescence cell of 10 mm path. The excitation wavelength was set to 540 nm (slit width 5 nm), and emission was monitored from 560–700 nm (slit 3.3. Synthetic Procedures width 5 nm). Column chromatography was conducted over silica gel (mesh 100–200). As shown in Scheme 3, compound 1 was synthesized using the reported procedure [34]. Compound 2 was synthesized in a facile way. 3.3. Synthetic Procedures Scheme 3. Synthesis route of RB-TU. Reagents and conditions: (a) CH OH, hydrazine hydrate, 80 C, Scheme 3. Synthesis route of RB-TU. Reagents and conditions: (a) CH3OH, hydrazine hydrate, 80 °C, 6 h; (b) DMF, 4-cyanophenyl isothiocyanate, r.t, 12 h. 6 h; (b) DMF, 4-cyanophenyl isothiocyanate, r.t, 12 h. Synthesis of compound 1: In a 100 mL flask, 1 mL hydrazine hydrate was drop-wise added to a As shown in Scheme 3, compound 1 was synthesized using the reported procedure [34]. solution of rhodamine B (1.67 mmol, 800 mg) in anhydrous CH OH (30 mL), the mixed solution was Compound 2 was synthesized in a facile way. then stirred and heated to 80 °C, and refluxed for 6 h. Pure water (30 mL) was added and the solvent Synthesis of compound 1: In a 100 mL flask, 1 mL hydrazine hydrate was drop-wise added to was extracted with EtOAc (3  60 mL), after the organic phase was dried with anhydrous MgSO a solution of rhodamine B (1.67 mmol, 800 mg) in anhydrous CH3OH (30 mL), the mixed solution and evaporated, orange solid compound 1 (356 mg, 48.8% yield) was obtained, and used without was then stirred and heated to 80 ℃, and refluxed for 6 h. Pure water (30 mL) was added and the further purification. solvent was extracted with EtOAc (3 × 60 mL), after the organic phase was dried with anhydrous The H NMR (400 MHz, DMSO-d6)  7.82 (d, J = 8.3 Hz, 1H), 7.58-7.48 (m, 2H), 7.04 (d, J = 8.0 Hz, MgSO4 and evaporated, orange solid compound 1 (356 mg, 48.8% yield) was obtained, and used 1H), 6.41 (d, J = 17.1 Hz, 6H), 4.32 (s, 2H), 3.36 (d, J = 7.0 Hz, 8H), 1.14 (t, J = 6.9 Hz, 12H) (Figure S7). without further purification. Synthesis of compound 2(RB-TU probe): To a solution of compound 1 (0.22 mmol, 100 mg) in The H NMR (400 MHz, DMSO-d6) δ 7.82 (d, J = 8.3 Hz, 1H), 7.58-7.48 (m, 2H), 7.04 (d, J = 8.0 1.5 mL DMF, a solution of 4-cyanophenyl isothiocyanate (0.31 mmol,50 mg) in 1.5 mL DMF was added, Hz, 1H), 6.41 (d, J = 17.1 Hz, 6H), 4.32 (s, 2H), 3.36 (d, J = 7.0 Hz, 8H), 1.14 (t, J = 6.9 Hz, 12H) (Figure the reaction mixture was stirred for 12 h at room temperature. After the solution evaporated, the S7). residue was purified by flash chromatography (EtOAc: hexane = 1:10) to afford RB-TU probe (36 mg, Synthesis of compound 2(RB-TU probe): To a solution of compound 1 (0.22 mmol, 100 mg) in 26.6% yield). 1.5 mL DMF, a solution of 4-cyanophenyl isothiocyanate (0.31 mmol,50 mg) in 1.5 mL DMF was The H NMR (400 MHz, Chloroform-d)  8.01 (d, J = 7.6 Hz, 1H), 7.72 - 7.57 (m, 3H), 7.44 (d, added, the reaction mixture was stirred for 12 h at room temperature. After the solution evaporated, J = 8.4 Hz, 2H), 7.34 (d, J = 8.5 Hz, 2H), 7.29 (d, J = 7.6 Hz, 1H), 6.97 (s, 1H), 6.47 (d, J = 8.8 Hz, 2H), 6.42 (d, J = 2.5 Hz, 2H), 6.29 (d, J = 2.6 Hz, 1H), 6.27 (d, J = 2.6 Hz, 1H), 3.32 (qd, J = 7.2, 2.5 Hz, 8H), 1.15 (t, J = 7.0 Hz, 12H) (Figure S8). HR-MS (C H N O S): Calcd.: 616.2620; Found: 617.2681(9.9 ppm) (Figure S9). 36 36 6 2 3.4. Reaction Kinetics Study The fluorescence spectra of RB-CT (1.0  10 M) were recorded after the addition of DCP in 4 4 4 3 the concentrations of 4.0  10 M, 5.0  10 M, 6.0  10 M, and 1.2  10 M over a 0–1200 s Molecules 2019, 24, 827 9 of 12 incubation period at room temperature. The observed rate constants k and corresponding half-life obs time t were determined according to Equations (1) and (2). 1/2 ln[(F -F )/F ] = k t (1) max t max obs t = ln2/k (2) 1/2 obs where F and F are maximum fluorescence intensity obtained after the reaction was completed and max t the fluorescence intensities at 583 nm at reaction time t. 3.5. Computational Methods Quantum chemical calculations were carried out by Gaussian 09 package [35]. Geometry was optimized using density functional theory (DFT) functional B3LYP [36–38] and 6–31G (d) basis set [39,40]. Time-dependent density functional theory (TD-DFT) calculation [41] was also performed at the same level of theory. Considering the effects of the environment on the calculated energies, solvation effects were considered throughout geometry optimizations via the self-consistent reaction field (SCRF) and conductor-like polarizable continuum model (CPCM) method [42]. 3.6. Interferents Selectivity and specificity tests were performed with different organophosphorus compounds (OPs), i.e., dimethyl methylphosphonate (DMMP), triphenyl phosphate (TPP), trimethyl phosphate (TMP), acephate, isocarbophos, and dimethoate (Scheme 1). To 2 mL reagent solution of the probe 5 1 molecule (1.0  10 M) in CH CN containing 3% Et N, 20 L stock solution of OPs (1.0  10 M) 3 3 was added. The solutions were incubated at room temperature for 5 min and the fluorescence spectra was recorded. 3.7. The Preparation of Test Kits of Real Nerve Agents in Liquid and Gas Phase The test kits toward GD in liquid and gas phase were produced by the following steps. For liquid phase, a waterman filter paper was soaked in the CH CN solution of RB-CT (1.0  10 M, 3% Et N), 3 3 after the solvent was air-dried, a “GD” logo was patterned by GD. For gas phase, a solution of RB-CT in CH CN (1.0  10 M, 3% Et N) was sprayed onto a filter paper to spell out “GD” first, after the 3 3 solvent was air-dried, the paper was exposed to GD vapor in a flask (40 ppm introduced as an aerosol) for 10 mins. The LCt and LD of Soman are estimated to be 100 mg min/m and 300 mg/individual, 50 50 respectively [43]. Safety Note: Only highly qualified and experienced personnel should work with CWAs employed here, as described in the experimental part below. Due to the high volatility and toxicity of the CWAs, all of the experiments in this part were carried out inside a safety fume hood and operated under efficient ventilation systems. To avoid any risk of CWA inhalation, respiratory protection of involved personnel was provided by protective breathing masks equipped with combined NBC filters. 4. Conclusions In conclusion, we have designed and synthesized a novel probe N-(rhodamine B)-lactam-2-(4-cyanophenyl) thiourea for chromogenic and fluorogenic detection of nerve agents. The probe undergoes an irreversible opening-ring reaction following the form of a seven-membered heterocycle adduct in the presence of DCP, accompanied by the obvious color change from colorless to pink, which has been confirmed by MS analysis and TD-DFT calculations. The response is that instantaneously half-life time is about 280 s at room temperature and the detection limit was found to be 2  10 M. Furthermore, we have demonstrated that the probe is applicable for rapid and facile in situ detection of GD with the naked eye in both liquid and vapor phase. Molecules 2019, 24, 827 10 of 12 Supplementary Materials: The following are available online. Figure S1: The limit of detection for naked eye. Figure S2: Calibration plots by using fluorescence intensity of RB-CT (1 M) as a function of reaction time t in different DCP concentrations. Figure S3: ESI-MS spectrum of RB-CT/DCP adduct. Figure S4: P NMR spectra of DCP and RB-CT with DCP in CD CN. Figure S5: HOMO-LUMO energy levels of TD-DFT optimized geometries of probe and predicted RB-CT/DCP adducts. Figure S6: The plot of pH versus fluorescence intensity of free 1 1 RB-CT and RB-CT+DCP. Figure S7: H NMR Spectrum of Compound 1. Figures S8 and S9: H NMR and ESI-MS Spectra of RB-CT. Author Contributions: Y.Z. and W.C. conceived the idea and designed the experiments; S.L. and Y.Z. performed the synthesis, characterization, and co-wrote the manuscript draft; S.L., H.Z., Y.C., and Z.Z. discussed the data and the mechanisms; M.Z. and C.Z. contributed to spectra analysis; C.Z., J.Z., M.Z., and W.C. edited and revised the manuscript; Y.Z. and M.Z. assisted in DFT and TD-DFT calculation; Authors S.L. and Y.Z. contributed equally. All authors discussed the results and commented on the manuscript. Funding: This research was funded by the National Key Research and Development Program of China (Grant No. 2017YFC0108500). Acknowledgments: The authors thank the financial support of the National Key Research and Development Program of China (Grant No. Grant No. 2017YFC0108500). The authors would like to thank Prof. Dr. Shi-Lu Chen for valuable guidance and discussion about the quantum chemistry calculation, and Dr. Xiang-Xi Meng for discussions in the synthesis and purification process of RB-CT. Conflicts of Interest: The authors declare no conflict of interest. References 1. Szinicz, L. History of chemical and biological warfare agents. Toxicology 2005, 214, 167–181. [CrossRef] [PubMed] 2. Gunderson, C.H.; Lehmann, C.R.; Sidell, F.R.; Jabbari, B. Nerve agents: A review. Neurology 1992, 42, 946–950. [CrossRef] [PubMed] 3. Lei, Z.; Yang, Y. A concise colorimetric and fluorimetric probe for sarin related threats designed via the "covalent-assembly" approach. J. Am. Chem. Soc. 2014, 136, 6594–6597. [CrossRef] [PubMed] 4. Russell, R.J.; Pishko, M.V.; Simonian, A.L.; Wild, J.R. Poly(ethylene glycol) hydrogel-encapsulated fluorophore-enzyme conjugates for direct detection of organophosphorus neurotoxins. Anal. Chem. 1999, 71, 4909–4912. [CrossRef] [PubMed] 5. Russell, A.J.; Berberich, J.A.; Drevon, G.F.; Koepsel, R.R. Biomaterials for mediation of chemical and biological warfare agents. Annu. Rev. Biomed. Eng. 2003, 5, 1–27. [CrossRef] [PubMed] 6. Kim, S.; Cho, B.; Sohn, H. Detection of nerve agent stimulants based on photoluminescent porous silicon interferometer. Nanoscale Res. Lett. 2012, 7, 527. [CrossRef] [PubMed] 7. Sorribes-Soriano, A.; de la Guardia, M.; Esteve-Turrillas, F.A.; Armenta, S. Trace analysis by ion mobility spectrometry: From conventional to smart sample preconcentration methods. A review. Anal. Chim. Acta 2018, 1026, 37–50. [CrossRef] [PubMed] 8. Maresova, E.; Tomecek, D.; Fitl, P.; Vlcek, J.; Novotny, M.; Fiser, L.; Havlova, S.; Hozak, P.; Tudor, A.; Glennon, T.; et al. Textile chemiresistors with sensitive layers based on polymer ionic liquids: Applicability for detection of toxic gases and chemical warfare agents. Sens. Actuator B-Chem. 2018, 266, 830–840. [CrossRef] 9. Walker, J.P.; Kimble, K.W.; Asher, S.A. Photonic crystal sensor for organophosphate nerve agents utilizing the organophosphorus hydrolase enzyme. Anal. Bioanal Chem. 2007, 389, 2115–2124. [CrossRef] [PubMed] 10. He, W.; Liu, Z.; Du, X.; Jiang, Y.; Xiao, D. Analytical application of poly{methyl[3-(2-hydroxy-3,4-difluoro)phenyl]propyl siloxane} as a QCM coating for DMMP detection. Talanta 2008, 76, 698–702. [CrossRef] [PubMed] 11. Kittle, J.D.; Fisher, B.P.; Esparza, A.J.; Morey, A.M.; Iacono, S.T. Sensing Chemical Warfare Agent Simulants via Photonic Crystals of the Morpho didius Butterfly. ACS Omega 2017, 2, 8301–8307. [CrossRef] [PubMed] 12. Roy, K.S.; Mazumder, A.; Goud, D.R.; Dubey, D.K. A simplistic designing of molecularly imprinted polymers for derivative of nerve agents marker using P-31{H-1}NMR. Eur. Polym. J. 2018, 98, 105–115. [CrossRef] 13. Nanda Kumar, D.; Satija, J.; Chandrasekaran, N.; Mukherjee, A. Acetylcholinesterase-based inhibition screening through in situ synthesis of gold nanoparticles: Application for detection of nerve agent simulant. J. Mol. Liq. 2018, 249, 623–628. [CrossRef] Molecules 2019, 24, 827 11 of 12 14. Costero, A.M.; Parra, M.; Gil, S.; Gotor, R.; Mancini, P.M.E.; Martinez-Manez, R.; Sancenon, F.; Royo, S. Chromo-fluorogenic detection of nerve-agent mimics using triggered cyclization reactions in push-pull dyes. Chem. Asian J. 2010, 5, 1573–1585. [CrossRef] [PubMed] 15. Goud, D.R.; Pardasani, D.; Tak, V.; Dubey, D.K. A highly selective visual detection of tabun mimic diethyl cyanophosphate (DCNP): effective discrimination of DCNP from other nerve agent mimics. RSC Adv. 2014, 4, 24645. [CrossRef] 16. Barba-Bon, A.; Costero, A.M.; Gil, S.; Martinez-Manez, R.; Sancenon, F. Selective chromo-fluorogenic detection of DFP (a Sarin and Soman mimic) and DCNP (a Tabun mimic) with a unique probe based on a boron dipyrromethene (BODIPY) dye. Org. Biomol. Chem. 2014, 12, 8745–8751. [CrossRef] [PubMed] 17. Kim, Y.; Jang, Y.J.; Lee, D.; Kim, B.-S.; Churchill, D.G. Real nerve agent study assessing pyridyl reactivity: Selective fluorogenic and colorimetric detection of Soman and simulant. Sens. Actuator B-Chem. 2017, 238, 145–149. [CrossRef] 18. Ali, S.S.; Gangopadhyay, A.; Maiti, K.; Mondal, S.; Pramanik, A.K.; Guria, U.N.; Uddin, M.R.; Mandal, S.; Mandal, D.; Mahapatra, A.K. A chromogenic and ratiometric fluorogenic probe for rapid detection of a nerve agent simulant DCP based on a hybrid hydroxynaphthalene-hemicyanine dye. Org. Biomol. Chem. 2017, 15, 5959–5967. [CrossRef] [PubMed] 19. Cai, Y.-C.; Li, C.; Song, Q.-H. Selective and visual detection of a nerve agent mimic by phosphorylation and protonation of quinolin oximes. J. Mater. Chem. C 2017, 5, 7337–7343. [CrossRef] 20. Royo, S.; Costero, A.M.; Parra, M.; Gil, S.; Martinez-Manez, R.; Sancenon, F. Chromogenic, specific detection of the nerve-agent mimic DCNP (a tabun mimic). Chemistry 2011, 17, 6931–6934. [CrossRef] [PubMed] 21. Pascual, L.; Sayed, S.E.; Martínez-Máñez, R.; Costero, A.M.; Gil, S.; Gaviña, P.; Sancenón, F. Acetylcholinesterase-Capped Mesoporous Silica Nanoparticles That Open in the Presence of Diisopropylfluorophosphate (a Sarin or Soman Simulant). Org. Lett. 2016, 18, 5548–5551. [CrossRef] [PubMed] 22. Sarkar, H.S.; Ghosh, A.; Das, S.; Maiti, P.K.; Maitra, S.; Mandal, S.; Sahoo, P. Visualisation of DCP, a nerve agent mimic, in Catfish brain by a simple chemosensor. Sci. Rep. 2018, 8, 3402. [CrossRef] [PubMed] 23. Chen, L.; Wu, D.; Yoon, J. Recent Advances in the Development of Chromophore-Based Chemosensors for Nerve Agents and Phosgene. ACS Sens. 2018, 3, 27–43. [CrossRef] [PubMed] 24. Royo, S.; Martínez-Máñez, R.; Sancenón, F.; Costero, A.M.; Parra, M.; Gil, S. Chromogenic and fluorogenic reagents for chemical warfare nerve agents’ detection. Chem. Commun. 2007, 4839–4847. [CrossRef] 25. El Sayed, S.; Pascual, L.; Agostini, A.; Martinez-Manez, R.; Sancenon, F.; Costero, A.M.; Parra, M.; Gil, S. A Chromogenic Probe for the Selective Recognition of Sarin and Soman Mimic DFP. ChemistryOpen 2014, 3, 142–145. [CrossRef] [PubMed] 26. Khan, M.S.J.; Wang, Y.-W.; Senge, M.O.; Peng, Y. Sensitive fluorescence on-off probes for the fast detection of a chemical warfare agent mimic. J. Hazard Mater. 2018, 342, 10–19. [CrossRef] [PubMed] 27. Ha, S.; Lee, M.; Seo, H.O.; Song, S.G.; Kim, K.S.; Park, C.H.; Kim, I.H.; Kim, Y.D.; Song, C. Structural Effect of Thioureas on the Detection of Chemical Warfare Agent Simulants. ACS Sens. 2017, 2, 1146–1151. [CrossRef] [PubMed] 28. Kumar, V.; Rana, H.; Raviraju, G.; Garg, P.; Baghel, A.; Gupta, A.K. Chromogenic and fluorogenic multianalyte detection with a tuned receptor: refining selectivity for toxic anions and nerve agents. RSC Adv. 2016, 6, 59648–59656. [CrossRef] 29. Yang, Y.K.; Keunjeong Yook, A.; Tae, J. A Rhodamine-Based Fluorescent and Colorimetric Chemodosimeter 2+ for the Rapid Detection of Hg Ions in Aqueous Media. J. Am. Chem. Soc. 2005, 127, 16760–16761. [CrossRef] [PubMed] 30. Goswami, S.; Manna, A.; Paul, S. Rapid ‘naked eye’ response of DCP, a nerve agent simulant: from molecules to low-cost devices for both liquid and vapour phase detection. RSC Adv. 2014, 4, 21984–21988. [CrossRef] 31. So, H.-S.; Angupillai, S.; Son, Y.-A. Prompt liquid-phase visual detection and low-cost vapor-phase detection of DCP, a chemical warfare agent mimic. Sens. Actuators B: Chem. 2016, 235, 447–456. [CrossRef] 32. Ramette, R.W.; Sandell, E.B. Rhodamine B Equilibria. J. Am. Chem. Soc. 1956, 78, 4872–4878. [CrossRef] 33. Gotor, R.; Gaviña, P.; Ochando, L.E.; Chulvi, K.; Lorente, A.; Martínez-Máñez, R.; Costero, A.M. BODIPY dyes functionalized with 2-(2-dimethylaminophenyl)ethanol moieties as selective OFF–ON fluorescent chemodosimeters for the nerve agent mimics DCNP and DFP. RSC Adv. 2014, 4, 15975–15982. [CrossRef] Molecules 2019, 24, 827 12 of 12 34. Yang, X.-F.; Guo, X.-Q.; Zhao, Y.-B. Development of a novel rhodamine-type fluorescent probe to determine peroxynitrite. Talanta 2002, 57, 883–890. [CrossRef] 35. Frisch, M.J.; Trucks, G.W.; Schlegel, H.B.; Scuseria, G.E.; Robb, M.A.; Cheeseman, J.R.; Scalmani, G.; Barone, V.; Mennucci, B.; Petersson, G.A.; et al. Gaussian 09, Revision D. 01; Gaussian. Inc.: Wallingford, CT, USA, 2009. 36. Lee, C.; Yang, W.; Parr, R.G. Development of the Colle-Salvetti correlation-energy formula into a functional of the electron density. Phys. Rev. B 1988, 37, 785–789. [CrossRef] 37. Becke, A.D. A new mixing of Hartree–Fock and local density-functional theories. J. Chem. Phys. 1993, 98, 1372–1377. [CrossRef] 38. Becke, A.D. Density-functional thermochemistry. III. The role of exact exchange. J. Chem. Phys. 1993, 98, 5648–5652. [CrossRef] 39. Hehre, W.J.; Ditchfield, R.; Pople, J.A. Self—Consistent Molecular Orbital Methods. XII. Further Extensions of Gaussian—Type Basis Sets for Use in Molecular Orbital Studies of Organic Molecules. J. Chem. Phys. 1972, 56, 2257–2261. [CrossRef] 40. Francl, M.M.; Pietro, W.J.; Hehre, W.J.; Binkley, J.S.; Gordon, M.S.; DeFrees, D.J.; Pople, J.A. Self-consistent molecular orbital methods. XXIII. A polarization-type basis set for second-row elements. J. Chem. Phys. 1982, 77, 3654–3665. [CrossRef] 41. Bauernschmitt, R.; Ahlrichs, R. Treatment of electronic excitations within the adiabatic approximation of time dependent density functional theory. Chem. Phys. Lett. 1996, 256, 454–464. [CrossRef] 42. Ming, W.W.; Wiberg, K.B.; Frisch, M.J. Solvent effects. 3. Tautomeric Equilibria of Formamide and 2-pyridone in the Gas Phase and Solution: an ab Initio SCRF Study. J. Am. Chem. Soc. 1992, 114, 1645–1652. [CrossRef] 43. Sadik, O.A.; Land, W.H., Jr.; Wang, J. Targeting Chemical and Biological Warfare Agents at the Molecular Level. Electroanalysis 2003, 15, 1149–1159. [CrossRef] Sample Availability: Samples of compounds 1–2 are available from the authors. © 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

Journal

Molecules – Multidisciplinary Digital Publishing Institute

Published: Feb 26, 2019

There are no references for this article.