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DOI: 10.1039/D0CC03985C (Communication) Chem. Commun., 2020, 56, 9308-9311

Selective DCP detection with xanthene derivatives by carbonyl phosphorylation†

Kanhu Charan Behera ab and Bamaprasad Bag *a
aMaterials Chemistry Department, CSIR-Institute of Minerals and Materials Technology, P.O.: R.R.L., Bhubaneswar-751013, Odisha, India. E-mail: bpbag@immt.res.in; Fax: +91 674 258 1637; Tel: +91 674 237 9254
bDepartment of Chemistry, Utkal University, Bhubaneswar-751004, Odisha, India

Received 7th June 2020 , Accepted 7th July 2020

First published on 7th July 2020

Abstract

Rhodamine derivatives (1–2) exhibited dual channel ‘turn-on’ photophysical signalling selectively with diethyl chlorophosphate (DCP) among various organophosphates (OPs), where the spiro-ring opening corresponds to the adduct formation through phosphorylation at their carbonyl O-spiro nucleophilic centres.

The vicious exploitation of chemical warfare nerve agents1 against mankind has increasingly raised safety concerns. Nerve agents, particularly organophosphates, inflict an inhibition effect on acetylcholine esterase leading to critical nervous system damage, thus they are considered to be highly toxic.2 The usage of organophosphates as pesticides and herbicides inflicts adverse effects3 on living organisms. Diethyl-chlorophosphate (DCP) is prominent among organophosphates due to its chemical reactivity based utility, its role as a reagent in hydroxyl activation in chemical reactions, its role as an intermediate in the synthesis of various insecticides, and because it is less toxic in comparison to organophosphate based pesticides; it is also used as a nerve agent stimulant due to its comparable reactivity. Therefore, selective detection and quantitative estimation of organophosphates and their degraded products, DCP in particular, have continued to be a highly desirable and indispensable objective.4 Various methodological explorations have been adapted for the detection of DCP; however, implementation of such techniques is jeopardized due to associated limitations. Analyte detection with suitable design of molecular probes responsive through fluorescent and colorimetric signals has already proven5 to overcome such limitations. The trending literature6–9 highlights that the analytical pathway for the detection of organophosphates and their degraded products primarily follows electrophilic phosphorylation depending upon their molecular structures, where cleavage of the phosphorus–halogen bond is facilitated by various nucleophilic reagents. Therefore, the design of such molecular probes usually comprises a nucleophilic receptor (as amino-, hydroxyl and oxime groups) that can selectively and sensitively recognize the targeted organophosphates through effective interaction and the recognition event is translated to the signaling subunit through perturbation of operative photophysical processes. Most of these ‘signaling subunit – recognition site’ ensembles for the detection of DCPs are reported to undergo phosphorylation at the hydroxyl6 or amino7 groups available on the receptor segment.

A few xanthene dye based chemosensors10 were also explored in the detection of DCP.8,9 Their methodological design incorporates a nucleophilic centre in the form of a hydroxyl (–OH) group8 or an amino-functionality9 primarily located at the substituents coupled to the signaling subunits. Their structural arrangements and the associated mechanism of DCP mediated spiro-ring opening led us to study the role of substituents as an alternative approach to probe design for such phosphorylation. Xanthene derivatives 1–3 were chosen as molecular probes in this study (Fig. 1), where the nucleophilic centres in their signaling units are the basis of their design and their photophysical spectral responses towards various pesticides were the signal monitoring window. The structural aspect of their design is such that the amino-phosphorylation could be avoided though non-availability of amino-protons on the substituents at their hydrazide end through unsaturated coupling of substituents as an imine linkage. Further, the substituent in the rhodamine-6G hydrazide derivative 1 did not contain a –OH group, whereas that in 2 contained an aromatic –OH group. In contrast, no hydroxyl group (–OH) was made available on the substituent attached to the fluorescein hydrazide derivative 3, rather it was present on the xanthene ring. On the basis of the present approach of probe design and with subsequent structure–function correlation, 1 is expected to show an abridged response on interaction with organophosphates, whereas 2 and 3 are arguably responsive through a preferential phosphorylation at the respective hydroxyl sites. In this investigation, in contrast, we report that 1 and 2 exhibited, and 3 failed to show, selective detection of DCP as reflected through the DCP-mediated opening of the spiro-ring, which translated into the corresponding photophysical signals with dual channel chromogenic and fluorogenic signaling as the monitoring window.


image file: d0cc03985c-f1.tif
Fig. 1 Xanthene dye based compounds (1–3) used in this study and the corresponding ORTEP diagram of their crystal structures. 40% ellipsoids; H-atoms are omitted for clarity.

The reaction of rhodamine-6G hydrazide with 4-diethylamino-benzaldehyde and 4-(diethylamino)salicylaldehyde resulted in 1 and 2,11 respectively, and a similar reaction between fluorescein hydrazide and trans-(diethyl amino)-cinnamaldehyde resulted in 3. These probes were characterized by using 1H-NMR, 13C-NMR and ESI-MS spectroscopy (ESI†). The molecular ion peaks ([m/z]+) observed at 588.48[M + 1]+, 604.50[M + 1]+, and 532[M + 1]+ in the ESI-MS spectra of 1, 2 and 3, respectively, established their formation, as also supported by the carbonyl stretching frequencies (∼1707 cm−1) in their FT-IR spectra. The characteristic quaternary carbon peak of the spirocyclic ring (C13) in their 13C-NMR spectra (δ ∼ 65.6 ppm, DMSO-d6) and structural elucidation of their single crystals‡ confirmed their existence in their spirocyclic forms (Fig. 1). No significant peak was observed in the 450–650 nm region in their absorption or fluorescence spectra, rationalized to their spirocyclic structure.

In order to investigate the guest-mediated photophysical signalling responses, various organophosphate as well as non-organophosphate based pesticides were individually added to the solution of 1 and 2 in EtOH–H2O (1[thin space (1/6-em)]:[thin space (1/6-em)]1 v/v, 0.1 M PBS, pH 7.1) and to a solution of 3 in DMSO (ESI†). Among all the analytes added, 1 exhibited (Fig. 2a) an absorption peak with a maximum at 533 nm in the selective presence of DCP (log[thin space (1/6-em)]ε[1+DCP] = 4.77) with a concurrent colour change of the solution (colourless to pink). Similarly, excitation of 1 led to the appearance of a fluorescence peak (λem = 553 nm) in the selective presence of DCP among all analytes under investigation (Fig. 2b). The observed dual mode colorimetric (colourless → pink, ε[1+DCP]/ε1 = 86) and fluorometric ((IF/I0)553 = 124, ϕF = 0.611, τav = 3.71 ns (96.7%)) signal transduction in 1 is assignable to that of its DCP induced ring-opened conformation. 2 also exhibits similar absorbance (ε[2+DCP]/ε2 = 55) and fluorescence ((IF/I0)553 = 113, ϕF = 0.547, τav = 3.23 ns (80%)) spectral amplification in the selective presence of DCP among all analytes. Except for DCP, the other analytes failed to induce any such photophysical spectral transformation in both 1 and 2.


image file: d0cc03985c-f2.tif
Fig. 2 (a) Absorption and (b) fluorescence spectra of 1 and (inset b) the FE factor in 1 and 2 (ESI†) with various analytes in buffered EtOH (1[thin space (1/6-em)]:[thin space (1/6-em)]1 v/v, 0.1 M PBS) medium. λex (fluo.) = 480 nm [probe] = 10 μM (abs.), 1 μM (flu.), [analyte] = 10–50 μM.

Similar DCP-induced spectral enhancements in the 450–650 nm region were not observed with 3. However, the addition of DCP to 3 caused (a) a decrease in the intensity of its characteristic absorption at 405 nm, and (b) an increase in the absorption at 345 nm with an isosbestic point at 360 nm. This indicates that the interaction of 3 with DCP, which failed to open its spiro-ring, still formed a non-fluorescent (ϕF ≤ 0.001 for 3, 3-DCP) adduct in a different coupling mode12 that exhibited a signalling response in a different output channel. Such photophysical responses of 3 were observed to be selective for DCP among the analytes studied.

The plot of absorbance of 1 or 2versus the mole fractions of the added DCP (Job's plot, λobs = 533[thin space (1/6-em)]nm) indicated a 1[thin space (1/6-em)]:[thin space (1/6-em)]1 (probe[thin space (1/6-em)]:[thin space (1/6-em)]DCP) complexation stoichiometry (ESI†). The absorption titration of 1 (10 μM) with DCP in EtOH–H2O (1[thin space (1/6-em)]:[thin space (1/6-em)]1 v/v, 0.1 M PBS, pH = 7.1) revealed that its DCP-induced A533 absorption peak gradually increased (Fig. 3a) with the gradual addition of DCP and remained constant thereafter, with a concurrent colour change in the solution. A similar trend in the spectral enhancement against the concentration of the added DCP was observed in its fluorescence spectra (Fig. 3b). Similar spectral patterns were observed for 2 on titration with DCP. The higher association constant (Ka) derived (ESI†) from the fluorescence titration of 1 (log[thin space (1/6-em)]Ka = 10.58) and 2 (log[thin space (1/6-em)]Ka = 10.71) with DCP inferred an effective probe–analyte interaction. The determined Ka values obtained from the absorption spectral titrations complement well those obtained from fluorescence titrations, and are correlated with a factor [{log[thin space (1/6-em)]Ka(fluo.)/log[thin space (1/6-em)]Ka(abs.)} = 1.27 (in 1) and 1.05 (in 2)] close to unity. The limit of detection of DCP with 1 and 2 was calculated to be 1.36 μM and 26 μM, respectively (ESI†), a concentration range adequate for their practical applications. Similarly, the respective limit of quantification of 1 and 2 was estimated to be 4.53 μM and 86.66 μM.


image file: d0cc03985c-f3.tif
Fig. 3 (a) Absorption and (b) fluorescence spectral patterns and (inset, a and b) the corresponding profiles of 1 as a function of added DCP in buffered EtOH (1[thin space (1/6-em)]:[thin space (1/6-em)]1 v/v, 0.1 M PBS). (Inset a): absorbance profile of 1 supplemented with DCP (1[thin space (1/6-em)]:[thin space (1/6-em)]1) versus time (s). Abs.: [1] = 10 μM; λobs = 533 nm; Flu.: [1] = 1 μM, λex = 480 nm, em./ex. (b) p = 5 nm.

The rate of DCP-mediated spiro-ring opening in 1 (k = 0.003 s−1) and 2 (k1 = 0.02 s−1, k1 = 0.0015 s−1) that followed first order kinetics inferred a faster response time of signalling. Interestingly, the A533 absorption in 2 on addition of DCP first increased and then decreased till saturation, indicative of different patterns of adduct formation in 1 and 2, respectively. The DCP did not open the spiro-ring in 3 but exhibited quenching of its A405 transition (k = 0.036 s−1), inferring formation of an 3 DCP adduct dissimilar to those of 1 and 2.

In general, rhodamine based probes are susceptible emitting false positive signals in analyte detection through proton mediated spiro-ring opening; therefore, the photophysical spectral profiles of 1 and 2 and those of their corresponding DCP adducts were studied at varying pH (ESI†). The spectral amplifications in both 1 and 2 were observed at a lower pH (2–5 pH range), attributed to their spirocyclic ring opening in an acidic environment. Their spectral patterns alone and in the presence of DCP at 6–10 pH inferred the stability of their DCP-mediated enhanced signals, without interference of protons. The control experiments with 1 and 2 upon the addition of trifluoroacetic acid (TFA) in varied proportions in buffered EtOH ruled out the proton mediated interference in their DCP selectivity (ESI†).

The influence of other analytes on the DCP selectivity of 1 was assessed by measuring the absorbance at 533 nm of solutions containing (a) 1 and individual pesticides followed by the addition of DCP, and (b) 1 and DCP followed by the addition of those analytes. Addition of DCP to individual solutions containing 1 and other analytes caused a strong absorption peak (A533) in each case to a comparable extent to that observed for 1 in the presence of DCP alone (ESI†). In contrast, addition of any of the interfering analytes to the solution containing 1 and DCP exhibited no or negligible changes to its DCP-induced absorption (A533), which ascertained that the detection of DCP is not affected by the presence of these analytes investigated here. The fluorescence spectra also showed a similar signaling pattern. The DCP induced phosphorylation at the carbonyl site in 1 and 2 may be susceptible to cross-selectivity toward compounds containing similar electrophilic centres. Therefore, the absorption spectral responses of both 1 and 2 were measured in the presence of p-toluene sulfonyl chloride (TsCl) and benzoyl chloride (BzCl). Addition of TsCl and BzCl to 1 (ε1+TsCl = 1154, ε1+BzCl = 5695 dm3 mol−1 cm−1) or 2 (ε2+TsCl = 10[thin space (1/6-em)]489, ε2+BzCl = 29[thin space (1/6-em)]894 dm3 mol−1 cm−1) was observed to result in electrophilic tosylation/benzoylation and show spiro-ring opening turn-on signalling; however, the extent of absorption spectral enhancements with TsCl and BzCl is significantly lower in comparison to that on addition of DCP (ε1+DCP = 58[thin space (1/6-em)]634, ε2+DCP = 97[thin space (1/6-em)]525 dm3 mol−1 cm−1) under similar experimental conditions (ESI†). These control experiments not only supported the DCP-mediated electrophilic phosphorylation at the carbonyl end of 1 and 2, but also ascertained their selectivity towards DCP over other interfering electrophilic compounds (TsCl and BzCl). Therefore, these probes are suitable enough to detect DCP selectively among interfering structurally related organophosphates and other electrophilic species. The effective detection of DCP with 1 was demonstrated in solutions of soil samples through naked-eye observation with a paper-strip/TLC plate methodology, where interaction of 1 (and 2 also) with DCP led to a significant colour change (ESI†) for visual detection.

The DCP induced down-field shifts of aromatic protons on the xanthene core of 1 and those of its imino-protons in its 1H NMR spectrum showed phosphorylated adduct formation. The quaternary carbon (C9) peak at 65.29 ppm in the 13C NMR (DMSO-d6) spectrum of 1 corresponding to the spirocyclic conformation disappeared for the in situ1-DCP complex. This confirmed the xanthene spiro-ring opening during 1-DCP formation. ESI-MS analysis with DCP revealed the phosphorylation reaction with the loss of HCl, and the formation of the diethyl phosphate derivative of 1 in a 1[thin space (1/6-em)]:[thin space (1/6-em)]1 (probe[thin space (1/6-em)]:[thin space (1/6-em)]DCP) stoichiometry. Further, the FT-IR peak corresponding to the spiro-cyclic carbonyl stretch of 1 was not observed in 1-DCP. The imino C[double bond, length as m-dash]N stretch of its amide form, P[double bond, length as m-dash]O stretch and P–O–C stretch frequencies were observed (ESI†), which ascertained the formation of the diethyl phosphate derivative 1 in its ring-opened conformation. The spectroscopic observations supported the DCP-induced spiro-ring opening in 1 (Fig. 4), and, therefore, complemented that observed through photophysical spectral responses for DCP detection.


image file: d0cc03985c-f4.tif
Fig. 4 Mechanism of phosphorylation in the formation of 1-DP and the observed colour change in the solution of 1.

The energy minimized structures of the probes and the analytes were compared in order to understand the probe's preferential adduct formation with DCP over various analytes used in this study (ESI†). The energies of the HOMO and the LUMO in 1 in the ground state were calculated to be −4.79 eV and −1.29 eV, respectively, estimating the energy of the HOMO–LUMO transition in 1 to be 3.49 eV. The HOMO–LUMO energy gap in the ground state corresponds to absorption transitions, and it was calculated at 354 nm in 1. Similarly, the HOMO (−4.77 eV)–LUMO (−1.20 eV) energy gap (3.56 eV) corresponds to an absorption transition at 347 nm in 2. The estimated absorption transitions in both 1 and 2 are in good agreement with the observed experimental absorption peaks (±25 nm). The absence of any HOMO → LUMO transition in the 400–600 nm region in the elucidated structures of 1 and 2 also inferred the existence of the rhodamine spirocyclic conformation. In the diethyl phosphate appended 1 (1-DP), the energy of the HOMO (−4.60 eV) was found to be slightly increased and that of the LUMO (−1.73 eV) was more stabilized in comparison to those in 1, due to the enhanced conjugation in the ring-opened conformation of the 1-DP adduct. As a result, the HOMO–LUMO energy gap (2.86 eV) in the 1-DP adduct corresponds to a red-shifted absorption transition at 433 nm. The formation of the diethyl phosphate derivative of ring-opened 1 was evidenced from the spectroscopic results. It is assumed that the reaction of 1 as a Lewis base with DCP as a Lewis acid undergoes reorganization prior to the formation of the 1-DP adduct where the P–O bond formation compensates highly their combined reorganization energies. The stability of the newly formed P–O bond in 1-DP depends predominantly on the interacting parameters of the two involved reactants, 1 and DCP. The stabilized energy of formation in the energy-minimized structure calculation of 1-DP ascertained that as well. In this context, the chemical potential and the HOMO–LUMO energy gap of the interacting molecules may be considered as the index of chemical reactivity and subsequent stability of the adduct formed. A correlation of various parameters observed (ESI†) for 1 and the analytes, despite being presumed to follow a pre-organizational approach prior to reacting for new adduct formation, predicted the reaction preferences of 1 towards DCP. (a) The HOMO–LUMO energy gap in DCP (8.06 eV) was observed to be higher than those of the other analytes whereas that of 1 (3.49 eV) was lower in comparison to those of all analytes studied. (b) The difference in calculated chemical potentials between 1 (3.04 eV) and DCP (4.29 eV) was found to be higher than those in the cases of the other analytes. (c) The ground state dipole moment (μg) was estimated to be higher in DCP (4.64 D) than in the other analytes and lower than that in 1 (4.98 D), which correlates to a higher affinity of DCP for adduct formation representing a lower difference in μg between 1 and DCP than those between 1 and the other analytes. (d) The Mulliken charge over the P-atom in DCP (1.1811e) was calculated to be higher than those over P-atoms in other organophosphate analytes, in correlation with the charge over the spiro-cyclic carbonyl O-atom (−0.5789e) in 1. Although theoretically calculated molecular behavior in gas phase would diverge with those in solutions, the correlations support the probe's preferences towards DCP as observed from their photophysical signals.

In summary, 1 and 2 were shown to exhibit selective and sensitive ‘turn-on’ signaling with DCP among various organophosphates, through their signature spectral features corresponding to the DCP-induced spiro-ring opening whereas 3 did not show such spectral patterns. The study also revealed the formation of diethyl phosphate derivatives of the probes (1 and 2) through phosphorylation at the carbonyl end on their spirolactam in their spiro ring-opened conformation, endorsing the methodology of probe design for DCP detection.

BPB wishes to thank the Director, CSIR-IMMT Bhubaneswar for requisite permissions and financial support through a CSIR grant (CSIR-IMMT-MLP-040).

Conflicts of interest

There are no conflicts to declare.

Notes and references

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Footnotes

† Electronic supplementary information (ESI) available: Detailed synthetic procedure, characterization of 1–3 and their DP-adducts (crystallographic, 1H, 13C NMR, ESI-MS, FT-IR), absorption and emission spectral data, theoretical calculations. CCDC 1963358, 1963360 and 1978815. For ESI and crystallographic data in CIF or other electronic format see DOI: 10.1039/d0cc03985c
‡ Crystallographic data: The CCDC no. for 1, 2 and 3 are 1963360, 1963358 and 1978815,† respectively.
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