Efficient Decontamination of Organophosphate-Based Pesticides and Nerve Agent Simulants Mediated by N‑Containing Nucleophiles

Abstract

There is a growing interest in developing safer and more effective decontaminating agents for organophosphate-based pesticides and nerve agents. In this study, we present an effective method for the nonaqueous decontamination of these compounds using small-molecule-based decontaminating agents under ambient conditions. Our approach utilizes aryl and heteroaryl carboxaldehyde hydrazones and hydrazides to effectively hydrolyze nerve agent simulants into their nontoxic degradation products. The effectiveness of this method was evaluated using a range of nerve agent simulants, including dimethyl 4-nitrophenyl phosphate (DMNP), dimethyl methylphosphonate (DMMP), and triphenyl phosphate (TPhP). In the presence of the heteroaryl hydrazone, the rate of hydrolysis was enhanced by 116-, 1930-, and 2490-fold relative to the uncatalyzed hydrolysis of TPhP, DMNP, and DMMP, respectively. Our findings demonstrate the potential of aryl carboxaldehyde hydrazones and hydrazides for the instantaneous and effective decontamination of nerve agents. These results are further substantiated by GIAO–DFT calculations. Additionally, the regioselectivity of the nucleophiles in the degradation of simulants to nontoxic products at alkaline pH (≥9.5) is elucidated.

Introduction

Nerve agents (NAs) are a class of highly toxic chemicals that pose grave threats to the central nervous system, inducing severe cholinergic effects that can lead to incapacitation or death. Over the last century, and particularly since World War I, organophosphorus (OP) compounds have been synthesized for both pesticide and chemical warfare applications. , Despite international laws prohibiting their production, accumulation, and use, organophosphorus nerve agents - such as Tabun (GA, N,N-dimethyl ethyl phosphoramidocyanidate), Sarin (GB, isopropyl methylphosphonofluoridate), Soman (GD, 3,3-dimethyl-2-butyl methylphosphonofluoridate), and VX (O-ethyl S-[2-(diisopropylamino)­ethyl] methylphosphonothioate) (Figure )continue to pose a global threat due to their extreme toxicity and limited treatment options. , This threat is exacerbated by the potential for accidental or deliberate release as chemical warfare agents (CWAs) against civilians and the military, as exemplified by recent incidents in Germany (2020), the U.K. (2018), Malaysia (2017), Syria and Iraq (2013), Japan (1995), and the Iraq-Iran war (1980–1988).

1.

Structures of organophosphorus G-type and VX nerve agents.

Nerve agents exert their devastating effects rapidly upon skin contact, inhalation, or ingestion due to their ability to chemically react with cholinesterase at its catalytic site through O-phosphorylation of the serine-203 residue in acetylcholinesterase (AChE). This inhibits AChE from promptly inactivating acetylcholine, leading to prolonged and dysregulated neurotransmission.

Pesticides are widely applied in agriculture due to their high efficacy and ease of production, substantially improving crop yield and quality. However, their misuse has led to persistent residues in the environment and food, raising serious concerns about ecological and human health. Thiram, a commonly used dithiocarbamate fungicide, is particularly notable for its environmental persistence, toxicity, and potential to cause developmental and immune system harm. While pesticides are generally less immediately toxic to AChE based on known LD₅₀ values in nonhuman animal models compared to nerve agents, their widespread use and insufficient monitoring remain significant concerns. Consequently, there is a growing need for sensitive, low-cost, and on-site detection methods to ensure food safety and environmental protection. In parallel, there is increasing interest in developing safer and more effective decontaminating agents for organophosphate-based pesticides and nerve agents to mitigate their impact and minimize the risk of secondary exposure, especially given the challenges in rapidly identifying a broad range of chemical warfare agents (CWAs).

Hydrolysis studies of nerve agents in hot-compressed water, in single surfactant solutions, , and in the presence of amines, − including long-chain and polymeric amines, , have been extensive, with the resulting hydrolytic products being less toxic than the parent agents. However, research in nonaqueous media with catalytic amounts of water remains scarce. Additionally, several studies have focused on binding materials that can adsorb and destroy airborne CWAs under ambient conditions, as well as those that can be used for treatment in contaminated environments, including activated carbon, metal oxides, − zeolites, and metal–organic frameworks, − to immobilize and destroy CWAs through dissociative adsorption. Reported results for solid-phase reagents have also shown their effectiveness in the adsorption and degradation of CWAs in the aerosol or vapor state; − however, their practicality in decontaminating concentrated CWA stockpiles is limited, prompting the exploration of small-molecule amines as catalysts for on-site hydrolysis. Nitrogen-containing bases, such as imidazole derivatives and oximate anions, have shown strong catalytic activity in hydrolyzing organophosphorus agents like Paraoxon, , and Diethyl 2,4-dinitrophenyl phosphate (DEDNPP) via P–O bond cleavage. Notably, oximate anions can accelerate hydrolysis rates by up to 10⁷-fold over spontaneous reactions, highlighting the potential of α-nucleophiles in developing broad-spectrum OP antidotes.

Due to the high toxicity of CWAs and legal restrictions on live agents, research on decontamination technologies often relies on the use of CWA simulants. These simulants are safer to use in the lab due to their lower toxicity and volatility and are also stable, crystalline salts that are easily prepared with longer half-lives. However, no single simulant can mimic all the chemical properties of an OP CWA due to varying chemical structures and hydrolysis conditions, necessitating careful selection based on chemical similarity and interaction strengths with decontaminants. Commonly used simulants for G-type and VX nerve agents include dimethyl 4-nitrophenyl phosphate (DMNP, 1), dimethyl methylphosphonate (DMMP, 2), and diethyl chlorophosphate (DCP, 4) (Figure ), which employ similar modes of action and target the central nervous system. −

2.

Structures of CWA simulants: dimethyl 4-nitrophenylphosphate (DMNP, 1), dimethyl methylphosphonate (DMMP, 2), triphenyl phosphate (TPhP, 3), and diethyl chlorophosphate (DCP, 4). (Simulants 1–3 have been used in this study.)

In our previous work, we demonstrated that DFT calculations and computational modeling align with the pH-dependent catalytic degradation of DMNPa CWA simulantin the presence of aminoguanidine-derived aldimines. Herein, we build on this prior work, exploring the design and synthesis of nontoxic small-molecule bases for the catalytic hydrolysis of CWA simulants DMNP (1), DMMP (2), and triphenyl phosphate (TPhP, 3) (Figure ) in nonaqueous media with catalytic amounts of water.

Our approach utilizes aryl and heteroaryl carboxaldehyde hydrazones and hydrazines to effectively hydrolyze the simulants into nontoxic degradation products. Hydrazones, in particular, are of pharmaceutical importance due to their diverse biological activities and therapeutic potential. Their roles as antimicrobial, anticancer, antitubercular, antioxidant, antiviral, and anti-inflammatory agents, among other properties, make them valuable in drug development for a wide range of diseases. − This broad spectrum of activities underscores the potential of hydrazones as versatile compounds in both pharmaceutical applications and chemical warfare agent decontamination strategies. It is hypothesized that hydrazones exhibit higher reactivity than hydrazides, hydrazines, and uncatalyzed reactions for CWA decontamination and that the electron-withdrawing substituents in the heteroaryl and aryl rings would enhance their hydrolytic reactivity. This is presumably due to an increase in the acidity of the hydrazone moiety, thereby rendering it more nucleophilic toward the phosphonyl group of pesticides or nerve agents.

In a comparative study, we conducted a detailed investigation of six different small-molecule bases (Figure ), each containing an active lone pair of electrons on a nitrogen atom, to facilitate the catalytic degradation and detoxification of CWA simulants DMNP, DMMP, and TPhP at a precise pH. These molecules were selected based on the presence of nucleophilic nitrogen centers, electronic effects of their aromatic systems, structural rigidity, basicity (as in the cases of DBN and DBU), potential for hydrogen bonding, and their practical availability and stability under field conditions. Through a combination of NMR and ultraviolet–visible (UV–vis) spectroscopic studies, along with DFT calculations, we found that hydrolysis in the presence of aryl hydrazones–particularly, 4-pyridine carboxaldehyde hydrazone (5c) - was significantly faster. The observed rate enhancement, calculated as the ratio of the half-lives of the uncatalyzed and catalyzed reactions, was on the order of 10²–10⁴-fold compared to uncatalyzed hydrolysis, as well as relative to nucleophilic counterparts with significantly greater basic strength.

3.

Structures of investigated bases/nucleophiles: DBN (5a), DBU (5b), 4-pyridine aldehyde hydrazone (5c), benzaldehyde hydrazone (5d), phenyl hydrazine (5e), and benzoic acid hydrazide (5f).

Our findings have significant implications for the development of tailored decontamination strategies against chemical warfare agents, specifically designed for various pH conditions and buffer systems. Moreover, the demonstrated efficacy of the tested aryl aldehyde hydrazones in degrading diverse CWA simulants in both aqueous and nonaqueous media highlights their potential applications across the defense, public safety, and environmental protection sectors, including their integration into chemical protective clothing.

Experimental Section

Materials

Dimethyl sulfoxide-d ₆ (DMSO-d ₆, ≥ 99.0%), 1,8-diazabicyclo[5.4.0]­undec-7-ene (DBU, >98.0%), 1,5-diazabicyclo[4.3.0]­non-5-ene (DBN, >98.0%), 4-pyridine carboxaldehyde (C₆H₅NO, ≥ 99.0%) were purchased from A2B Chemicals (America) and used without further purification. The following chemicals were also purchased from commercial vendors: dimethyl 4-nitrophenyl phosphate (DMNP, C₈H₁₀NO₆P, ≥99.0%, TCL, America), dimethyl methyl phosphonate (DMMP, C₃H₉O₃P, >98.0%, SynQuest Laboratories), triphenyl phosphate (TPhP, C₁₈H₁₅O₄P, >99.0%, TCL, America), hydrazine hydrate (C₂H₈N₄O₃, 64 wt % in H₂O, Fisher Scientific), dichloromethane (CH₂Cl₂, ≥98.0, Fisher Scientific), ethanol (C₂H₆O, ≥ 98.0%, Fisher Scientific), glycine (C₂H₅NO₂, ≥99.0%, Fisher Scientific), sodium hydroxide (Sigma-Aldrich), sulfuric acid (H₂SO₄, ≥99.5%, Fisher Scientific), chloroform-d (CDCl₃, ≥99.0%, Fisher Scientific), magnesium sulfate anhydrous (MgSO₄, ≥99.9%, Macklin), 2-amino-2-methyl-1,3-propanediol (C₄H₁₁NO₂, ≥98.0%), boric acid (BH₃O₃, ≥99.5%, Fisher Scientific), phenyl hydrazine (C₆H₈N₂, ≥97%, Sigma-Aldrich), benzaldehyde (C₇H₆O, ≥99.0%, Fisher Scientific), and benzoic acid (C₇H₆O₂, ≥99.0%, Fisher Scientific).

Methods

4-Pyridine carboxaldehyde hydrazone (5c), benzaldehyde hydrazone (5d), and benzoic acid hydrazide (5f) were synthesized according to reported methods with some modifications. −

Synthesis of 4-Pyridine Carboxaldehyde Hydrazone (5c)

A mixture of 4-pyridine carboxaldehyde (0.5 g, 4.7 mmol) and hydrazine hydrate 64% (0.61 g, 12.2 mmol) in dichloromethane (10 mL) was added to 10% (v/v) hydrochloric acid (1.0 mL) at room temperature. The mixture was refluxed (oil bath at 70 °C) with continuous stirring for 6 h. The obtained solution was cooled to room temperature and neutralized with 0.1 M NaHCO₃. The crude mixture was concentrated under vacuum; water was then added to the residue and extracted with dichloromethane (3 × 10 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered and the solvent was evaporated under vacuum to give to give a pale-yellow liquid (1.05 g, 8.67 mmol, 87%). The ¹H and ¹³C NMR spectra are given in the Supporting Information (Figure S1).

Synthesis of Benzaldehyde Hydrazone (5d)

To a mixture of benzaldehyde (0.86 g, 8.11 mmol) and hydrazine hydrate (0.61 g, 12.2 mmol) in dichloromethane (20 mL) at 0 °C was added 10% (v/v) HCl solution in water (0.87 g, 11 mmol) dropwise with stirring over 2 min. The mixture was refluxed (oil bath at 70 °C) with continuous stirring for 6 h. The obtained solution was cooled and concentrated under vacuum. Then approximately 10 mL water was added to the residue and extracted with ethyl acetate (3 × 10 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered and the solvent was evaporated under vacuum to give a yellow-tinged liquid (1.09 g, 9.06 mmol, 91%). The ¹H and ¹³C NMR spectra are given in the Supporting Information (Figure S2).

Synthesis of Benzoic Acid Hydrazide (5f)

To a mixture of benzoic acid (1.22 g, 10 mmol) and hydrazine hydrate (0.61 g, 12.2 mmol) in 25 mL of water–ethanol mixture (1:5 v/v) was added acetic acid (0.6 mL, 10 mmol) at room temperature. The mixture was refluxed with continuous stirring for 6 h. The obtained solution was cooled and neutralized with 0.1 M NaHCO₃. The crude mixture was concentrated under vacuum; water was then added to the residue and extracted with dichloromethane (3 × 10 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered and the solvent was evaporated under vacuum to give a yellowish solid (1.22 g, 8.94 mmol, 89%). The ¹H and ¹³C NMR spectra are given in the Supporting Information (Figure S3).

Material Characterization

NMR Spectroscopy

¹H, ¹³C, and ³¹P NMR spectra were acquired using a Bruker Avance III 400 MHz spectrometer. ¹H and ¹³C NMR spectra for aryl hydrazones and hydrazides were recorded in CDCl₃ and DMSO-d ₆, respectively. All spectra were referenced to the residual solvent signals in the deuterated solvents.

Preparation of Buffer Solutions

Buffer solutions were prepared at the desired pH levels using deuterated organic solvents and deionized water. The pH values were measured with an Orion Star A211 pH meter (Thermo Scientific) with an accuracy of ±0.01 units. The pH meter was calibrated at 25 °C using the three-point calibration method with commercially available standard buffer solutions from Thermo Scientific at pH 4.00, 7.00 and 10.00.

Preparation of 1 M Glycine/NaOH-DMSO Buffer (pH 9.5)

1.5 g of glycine and 0.36 g of NaOH were dissolved in a 20 mL mixture of water and DMSO-d ₆ in a 1:4 v/v ratio. The pH was subsequently adjusted to 9.5 using a dilute NaOH (0.2 M) solution. The buffer solution was sealed tightly and stored at room temperature.

Catalytic Hydrolysis of Chemical Warfare Agent Simulants by α-Nucleophiles

A specified quantity of catalysts was accurately weighed and subsequently dissolved in a buffer solution at the corresponding pH to achieve a final concentration of 50 mM (5 equiv relative to DMNP). Similarly, DMNP (3.6 μL), DMMP (4.2 μL) and TPhP (0.0167 g) were dissolved in 2 mL of the buffer solution at the designated pH separately to prepare a 10 mM solution. The hydrolysis profiles of dimethyl 4-nitrophenylphosphate (DMNP), dimethyl methylphosphonate (DMMP) and triphenyl phosphate (TPhP) were investigated in situ using liquid-state ³¹P NMR spectroscopy in DMSO-d ₆. All reactions were conducted at ambient temperature. In a typical experiment, 0.3 mL of the 50 mM solution of a catalyst (α–nucleophile) was transferred into a 5 mm diameter, 8-in.-long NMR tube followed by addition of 0.3 mL of the 10 mM nerve agent simulant and the mixture was shaken for 10 s. The sample was then analyzed using ³¹P­{¹H} NMR (proton decoupled) at various time intervals. Spectra were recorded at 25 °C with 32 scans and relaxation delay of 2 s until complete hydrolysis of DMNP, DMMP and TPhP was achieved. The relative rates of hydrolysis of DMNP, DMMP, and TPhP to dimethyl phosphate (DMP), monomethyl methylphosphonate (MMP)/methylphosphonic acid (MPA), and diphenyl phosphate (DPhP) respectively, was determined via ³¹P­{¹H} NMR spectroscopy, based on the relative integrations of their characteristic peaks.

Degradation Kinetics of DMNP, DMMP, and TPhP

The degradation kinetics of DMNP, DMMP, and TPhP hydrolysis, catalyzed by nucleophiles under various conditions, including the control without using any catalyst, were evaluated as previously reported. , Liquid-state ³¹P NMR spectroscopy was performed on a Bruker Avance III HD 400 spectrometer operating at 161.9 MHz. The reaction conversion (denoted as R) at different time points was determined by analyzing the ratio of the integrated signal of hydrolysis products to the total integrated ³¹P signals of both reactants and products, as expressed in eq

$$R={\mathrm{\Sigma }I}_{\mathrm{p}}/({\mathrm{\Sigma }I}_{\mathrm{r}}+{\mathrm{\Sigma }I}_{\mathrm{p}})$$ 1

where ∑I _r and ∑I _p represent the sums of signal integrations corresponding to the reactants (CWA simulants) and their respective hydrolysis products, respectively.

The reaction kinetics followed pseudo-first-order behavior, described by eq

$$\ln [1-R]=-k_{\mathrm{obsd}}·t$$ 2

The observed rate constant (k _obsd) was obtained from the initial slope of ln­[1-R] vs time (t, min) linear plots. Each k _obsd value represents the mean of three independent measurements, with a relative standard deviation (RSD) not exceeding 2.5%.

The reaction half-life (t _1/2) was determined using eq

$$t_{1/2}=\ln (2)/k_{\mathrm{obsd}}$$ 3

UV–Vis Spectroscopic Studies

A Varian Cary 50 UV–visible Spectrophotometer equipped with Peltier temperature-controlled 10 mm quartz cells was used for all UV–vis analyses. The experiments were conducted with a substrate-to-nucleophile concentration ratio of 1:50, specifically 2 × 10^–3 M (substrate) and 100 × 10^–3 M (nucleophile) in a glycine-DMSO buffer at pH 9.5. For each experiment, 0.4 mL of the substrate-nucleophile mixture was transferred into a 1 cm cuvette and promptly placed in the spectrophotometer. The reaction progress was monitored by the decrease in DMNP absorbance at 280 nm and increase in p-nitrophenol/ate anion (4-NP) absorbance at 400 nm. The data was baseline-corrected using pure DMSO-glycine buffer (1.0 M, pH 9.5) as reference.

DFT Calculations

GIAO NMR chemical shift calculations for DMNP and its hydrolyzed products were performed in the gas phase using Gaussian-16 software. These calculations were carried out at the GIAO-B3LYP/6–31G­(d)//B3LYP/6–31G­(d) theoretical level. To validate the optimized structures, frequency calculations were conducted at the same level of theory, ensuring that all vibrational frequencies were positive.

Results and Discussion

The phosphorus center of organophosphate (OP) compounds, being electron-deficient, serves as a favorable target for nucleophilic attack during the hydrolysis or enzymatic degradation of nerve agents (NAs). The outcomes of these hydrolytic reactions depend on various factors, including the substrate’s structure, the type and basicity of the nucleophile, the properties of the leaving group, and solvation effects. − Additionally, the ionization state of the nucleophile determined by the medium’s pH and the nucleophile’s pKa plays a crucial role in these reactions. These considerations guided our selection of nerve agent simulants: DMNP (1), DMMP (2), and TPhP (3) for hydrolysis investigations with pharmaceutically relevant hydrazones and various small-molecule nucleophiles exhibiting the α-effect. These nucleophiles acted as catalysts to effectively hydrolyze the simulants into nontoxic degradation products (Scheme ).

1. Reaction of OP Simulants with Bases or Nucleophiles (e.g., 4-Pyridine Carboxaldehyde Hydrazone).

The reactivity of simulants 1–3 was assessed by reacting them with 5 equiv of bases (nucleophiles) under ambient conditions in a glycine buffer solution (pH 9.5) in DMSO-d ₆ (1:4 v/v). These conditions were optimized as part of the current study to provide a nonaqueous reaction environment with only a catalytic amount of water to promote hydrolysis. Several buffer-to-DMSO-d ₆ ratios were explored; however, deviations from the 1:4 ratio led to precipitation or phase separation, limiting their suitability for homogeneous reaction monitoring by NMR spectroscopy. The selected solvent system ensured sufficient solubility of reactants and allowed reproducible detection of degradation products. To evaluate the impact of nucleophile concentration, we varied the amount of base from 1 to 20 equiv and monitored the resulting ³¹P­{¹H} NMR spectra over a 60 min period. A range of 5 to 20 equiv exhibited similar catalytic effects, efficiently degrading the simulants at pH 9.5, whereas a 1:1 mol/mol base-to-substrate ratio resulted in slower hydrolysis. Substrates and reaction products were clearly distinguished by ³¹P NMR (Table ), and reaction kinetics were monitored by integrating the corresponding peaks in the NMR spectra. The results of the degradation experiments for each agent are presented in individual graphs, showing the conversion profiles of the six nucleophiles and the glycine buffer alone (control) with DMNP, DMMP, and TPhP in glycine–DMSO buffer at pH 9.5. These results are discussed in detail in subsequent sections. Notably, the non-nucleophilic glycine buffer showed no significant background hydrolysis of the CWA simulants after 24 h (Figure S4). The data reveal distinct differences in reactivity and efficiency among the compounds in degrading the simulants.

1. ³¹P NMR Chemical Shifts of the Signals Used to Monitor the Hydrolysis Reaction.

	CWA simulants			degradation products
compound	DMNP	DMMP	TPhP	DMP	M4NP	MMP	MPA	DPhP
chemical shift (ppm)	–4.9	32.9	–17.3	1.3–2.7	–5.6	35.1	22.4	–12.0 to −11.5

DMNP Hydrolysis by Nucleophiles

The hydrolysis of DMNP, a phosphorus acid ester with a good leaving group (p-nitrophenolate anion), occurs through the cleavage of the P-OAr bond. This reaction yields nontoxic dimethyl phosphate (DMP) as the major product (Scheme S1, path a). A side reaction involves attack at the carbon atom of the methyl group, leading to the cleavage of the PO–CH₃ bond (S_N2), which produces the undesired toxic vesicant methyl 4-nitrophenyl phosphate (M4NP) as the major product ,− (Scheme S1, path b). These mechanisms mimic the hydrolysis chemistry of nerve agents like Tabun and the V series agents, depending on the type of nucleophile and the pH of the medium.

As shown in Figure a and Table , DMP and M4NP exhibit ³¹P NMR peaks at 2.4 and −5.5 ppm, respectively, while DMNP has a peak at −4.9 ppm in DMSO-d ₆. The splitting patterns of these peaks, resulting from phosphorus-proton coupling (J_PCH 11.0 Hz), are consistent with previously reported data. , The results of DMNP conversion over time in the presence of various nucleophilic catalysts are depicted in Figure b. 4-Pyridine carboxaldehyde hydrazone (5c) and benzaldehyde hydrazone (5d) exhibited the fastest reactivity, rapidly hydrolyzing 82 and 75% of DMNP to the nontoxic product DMP, respectively, within 3 min. Both achieved complete conversion in less than 60 min, indicating their high effectiveness in neutralizing the CWA simulant under the tested conditions (Figures S5 and S6). In contrast, DBN (5a), DBU (5b), and phenyl hydrazine (5e), required significantly more time to achieve complete hydrolysis. Specifically, these compounds managed to convert 40, and 45, and 68% of the simulants into nontoxic products within 3 min, respectively, achieving complete conversion in 90 min (Figures S7–S9 and Table S1). This extended reaction time suggests a somewhat reduced, but still significant, reactivity compared to 5c and 5d. Benzoic acid hydrazide (5f) demonstrated the lowest reactivity among the nucleophiles examined, despite not being the least basic. This reduced reactivity can be attributed to intramolecular hydrogen bonding and resonance stabilization between the hydrazine moiety and the adjacent carbonyl group, which can delocalize the lone pair on the nucleophilic nitrogen. These interactions reduce the availability of the nitrogen lone pair for nucleophilic attack, effectively diminishing its reactivity despite its inherent basicity. The complete degradation of DMNP using this nucleophile took over 10 h, resulting in a mixture of products, including DMP and M4NP, at pH 9.5 (Figure S10).

4.

(a) Representative ³¹P NMR spectrum of DMNP indicating the chemical shift of the substrate (DMNP, δ³¹P −4.9 ppm) and degradation products (DMP, δ³¹P 2.4 ppm and M4NP, δ³¹P −5.5 ppm). (b) conversion profile of DMNP to products by the six nucleophiles.

Overall, these results suggest that nucleophiles capable of stabilizing a delocalized anionic form and minimizing steric barriers are better suited for rapid and selective degradation of nerve agent simulants under buffered conditions, regardless of their absolute basicity. Thus, the observed reactivity trend is largely consistent with the α-effect, where nucleophiles bearing adjacent lone-pair-containing atoms exhibit enhanced reactivity. However, this trend is disrupted for compound 5f, where the presence of a nearby electron-withdrawing carbonyl group diminishes its reactivity as a nucleophile.

Proposed DMNP Detoxification Mechanism

The degradation of DMNP at pH 9.5 follows a well-defined mechanism, emphasizing the interplay between nucleophilicity and the acid–base properties of the studied nucleophiles in determining reactivity.

Mechanistic Pathway

In an alkaline environment (pH ∼ 9.5), the reaction begins with the nucleophile attacking the electrophilic phosphorus center in DMNP via an S_N 2 mechanism, effectively displacing the leaving group. At pH 9.5, p-nitrophenol (pKa 7.15) exists predominantly in its deprotonated anionic form, which serves as a superior leaving group compared to its neutral counterpart. The stability and reduced basicity of anionic p-nitrophenol facilitate its substitution over the methoxy group (CH₃O^–, pKa ∼ 15.4). The nucleophilic attack is proposed to generate a transient nucleophile–organophosphorus (Nu–OP) adduct, which may then undergo a second displacement by hydroxide ion present in the mildly basic DMSO–glycine buffer (pH 9.5). This hydrolysis step would release the catalytic residue (hydrazinium or hydroxonium moiety) and yield the final, nontoxic phosphate product (Figure A). In addition to the primary pathway involving attack at phosphorus and P–OCH₃ bond cleavage, a side pathway involving nucleophilic attack at the methyl group is also plausible, particularly for strong N-nucleophiles. Although such methylation pathways have been reported in related systems, we did not observe methylated nucleophile products under our conditions.

5.

Mechanism of DMNP degradation at pH ≥ 9.5 (A) and at pH ≤ 8.5 (B). Mechanism B in part is reproduced from ref . Copyright 2025, American Chemical Society.

It is noteworthy that, unlike classical α-nucleophiles such as oximes, which typically act as suicide nucleophiles and are consumed during organophosphate degradation, − the hydrazones and related nucleophiles used in this study appear to be regenerated during the reaction, thus maintaining the efficiency of the degradation process (Figure A). This behavior is atypical for α-nucleophiles and suggests a potentially catalytic role under the employed conditions.

At pH ≤ 8.5, we propose that the ionization of the phenolate ion is suppressed, favoring reversible protonation of the methoxy group, which enhances its ability to act as a leaving group. Consequently, the S_N2 mechanism is more likely to proceed via cleavage of the P–OCH₃ bond rather than the P–OAr bond, leading to the formation of M4NP (Figure B). While some studies report that PO–CH₃ bond cleavage is more plausible– attributed to nucleophile methylation– our prior computational studies in the same medium indicate that P–OCH₃ cleavage is thermodynamically feasible. Moreover, no methylated products were observed experimentally, suggesting that nucleophile methylation does not play a significant role in the degradation pathway. In less alkaline or neutral environments, the reduced concentration and nucleophilicity of hydroxide ions likely further diminish overall reactivity.

UV–Vis Spectroscopic Evidence

The proposed mechanism is supported by UV–vis spectrophotometric studies of the reaction between DMNP and 4-pyridine carboxaldehyde hydrazone (5c), as shown in Figure . As the reaction progresses, a decrease in the absorbance of DMNP at 280 nm (Figure a), coupled with the emergence of the peak at 414 nm corresponding to the p-nitrophenoxide ion (Figure c,e), confirms its role as a leaving group in the reaction, aligning with the proposed S_N2 mechanism. This understanding of the degradation pathway can be instrumental in optimizing conditions for the effective detoxification of similar nerve agent simulants.

6.

UV–vis spectra of (a) DMNP; (b) 4-pyridine carboxaldehyde hydrazone; (c) the reaction mixture at different times during hydrolysis of DMNP in the presence of 4-pyridine carboxaldehyde hydrazone showing an increase in absorbance of p-nitrophenoxide ion, at pH ≥ 9.5; (d) the reaction mixture at different times during hydrolysis of DMNP in the presence of 4-pyridine carboxaldehyde hydrazone at pH ≤ 8.5 showing the absence of p-nitrophenoxide ion. Reaction conditions: [DMNP] = 10 mM, [4-pyridine carboxaldehyde hydrazone] = 50 mM, 25°. (e) UV absorption spectrum of nitrophenoxide at pH 8.5 and 10; (f) ³¹P NMR stacked spectra for the reaction of DMNP at pH ≤ 8.5.

A previous study reported on the pH dependence of aminoguanidine-derived aldimines in the degradation of DMNP. Similarly, in this study, at pH values below 8.5, a distinct ³¹P NMR peak at −5.5 ppm (Figure a), exhibiting a quartet phosphorus-proton splitting pattern, was observed during the hydrolysis reaction in the presence of 4-pyridine carboxaldehyde hydrazone in DMSO-glycine buffer. This peak is attributed to the moderately toxic product methyl 4-nitrophenyl phosphate (M4NP), resulting from the substitution of the methoxy group on DMNP. M4NP shares a similar mode of action with DMNP on acetylcholinesterase.

The proposed mechanism for DMNP degradation at pH ≤ 8.5 was further elucidated by UV–vis spectrometric studies. Figure d illustrates the UV–vis absorption characteristics of DMNP in the presence of 4-pyridine carboxaldehyde hydrazone (5c) at this pH. Initially, the UV absorption peak for 5c is observed at 355 nm (Figure b). As the reaction progresses, this peak becomes increasingly prominent, indicating the continued presence and stability of 5c throughout the reaction. Simultaneously, the expected UV absorption peak of p-nitrophenol (4-NP) at 414 nm does not significantly appear in the spectrum. This absence aligns with the observation that there is no substantial cleavage of 4-NP under these conditions. M4NP is proposed as the final hydrolysis product under these conditions, as supported by its diagnostic ³¹P NMR signal (δ³¹P – 5.6 ppm in DMSO-d ₆, Figure f and Table ). Although M4NP was not isolated for independent UV–vis characterization, the reaction mixture exhibits an absorption profile that overlaps with that of the nucleophile (e.g., 5c), making spectral distinction challenging. As reported, M4NP analogs absorb within this same region (300–355 nm), and their spectra may overlap with that of the nucleophile, making the two indistinguishable under these conditions. Nevertheless, the combined NMR and UV data are consistent with the formation of M4NP in situ.

As previously reported, the structural assignments of the hydrolysis products from DMNP degradation under various pH conditions were further validated through gauge-including atomic orbital (GIAO) ³¹P NMR chemical shift calculations. The calculated GIAO-B3LYP-6–31G­(d)//B3LYP-6–31G­(d) chemical shifts for M4NP (δ³¹ P = −5.5), DMP (δ³¹ P = 3.6), and DMNP (δ³¹ P = −3.06) (Figure ) closely align with the experimentally observed values of δ³¹ P = −5.4 to −5.6, 1.3 to 2.5 (NMR solvent-dependent), and −4.9, respectively. Additionally, our measured δ³¹P values for these compounds correlate well with previously reported chemical shifts. , Notably, at this theoretical level, the GIAO-calculated δ³¹P for DMNP (−3.06 ppm) deviates from the observed shift (δ³¹P = −4.9) by approximately 1.8 ppm. In contrast, the discrepancies between theoretical and experimental values for DMP and M4NP are minimal. The DFT-calculated δ³¹P NMR chemical shifts for all intermediates and products except methylphosphonate were previously reported by our group. These values are reproduced here to enable comparison with the newly calculated chemical shift for methylphosphonate and to maintain consistency in the discussion of spectral assignments. We opted not to use implicit solvation models in our GIAO calculations, as the gas-phase NMR chemical shifts demonstrated strong agreement with the experimental results.

7.

DFT-Calculated δ³¹P NMR (GIAO/B3LYP/6–31G*//B3LYP/6–31G*) chemical shifts (ppm) for hydrazone reaction with OP simulant DMNP. The chemical shift for methylphosphonate (in blue) is newly reported in this work. All other calculated values were previously published in ref ., Copyright 2025, American Chemical Society, and are reproduced here for comparative purposes.

DMMP Hydrolysis by Nucleophiles

G-type nerve agents, such as Sarin (GB) and Soman (GD), which are organophosphonates, can be effectively simulated with less toxic organophosphonates like DMMP. The global hydrolysis reaction of DMMP in an aqueous medium involves the cleavage of the P–O bond, yielding stable products: methyl phosphonic acid (MPA) and methanol. This reaction mirrors the hydrolysis of nerve agents, where the more labile P–F bond is cleaved to produce nontoxic byproducts (Scheme S2). It is important to note that the hydrolysis of DMMP follows a two-step process. This process involves the sequential substitution of the methoxy groups with hydroxyl groups via P–O bond cleavage, leading to the formation of methyl methylphosphonate (MMP) as an intermediate, and methyl phosphonic acid (MPA) as the final hydrolysis product.

As depicted in Figure a and Table , the sequential degradation products of DMMPMMP (intermediate) and MPA (final) exhibit ³¹P NMR peaks at approximately 35.1 and 24.1 ppm, respectively, while DMMP has a ³¹P NMR peak at 32.9 ppm in DMSO-d ₆. Our NMR experimental results reveal that the phosphorus-proton coupling splitting patterns of these peaks for DMMP (Figure S11), MMP, and MPA are observed as multiplet, reduced multiplet, and quartet, respectively.

8.

(a) Representative ³¹P NMR spectra of DMMP indicating the chemical shift of the substrate (DMMP) 32.99 ppm, and degradation products MMP δ³¹P 35.1 ppm and MPA, δ³¹P 24.0 ppm in DMSO-d ₆, at 25 °C. (b) Conversion profile of DMMP by the six nucleophiles.

Figure b, represents the conversion profile of DMMP by nucleophiles over time, 4-pyridine carboxaldehyde hydrazone (5c) and benzaldehyde hydrazone (5d) demonstrated the highest catalytic reactivity, achieving nearly 90% and 75% conversion of DMMP to the nontoxic product MPA within just 3 min (Figures S12, S13 and S14). Our NMR experiments indicate that intermediate formation occurs very rapidly, within seconds. Both 5c and 5d completed the conversion of DMMP in under 90 min, highlighting their strong catalytic efficiency in detoxifying the CWA simulant under the tested reaction conditions (Table S3).

Conversely, DBN (5a), DBU (5b), and phenylhydrazine (5e), while still effective in degrading the DMMP, required significantly more time to achieve complete hydrolysis of the simulant. Specifically, 5a, 5b, and 5e, decomposed 38, 42, and 64% of the simulant into their nontoxic products within 3 min, respectively, and achieved complete conversion in 90 min (Figures S15 and S16). This longer reaction time compared to 5c and 5d indicates a somewhat reduced, yet still notable, level of reactivity among these nucleophiles.

The enhanced reactivity of phenylhydrazine (5e, pK _a ∼ 28.8 in DMSO), , an α-nucleophile, compared to other sterically hindered nucleophiles such as DBN (5a) or DBU (5b, pK _a ∼ 13.9) in DMSO can be attributed not only to its higher basicity, but also its structural features particularly the N atom next to the amino group, which increases nucleophilicity and promotes efficient attack on electrophilic phosphorus centers.

Benzoic acid hydrazide (5f), with a pK _a of approximately 18.9 in DMSO, , exhibited the lowest reactivity among the nucleophiles tested due to the diminished α-effect based on the nearby carbonyl group. Although 5f is a stronger base than DBU, based on pK _a values, it is less reactive than DBU. Complete degradation of DMMP with 5f required over 10 h under the given conditions (Tables S3 and S4). This reduced reactivity can be attributed to its relatively lower basicity and nucleophilicity compared to other nucleophiles such as DBU and DBN. The electron-withdrawing carbonyl group in 5f likely decreases electron density at the nucleophilic center and may introduce steric hindrance, further impairing its ability to effectively attack electrophilic phosphorus centers.

The proposed mechanism for the degradation of DMMP by α-nucleophiles such as (5c), (5d), (5e), and (5f) begins with a direct nucleophilic attack at the electrophilic phosphorus center by the α-nucleophile. This attack forms a pentacoordinate intermediate stabilized by the nucleophile. Subsequently, hydroxide ions (OH^–), generated from proton abstraction of the small amount of water present in the reaction mixture, facilitate the displacement of the nucleophile and breakdown of the intermediate. The process releases CH₃O^– and forms methyl methylphosphonate (MMP). A second nucleophilic attack, likely assisted by additional hydroxide ions formed through further proton abstraction by the α-nucleophile, leads to complete hydrolysis, yielding methyl phosphonic acid (MPA) as the final product (Figure ).

9.

Mechanism of DMMP degradation by α-nucleophiles.

TPhP Hydrolysis by Nucleophiles

In our study, triphenyl phosphate (TPhP) was employed as a CWA simulant alongside DMNP and DMMP to evaluate the efficiency of aryl hydrazones and various nucleophiles as decontaminating agents for nerve agent analogs that are less soluble in aqueous and nonaqueous media and have lower volatility. TPhP is nonvolatile and exhibits reduced solubility under our reaction conditions, providing a challenging test case for nucleophilic degradation. TPhP has a ³¹P NMR chemical shift (δ³¹P) of approximately −17.3 ppm (Figure S17), while nucleophilic attack on TPhP results in degradation products with a δ³¹P shift of −12.0 to −11.5 ppm (Table , and Figure a). As expected, the results revealed a consistent trend in the degradation efficiency of the nucleophiles across the different nerve agent simulants. However, the hydrolysis of TPhP proceeded at a slower rate compared to DMNP and DMMP. Specifically, compounds 4-pyridine carboxaldehyde hydrazone (5c) and benzaldehyde hydrazone (5d) achieved only 70 and 56% degradation in 1 h, respectively (Figures S18–S20), with complete hydrolysis of TPhP occurring in approximately 2 h (Table S5). Conversely, DBN (5a) and DBU (5b) required about 5 h, while phenylhydrazine (5e) and benzoic acid hydrazide (5f) took nearly 24 h to achieve complete degradation of TPhP (Figure b and Table S5).

10.

(a) Representative NMR spectra indicating the chemical shift of the substrate TPhP (−17.2 ppm) and degradation product DPhP (δ³¹P −12.0 to −11.5 ppm) in DMSO-d _6; (b) Reaction progress with the six nucleophiles on DMMP.

This prolonged reaction time for TPhP suggests its reduced reactivity compared to DMNP and DMMP. While strong nucleophiles typically favor S_N2-type mechanisms, TPhP degradation proceeded relatively slowly and selectively, which we attribute to significant steric hindrance from the bulky aryl groups surrounding the electrophilic phosphorus center. This steric congestion may inhibit direct backside attack, rendering a stepwise, possibly dissociative (S_N1-like) pathway more favorable under our conditions. The reaction rate may be limited by the slow formation of the phosphonium ion, which is influenced by negative solvation effects and the limited solubility of TPhP in the cosolvent. Nonetheless, we cannot fully exclude the possibility of a concerted mechanism.

The α-nucleophiles displayed a consistent trend in degradation efficiency, indicating that their reactivity profiles remain stable across different simulants.

A notable observation from these experiments is the 24–116-fold increase in decontamination efficiency, of the nucleophiles for TPhP compared to the uncatalyzed hydrolysis condition (t _1/2 = 3121 min).

Kinetic Studies

We applied both first-order and second-order kinetic models to the experimental data to determine the best-fit model for quantitatively assessing the degradation half-lives of DMNP, DMMP, and TPhP by the nucleophiles. The kinetic plots, shown in Figure , indicated that the reactions involving various nucleophiles and nerve agent simulants follow first-order kinetics. This conclusion is supported by the calculated half-life values and the linearity of the plots, which exhibit high correlation coefficients (R ² > 0.85). Therefore, under pseudo-first-order kinetic conditions, we used eqs and to generate linear plots of ln­[1-R] versus time, which were employed to quantitatively assess the degradation half-lives (t _1/2, min).

11.

Representative first-order kinetic plots for the degradation of CWA simulants (10 mM): (a) DMNP; (b) DMMP; and (c) TPhP, catalyzed by the nucleophiles (50 mM) along with blank (buffer solution) as control in 1.0 M glycine-DMSO (pH 9.5) at 25 °C.

As shown in Table , the heteroaryl and aryl hydrazones 5c and 5d exhibited higher k _obsd values and shorter half-lives across all three simulants, indicating greater reactivity. They demonstrated 1930-fold and 2490-fold rate increase compared to uncatalyzed hydrolysis for DMNP and DMMP, respectively, in the same medium. The rapid degradation kinetics observed with heteroaryl, and aryl hydrazones are believed to result from two main factors: the basic catalytic effect of amino groups (homogeneous catalysis) and the stereoelectronic properties of the heteroaryl and aryl rings surrounding the hydrazone moiety. These properties likely facilitate nucleophilic catalysis toward the phosphonyl group of the pesticide or nerve agent. The half-lives of the reactions, presented in Table , illustrate the differential reactivity of the nucleophiles, providing valuable insights for optimizing their use in practical decontamination scenarios.

2. Kinetic Data for the Hydrolysis of Nerve Agent Simulants with Various Nucleophiles.

	DMNP			DMMP			TPhP
nucleophile	k obsd (min–1)	t 1/2 (min)	R 2	k obsd (min–1)	t 1/2 (min)	R 2	k obsd (min–1)	t 1/2 (min)	R 2
DBN (5a)	0.0251	27.609	0.955	0.0289	23.979	0.932	0.0074	93.648	0.994
DBU (5b)	0.0293	23.651	0.956	0.0312	22.148	0.956	0.0078	88.732	0.995
4-Pyridine carboxaldehyde hydrazone (5c)	0.4284	1.617	0.997	0.5529	1.253	0.982	0.0258	26.808	0.937
Benzaldehyde hydrazone (5d)	0.2496	2.776	0.944	0.2140	3.236	0.966	0.0178	38.867	0.915
Phenyl hydrazine (5e)	0.0356	19.444	0.893	0.0888	7.797	0.897	0.0094	73.024	0.903
Benzoic acid hydrazide (5f)	0.0101	68.410	0.974	0.0107	64.465	0.977	0.0053	129.05	0.994
Blank	0.0002	3121.6	N/A	0.0002	3121.6	N/A	0.0002	3121.6	N/A

We evaluated the catalytic efficiency of the nucleophiles through a second-cycle experiment, in which one equivalent of DMNP was added after its complete degradation in the first cycle. Notably, the initial reaction rates decreased, with DMNP conversion dropping to 45% for aryl hydrazones and averaging 32% for other nucleophiles. All nucleophiles showed less than 45% conversion of DMNP exclusively to the toxic product M4NP within 1 h. Despite an excess of nucleophiles, the reduced efficiency and altered product distribution were attributed to a significant pH drop from an initial 9.5- due to accumulation of the acidic degradation product, 4-nitrophenol (4-NP). This acidification shifted the medium out of the optimal alkaline range required for efficient nucleophilic attack on the phosphorus center. These findings highlight the critical role of pH control in sustaining nucleophile activity during successive degradation cycles, consistent with Wilson et al., who reported similar pH-dependent behavior in nitrogen-based DMNP decontaminants.

Conclusions

In this study, we investigated the reactivity of DMNP, DMMP, and TPhP with a series of hydrazone, hydrazine, and hydrazide α-nucleophiles, focusing on their interactions in a glycine-DMSO buffer at pH 9.5. Our findings indicate that these reactions predominantly followed an alkaline hydrolysis pathway, as evidenced by UV–vis and ³¹P NMR data. This pH-dependent mechanism calls attention to the importance of reaction conditions in influencing the degradation of chemical warfare agents (CWAs) and their simulants, emphasizing the need for precise control in practical detoxification applications.

Kinetic analysis revealed a significant rate enhancement when the simulants reacted with hydrazones, suggesting that the electronic and structural features of the hydrazone moiety play a key role in determining reactivity. Among the α-nucleophiles studied, heteroaryl and aryl hydrazones (5c and 5d) exhibited the highest rate accelerations, followed by phenylhydrazine (5e), while benzoic acid hydrazide (5f) showed the lowest reactivity. Overall, the observed reactivity trend is largely consistent with the α-effect, with the exception of compound 5f, where the presence of a nearby electron-withdrawing carbonyl group diminishes its reactivity as a nucleophile.

Interestingly, our results reveal a phenomenon of nonsynchronicity in bond formation at the phosphorus electrophilic center. In the presence of nucleophiles with varying basicity and nucleophilicity we observed a reactivity leveling effect among hydrazine-derived and amidine/guanidine-type nucleophiles in the S_N2­(P) reaction pathway. This pathway displayed two distinct reactivity patterns at different pH levels (∼9.5 and ≤ 8.5), suggesting the involvement of concurrent mechanisms in nucleophilic displacement at phosphorus.

Nucleophilic substitution of TPhP proceeded relatively slowly and, presumably, via an S_N ¹ pathway, likely due to steric hindrance from bulky aryl groups surrounding the electrophilic phosphorus center.

In conclusion, this study highlights the complex interplay of structural and electronic factors influencing nucleophilic reactivity in the degradation of CWA simulants. These insights can guide the development of more effective nucleophilic agents for detoxification applications, emphasizing the value of mechanistic understanding. Future efforts will focus on grafting these potent decontaminants onto engineered fabrics and surfaces for practical deployment.

The data underlying this study are available in the published article and its Supporting Information.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.5c02810.

• NMR spectra for characterization purposes and for monitoring reactions, schemes and methods of hydrolysis of simulants with various nucleophiles as catalysts (PDF)

The authors declare no competing financial interest.

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