Nerve Agent Destruction by Recyclable Catalytic Magnetic

Ind. Eng. Chem. Res. 2005, 44, 7991-7998

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Nerve Agent Destruction by Recyclable Catalytic Magnetic Nanoparticles Lev Bromberg and T. Alan Hatton* Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139

Organophosphorus pesticides and warfare agents are not readily hydrolyzed in aqueous media without applying extremes of pH, heat, or bleach. We show that suspensions of magnetite (Fe3O4) nanoparticles modified with a common antidote, 2-pralidoxime (PAM) or its polymeric analogue, poly(4-vinylpyridine-N-phenacyloxime-co-acrylic acid), catalyze the hydrolysis of an organophosphate (OP) compound serving as a model of the warfare nerve agents, at neutral pH. The oximemodified magnetite particle serves as a nanosized particulate carrier with a powerful R-nucleophile, the oximate group, immobilized on its surface. The rates of OP hydrolysis by the PAM-modified magnetite are comparable to those of the most potent copper-based catalysts. The oxime-modified magnetite nanoparticles are colloidally stable at neutral pH and are readily recovered for reuse from the aqueous milieu by high-gradient magnetic separation methods with no loss of catalytic activity. Introduction The presence of organophosphates (OP) in industrial and agricultural drain waters, spills, runoffs, and drifts, as well as OP agent-based chemical munitions that can be released in case of warfare or terrorist attack, pose great risks to human health and the environment. The number of intoxications with OP pesticides and insecticides is estimated at some 3 000 000/year, and the number of deaths and casualties over 300 000 per year worldwide.1 Numerous OP pesticides and insecticides and warfare agents such as sarin, soman, and VX are nerve poisons and may cause cumulative damage to the nervous system and liver as well as being carcinogenic. The primary mechanism of action of the OP esters is irreversible inhibition of acetylcholinesterases and accumulation of the neurotransmitter acetylcholine at nerve synapses. Structures of the nerve poisons sarin, soman, and their model analogue, diisopropyl fluorophosphate (DFP, used in the present work), are given in Chart 1. Among the various approaches to OP decontamination,2 catalytic destruction (CD) has become an important area of focus.3 Its specificity and high catalytic rates make CD appropriate for decommissioning nerve agent stockpiles, counteracting nerve agent attacks, and remediating organophosphate spills. There has been considerable effort directed toward development of biocatalysts such as OP-degrading enzymes that are isolated from a variety of living organisms or produced by means of gene expression, for the CD of wastewaters and nerve agent stockpiles.3 Lack of availability of the OP-degrading enzymes in sufficient quantities and relatively low enzyme stability in the environment have thus far caused the majority of the practical CD technologies to focus on acid- or base-catalyzed hydrolysis4-6 or nucleophile-aided hydrolysis.7 In this regard, R-nucleophiles such as hydroperoxides,8 hypochlorite,7 iodosocarboxylates,9 and oximates10 have been investigated alone or in concert with surfactants. Also, there has been some * To whom correspondence should be addressed. Tel.: (617) 253-4588. Fax: (617) 253-8723. E-mail: [email protected].

Chart 1. Chemical Structures of Sarin, Soman, and Diisopropyl Fluorophosphate (DFP).

interest in the use of lanthanide (Ln) cations to facilitate the hydrolysis of phosphodiesters.11,12 Mechanistically, the Ln cations serve both as Lewis acids to bind and charge-neutralize the phosphodiester’s P-O-, while simultaneously providing for a metal-bound OH nucleophile to attack the substrate’s phosphonyl group. Increasing the metal cation’s charge density enhances both its strength as a Lewis acid and the acidity of its hydration water. Hence, particular attention centered on Ce(IV), the only lanthanide with a readily available +4 oxidation state.11 Unfortunately, above pH 4, the formation and precipitation of Ce(IV)-hydroxide gels hinders the kinetics of Ce(IV)-mediated phosphodiester hydrolyses.11 No scaleable Ln-mediated hydrolysis of the OP nerve agents has been reported. Thus, very few reagents are currently available that are inexpensive and nontoxic and, at the same time, exhibit catalytic rather than stoichiometric dephosphorylating activity, at neutral pH.13 Notable exceptions include micellar iodosobenzoate and related derivatives9 and micelleforming metallocomplexes.14 In the present work, we have studied a catalytic decomposition agent, friendly to the environment and inexpensive, which could be readily dispersed in an aqueous medium, be it an aqueous reservoir or bodily fluid, could maintain its ability to decompose OP at neutral pH, and could be capable of being efficiently removed from the medium when necessary and then reused at will. We thus designed a nanosized particulate carrier with a powerful R-nucleophile, an oximate group, immobilized on its surface as a CD agent. The carrier comprised iron oxide (Fe3O4) that is superparamagnetic and thus can be separated from an aqueous suspension by high-gradient

10.1021/ie0506926 CCC: $30.25 © 2005 American Chemical Society Published on Web 09/10/2005

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Ind. Eng. Chem. Res., Vol. 44, No. 21, 2005

Chart 2. Chemical Structures of the Oxime-Containing Species Employed for Magnetite Particle Modification in the Present Work.

magnetic separation (HGMS).15 We have designed a single-step synthetic procedure yielding nanosized magnetite particles complexed with oximate-containing moieties such that the resulting nanoparticles are colloidally stable in aqueous environment at neutral pH. Yet, the particles were of sufficient size (hydrodynamic diameter, ∼100 nm) to be effectively removed from water by a portable HGMS device for reuse. It appeared that the novel nanoparticles were as effective as well-known, nonmagnetic copper(II) chelates16 in hydrolyzing a model OP nerve agent, diisopropyl fluorophosphate (DFP). Experimental Section Chemicals. Iron(II) chloride tetrahydrate (99%), iron(III) chloride hexahydrate (98%), acrylic acid (99%), 4-vinylpyridine (95%), 2-bromoacetophenone (phenacyl bromide, 98%), 2-pyridinealdoxime methiodide (PAM, 99%), diisopropyl fluorophosphate (DFP, 99%), and 2,2′azobisisobutyronityrile (AIBN, 98%) were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO) and used as received. All other chemicals, solvents, and gases were obtained from commercial sources and were of highest purity available. Polymer Synthesis. A copolymer of acrylic acid (AA) and 4-vinylpyridine (4-VP) was synthesized by freeradical copolymerization using AIBN as an initiator. A vial containing a solution of 0.1 mol (10 mL) of 4-VP, 0.1 mol (6.9 mL) of AA, and 0.2 g of AIBN in N,Ndimethylformamide (10 mL) was deaerated by nitrogen purge, sealed, and kept at 70 °C overnight. The resulting viscous copolymer [p(VP-AA)] solution was repeatedly washed by acetone and methanol and precipitated by hexane followed by drying under vacuum, dissolution in deionized water, and exhaustive dialysis (membrane MW cutoff (MWCO), 3500) against DI water. The purified p(VP-AA) samples were lyophilized and stored dry at 2-8 °C until further use. (C12H17NO2)x, found (calcd): C, 69.26 (69.54); H, 8.29 (8.27); N, 7.74 (6.76). 1H NMR (400 MHz, CD OD): δ 1.7 (m, 2 H, CH - in 3 2 the main chain), 2.7 (m, 1 H, CH- in the main chain), 7.2 (m, 3H, pyridine), 8.45 (m, 2 H, pyridine). Weightaverage MW by GPC, 63 000; polydispersity index, 1.9. The p(VP-AA) was further modified to yield its oximated analogue, abbreviated p(VPOx-AA) (Chart 2). A solution of p(VP-AA) copolymer (3.75 g, 18 mmol) and 4.0 g (19 mmol) of 2-bromoacetophenone (phenacyl bromide) in 150 mL of absolute ethanol was refluxed in a round-bottom flask at 70 °C under stirring for 48 h.

Then the solvent was vacuum-evaporated, and the contents of the flask were resuspended in 150 mL of anhydrous methanol. After addition of hydroxylamine hydrochloride (2.5 g, 36 mmol) and sodium hydroxide (1.4 g, 36 mmol), the resulting suspension was kept at 70 °C under reflux with stirring for 48 h. Then the solvent was evaporated, and the contents of the flask were repeatedly washed by diethyl ether on a filter and dried. The residual solids were dissolved in 100 mL of 15% aqueous ethanol, and the solution was dialyzed against 50% aqueous ethanol followed by exhaustive dialysis against deionized water (membrane MWCO, 3500). The resulting copolymer was freeze-dried and stored at -20 °C. (C20H25N2O3)x, found (calcd): C, 69.64 (70.36); H, 7.17 (7.38); N, 7.95 (8.20). 1H NMR (400 MHz, CD3OD): δ 1.75 (m, 2 H, CH2- in the main chain), 2.38, 3.28 (m, 1 H, CH- in the main chain), 6.7, 7.0, 7.3 (m, 6 H, benzyl), 8.3 (m, 5H, pyridine). Particle Synthesis. Magnetic nanoparticles were produced by chemical coprecipitation of iron(II) and iron(III) chlorides. Namely, 1.88 g (7.0 mmol) of FeCl3‚ 6H2O and 0.69 g (3.5 mmol) of FeCl2‚4H2O were added to 40 mL of deionized water, the solution was deaerated by nitrogen purge in a stirred 250 mL three-necked flask, and the temperature of the flask contents was brought to 80 °C. An aqueous solution of a stabilizing compound (2.6-2.8 g of compound in 40 mL of water; pH adjusted to 6) was added to the flask, and the resulting mixture was equilibrated at 80 °C while being stirred under nitrogen purge. Then the nitrogen purge was ceased, the contents of the flask were at once added to 80 mL of a 28% ammonium hydroxide, and the mixture, which rapidly turned black, was vigorously stirred for 5-10 min. The resulting precipitate possessed strong magnetic properties and was thus separated from the liquid by decantation using a Franz Isodynamic magnetic separator (Trenton, NJ). The precipitate was dried in an oven at 60 °C until constant weight and resuspended in deionized water by sonication for 30 s with a Branson sonifier 450 at an output of 40%. The suspension was dialyzed against excess deionized water (membrane MW cutoff, 3500) and lyophilized. The compositions of these oxime-containing particles were assessed by elemental analysis as follows. PAM-modified magnetite, [C7H9N2O (Fe3O4)2]x, found (calcd): C, 13.43 (14.01); H, 1.66 (1.51); Fe, 54.80 (55.82); N, 3.97 (4.67); oxime group content, 1.7 mmol/g. p(VPOxAA)-modified magnetite, [C38H44N4O6 (Fe3O4)3]x, found (calcd): C, 33.34 (33.87); H, 3.48 (3.29); Fe, 37.38 (37.30); N, 5.16 (4.16); oxime group content, 1.5 mmol/g. Particle Characterization. Dynamic light scattering (DLS) experiments were performed with a Brookhaven BI-200SM light scattering system at a measurement angle of 90°. Volume-average particle size distributions were obtained using the built-in software, and the reported particle hydrodynamic diameters are the average of five measurements. All samples were filtered with a 0.45 µm syringe filter prior to the DLS tests. Superconducting quantum interference device (SQUID) experiments were conducted using a Model 32 kG Gaussmeter (Digital Measurement Systems) to determine the magnetization of the particles in an applied magnetic field. Particle suspensions weighing 40-80 mg were placed in an airtight sample cell, and the exact mass of the samples was determined following the SQUID measurement. All SQUID measurements were performed at 300 K over a -1 to +1 T range.

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Kinetic Measurements. Kinetics of the DFP decomposition were measured at 25 °C with an Orion 96-09 combination fluoride electrode (Thermo Electron Corp., Beverly, MA) and a Model 45 dual display multimeter (Fluke Corp., Everett, WA) connected to a PC with FlukeView Forms software for data processing. The electrode potential-time output was recorded continuously, and each datum point represents an average of the voltage amplitude within a 1 s interval. The electrode was immersed in a 9 mL aqueous sample, and the output was allowed to equilibrate for about 10 min. A known volume of DFP was added into the sample at once via a precision sampling syringe (Valco Instruments Co. Inc, Houston, TX) and the time commenced. Samples containing no particles were stirred by a small magnetic bar, whereas suspensions with particles sized at or above 100 nm were stirred using nitrogen bubbling. No particle sedimentation or precipitation was observed in any of the experiments. The pH was measured in each sample upon completion of the kinetics measurements. No significant changes in pH, set initially at 7.0, were observed in any of the runs. The electrode was calibrated in independent series of experiments using aqueous solutions of sodium fluoride with or without magnetic particles suspended at various concentrations. Magnetic Separation and Reuse. High-gradient magnetic separation (HGMS) experiments were performed using a cylindrical glass column with an internal diameter of 7 mm and a length of 22 cm (a volume of 8.46 mL) packed with 3.6 g of type 430 fine-grade stainless steel wool (40-66 µm diameter) supplied by S. G. Frantz Co., Inc. (Trenton, NJ) placed inside a quadrupole magnet system comprising four nickelplated neodymium iron boron 40 MGOe permanent magnets sized 18 × 1.8 × 1.8 cm each (Dura Magnetics, Inc., Sylvania, OH). The flux density generated inside the packed column was ca. 0.73 T. Magnetic washing of the particles was performed by passing 9 mL of a sample containing 1 mg/mL particles suspended in 10 mM Tris equilibrated with 1.33 or 4.0 mM DFP solution through the column at a flow rate of 1.5 mL/min controlled by a peristaltic pump. Then the column was removed from the magnet, and 18 mL of deionized water (pH adjusted to 7.0) was passed through the column to collect the washed particles. Several such samples were recovered to collect a sufficient quantity of particles for reuse. The particle suspension was evaporated to dryness at 50 °C, and the residual solid was weighed and resuspended in 10 mM Tris buffer by sonication to result in an effective solid concentration of 1 mg/mL. The resulting suspension was subjected to the kinetic experiment using electrode detection of the fluoride ions generated by the DFL decomposition. The process of particle recovery and reuse was repeated in two sequential cycles. Results and Discussion Particle Design. Catalytic magnetic particles were synthesized with or without a stabilizing compound by the coprecipitation of iron(II) and iron(III) chlorides by ammonia. The Fe2+/Fe3+ molar stoichiometry of 1:2 was chosen such that under nonoxidizing conditions magnetite (Fe3O4) was formed:17

2FeCl3 + FeCl2 + 8NH3 + 4H2O f Fe3O4 V + 8NH4Cl

Prior to the precipitation, the iron ions in the aqueous mixture in most experiments were complexed with either 2-pyridinealdoxime (PAM) or poly(4-vinylpyridine-co-acrylic acid) [p(VP-AA)] or the same polymer that had undergone oximation [p(VPOx-AA)] (Chart 2). The molar ratio between iron ions and oxime or carboxyl groups capable of complexing with iron in our synthetic procedure was chosen to be 1:1. PAM is a potent reactivator of the acetylcholinesterase inhibited by OP nerve agents and its administration, together with atropine, comprises standard organophosphate antidotal therapy.1,18 Studies have shown that PAM decomposes the OP poisons via nucleophilic attack where the oximate ion is an active species.19 Chelation of PAM with metal ions such as Cu2+ and Ni2+ is well-known, and the resulting complexes catalyze the decomposition of the OP compounds such as DFP and sarin,19 albeit less vigorously than do some Cu(II)dipyridyl and Cu(II)-imidazole complexes.20 Copper chelates with a 1:1 molar ratio of the Cu2+ ion to ligands such as R-amino acids or diamines were reported to be in a class on their own as catalysts for the hydrolysis of DFP and sarin, while analogous iron(II) and iron(III) complexes were devoid of catalytic activity.16 Interestingly, despite the fact that complexes of PAM and iron have been known for a long time,21 to the best of our knowledge no data on their activity in OP decomposition has been gathered, despite the observation of a high activity of the analogous Cu2+, Ni2+, and Zn2+ complexes with oximes.19,22 PAM and Fe2+/Fe3+ have been described to form, in aqueous solution, a low-spin octahedral complex in which iron is bonded to six nitrogen atoms.23 We reasoned that the iron-PAM complex molecule, with its three polar oxime-OH groups, might remain on the surface of the magnetite particles upon coprecipitation of the iron ions, and thus the resulting nucleophile-modified magnetite would be active in the OP decomposition. It has been demonstrated24 that the complexation of transition metal ions with PAM does not lower the nucleophilicity of the latter. An analogous rationale applies to the polymeric oxime, p(VPOx-AA), which was designed to model PAM by having a quaternized pyridinium group in close proximity to the aldoxime group, which can enhance the hydrolytic properties of the latter.25 The p(VPOx-AA) can be viewed as a polymeric analogue of phenyl-2pyridylketoxime, which complexes strongly with iron oxides.26 Furthermore, the presence of numerous carboxyls that can chelate with iron ions embedded in the magnetite structure15 and on the other hand impart charge to the magnetite surface was thought to improve the particles’ stability. Figure 1 shows the effects of pH on the ζ-potential and hydrodynamic diameter of the magnetite particles modified by PAM or p(VPOx-AA) copolymer. The volume-average hydrodynamic diameters of the PAM/M and p(VPOx-AA)/M particle species were approximately 100 and 150 nm, respectively, in the pH ranges from 1.5 to 8 and from 4 to 9, respectively. The PAM/M particles maintained a weak positive charge due to the ionization of the 1-methylpyridinium groups until pH 8, above which point the PAM was no longer ionized and the particles aggregated. The appearance of the negative ζ-potential on the aggregates at pH >8 can be attributed to the exposed magnetite surfaces, which are negatively charged at these pHs.17 The observed

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Figure 2. Potential-time response of fluoride-selective electrode to various DFP concentrations in the absence and the presence of 1 mg/mL magnetite particles modified with PAM. T ) 25 °C; 10 mM Tris buffer, pH 7.0.

Figure 1. Effect of pH on (a) ζ-potential and (b) hydrodynamic diameter of magnetite particles modified with 2-pralidoxime (PAM/ M) and poly(4-vinylpyridine-N-phenacyloxime-co-acrylic acid) [p(VPOX-AA)/M]. T ) 25 °C; 10 mM Tris buffer with pH adjusted by 1 M NaOH or HCl.

electrokinetic mobility pattern of the PAM/M particles corresponds well with the pKa ) 7.8 of PAM.24 The pH dependencies of the electrokinetic mobility of the polymer-coated particles, p(VPOx-AA)/M, indicate an effect of the amphoteric nature of the copolymer, with its N-substituted 4-vinylpyridinium moieties positively charged at pH < 8 and carboxyls of acrylic acid negatively charged at pH 4.6. That is, the particles maintained an overall negative charge at pH 5, below which the carboxylic groups lost ionization, which led to the particle aggregation. However, at pH > 8, the particles became more negatively charged overall, which can be attributed to both the loss of ionization of the 4-vinylpyridinium groups and the increased degree of ionization of the carboxyls. Electrostatic complexation between the carboxyls and 4-vinylpyridinium groups in the pH range 5-8 is likely. The variation in the magnetization of the oximemodified magnetite particle suspensions with applied magnetic field, as measured in the SQUID experiments, was used to determine the size distribution of the Fe3O4 particles as described elsewhere.15 An analysis of the magnetic susceptibility data yielded primary Fe3O4 particle diameters of 7-8 nm, in accord with the previously reported data on Fe3O4 modified by poly(acrylic acid) derivatives.15 These core sizes are significantly smaller than the average diameters determined using dynamic light scattering (see above), however, which suggests that individual magnetite nanoparticles aggregate to form small clusters in suspension. Catalytic Destruction of Nerve Agent. Hydrolysis of DFP to produce the fluoride ion was monitored by the ion-selective fluoride electrode. Typical electrode response curves are depicted in Figure 2. The electrode response time is on the order of several seconds, whereas the generation of the fluoride ion as a byproduct of the DFP decomposition is manyfold slower, and thus the inherent response time of the fluoride ion-selective electrode has no apparent influence on the determination of the hydrolysis rate.27 For

the DFP solution in 10 mM Tris buffer, at a constant pH 7.0, the electrode potential was stable for at least 16 h in the absence of the catalytic particles, indicating negligible accumulation of the fluoride ions. Addition of DFP to a suspension of oxime-containing particles, however, resulted in the rapid appearance and accumulation of the fluoride ions, as is seen from the rather dramatic response of the ion-selective electrode. To convert the electrode potential to the time-dependent fluoride concentration (Ct) readings, we developed electrode calibration curves in sodium fluoride solutions in the presence of the 1 mg/mL suspensions of the oximemagnetite particles at pH 7.0 in 10 mM Tris buffer. The electrode potential readings yielded almost ideally Nernstian dependences with [F-] ranging from 10 µM to 0.1 M in the particle suspensions, and thus the developed calibrations allowed for computation of the Ct values with a high degree of confidence. No interference from magnetite, PAM, or the polymers under study was observed. At low initial concentrations, the rate of metal chelate- and oxime-catalyzed hydrolysis of DFP and sarin to produce the fluoride ion has been established to be pseudo-first-order with respect to the total concentration of unreacted OP agent:16,20,27-29

ν

d[DFP] dCt ) ) νo ) kobs[DFP] dt dt

(1)

where kobs is the observed pseudo-first-order rate constant. The initial slope of the Ct vs t kinetic curves corresponds to the initial rate of the DFP hydrolysis (νo). The observed rate constant for the DFP hydrolysis is obtained from the experimental data using the integrated form of eq 1:

- ln(1 - Ct/[DFP]o) ) kobst

(2)

The observed rate of spontaneous DFP hydrolysis in 10 mM Tris buffer at pH 7.0 in the absence of particles or of PAM was negligibly small (kobs ) 7 × 10-7 s-1; see curve 1 in Figure 2). Under identical solution conditions but in the presence of up to 10 mg/mL of either bare magnetite or the p(VP-AA)-modified magnetite nanoparticles; i.e., in the absence of oximes, the hydrolysis occurred at rates exceeding that of the spontaneous hydrolysis by up to 10-fold (see Figure 3). Some hydrolytic activity of magnetite surfaces toward organophosphates has been reported previously,30 possibly related to the ability of the iron hydroxide groups present on the magnetite surface to participate in the


Figure 3. Dependence of the observed DFP hydrolysis kinetic constants (kobs) on the effective concentration of additives magnetite (M), 2-pralidoxime (PAM), magnetite modified with pralidoxime (PAM/M), poly(4-vinylpyridine-co-acrylic acid) (p(VP-AA)/ M), and poly(4-vinylpyridine-N-phenacyloxime-co-acrylic acid) (p(PVOx-AA)/M). The broken line shows kobs for the spontaneous hydrolysis of DFP in buffer solution alone. T ) 25 °C; pH 7.0, 10 mM Tris buffer.

Figure 4. Initial rate of DFP hydrolysis (νo) vs concentration of the oxime groups ([Ox]o) in suspensions of magnetite modified with PAM or p(VPOX-AA) or solutions of PAM. [DFP]o ) 4 mM; T ) 25 °C; 10 mM Tris buffer, pH 7.0.

Scheme 1. Catalytic Hydrolysis of DFP by Oxime-Modified Magnetite Particles or PAM

nucleophilic substitution reactions.31 The presence of oxime groups on the nanoparticle surfaces dramatically enhanced the nucleophilicity of the magnetite surface, however, with the rates of the DFP hydrolysis with PAM-modified magnetite being 2-3 orders of magnitude faster than those of spontaneous hydrolysis (Figure 3). The hydrolysis of OP agents catalyzed by transition metal complexes has been most frequently characterized by the half-life (t1/2 ) 0.693/kobs) of the substrate and the apparent second-order hydrolysis rate constant, k ) νo/[catalyst]o[substrate]o.16,20,32-34 For comparison, in Table 1 we collected the t1/2 and k values for DFP hydrolysis by the oxime-containing species of the present work as well as literature data for DFP hydrolysis conducted under comparable conditions with several copper(II)-chelate complexes. As is seen, at excess catalyst the DFP decomposition in the presence of the PAM/M particles was more rapid than with the copper(II)-bipyridyl complexes, either monomeric or polymeric, which have thus far been reported to be most catalytically active,20,27,32,33 indicating the potential utility of PAM/M for nerve agent decomposition. Although much less active than the PAM/M, the polymer-modified magnetite, p(VPOx-AA)/ M, appeared to be a capable catalyst. We thus embarked next to a more detailed study of the catalytic mechanism using the oxime-containing particles introduced herein. The hydrolysis of DFP or sarin by metal chelates or oximates proceeds via the formation of complexes that are unstable and easily hydrolyzed in water, producing water-soluble phosphoric acids and fluoride ions.16,35,36 Hence, the catalytic reaction between DFP and oxime-

modified magnetite particles or PAM can be presented as shown in Scheme 1. The corresponding rate law is given by

ν)

dCt kcat[Ox][DFP] ) dt KM + [DFP]

(3)

where KM ) k-1 + kcat/k1 is the Michaelis constant, kcat is the catalytic rate constant, and [Ox] is the concentration of the catalytic (oxime) groups in the system. We examined dependencies of the initial rate of the DFP hydrolysis on initial concentration of the catalyst and substrate ([Ox]o and [DFP], respectively) using PAM and PAM- and p(VPOX-AA)-modified magnetite, and the experimental results are depicted in Figures 4 and 5. Linear fits in the initial rate vs catalyst concentration plots for constant substrate concentration or reciprocal linear rate vs reciprocal substrate concentration for constant catalyst concentration were obtained in all cases (R2 > 0.97), supporting the Michaelis-Menten

Table 1. Conditions, Half-Life (t1/2), and Apparent Second-Order Rate Constant (k) for the Catalyzed Hydrolysis of DFP catalyst PAM/M PAM P(VPOx-AA)/M Cu(II) complex with poly(4-vinylpyridine) quaternized with ethyl bromide and 4chloromethyl-4′-methyl-2,2′-bipyridine solution in 4-morpholine sulfonic acid buffer

copper(II)-N,N′-dimethylenediamine (1:1) complex in bicarbonate-CO2 buffer copper(II)-2,2′-dipirydyl chelate (1:1) in bicarbonateCO2 buffer

pH

T (°C)

t1/2 (min) ([catalyst]/ [DFP], M/M)

k (M-1 s-1)

ref

7.0 7.0 7.0 7.0

25 25 25 25

6.4 (4.3) 58 (1.8) 196 (3.8) 64 (13.9)

0.29 ( 0.088 0.075 ( 0.051 0.057 ( 0.018 0.025

present work present work present work 30

7.0 7.0 7.0 6.8

25 37 37 25

10 (33.9) 37 (16.1) 30 (17) 690 (125)

0.023 0.05 0.063 0.06

30 30 30 25

7.6

38.5

42 (2)

18

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Figure 5. Lineweaver-Burk plots of the reciprocal initial rate of DFP hydrolysis (1/νo) vs reciprocal initial DFP concentration (1/[DFP]o) in suspensions of magnetite modified with PAM or p(VPOX-AA) or solutions of PAM. Initial oxime concentrations were 1.7, 7.3, and 1.5 µM with PAM/M, PAM, and p(VPOx-AA)/M species, respectively. T ) 25 °C; 10 mM Tris buffer, pH 7.0.

scheme and enabling computation of the constants kcat and KM , which are given in Table 2. From the results in Table 2 it is evident that the catalytic efficiency, kcat/KM, of the PAM-modified magnetite suspensions was about 2.5-fold higher than that of the PAM solutions in the absence of magnetite. This interesting finding clearly points to the higher catalytic activity of the iron chelate compared to the oxime itself, an effect that has not been previously reported with magnetite-complexed PAM. Enhanced hydrolytic activity of Cu2+, Ni2+, or Zn2+ complexes with oximes compared to the oximes themselves has been described previously.22,24 Interestingly, the hydrolysis catalyzed by the polymermodified particles, p(VPOx-AA)/M, proceeded with 17fold and 7-fold lower catalytic efficiency than when catalyzed by the PAM/M particles or PAM solutions, respectively. Catalytic activity of the oxime groups, which depends on their ability to generate the oximate anion (an active species in the nucleophilic attack on the phosphorus electrophilic centers), tends to increase with the pKa of the oxime compound.37 That is, the greater the affinity of the oximate anion for a proton, the greater its reactivity with the phosphoryl center of the OP. In this regard, the oximate activity in p(VPOxAA)/M particles, with their apparent pKa below 5, should be expected to be lower than in PAM/M, with their pKa at or above 8.0. In addition, the oximate accessibility toward the substrate (DFP) can be lower in the layers of the p(VPOx-AA) polymer compared to the low molecular weight compound (PAM). Nanoparticle Recovery and Recycle. A primary motivation for the use of the magnetite particles was that, if needed, we could exploit their superparamagnetic properties to facilitate their recovery for reuse by high-gradient magnetic separation. These superparamagnetic properties are not available with the other metal chelates that catalyze nerve agent decomposition. We have demonstrated that we can separate the magnetite-based catalyst from the reaction medium and reuse it multiple times with negligible loss of catalytic activity for the effective degradation of organophosphates.

Figure 6. Catalytic stability of magnetite nanoparticles modified by PAM and p(VPOx-AA) indicated by the essentially unchanged kinetic profiles observed for DFP hydrolysis when the particles are recovered, washed, and reused. T ) 25 °C; pH 7.0, 10 mM Tris; initial DFP concentration in all cycles, 4 mM (PAM) or 1.33 mM (p(VPOx-AA)). Cycle 1 denoted the first use of the particles, cycle 2 the first magnetic recovery and resuspension of the particles at 1 mg/mL, and cycle 3 the second magnetic recovery and resuspension of the particles at 1 mg/mL.

Recovery of the nanoparticles from the aqueous solutions was achieved in a series of magnetic filtration experiments in each of which, following the successful catalytic hydrolysis of DFP by the oxime-coated nanoparticles, the suspension of particles was passed through the HGMS filter placed inside the quadropole magnet device. The magnetic particles were trapped in the filter and subsequently recovered by removing the steel-woolpacked column from the magnetic environment and passing fresh water through the filter; these particles were then reused in the hydrolysis of a fresh batch of DFP solution. This cycle of the DFP hydrolysis and particle filtration and collection was repeated twice as described in the Experimental Section. In all cases essentially complete recovery and reuse of the particles was possible. The effect of such a recycling on the ability of the PAM/M and p(VPOx-AA)/M particles to catalyze the DFP hydrolysis is depicted in Figure 6. The observed initial rate kinetic constants for DFP hydrolysis with the PAM/M and p(VPOx-AA)/M particle species were determined in three cycles to be unchanged at (4.5 ( 0.31) × 10-4 and (5.2 ( 0.36) × 10-5 s-1, respectively. In addition, the magnetization curves of the recovered particles determined by SQUID were essentially identical to those of the original particles; these results indicate that the catalytic particles suffered no degradation and were chemically stable under the hydrolysis conditions used in these studies. Note that the initial kinetics for the two cases shown above were substantially different but that the rate of hydrolysis with PAM/M slowed and was characterized by a slower first-order process with the same kinetic rate constant (same slope on a semilogarithmic plot) as observed for p(VPOx-AA)/M over the entire experimental range. Such behavior was observed in all cases when the substrate was in stoichiometric excess relative to the catalyst and is consistent with other observations reported in the literature.38 A more detailed study is underway to elucidate the reasons for this behavior; this current study has focused on the using initial rate kinetics as the metric for catalytic efficiency, in accord with customs established by earlier workers in this field.

Table 2. Michaelis Constant (KM), Catalytic Rate Constant (kcat), and Catalytic Efficiency Found for the DFP Hydrolysis in the Presence of Oxime-Containing Species, in 10 mM Tris Buffer at pH 7.0 species

KM × 103 (M)

kcat × 104 (s-1)

catalytic efficiency

PAM/M PAM p(VPOx-AA)/M

2.5 ( 0.12 1.2 ( 0.057 0.83 ( 0.040

6.1 ( 0.37 1.2 ( 0.073 0.12 ( 0.006

0.24 0.10 0.014


Previous study32 found a dramatic decline in the hydrolysis rate of soman when a solution of a polymeric amine-copper(II) complex was used in the presence of a polymeric sorbent, due to the strong adsorption of the substrate onto the hydrophobic polymeric resin. Modification of such polymeric sorbents with the catalytic magnetic nanoparticles of the present work should be effective in alleviating these problems and aid in the more efficient degradation of the OP agents.39 The catalytic capacity of the particles and their ability to be captured and recycled for reuse should aid in applications such as treatment of surface water and groundwater, where it is undesirable to leave the catalysts and the degradation products dispersed in the large volumes of treated contaminated aqueous supplies; magnetite nanoparticles tailored to have ion-exchange characteristics to capture the phosphoric acids resulting from the OP decomposition can be introduced with the catalytic nanoparticles to ensure that the degradation products can also be removed. These topics are currently under study. Concluding Remarks Most of the OP pesticides in current worldwide use as well as combat OP nerve agents are quite stable toward aqueous hydrolysis at neutral pH. In this regard, the development of cost-effective catalytic destruction technologies for the OP agents that do not involve the application of extremes of pH, heat, or toxic chemicals, becomes extremely attractive. In this work, we have reported that magnetite nanoparticles modified by oxime-containing moieties catalyze the hydrolysis of the OP nerve agent, DFP, at neutral pH. The observed rates of DFP hydrolysis by the modified magnetite were comparable to those with toxic copper(II) chelates. The ready availability of magnetite and antidotal oxime drugs or their polymeric analogues, the simplicity of the developed synthetic route toward the oxime-modified particles, and the possibility of particle recovery by magnetic separation demonstrated herein suggest that the newly developed nanoparticles show much promise as a novel addition to the family of catalytic destruction technologies for organophosphate compounds. Literature Cited (1) Eyer, P. The role of oximes in the management of organophosphorus pesticide poisoning. Toxicol. Rev. 2003, 22 (3), 165190. (2) Chiron, S., Fernandez-Alba, A.; Rodriguez, A.; Garcia-Calvo, E. Pesticide chemical oxidation: State-of-the-art. Water Res. 2000, 34 (2), 366-377. (3) 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. (4) Magee, R. S.. U. S. chemical stockpile disposal program: The search for alternative technologies. In Effluents from Alternative Demilitarization Technologies; Holm, F. W., Ed.; Kluwer: Dordrecht, The Netherlands, 1998; Vol. 22, p 112. (5) Amos, D.; Leake, B. Cleanup of chemical agents on soils using simple washing or chemical treatment processes. J. Hazard. Mater. 1994, 39, 107-117. (6) Yang, Y. C. Chemical detoxification of nerve agent. Acc. Chem. Res. 1999, 32, 109-115. (7) Yang, Y. C.; Baker, J. A.; Ward, J. R. Decontamination of chemical warfare agents. Chem. Rev. 1992, 92, 1729-1743. (8) Wagner, G. W.; Yang, Y.-C. Rapid nucleophilic/oxidative decontamination of chemical warfare agents. Ind. Eng. Chem. Res. 2002, 41, 1925-1928. (9) Moss, R. A.; Chung, Y. C. Immobilized iodosobenzoate catalysts for the cleavage of reactive phosphates. J. Org. Chem. 1990, 55, 2064-2069.

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Received for review June 13, 2005 Revised manuscript received July 31, 2005 Accepted August 10, 2005 IE0506926

Nerve Agent Destruction by Recyclable Catalytic Magnetic

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