MAY/JUNE 1997 Volume 8, Number 3 © Copyright 1997 by the American Chemical Society
ARTICLES Syntheses of Haptens Containing Dioxaphosphorinan Methoxyacetic Acid Linker Arms for the Production of Antibodies to Organophosphate Pesticides Wolter ten Hoeve,† Hans Wynberg,† William T. Jones,*,‡ Dawn Harvey,‡ Gordon B. Ryan,‡ and Paul H. S. Reynolds‡ Syncom, University of Groningen, Groningen, The Netherlands, and Plant Improvement Division, The Horticulture and Food Research Institute of New Zealand, Private Bag 11 030, Palmerston North, New Zealand. Received October 16, 1996X
Four generic heterobifunctional reagents, namely 2-(2-chloro-5-methyl-1,3,2-dioxaphosphorinan-5yl)methoxyacetic acid methyl ester, p-sulfide, 2-(2-chloro-5-methyl-1,3,2-dioxaphosphorinan-5-yl)methoxyacetic acid methyl ester, p-oxide, 2-(2-mercapto-5-methyl-1,3,2-dioxaphosphorinan-5-yl)methoxyacetic acid bispotassium salt, p-sulfide-, and (2-methoxy-5-methyl-1,3,2-dioxaphosphorinan5-yl)methoxyacetic acid, methyl ester, have been synthesized and used to prepare organophosphate, thiophosphate, and dithiophosphate haptens containing a functional carboxyl group which can be used to conjugate the haptens to proteins. These hapten-protein conjugates have been used as antigens for preparing polyclonal sera against all classes of organophosphate pesticides. The eight examples used protein-hapten conjugates of chlorpyrifos, parathion, diazinon, paraoxon, azinphos, dimethoate, demeton, and dichlorvos. These were all immunogenic and resulted in sera containing antibodies that recognized the corresponding parent pesticide with high specificity.
INTRODUCTION
Residues of pesticides used for protection of horticultural crops against insect infestations need to be monitored from a human health as well as an economic perspective. Legal maximum residue limits are stipulated by both national and international regulatory agencies for many of these compounds in most food crops * Address correspondence to Dr. W. T. Jones, The Horticulture and Food Research Institute of New Zealand, Batchelar Research Centre, Private Bag 11 030, Palmerston North, New Zealand. Telephone: +64-6-356-8080. Fax: +64-6-351-7031. † University of Groningen. ‡ The Horticulture and Food Research Institute of New Zealand. X Abstract published in Advance ACS Abstracts, April 1, 1997.
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and products. Increasingly, levels of such residues are being monitored by the international and domestic agencies. Accordingly, there is a need for internationally acceptable, rapid, reliable, sensitive, and cost-effective assay systems for determining the presence of these compounds. Suitably designed immunoassay-based tests can fulfill all these requirements. Modern immunoassays are based on two important phenomena: (i) the extraordinary discriminatory power of antibodies and (ii) detection systems that allow the reaction of the antibody with its hapten to be quantified at low concentrations of the reactants (antibody and hapten). The use of enzyme immunoassays (EIAs) and solid phase technology has brought about widespread use of these techniques. An excellent review of enzyme immu© 1997 American Chemical Society
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ten Hoeve et al.
Figure 1. Synthetic scheme for the production of generic intermediates for synthesis of organophosphate, organothiophosphates, and organodithiophosphate haptens.
noassay is provided by Tijssen (1990). A large number of assays have been developed for aflatoxins (Chu et al., 1991) and herbicides and insecticides (Aston et al., 1992). Residues are small molecules (Mr < 1000) and are usually unable to invoke an immune response when injected into animals. These molecules therefore have to be conjugated to larger immunogenic carrier molecules to produce the desired response. The organophosphate group of pesticides, being small molecules, are not immunogenic, nor can the majority of them be readily conjugated to a suitable carrier protein to render them antigenic. Until recently, antibodies have been raised only to those organophosphates that could easily be modified to allow coupling of the modified pesticide to the carrier protein (parathion, Ercegovich et al., 1981; paraoxon, Brimfield et al., 1985; Heldman et al., 1985). Heldman et al. (1985) produced antibodies to paraoxon by conjugation via a β-alanine linker attached through the phosphate atom. McAdam et al. (1992) described the production of haptens for preparing antibodies to fenitrothion. This entailed coupling the hapten through the phosphate group to the carrier protein and produced haptens resembling those of Heldman et al. (1985). This method of coupling through the phosphate group produced the best antibodies (Hill et al., 1992). McAdam and Skerritt (1993) described the use of tert-butyl-3[[chloro(methoxy)phosphorothioyl]amino]propanoate as a generic intermediate in preparing haptens of the organothiophosphate pesticides and successfully raised antibodies to fenitrothion, chlorpyrifos-methyl, and pyrimiphos-methyl. This work describes the synthesis of new generic heterobifunctional intermediates containing a dioxaphosphorinan ring which can be used to synthesize organophosphate, thiophosphate, and dithiophosphate haptens containing a functional carboxyl group to conjugate to proteins. These hapten-protein conjugates can be used as antigens for preparing antibodies for use in the development of specific immunoassays for the detection of specific pesticides. RESULTS
Synthesis of Haptens and Conjugates. Access to haptens of some common organophosphate pesticides was investigated in the first instance through replacement of the methoxy or ethoxy group in these pesticides by another alkoxy group which would contain an amino or carboxylic acid function. The difficulties encountered in attaining such unsymmetrical compounds in a pure state
led to the investigation of an approach in which both methoxy and ethoxy groups were replaced by a group that would lead to a more symmetrical compound. Such a compound would presumably be easier to purify. On this basis, a 1,3-propanediol was chosen which contained a group at position 2 with a protected carboxylic acid function. 2-(Hydroxymethyl)-1,3-propanediol is commercially available and can be converted easily to 5-(hydroxymethyl)-2-methyl-1,3-dioxane (Gash, 1972). We reasoned that functionalization of the free hydroxy group leading to a group containing a carboxylic ester function, followed by coupling of the deprotected diol part to the phosphorus atom of pesticides, and finally selective hydrolysis of the carboxylic ester should lead to the desired haptens. In practice, this approach (shown in Figure 1) appeared to be useful for the preparation of a series of haptens. Treatment of 5-(hydroxymethyl)-2-methyl-1,3-dioxane 1 with 2 equiv of sodium hydride in DMF followed by the addition of bromoacetic acid gave the sodium salt of the coupled product. This was directly reacted with dimethyl sulfate to furnish the methyl ester 2. In order to gain access to the desired haptens, it was necessary to transform the acetal moiety into a diol, while maintaining the ester moiety intact. Common acidic cleavage methods led to the loss of the ester group, but under mild conditions, using pyridinium p-toluenesulfonate, the acetal was preferentially cleaved. The resulting crude diol 3 was used in the following transformations to prepare a set of generic intermediates useful for syntheses of organophosphate, organothiophosphate, and organodithiophosphate haptens. (1) Treatment with thiophosphoryl dichloride and triethylamine in toluene gave a mixture of two isomeric (chlorine atom cis or trans, respectively) thionodioxaphosphorinanes 4, the major isomer of which could be isolated as a crystalline compound. (2) Treatment with phosphoryl chloride and triethylamine in toluene gave a mixture of two isomeric dioxaphosphorinanes 5 which, in our hands, were inseparable. (3) Heating with phosphorus pentasulfide in toluene followed by treatment with potassium hydroxide resulted in the precipitation of the bispotassium salt 6. (4) Stirring with trimethylphosphite and trimethylamine (Edmundson et al., 1985) gave the distillable cyclic phosphite 7. The precursors 4-7 were then used for the preparation of the following haptens (Figure 2).
Haptens for Production of Antibodies to Organophosphates
(a) Chlorpyrifos. The sodium salt of trichloropyridinol (obtained from the pyridinol with sodium hydride in DMF) was reacted with the crystalline isomer of chloride 4 in DMF at room temperature (RT). The resulting crystalline ester 8 could be hydrolyzed, under very mild basic conditions, viz., potassium carbonate in water, methanol, and THF, to the hapten, crystalline acid 9. (b) Parathion. 4-Nitrophenol was coupled to chloride 4 as described for chlorpyrifos to yield crystalline ester 10. The ester was hydrolyzed with lithium hydroxide, resulting in crystalline acid 11. (c) Diazinon. The sodium salt of isopropylmethylpyrimidinol was reacted with chloride 4 to furnish crystalline ester 12. Hydrolysis with potassium carbonate gave crystalline acid 13. (d) Paraoxon. 4-Nitrophenol was reacted with the mixture of isomeric chlorides 5, resulting in two isomeric esters 14, which could be separated by recrystallization. The paraoxon hapten 15 was prepared by potassium carbonate hydrolysis of the methyl ester. The use of lithium hydroxide, as for parathion, led to hydrolysis of the phosphate ester. (e) Azinphos. The bispotassium salt 6 was treated with 1 equiv of hydrochloric acid to protonate the weaker carboxylic acid function and reacted with 3-(chloromethyl)-1,2,3-benzotriazin-4(3H)-one in acetone to give hapten 16 which was purified through acid-base separation and by crystallization. (f) Dimethoate. This was prepared as for azinphos. N-Methylchloroacetamide was reacted with bispotassium salt 6 to give crystalline acid 17. (g) Demeton. Access to hapten 19 was possible by making use of the known O-S rearrangement (Sasse, 1964). Ethylthioethanol was reacted with butyllithium followed by addition of chloride 4. The resulting ester 18 slowly underwent O-S rearrangement. However, the product could not be hydrolyzed to acid 19 without completely destroying the molecule. Therefore, ester 18 was hydrolyzed before rearrangement took place. The resulting crude acid slowly underwent O-S rearrangement, and hapten 19 was obtained in low yield as a crystalline solid. (h) Dichlorvos. Phosphite 7 was reacted with anhydrous chloral by the Perkow reaction (Gallenkamp, 1982) to yield crude ester 20 which was hydrolyzed with potassium carbonate to the crystalline acid 21 together with an unknown acid. The desired hapten 21 was purified from the unknown acid by washing with ether and recrystallization from water/methanol. None of the haptens inhibited cholinesterase activity at 200 ppm when tested with a commercial kit (Enzytec Inc.). This contrasts with the parent pesticides which gave 100% cholinesterase inhibition at concentrations ranging from 0.3 to 2.0 ppm. No deterioration has been observed when haptens were stored dry at 4 °C for 2 years. Production of Antibodies to Haptens. The carboxyl function on haptens 9, 11, 13, 15-17, 19, and 21 was activated to a succinimidyl ester (Langone and Vanakis, 1975) and coupled to free amines on ovalbumin (OVA) or mouse serum albumin (MSA) to produce antigens with which to immunize mice or to use as plating antigens for enzyme-linked immunoassays (ELISAs), respectively. Using this procedure, conjugates were prepared with the degree of coupling between being 8 and 20 mol of hapten per mole of OVA and 12-30 mol of hapten per mole of MSA. OVA immunoconjugates of haptens were injected into mice, and the resulting antisera were assessed in an ELISA format as described in Materials
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and Methods. In all cases, immunization resulted in the production of antisera recognizing haptens conjugated to MSA. No reaction was observed when preimmune sera were reacted with MSA-haptens or when sera from immunized mice were tested against MSA. Thus, a specific reaction to the hapten was apparent. Optimal plating concentrations of MSA-hapten and sera dilution of individual mice to give an absorbance at 492 nm of 0.8-1.2 were established for each antigen by checkerboard titration. Competition, defined as the inhibition of binding of antibody to microwell plates as a result of incubation with organophosphate pesticide or hapten, was observed in all experiments for the pesticide resembling the hapten (i.e., chlorpyrifos for the chlorpyrifos hapten) used to immunize mice. Within a particular assay, variation was observed between mice in the I50 and in the useful range (I20-I80) for measuring of organophosphate, with specific results as follows. (a) Chlorpyrifos Immunoconjugate. The optimal dilutions of MSA-hapten were 25, 90, 50, and 25 ng/mL and of sera 1/32000, 1/64000, 1/32000, and 1/32000 for mouse 1-4, respectively. I50, I20, and I80 for chlorpyrifos using mouse 1 and 2 sera were 300 ng/mL, 60 ng/mL, and 1.5 µg/mL and mouse 3 and 4 sera 150, 25, and 860 ng/mL, respectively. Competition was not measurable at concentrations of paraoxon, parathion, and azinphos-methyl up to 200 µg/mL. The chlorpyrifos hapten had an I50 of 20 ng/mL; I20 ) 1 ng/mL and I80 ) 100 ng/mL for all mice. (b) Parathion Immunoconjugate. The optimal dilutions of MSA-hapten were 25, 25, 50, and 50 ng/mL and of sera 1/32000, 1/64000, 1/32000, and 1/64000 for mouse 1-4, respectively. I50, I20, and I80 for parathion using mouse 1 serum were 1.2 µg/mL, 80 ng/mL, and 16 µg/ mL, using mouse 2 serum were 800 ng/mL, 60 ng/mL, and 11 µg/mL, using mouse 3 serum were 4 µg/mL, 300 ng/mL, and 31 µg/mL, and using mouse 4 serum were 2.1 µg/mL, 350 ng/mL, and 33 µg/mL, respectively. For all mouse sera, no competition was observed with chlorpyrifos and azinphos. Paraoxon showed cross reactivities of 0.6 and 0.4% for mouse 1 and 2, respectively, and no cross reaction for mouse 3 and 4 sera. Competition with parathion hapten gave an I50 of 20-25 ng/mL, an I20 of 1-2 ng/mL, and an I80 of 80-100 ng/mL. (c) Paraoxon Immunoconjugate. The optimal dilutions of MSA-hapten were 200, 150, 25, and 25 ng/mL and of sera 1/16000, 1/32000, 1/8000, and 1/16000 for mouse 1-4, respectively. I50, I20, and I80 for paraoxon using mouse 1 serum were 8.1, 1.2, and 46 µg/mL, using mouse 2 serum were 2.2 µg/mL, 710 ng/mL, and 105 µg/mL, using mouse 3 serum were 8, 1.2, and 43 µg/mL, and using mouse 4 serum were 2 µg/mL, 200 ng/mL, and 23 µg/mL, respectively. No cross reaction was observed for any sera from mice injected with paraoxon-hapten immunoconjugates, with parathion, chlorpyrifos, or azinphos-methyl or -ethyl. The paraoxon hapten gave an I50 of 20 ng/mL, an I20 of 2 ng/mL and an I80 of 100 ng/mL. (d) Azinphos Immunoconjugate. The optimal dilutions of MSA-hapten were 25, 12.5, 25, and 25 ng/mL and of sera 1/64000 for mouse 1-4, respectively. I50, I20, and I80 for azinphos-methyl using mouse 1 and 3 sera were 4 µg/mL, 400 ng/mL, and 30 µg/mL, using mouse 2 serum were 20 µg/mL, 800 ng/mL, and 120 µg/mL, and using mouse 4 serum were 800 ng/mL, 20 ng/mL, and 34 µg/ mL. Chlorpyrifos and paraoxon showed no cross reaction. Parathion showed cross reaction at 1-2% azinphosmethyl. Azinphos-ethyl was slightly more competitive than azinphos-methyl. For the azinphos hapten, I50 ranged from 6 to 10 ng/mL, I20 from 1 to 2 ng/mL, and I80 from 60 to 100 ng/mL. (e) Demeton Immunoconjugate. The optimal dilutions
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Figure 2. Overall synthetic scheme for organophosphate haptens.
of MSA-hapten were 25, 200, 50, and 50 ng/mL and of sera dilution of 1/32000, 1/64000, 1/128000, and 1/32000 for mouse 1-4, respectively. I50, I20, and I80 for demeton using mouse 1 and mouse 4 sera were 20 µg/mL, 800 ng/ mL, and 120 µg/mL, using mouse 3 serum were 66, 7, and >200 µg/mL, and using mouse 2 serum were 20.0, 1.8, and 80.0 µg/mL, respectively. For the demeton hapten, I50 ranged from 10 to 20 ng/mL, I20 from 2 to 6 ng/mL, and I80 from 80 to 150 ng/mL.
(f) Dimethoate Immunoconjugate. The optimal dilutions of MSA-hapten were 500, 250, 500, and 500 ng/ mL and of sera dilution 1/32000, 1/10000, 1/64000, and 1/64000 for mouse 1-4, respectively. I50, I20, and I80 for dimethoate using mouse 1-3 sera were 45, 6, and 180 µg/mL and using mouse 4 serum were 66, 7, and >200 µg/mL, respectively. For the dimethoate hapten, I50 ranged from 20 to 30 ng/mL, I20 from 4 to 10 ng/mL, and I80 from 90 to 150 ng/mL.
Haptens for Production of Antibodies to Organophosphates
(g) Dichlorvos Immunoconjugate. The optimal dilutions of MSA-hapten were 90, 250, 500, and 500 ng/mL and of sera 1/32000 for mouse 1-4, respectively. I50, I20, and I80 for dichlorvos using mouse 1, 2, and 4 sera were 80, 12, and >200 µg/mL and using mouse 3 serum were 200, 35, and >200 µg/mL, respectively. For the dichlorvos hapten, I50 ranged from 20 to 40 ng/mL, I20 from 5 to 15 ng/mL, and I80 from 90 to 180 ng/mL. (h) Diazinon Immunoconjugate. The optimal dilutions of MSA-hapten were 250, 500, 250, and 250 ng/mL and of sera 1/25000, 1/12800, 1/25000, and 1/25000 for mouse 1-4, respectively. I50, I20, and I80 for diazinon using mouse 1 serum were 2.1 µg/mL, 250 ng/mL, and 15 µg/ mL, using mouse 2 serum were 1.4 µgmL,
