Chem. Res. Toxicol. 1991,4, 517-520
517
Comparative Anticholinesterase Potency of Chiral Isoparathion Methyl Seungmin Ryu, Jing Lin, and Charles M. Thompson* Department of Chemistry, Loyola University of Chicago, Chicago, Illinois 60626 Received June 26, 1991
Introduction Organophosphates remain a major class of compounds used for the control of insect pests. In most instances, metabolic oxidation of a thionate (PES) to an oxon (P=O) moiety is first required for their toxic action (1). The toxicity of the oxon compounds is attributed to the ability of these molecules to inactivate acetylcholinesterase ( 2 ) ) the enzyme responsible for the hydrolysis of the neurotransmitter acetylcholine (3))by formation of a covalent bond between an active-site serine residue and the phosphorus atom synchronous with the ejection of one of the organophosphate ligands. In eq 1, X is an appropriate Enz-OH
+
OP-X
Kd e [Enz-OH'OP-X] ki
-t kP
Enz4P
+ Xe
(1)
leaving group in the organophosphorus ester, Kd is the dissociation constant between reactants and complex, k, is the phosphorylation constant, and ki is the bimolecular inhibition constant and is equal to kp/Kd.Kd is regarded as a measure for binding and depends upon the structural features of the molecule, and k , reflects the effect of change in the reactivity of the ester. Whereas the electronic properties of the phosphorus atom play a major role in inhibitory potential ( 4 ) )more subtle atomic changes in the organophosphorus ester also may lead to differences in these kinetic parameters. For example, a center of asymmetry a t the phosphorus of an organophosphate can have a significant effect on the biological activity of the ester (5),especially its ability to inhibit acetylcholinesterase and related esterases (6, 7). These representative investigations of enzyme inactivation by the enantiomers of several important organophosphates have helped define the influence of asymmetry upon the mechanism of action and metabolism. In a prior report, we showed that phosphorothiolate impurities (isomerides) are significantly better anticholinesterases than the phosphorothionates from which they are formed (8) (eq 2). The mechanism of cholin-
7i MeO-P-X I
OMe phosphorothionate
-
0
II
MeS-P-X
I
(2)
OMe phosphorothidate
esterase inactivation by the isomerides presumably proceeds by displacement of an established leaving group X. Therefore, most phosphorothiolates react with cholinesterases to form an 0,s-dimethyl phosphorothiolated serine hydroxyl, and chirality a t the phosphorothiolate phosphorus atom also would be expected to affect the nature of this inhibition. In light of this discussion and the common structural relationship among the phosphorothiolate impurities, we decided to seek evidence that would support the importance of stereochemistry at phosphorus upon the inactivation of various cholinesterases. In this communication, we report this evidence,
namely, the relative inhibitory potency of the chiral antipodes of isoparathion against four different cholinesterases.
Materials and Methods Caution: Organophosphorus esters are hazardous and should be handled in a well-ventilated hood. The phosphorothiolates described herein are readily destroyed by reaction with 6 N NaOH solution at room temperature. 31PNMR chemical shifts are relative to phosphoric acid (H$04 in CDCl,). Optical rotations were conducted in CH3CNor CH30H at the concentrations indicated. Capillary gas chromatography (GC)was performed on a 15-m,DB-1 capillary column at gas flow rates of 300 mL/min (air), 30 mL/min (hydrogen),and 15mL/min (helium). The injector and detector temperatures were 250 and 275 "C, respectively. Ramped oven temperatures of 50-250 "C at 20 "C/min were used. High-performanceliquid chromatography (HPLC) was conducted on a 10-pm octadecylsilane (ODS; 30-cm) reversed-phasecolumn using methanol-water as solvent with detection at 270 nm. All solvents and reagents were purified when necessary by standard literature methods. Racemic isoparathion methyl was available from a prior study (8). (+I- and (-)-0-(4-nitrophenyl) 0-methyl S-methylphosphorothioate (isoparathionmethyl) were prepared either through diastereomeric amides derived from proline (9) or by fractional crystallization of the (-)-strychnine thioacid salts (IO) (Scheme I). Human erythrocyte, bovine erythrocyte, electric eel, and horse serum cholinesterases were obtained from Sigma Chemical Co., St. Louis. Male rats with a body weight range of 175-250 g were sacrificed via decapitation and the brains excised, rinsed of excess blood, stored in 0.01 M potassium phosphate buffer (pH 7.6), and chilled to 0 "C ifstored. Crude and solubilized rat brain acetylcholinesterasewere obtained as follows. Excised rat brains (previously stored at 0 "C) were rinsed with 0.01 M potassium phosphate buffer (pH 7.6) and homogenized at 0 "C in 10 mL of phcephate buffer. For solubilized protein, Triton X-100 (10 mL; 1%solution) was added, mixed gently for 15 min, and centrifuged for 1 h at 1OOOOOg. The supernatant containing the solubilized AChE was removed and stored at 4 "C. Details of the ki determinations using varied inhibitor incubation times were summarized in a prior report (8). To more precisely evaluate the kinetic components Kd and k, (Table 111), measurements of the remaining cholinesterase activity after incubation of the enzyme with a progression of inhibitor concentrations was conducted. In a typical experiment, six test tubes containing suitably diluted enzyme solution (990 pL) were each treated with 10 pL of a progression of inhibitor concentrations. The inhibition was permitted to progress for 20 min, and the remaining enzyme activity was determined over a period of 30 min (1-min intervals). Plots of 1/[I] versus l/slope (at the individual inhibitor concentration) were generated and the kinetic parameters obtained (11). All kinetic assays depend on the cholinesterase-catalyzedhydrolysis of acetylthiocholineand the estimation of thiocholine produced (12).
Results Chiral isoparathions 3a/3b were prepared by boron trifluoride promoted methanolysis of the corresponding diastereomeric proline amides (Scheme I) (9). Prior to methanolysis, the proline amides 2a/2b were purified to greater than 99% as determined by HPLC and 31PNMR
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518 Chem. Res. Toxicol., Vol. 4, No. 5, 1991
compd 1 2a 2b 3a 3b 4a 4b
[(r]26D
-70.8" -49.7" +30.5" -30.5" +23.0° -22.9"
(concn)
(3.25, MeOH) (1.35, MeOH) (1.15, MeOH) (1.85, MeOH) (0.10, MeCN) (0.45, MeCN)
Communications
Table I. Physical and Spectral Data P P ) , ppm GC, min 38.5 3.1 30.7 13.2 30.5 13.2 27.9 8.8 27.9 8.8 51.0 51.0
HPLC, min
mp, "C oil
18.7O 19.9 8.5b 8.5b
WaX
oil oil oil 214-216 (needle) 219-220 (prism)
'55:45 MeOH/H,O, flow rate = 1.5 mL/min. b50:50 MeOH/H,O, flow rate = 2.0 mL/min.
Table 11. IsoDarathion Methyl Enzyme Inhibition Constants (k,:M-' m i d ) difference racemic, enzyme (+)O,b (-)a,b (-)/(+) calcd racemic, found rat brain AChE (solubilized) 25 "C 2.10 X lo4 (*4.1%) 3.26 X lo5 (*2.5%) 15.5 1.74 X lo5 1.90 X lo5 (4Z2.770) 6.34 X lo5 (4Z4.0%) 8.3 3.55 X lo5 4.16 X 106 (*LO%) 37 "C 7.68 X lo' (*2.3%) rat brain AChE (homogenate) 25 "C 2.06 X lo4 (&6.7%) 3.15 X lo5 (4~5.7%) 15.3 1.68 X lo5 1.82 X lo5 (*2.2%) 37 "C 7.14 x 104 (*5.3%) 4.34 x 105 (4~4.1%) 6.1 2.53 X lo5 2.91 X lob (&2.0%) human erythrocyte AChE 37 "C 2.24 X 10' (&9.7%) 6.15 X lo' (4Z6.0%) 2.7 4.20 X lo' 4.38 X lo' (f7.4%) bovine erythrocyte AChE 37 "C 6.18 X lo3 (&1.7%) 4.35 X lo' (*0.9%) 7.0 2.48 X lo4 2.30 X lo' (4Z2.8%) horse serum BChE 1.21 x 104 ( h 0 . m ) 6.81 x 104 (4~1.9%) 5.6 4.01 X 10' 4.20 X lo4 (&4.8%) 37 "C electric eel AChE 25 "C 6.42 X lo2 (*0.5%) d
dev, %' 9.2 17.2 8.3 15.0 1.6 7.3 4.7
"Coefficient of standard deviation = (SD/r) X 100%. b t test confidence level of 99.9%. 'Deviation % = [(calcd - found)/calcd] X 100%. Not determined.
(Table I). As a further check of purity, chiral isoparathion methyl antipodes also were obtained by alkylation of the diastereomeric strychnine salts 4a/4b (IO). The rotations of the enantiomers prepared by both methods were equal. The (-)-isomer probably corresponds to the S, configuration, relative to that assigned for sulprophos (5). The ki values for (+)-, (+, and (-)-isoparathion methyl were determined for four cholinesterases (Table 11) including rat brain acetylcholinesterase a t 25 and 37 "C. Studies a t different temperatures were undertaken to assess this particular contribution to the inhibitory potential of the stereoisomers and to correlate prior studies conducted at both temperatures. The (-)-isomer showed 315-fold greater inhibitory potency than the (+)-isomer. All inhibition constants within a stereoisomer set were shown to be significantly different between the isomers and racemate (t test confidence level = 99%). Racemic isoparathion methyl showed inhibitory potency that was approximately the average (calculated,value) of the individual antipodes. Slight deviations from the calculated value were noted in some cases. An interesting pattern was revealed in the sensitivity of the varied enzyme sources to chiral isoparathion with a range in ki of 60-fold for the (+)-isomer and about 15-fold for the (-)-isomer. Of these, the rat brain enzyme source was the most sensitive and electric eel the least. However, we were unable to obtain an accurate ki value for the (-)-isomer against electric eel acetylcholinesterase (EEAChE). Spontaneous reactivation may be responsible, in part, for this difficulty since longer incubation times with the inhibitor led to increased enzyme activity. The stereoisomers showed similar ki differences with solubilized and homogenate rat brain cholinesterases. A marked discrimination for the (-)-isomer at 25 "C was found for both enzyme preparations. Most of the enzymes distinguish similarly between the isomers a t 37 OC, with the exception of human erythrocyte cholinesterase. Further kinetic analysis of antipode action on solubilized rat brain cholinesterase was undertaken (Table 111). The stereoselective inhibitory action was found to be linked to
Table 111. Inhibition Parameters for Chiral Isoparathion Methyl against Rat Brain AChE (Solubilized) at 37 OC" isomer ki, M-' min-' Kd, mM k,, min-' (-4 6.30 X lo5 2.70 X lo4 0.17 (i8.2%) (*) 4.13 X lo6 4.50 X lo-' 0.19 (&10.2%) (+) 7.82 X lo4 2.73 X 0.21 (k6.170) "Kinetic data were measured with a progression of inhibitor concentrations.
the dissociation constants (Kd) with (-)-isoparathion methyl. The phosphorylation (k ) values were not significantly different. The bimolecufar inhibition constants obtained by variation in the inhibitor concentrations were 8-fold in favor of the (-)-isomer (Table 111) and differed slightly from the varied incubation time method (Table 11).
Discussion Chiral isoparathion mgthyl was chosen to represent the general class of 0,s-dimethyl phosphorothiolates, compounds that are found as impurities in commercial organophosphate formulations. Since the p-nitrophenol moiety is an excellent leaving group, we postulated that any stereoselective action found for isoparathion methyl would at least minimally represent phosphorothiolates bearing similar or weaker leaving groups (X). The in vivo toxicity of chiral isoparathion methyl had previously been determined to favor the (-)-isomer by 5-fold (10) and is in good agreement with the in vitro inhibition data presented here. Yet, there are some further interesting observations that emerged in this study. First, the human erythrocyte cholinesterase was less able to discriminate the chiral isomers than the rat brain acetylcholinesterase. Human erythrocyte cholinesterase may be less stereoselective owing to more facile esteratic site access. In any event, a 6-15-fold difference in inhibitory action against the cholinesterases by the (-)-stereoisomer is significant since virtually no branching in the molecule
Communications
Chem. Res. Toxicol., Vol. 4, No. 5, 1991 519 Scheme I. Synthesis of Chiral Isoparathion Methyl
i
CH3S-
1P -C1 I
c1 1
1. t - p r o l i n e e t h y l ester
h
i
2a
3a
2. p - n i t r o p h e n o l
3. s e p a r a t e
0
0
CH3S- II
-N?C0,Et
BF3-MeOH
C H 3 e It P -*1*111 SCH 3
I
0
is present. Second, in consideration of the strong inhibitory potency of isoparathion methyl, the differences in rat brain kiare somewhat larger than expected. Third, the formation of the enzyme-inhibitor complex, as reflected by the dissociation constant Kd, is limited by the stereoisomer configuration, possibly reflecting the finite dimensions of the esteratic locus of rat brain AChE. A more “tightly” defined active site also could adversely affect subsequent access to the phosphorylated serine by reactivation agents (oximes), suggesting that nervous tissue based AChE could possibly remain poisoned longer than other cholinesterase sources. Last, once the irreversible complex between enzyme and inhibitor is formed, the phosphorylation (k,; Table 111) proceeds rapidly and is independent of the chirality of the inhibitor. This observation is consistent with the fact that the electronegativity a t phosphorus is identical for both stereoisomers, leading to comparable k, values, whereas the chirality affects the three-dimensional orientation and is consistent with the different Kd values observed for all enzymes examined. Doubtless, organophosphorus-based inhibition of cholinesterase is a well-established phenomenon and is responsible for a majority of the toxic action by these materials to many target and nontarget organisms. The investigation of enzyme inactivation by organophosphates can provide critical information about essential atomic features of the inhibitor that play a role in the intoxication event. The use of a chiral inhibitor has provided more
precise information about the enzyme’s ability to accommodate certain three-dimensional structures (13). Several phosphorothiolates acting either as insecticides or as impurities in insecticides have been shown to be potent anticholinesterase agents (14). Phosphorothiolates that bear a center of asymmetry a t phosphorus differ in their inhibitory potency (5). Of particular concern is the finding that acetylcholinesterase in mammalian brain tissue is highly susceptible to inhibition by these stereoisomers. This study reinforces the importance of stereochemistry at phosphorus and species specificity as essential determinants in the evaluation of the intoxication mechanism.
Acknowledgment. We thank the National Institutes of Health (ES04434)for kind financial support of this project. We also express our gratitude to Loyola University of Chicago for the purchase of the Varian VXR 300-MHz NMR used in this study and additional support administered through the BRSG. Kind technical assistance was provided by Kwang C. Chung (Northwestern), Amy Larsen, Debra Quinn, and Prof. Diane Suter (Biology Department, Loyola) for which we are grateful.
References (1) Eto, M.(1974)Organophosphorus Pesticides; Organic and Biological Chemistry, CRC Press, FL. (2) Koelle, G. B., Ed. (1963)in Cholinesterases and Anticholinesterase Agents. Handbuch der Expenmentellen Pharmakoligie, Vol. 15, Springer-Verlag, Berlin.
Chem. Res. Toxicol. 1991, 4 , 520-524
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(3) Quinn, D. (1987) Acetylcholinesterase. Enzyme structure, reaction dynamics, and virtual transition states. Chem. Reu. 87, 955-979. (4) Wallace, K. B., and Kemp, J. R. (1991) Species specificity in the chemical mechanism of organophosphorus anticholinesterase activity. Chem. Res. Toxicol. 4, 41-49. (5) See, for example: Hirashima, A., Leader, H., Holden, I., and Casida, J. E. (1984) Resolution and stereoselective action of sulprofos and related S-propyl phosphorothiolates. J. Agric. Food Chem. 32, 1302-1307. (6) Benshop, H. P., and DeJong, L. P. A. (1988) Nerve agent stereoisomers: Analysis, isolation and toxicology. Acc. Chem. Res. 21, 368-374. (7) Jaw, J. (1984) Stereochemical aspects of cholinesterase catalysis. Bioorg. Chem. 12, 259-278 and references therein. (8) Thompson, C. M., Frick, J. A., Natke, B. E., and Hansen, L. K. (1989) Preparation, analysis and anticholinesterase properties of 0,O-dimethyl phosphorothiolate isomerides. Chem. Res. Toxicol.
2, 386-391. (9) Ryu, S., Jackson, J. A,, and Thompson, C. M. (1991) Methanolysis of phosphoramidates with boron trifluoride-methanol complex. J. Org. Chem. (in press). (IO) Hilgetag, V. G., and Lehmann, G. (1959) Optisch aktive thiophosphate. J. Prakt. Chem. 4, 224-234. (11) Dixon, M. (1953) The determination of enzyme inhibitor constants. Biochem. J. 55, 170-171. (12) Ellman, G. L., Courtney, K. D., Andres, V., and Featherstone, R. M. (1961) A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem. Pharmacol. 7, 88-95. (13) Berman, H. A., and Decker, M. M. (1989) Chiral nature of covalent methylphosphonyl conjugates of acetylcholinesterase. J. Biol. Chem. 264, 3951-3956. (14) Thompson, C. M. (1991) Preparation, Analysis and Toxicity of Phosphorothiolates. In Organophosphorus Insecticides: Chemistry, Fate and Effects (Chambers, J . , and Levi, P., Eds.) Academic Press, San Diego (in press).
Articles High-Performance Liquid Chromatography with Electrochemical Detection for Determination of the Major Malondialdehyde-Guanine Adduct Yukihiro Godat and Lawrence J. Marnett* Departments of Biochemistry and Chemistry, Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232 Received March 5, 1991
A method is described for quantitative analysis of the pyrimidopurinone adduct (MIG) arising from reaction of malondialdehyde (MDA) with DNA. DNA samples treated with MDA are reduced with sodium borohydride and hydrolyzed with 0.1 N HCl. The bases released are isolated by solid-phase extraction and analyzed by high-performance liquid chromatography with electrochemical detection. 5,6-Dihydro-MIG (HzMIG) is detected with a glassy carbon electrode a t an applied potential of 700 mV vs Ag/AgCl. At this potential, interference from normal deoxynucleoside bases is low. The limit of detection of HzMIG eluted from a n HPLC column is 100-200 fmol. T h e method described should be useful for quantitation of MIG in a variety of DNA samples and biological fluids. Introductlon Malondialdehyde (MDA)' is a product of lipid oxidation widely produced in the plant and animal kingdom (1). It is mutagenic to bacteria and mammalian cells and carcinogenic to rats (2-6). Since it is produced as a result of normal metabolic processes in human beings, it may have importance as an endogenous human carcinogen. A structure-activity study on MDA and related compounds suggests that both carbonyl groups must react to induce frameshift mutations in Salmonella typhimurium hisD3052 (7). Several adducts have been identified as a result of the reaction of MDA with deoxynucleosides (8-13). One of these, a pyrimidopurinone abbreviated MIG, has been proposed to account for the ability of MDA to induce frameshift mutations (11). MIG arises via reaction of MDA with deoxyguanosine and is anticipated to Present address: National Institute of Hygienic Sciences, Kamiyoga 1-chome, Setagaya-ku, Tokyo 158, Japan.
be a major product of reaction of MDA with DNA (eq 1). However, sensitive and specific analytical methods are not available with which to quantitate MDA-DNA adduction.
MDA
M9G
Quantitation of MDA-DNA adducts, either as the deoxynucleosides or as the bases, has taken on added significance with the recent report that the free base MIG is detectable in normal human urine (14). This finding raises the possibility that MDA-DNA adduction occurs Abbreviations: MIG, pyrimido[l,2-a]purin-10(3H)-one;dR, deoxyribose; R, ribose; MDA, malondialdehyde; NaMDA, sodium malondialdehyde; TEP, tetraethoxypropane; H2MIG, 5,6-dihydropyrimido[1,2a]purin-l0(3H)-one;HPLC-EX, high-performance liquid chromatography with electrochemical detection.
0893-228x/91/2704-0520$02.50/00 1991 American Chemical Society
