Structure and Mode of Action of Organophosphate Pesticides:

A Computational Study Lasantha K. Rathnayake and Scott H. Northrup

School of Environmental Studies and Department of Chemistry, Tennessee Technological University, Cookeville, TN, USA

An important class of synthetic chemicals actively released into the environment is the

organophosphates (OPs) [Gupta, 2006]. The primary mechanism of OP toxicity is inhibition of

AChE (acetylcholinesterase) in the central and peripheral nervous system [Mileson, et al, 1998].

Most well-known of these are pesticides (e.g. parathion and diazinon). OP compounds also include

chemical warfare nerve agents (e.g. sarin, soman, and VX). Presence of OP-containing pesticides

in the environment is ubiquitous. Pesticide exposure arises from living next to treated areas or in

agricultural regions, as well as from house and yard pesticide treatment.

Introduction

Explore the equilibrium structures and reactivities of six to ten OPs widely used as pesticides.

Explore pathways of reaction of OPs with nucleophiles mimicking their target enzyme AChE.

Use advanced knowledge-based ligand-docking algorithms and molecular dynamic (MD)

simulations to predict the docking of OPs to AChE and subsequent pathways for binding.

Perform a quantitative structure-activity relationships (QSAR) study to understand the toxic

effect of different functional groups of the OPs.

Project Goals

Figure 1: Pesticide transportation paths to untargeted organism (EPA 2001) (left); Human AChE (right)

OPs interfere with the catalytic mechanism of AChE by rapidly phosphorylating the catalytic

serine residue producing a phosphonyl adduct [Ekstrom, et al, 2006].

Figure 2: Inhibition activity of OP in active site of AChE (left), Crystal structures of mouse AChE with covalently

bonded tabun: nonaged (center) and aged (right)

Although a basic understanding exists as to how these compounds function and their toxicological

effects, much remains to be understood about how details of their molecular structure relate to

toxicity and species specificity.

What makes one OP a good candidate for a household insecticide while another OP is a

chemical warfare agent?

What structural features give rise to specificity for toxicity in different organisms?

What are the fates of these compounds in the environment?

Are there ways to more effectively remediate or reverse their detrimental effects on organisms

in the environment?

Better understanding of the thermodynamics/kinetics of the reactions [Bock, et al, 2009] and

structural properties of OPs could lead to following:

Design of superior pesticides with greater margins of safety.

Development of preventative strategies for bodily exposure to chemical warfare agents.

Reversal of detrimental effects of exposure of OPs to humans and the environment.

Remarks

Gaussian 03 / 09

Hybrid density functional theory (HDFT) method of mPW1B95-44 in conjunction with

6-31+G(d,p) basis set.

Rosetta / MOE / NAMD/ Gromacs (work in progress)

QSAR and MD (work in progress)

Computational Methodologies

OP compounds of interest

Results and Discussion

Results and Discussion cont…

Atom colors

• C - dark green

• O - red

• Cl - light green

• N - blue

• H - gray

• S - yellow

• P - orange

Potential reaction mechanisms of dichlorovos (DCV) in the active site of AChE

Methanol mimics the activity of serine residue in active site of AChE

EVSC Ph.D program; Dept. of Chemistry; TTU Faculty Research Grant – S. H. Northrup; TTU

Dept. of Chemistry Student Research Grant – L. Rathnayake

Acknowledgement

Bock, C.W., Larkin, J.D., Hirsch, S.S., Wright, J.B., Nucleophilic destruction of organophosphate toxins: A computational investigation, Journal of Molecular Structure:THEOCHEM 915(2009)11-19

Ekstrom, F., Akfur, C. Tunemalm, A-K, Lundberg, S., Structural Changes of Phenylalanine 338 and Histidine 447 Revealed by the Crystal Structures of Tabun-Inhibited Murine Acetylcholinesterase,

Biochemistry 2006, 45, 74-81

Gupta, R.C., Toxicology of Organophosphate & Carbamate Compounds, Acad. Press, 2006

Mileson, B. E.; Chambers, J. E.; Chen, W. L.; Dettbarn, W.; Ehrich, M.; Eldefrawi, A. T.; Gaylor, D. W.; Hamernik, K.; Hodgson, E.; Karczmar, A. G.;.; Sultatos, L. G.; Wallace, K. B. Common

mechanism of toxicity: a case study of organophosphorus pesticides. Toxicol. Sci. 1998, 41, 8−20

Wright, J.B. and White, W.E., J. Molecular Structure (Theochem) 454 (1998) 259-265

Reference

Reactivity of OPs with methanol, which mimics AChE, was studied in different perspectives.

In direct mechanisms, the proton transfer from methanol to leaving group of OPs are more

likely to proceed through the assistance by one water molecule and the phosphinyl oxygen. In

indirect mechanisms, the proton transfer is most favorable in the water-assisted Wright

mechanisms. The next most favorable proton transfer is shown in water and histidine assisted

Wright mechanisms. These evidences suggest that the presence of phosphinyl-oxygen, water

and/or histidine can accelerate particular reactions.

The most favorable leaving group in terms of ΔG°rxn for any OPs of interest is L1, which has

the highest molecular weight and contains electron withdrawing functional groups .

Also, the leaving group is determined by the reaction mechanism and orientation of supporting

molecules. E.g. if the reaction goes through a modified Wright type mechanism, the larger

group has more potential to leave, whereas, if the reaction goes through direct-water-

phosphinyl-oxygen mechanism (D-WtPn), the leaving group can be the nearest functional

group to the serine residue and the other supporting molecules.

A favorability sequence can be established for mechanisms of DCV-L1 as follows: D-WtPn ≈

W-WtWt-PCM ≈ W-WtWt > W-S-PCM ≈ W-HisWt > W-S ≈ D-Wt > D-S ≫ D-His.

The results give strong evidence for the reaction rate being increased by the catalytic effect (in

this case water and histidine assistance) for the reactions between OPs and AChE.

Conclusions

Figure 3: Some conformations (optimized) of the OP compounds of interest

D1

D4D3

D2

W1

W2

W3

Figure 4: Potential reaction mechanisms of the reaction

between DCV and methanol. D1 to D4 and W1

to W3 are direct (D) and indirect (Wright type

(W) [Bock et. al., 2006]) hydrogen transfer

mechanisms from methanol to the leaving group

(L1 or L2) of DCV. [S, Wt, His, WtPn, HisWt,

WtWt are straight (no assistance) water, histidine,

water and phosphinyl O, histidine and water, and

two water molecule assistance respectively. R, P,

Ts (Ts, Ts1, Ts2, Ts3), Pr, In (In1, In2), and Po,

and reactants, products, transition state/s, pre-,

intermediate-, and post-complexes, respectively.]

Figure 5: P1: Comparison of effective activation energies

towards leaving of L1, methanol and ammonia for

selected OPs. P2: Comparison of effective

activation energies of different mechanisms for the

reaction between OPs and methanol towards the

leaving group L1. P3: Reaction profiles of the

different potential mechanisms. PCM – polarized

continuum model (water as solvent)

Table 1: Thermodynamic and kinetic parameters for the direct and indirect hydrogen transfer mechanism for

the reaction between OP (DCV) and methanol

Reaction path ΔGºrxn Keq keff(f) Ea(f) keff(r) Ea(r)

DCV-L1-D-S -7.29 2.20x1005 2.88x10-18 41.97 1.31x10-23 49.26

DCV-L2-D-S 0 1 2.88x10-21 46.07 2.88x10-21 46.07

DCV-L1-D-His -7.52 3.26x1005 9.60x10-35 64.45 2.94x10-40 71.98

DCV-L2-D-His 0 1 1.17x10-22 47.96 1.17x10-22 47.96

DCV-L1-D-Wt -7.07 1.53x1005 2.00x10-13 35.37 1.31x10-18 42.44

DCV-L1-D-WtPn -7.07 1.53x1005 6.77x10-02 19.64 4.43x10-07 26.71

DCV-L2-D-WtPn 0 1 1.87x10-03 21.69 1.87x10-03 21.69

DCV-L1-W-S -6.37 4.64x1004 4.52x10-13 34.89 9.74x10-18 41.25

DCV-L2-W-S 0 1 1.79x10-12 34.04 1.79x10-12 34.04

DCV-L1-W-WtWt -11.16 1.51x1008 8.77x10-04 22.21 5.80x10-12 33.37

DCV-L1-W-HisWt -14.64 5.34x1010 1.82x10-11 32.70 3.41x10-22 47.33

DCV-L1-W-S-PCM -19.33 1.47x1014 2.15x10-10 31.22 3.21x10-17 40.53

DCV-L1-W-WtWt-PCM -13.17 4.53x1009 3.02x10-02 19.82 6.67x10-12 32.99

ΔGºrxn and Ea are in kcal/mol. keff(f), keff(r) are in s-1. Reaction temp 298.15 K

P3

P2P1