1 Malathion Impurities Dr. Yehia Ibrahim 6/26/2012 2 Malathion Impurities Dr. Yehia Ibrahim 6/26/2012 3 Malathion Impurities Dr. Yehia Ibrahim 6/26/2012 Organophosphates and Germany • 1820s: investigations into organophosphate (OP) chemistry began • Early 1900s: several OP compounds synthesized • 1930s: toxicity of OPs becoming recognized • 1940s: insecticidal action observed by Germany during WWII 4 Malathion Impurities Dr. Yehia Ibrahim 6/26/2012 Organophosphates and Germany • Group led by Gerhard Schrader searching for substitutes to nicotine as an insecticide – Nicotine in short supply during WWII – Developed a number of incredibly toxic nerve agents Sarin Soman Tabun 5 Malathion Impurities Dr. Yehia Ibrahim 6/26/2012 Organophosphates and Germany • Schrader’s group also created some of the first commercial OP insecticides Dimefox TEPP Schradan Parathion 6 Malathion Impurities Dr. Yehia Ibrahim 6/26/2012 After WWII • Schrader’s research records were captured by Allied forces – Led to massive increase in interest in OP insecticides • Early OPs – very effective against insects – Much more toxic to vertebrates than organochlorine insecticides – Nonpersistent and chemically unstable 7 Malathion Impurities Dr. Yehia Ibrahim 6/26/2012 Malathion • First produced by American Cyanamid in 1950 • Very safe due to its low vertebrate toxicity • Used on most fruits, vegetables and forage crops • Works on a wide range of insect pests 8 Malathion Impurities Dr. Yehia Ibrahim 6/26/2012 9 Malathion Impurities Dr. Yehia Ibrahim 6/26/2012 Diethyl 2-[(dimethoxyphosphorothioyl)sulfanyl]butanedioate 10 Malathion Impurities Dr. Yehia Ibrahim 6/26/2012 11 Malathion Impurities Dr. Yehia Ibrahim 6/26/2012 O Diethyl Maleate C CH O CH CH 2 3 S + CH O CH CH 2 3 CH O P O CH C O 3 3 S C S O CH CH O CH CH O P S 2 3 H O 3 O CH O CH C 2 2 CH + CH C 3 CH O CH 2 CH 3 Malathion 3 O CH 3 CH 2 O CH C O Diethyl Fumarate 12 Malathion Impurities Dr. Yehia Ibrahim 6/26/2012 Very Simple Synthetic Reaction Michael Addition during Condensation Reaction Nucleophilic addition of an SH-group to the double bond of diethyl maleate or diethyl fumarate. 13 Malathion Impurities Dr. Yehia Ibrahim 6/26/2012 Maleic Acid Fumaric Acid 14 Malathion Impurities Dr. Yehia Ibrahim 6/26/2012 Carboxylic Acid, Hydrogen Bonds The carbonyl group is an electron withdrawing group when conjugated with a double bond. This decreases the electron density and activates the double bond to nucleophilic addition. H O 2 C C C C C C C C + RSH O O O O RS H S CH O OCH -d 3 P 3 -d .. O O .. S H H C OC 2 H 5 C OC 2 H 5 H +d C +d C C C H C 2 H 5 O C H C OC 2 H 5 O O Diethyl Fumarate O Diethyl Maleate S C CH O P S CH O C 2 H 5 3 O CH 2 O C 2 H 5 CH C 3 O Malathion CH 3 O CH 3 O P SH CH 3 O CH 3 S CH 3 O CH 3 O P SH S P SH O O Diethyl Maleate or Diethyl Fumarate Malaoxon Malathion Isomalathion 18 Malathion Impurities Dr. Yehia Ibrahim 6/26/2012 1. Malathion is considered to be one of the least toxic (i.e., safest) insecticides to mammals. 2. The above statement is conditional to the absence of any highly toxic and or strong potentiating impurities. 3. Malathion is not protected by any patent and can, therefore, be synthesized by any manufacturer. 19 Malathion Impurities Dr. Yehia Ibrahim 6/26/2012 4. Most malathion impurities are more potent than malathion itself and some of them strongly potentiate its mammalian toxicity. 5. Furthermore, some impurities act similarly and dissimilarly to malthion mode of action. 6. The above statement tremendously complicates the risk of formulated malathion. 20 Malathion Impurities Dr. Yehia Ibrahim 6/26/2012 7. Malathion safety will be in jeopardy if manufacturing companies irresponsibly produce it without assuring the absence of any detrminntal levels of its impurities. 8. Regulatory authorities should make sure that registered malathion in their regions be analyzed to ensure the absence of malathion risk-induced impurities. Yehia A. Ibrahim (June 2012) 21 Malathion Impurities Dr. Yehia Ibrahim 6/26/2012 Test Material % a.i. LD (mg/kg) 50 Malathion TC 94.6 1650 Malathion TC, containing 0.44% Isomalathion 96.2 1850 Malathion TC, containing 0.20% Isomalathion 96.2 2400 Malathion TC 96.8 5100 Malathion TC 96-98 5550 22 Malathion Impurities Dr. Yehia Ibrahim 6/26/2012 Safety Margin Above 97.5% 23 Malathion Impurities Dr. Yehia Ibrahim 6/26/2012 24 Malathion Impurities Dr. Yehia Ibrahim 6/26/2012 • Cholinergic Neurotoxicity (AChE Inhibition) • Organophosphorus Ester-Induced Delayed Neurotoxicity (OPIDN) • Organophosphorus Ester-Induced Chronic Neurotoxicity (OPICN) 25 Malathion Impurities Dr. Yehia Ibrahim 6/26/2012 • Acetylcholinesterase (AChE) is an enzyme that degrades the neurotransmitter acetylcholine, producing choline and an acetate group. It is mainly found at neuromuscular junctions and cholinergic synapses in the central nervous system, also in red blood cell membranes. 26 Malathion Impurities Dr. Yehia Ibrahim 6/26/2012 • first neurotransmitter identified (Otto Loewi, 1921) • important role in movement: causes muscle contraction • also found in brain: important role in attention, learning & memory 6/26/2012 Malathion Impurities Dr. Yehia Ibrahim 27 28 Malathion Impurities Dr. Yehia Ibrahim 6/26/2012 6/26/2012 Malathion Impurities Dr. Yehia Ibrahim 29 ACh is broken down in the synaptic cleft by the enzyme acetylcholinesterase (AChE) 30 Malathion Impurities Dr. Yehia Ibrahim 6/26/2012 • An esteratic site – The amino acid serine • An anionic site – The amino acids Tyrosine (3 of them), Aspartic Acid, and Tryptophan Serine Tyrosine Aspartic Acid Tryptophan 31 Malathion Impurities Dr. Yehia Ibrahim 6/26/2012 O O H H H Reaction Mechanism H Acetylcholine – CH H C 3 O 3 O CH H C 3 + CH 3 N 3 + CH H C O N 3 3 H C O O 3 R2 - R1 N – - O H O - R2 CH H C 3 R1 Anionic Site 3 N + CH H O N 3 HO Serine - – Choline O O - O – H C O HH O H H C OH 3 H 3 O H C Acetate 3 OH O – R2 - O H R1 R1 R2 R2 N - R1 - N H O H - N H Serine O Anionic Site O 32 Malathion Impurities Dr. Yehia Ibrahim 6/26/2012 • Inhibition of Acetylcholinesterase (AChE), an enzyme essential for life. 33 Malathion Impurities Dr. Yehia Ibrahim 6/26/2012 • AChE inhibitors — block the function of AChE and thus cause excessive acetylcholine to accumulate. The excess acetylcholine causes neuromuscular paralysis (i.e. interminable muscle contractions) throughout the entire body, leading to death by asphyxiation. 34 Malathion Impurities Dr. Yehia Ibrahim 6/26/2012 • Phosphates are biologically active as acetylcholinesterase (AChE) inhibitors, whereas phosphorothioates need bioactivation to their phosphate analogues (oxons) to become biologically active. 35 Malathion Impurities Dr. Yehia Ibrahim 6/26/2012 S O +d CH O +d P S X CH O P S X 3 3 O O Malathion CH Malathion-Oxon CH 3 3 36 Malathion Impurities Dr. Yehia Ibrahim 6/26/2012 (b) acyl pocket Peripheral binding site (d) Trp 286 phe 297 Asp 74 phe 295 O N O- Glu 334 H Trp 86 Choline N d+ N (c) binding H d- O site His 447 (a) Catalytic triad of AChE Ser 203 Active site gorge of AChE 37 Malathion Impurities Dr. Yehia Ibrahim 6/26/2012 Malathion Malathion-Oxon (Malaoxon) AChE Inhibition ACh Accumulation Cholinergic Neurotoxicity 38 Malathion Impurities Dr. Yehia Ibrahim 6/26/2012 Peripheral binding site acyl pocket (d) (b) Trp 286 phe 297 Asp 74 phe 295 O Trp 86 O N Choline O d+ (c) binding Glu 334 - H P O N site Nd+ H Od- (a) His 447 Catalytic triad of AChE Ser 203 MalaoxonSARIN phosphonyl-AChE Complexenzyme 39 Malathion Impurities Dr. Yehia Ibrahim 6/26/2012 Peripheral binding site acyl pocket (d) (b) Trp 286 phe 297 Asp 74 phe 295 O Trp 86 O N Choline O d+ (c) binding Glu 334 - H P O N site Nd+ H Od- (a) His 447 Catalytic triad of AChE Ser 203 DimethylSARIN phosphonyl Phsphorylenzyme AChE 40 Malathion Impurities Dr. Yehia Ibrahim 6/26/2012 Peripheral binding site acyl pocket (d) (b) Trp 286 phe 297 Asp 74 phe 295 O Trp 86 O N Choline O d+ (c) binding Glu 334 - H P O N site Nd+ H Od- (a) His 447 Catalytic triad of AChE Ser 203 AgedSARIN Phosphoryl phosphonyl enzymeAChE 41 Malathion Impurities Dr. Yehia Ibrahim 6/26/2012 42 Malathion Impurities Dr. Yehia Ibrahim 6/26/2012 O CH 3 CH 2 O C S O CH CH 3 2 P CH S O CH 3 C CH 3 CH 2 O O Malathion 43 Malathion Impurities Dr. Yehia Ibrahim 6/26/2012 Oxidase O CH 3 CH 2 O C S O CH CH 2 3 Carboxylesterase P Methyl Transferase CH S O CH 3 C CH 3 CH 2 O O Organophosphorus Hydrolase 44 Malathion Impurities Dr. Yehia Ibrahim 6/26/2012 O O CH CH 3 C 2 CH O S 3 CH P 2 CH 3 O S CH C Malathion O O CH 2 CH 3 O O H O O CH CH 3 C C 2 CH O S CH O O 3 CH 2 3 CH P P 2 CH 3 O S CH CH 3 O S CH C C O O H O O CH 2 CH 3 Malathion Diacid Malaoxon 45 Malathion Impurities Dr.