Alex.Greene

=A Review of the Discovery, Pharmacology, Treatment and Japanese Terrorist Attacks involving Isopropyl methylphosphonofluoridate (Sarin)=

Abstract
Sarin is a G type nerve agent which was discovered in the 1930's by German scientists, who after an accident in the laboratory realized its deadly potential [Center For Diease Control 2013] [Szinicz 2005]. Sarin can be synthesized many ways, but the preferred methods are simple and give pure products with small amounts of side product [Bryant 1960]. Sarin attacks the enzymes in the body which breakdown neurotransmitters and this inhibition can lead to many physiological problems [Goodsell 2004] [Brown 2006]. There are anti-dotes for Sarin available and research on more potent anti-dotes is underway [Dolgin 2013] [Abu-Qare 2002]. These anti-dotes are needed because the potential for Sarin to be used as a chemical weapon as seen in the Japanese Sarin Terror attacks [Okumura 1996] [Nakajima T 1997].

Introduction
Isopropyl methylphosphonofluoridate (compound 1) or more commonly known as Sarin is chemical weapon [Center For Diease Control 2013] [Holstege 1997] [Abu-Qare 2002]. Sarin gas is considered a nerve agent because it attacks interactions of the nervous system [Center For Diease Control 2013] [Munro 1999]. All nerve agents are organophosphates which contain a fluorine or a cyanine group, and they are very similar chemically to insecticides and pesticides [Reutter 1999]. Nerve agents are the most toxic and potentially lethal type of chemical weapons [Reutter 1999] Sarin gas is further classified as a G-type nerve agent, along with Tabun and Soman [Center For Diease Control 2013]. G-type nerve agents are miscible in water and many other organic solvents [Center For Diease Control 2013]. Of all the G-type nerve agents, Sarin is the most volatile of the G-type nerve agents and has a similar evaporation rate to that of water [Munro 1999]. Sarin is consider to be non-persistent because of the fact that it ready hydrolyses with acid or base [Munro 1999]. Some of the characteristics of Sarin gas are clear, colorless, tasteless, and odorless, which make detecting Sarin difficult until exposure has already occurred [Center For Diease Control 2013].

Figure 1 Structure of Sarin (Compound 1) (2D and 3D) retrieved from Chemspider.com

History of Sarin
The development of Sarin and other G-type nerve agents were mainly the result of German scientists in the 1930’s working on new pesticides [Holstege C P 199]. Originally it was Lange and his student Von Krueger who at the University of Berlin synthesized organophosphates in 1932 [Dheraj K 2000]. Lange knew of the possibility of the use of the organophosphates as pesticides, but only made a small comment of this in his work [Dheraj K 2000]. However this small note caught the interest of the German Military and Gerhard Schrader in 1936 [Szinicz 2005]. Soon after Schrader’s research began into organophosphates to be used as pesticides, an accident in the lab revealed the high toxicity of the organophosphates [Holstege C P 1997]. One drop of tabun was spilled in the laboratory and soon the entire research team showed signs of poisoning. Experiments on animals showed that exposure to the gaseous vapor caused rapid death [Holstege C P 1997]. In 1935, an official policy by the German military required all inventions with the possibility of military worth to be reported to the German Ministry of War [Szinicz 2005]. In 1938, Schrader and his team generated sarin, and the name sarin comes from the names of the integral members of the research group: “S chrader, A mbrose, R udringer, and V an der L in de” [Holstege C P 1997]. In accordance with the law Schrader sent samples of tabun and sarin to the German Ministry of War, and the potential of both compounds was quickly recognized and all patents regarding the substances were classified [Szinicz 2005].

Preparation of Sarin
There are various ways to prepare sarin, however most ways are either tedious or low yielding [Bryant 1960]. This being said there are some ways which are simple and produce a pure product [Bryant 1960]. The first procedure is to start with di-isopropyl-methylphosphonate (compound 2) and react with carbonyl chloride (compound 3). This reaction replaces one of the isopropoxy groups attached to the phosphorous with a chlorine from the carbonyl chloride to give isopropyl-methlyphosphonochloridate (compound 4) [Bryant 1960]. Once the compound 4 is synthesized the chlorine is replaced by a fluorine when the compound 4 is reacted with sodium fluoride (compound 5). The fluorine replaces the chloride giving sarin (compound 1). [Bryant 1960].

The second reaction sequence beings with 2-propanol (compound 6) and then reacting compound 6 with equal moles of methylphosphonic diflouride (compound 7) and methylphosphonic dichloride (compound 8) [Bryant 1960]. Once the three regents are combined in an inert solvent they react to give compound 1 [Bryant 1960]. One last way to synthesize Sarin is to add tetra(isopropoxy)silane (compound 9) to compound 7 to give Sarin plus tetrafluorosilane (compound 10) and isopropyl methylphosphonate (compound 2) [Muller 1998]. A common trait of all these synthesis reactions is the simplicity of the synthesis which is desired when working with such potentially deadly product.

Figure 2. The 3 discussed synthesis methods for Sarin. (Structures retrieved from Chemspider.com)

Physiological Effects of Sarin
Sarin affects both the central and peripheral systems [Abu-Qare 2002]. Sarin can enter the body through many different ways, it can be inhaled, absorbed through the gastrointestinal tract, skin or tear ducts [Abu-Qare 2002]. When Sarin is absorbed through the skin the affect is delayed but also lengthened [Eason 2013]. When Sarin enters into the body it binds to the enzyme acetylcholinesterase (AChE) at the neuron junction and when this binding occurs the AChE enzyme can no longer break down the acetylcholine, thus building up in the junction [Reutter 1999]. Acetylcholine (compound 11) is one of the main neurotransmitters of the central nervous system, which means that it is responsible for sending chemical information from the brain to the rest of the body [Brown 2006]. Specifically the role of compound 11 is to send messages to the muscles from the brain [Goodsell 2004]. The breakdown of compound 11 allows the signal to the muscles to be rest [Goodsell 2004]. This needs to be done quickly in which is why it is good that AChE has one of the fastest reaction rates in the human body, with a single AChE breaking down a molecule of acetylcholine every 80 milliseconds [Goodsell 2004]. The nervous system relies on quick impulses of information and for acetylcholine to be a viable neurotransmitter it must be readily and quickly broken down, this breakdown is facilitated by AChE [Brown 2006].

Figure 3. Structure retrieved from Chemspider.com

Figure 4. Acetylcholinesterase (AChE) Protein DataBank [David 2004]

Normally functioning AChE will catalyze the breakdown of a carboxylate ester, including acetylcholine which is a carboxylate ester [Millard 1999]. First, AChE attacks the ester which forms an unstable tetrahedral intermediate [Millard 1999]. The tetrahedral intermediate degrades into an acetyl-enzyme. This acetyl enzyme undergoes a nucleophilic attacked by water, which forms another unstable tetrahedral intermediate and then the reformed AChE enzyme is released as the leaving group [Millard 1999]. When AChE binds with Sarin the complex that forms is a hemisubstrate which holds the enzyme in the tetrahedral intermediate. The difference between the Sarin complex and the carboxylate ester is that the Sarin complex tetrahedral structure does not break down as fast as the carboxylate ester. The Sarin complex can stay in inhibited with the AChE enzyme for multiple hours or even days [Millard 1999]. The reason for this slow breakdown is that the active site of the histidine (H440 site) is hindered and the water molecule cannot reach the right face of the phosphorous for the nucleophilic attack [Millard 1999]. Another possible explanation is the H440 site becomes ineffective because the imidazolium form hydrogen bonds with the oxygen of the sarin [Millard 1999]. The final step in deactivating the AChE enzyme is called aging and causes irreversible inhibition [Millard 1999]. Only branched inhibitors undergo ageing, such as Sarin, and it is this branched alkyl group which is dealkyated by a carbonium ion and adds a negative formal charge to the active site of the phosphylated AChE [Millard 1999]. The addition of the formal charge makes the nucleophilic attack of the water unlikely to occur and prevents the AChE from being released from the sarin molecule.

Excess acetylcholine affects nicotinic and muscarinic receptors [Eason 2013]. The symptoms that normally result from excess acetylcholine include wheezing, nausea, diarrhea, vomiting, sweating, seizures, and skeletal muscle twitches [Eason 2013]. The most dangerous interaction is blocking the nicotinic receptors because this can lead to paralysis of respiratory muscles [Thiermann 2013]. In addition to the short term high dose effects of Sarin there are also low dose long term effects of all organophosphate esters (OP), not just Sarin [Oswal 2013]. OP insecticide and pesticides in addition to chemical weapons can cause these long term effects [Oswal 2013]. One clear example is Gulf War Illness which is categorized by long lasting weakness, memory loss, fatigue, headaches, and increased susceptibility to infections [Oswal 2013]. It is still not clear as to the cause of the long term symptoms and the effects of the Sarin on the body’s long term physiological processes [Oswal 2013].

It is also important to noted that Sarin is a chiral molecule, thus having a + and - stereoisomer, and it is the – isomer which is stronger inhibitor of AChE [Spruit 2000]. In electric eels, the inhibition of AChE by – Sarin is four orders of magnitude greater than +Sarin and the LD50 of –Sarin is half as much as compared to ±sarin. [Spruit 2000]. In a study done on mice which were given ±Sarin by intravenous bolus, there was no +sarin detected in the blood of the mice at a detection limit of 5pg/mL [Spruit 2000]. This is important because this shows –Sarin the more active of the two stereoisomers and one could make a more potent chemical weapon by selectively synthesizing –Sarin.

Treating Sarin Exposure
The best treatment for the exposure to sarin is preventative measures such as proper physical garments including masks, gloves, and body suits [Thiermann 2013]. Sarin gas can stay trapped on and within clothing which is why proper and prudent measures should be taken when assisting possible Sarin victims [Thiermann 2013]. If sarin exposure is suspected there are approved antidotes which counteracts and lessen the effects of sarin on the body [Thiermann 2013]. The cocktail of atropine (compound 12), 2-PAM (compound 13) and diazepam (compound 14) is considered to be the best antidote for sarin exposure [Dolgin 2013]. This is the cocktail which is contained in autoinjectors mostly used by military personnel in combat areas [Dolgin 2013]. Compound 12 works to displace the acetylcholine from the receptors and thus allowing it to be broken down [Thiermann 2013]. Compound 13 is an oxime and their mechanism is to reactivate the AChE and separate it from the nerve agent [Thiermann 2013]. Compound 14 works to prevent seizures in the victim which prevents additional brain damage [Thiermann 2013]. Some possible replacement drugs have been identified, such as lorazepam (compound 15) and midazolam (Compound 16) which are both benzodiazepines like compound 14, but offer greater seizure protection [Dolgin 2013]. RS194B (compound 17) is a possible replacement for compound 13 because it functions much like 2-PAM to restore the enzymatic activity of AChE but RS194B can cross the blood/brain barrier and restore function in the central and peripheral nervous system [Dolgin 2013]. Finally, a potential replacement for compound 12 is scopolamine (compound 18) which is more fat soluble than compound 12 and can therefor cross the blood/brain barrier easier [Dolgin 2013]. While more drugs are being developed for treating Sarin they still have to be administered, however if there was a way to have an sarin defense system already in the body this could save many lives. One possibility is the PON1 enzyme, PON1 is the only enzyme in the body which hydrolyzes the phosphours-flourine bond in sarin [Davies 1996]. The level of PON1 naturally varies in the human population which explains why some people are more affected by sarin than others [Davies 1996]. There is interest a way to increase the normal amount of PON1 in individuals so that this could offer a constantly armed anti-sarin defense measure in the body.

Figure 5 Drugs and other Treatments for Sarin Exposure (Retreived from Chemspider.com and Protein DataBank)

Japan Terror Attacks
During the night June 27 1994 a religious cult released Sarin in the city of Matsumoto, Japan through a fan from a truck [Nakajima 1997]. 7 people died as a result of the attack and about 600 were affected. There were 52 rescuers who aided individuals during the attack and none of them thought the release of sarin was intentional and therefore did not take any precautions [Nakajima 1997]. The first responders did not wear any gloves, masks, or protective clothing and as a results 34.6% of them experienced Sarin poisoning [Nakajima 1997]. This attack showed the need for clear protocol to protect the victims as well as first respondents in response to a chemical attack. The lessons learned in 1994 would eventually save lives in the coming year.

On the morning of March 20 1995, the same group of religious terrorists as the 1994 attack again released Sarin in 5 separate subway cars on 3 line in the subway system of the Japanese city of Tokyo [Okumura 1996]. Authorities quickly identified the substance as Sarin. As victims arrived medical professionals preformed triage to place the patients into three groups: mild, moderate and severe based on symptoms [Okumura 1996]. The mild group only had ocular symptoms, the moderate group had systemic symptoms such as wheezing, and the severe group required respiratory support [Okumura 1996]. Overall the response by the emergency personnel of Tokyo was exceptional in preventing the additional loss of life by quickly identifying the substance, separating and decontaminating victims, and administering proper antidotes.

Conclusion
Overall, Sarin is a potent neurotransmitter disruptive agent that attacks the nervous system which can be deadly if the exposure is severe enough. The possible simplicity of Sarin synthesis should be noted because the use and potential use by terrorist and other violent organizations. The ease of synthesis should provide motivation for continued Sarin anti-dote research, not only in reactionary anti-dotes but also precautionary anti-dotes such as PON1, which could increase protection not only from Sarin exposure but also commonly used insecticides and pesticides which are chemically similar.