Daniel Antonio Shimizu Kitagawa1, Sabrina Teixeira Martinez2, 3, Erick Braga Ferrao Galante2, Tanos Celmar Costa Franca2, 4
Abstract: Chemical agents represent a serious threat to the modern world. Among them, they stand out nerve agents because of its high lethality and dangerousness. They are typically organophosphate compounds, which act by inhibiting acetylcholinesterase, a key enzyme in the transmission of nerve impulses process. There are several forms of treatment for organophosphate poisoning, and pralidoxime (2-PAM) is the drug most used as reactivator of acetylcholinesterase. In this work, we developed the first three steps for the synthesis of 2-PAM, with the objective of obtaining data to calculate the kinetic parameters of these steps. These parameters may be used for the manufacture of 2-PAM in semi-pilot scale. Through the studies conducted it has been found that the preparation of the oxime has very rapid kinetics.
Keywords: Chemicals warfare agents, Organophosphates compounds, Acetylcholinesterase, Oximes, Pralidoxime
1.Introduction
Chemical agents are a major threat to the modern world. One of its features is its danger, where small amounts can cause numerous poisonings. Moreover, they are easy to obtain and does not require many resources to manufacture. Thus, the use of chemical agents can be a dangerous alternative for organizations with limited resources, as underdeveloped countries and terrorist factions [1].
Among the chemicals the nerve agents stand out due to its high lethality. The neurotoxic substances are organophosphates (OP) which, in addition to its use as weapons, can also be used as pesticides. These compounds are inhibitors of acetylcholinesterase (AChE), an enzyme of great importance in controlling the transmission of nerve impulses.
A number of drugs are used in the treatment of poisoning by OP. Among these drugs are compounds containing the functional group oxime, whose function is to promote the reactivation of AChE. One of the most used oxime is pralidoxime or 2-PAM [(E) -2 - [(hydroxyimino) methyl] – pyridine] [1].
This work aimed to raise kinetic parameters of the reaction of synthesis of 2-PAM. The collection of kinetic parameters is a very important activity for the laboratorial chemical engineering, since it consists on the initial stage of semi pilot plant design for the manufacture of a chemical like 2-PAM.
2.Chemical Agents and Neurotoxics
There is a variety of chemical agents, each with different toxicological properties. According to the field manual C 3-40, chemical agent is any substance that through its chemical activity, produces, when used for military purposes, a toxic effect, smoke or fire. Chemical agents that produce a toxic effect may be classified as disabling, choking, vesicants, nerve and blood agents [2].
Incapacitating agents are those that cause temporary physiological and mental effects, preventing victims of combating. The most used are o-chlorobenzylmalonitrile (CS - cause eye irritation) and adamsite (DM - causes vomiting). Pulmonary toxicants, whose main agent is phosgene, act in the respiratory tract, causing lesions in capillaries and stroke in pulmonary alveoli, leading to suffocation. Vesicants are those that cause irritation and blistering of the skin and mucous membranes, and its most important agent is mustard gas. Blood agents, the most important being hydrogen cyanide, act interfering in tissue oxygenation, causing them quickly the state of necrosis and subsequent death. The neurotoxic, or nerve agents, are those that affect the nervous system, specifically the role of AChE [1].
The neurotoxics are highly toxic, both in vapor and in liquid form, because they are absorbed by the body through the skin or respiratory system. Symptoms include distress, loss of coordination and seizures leading to death. These compounds cause the collapse of the central nervous system (CNS) [3].
2.1.Neurotransmission
Neurotransmission or synaptic transmission is the transfer of nerve impulses from one neuron to another. Nerve impulses elicit responses in muscles, glands and postsynaptic neurons [4]. The neurotransmission can be divided into the four steps illustrated in Figure 1.
Figure 1. Nerve impulse transmission [5].
The first step is the release of a neurotransmitter, which is a substance stored inside the synaptic vesicles. These vesicles are located in the termination of the axon, which are the branches of neurons. Arriving at the termination of the axon, the nerve impulse causes the fusion of synaptic vesicles with the pre-synaptic membrane, releasing the neurotransmitter into the synaptic cleft.
The next step is the combination of the transmitter with the neurotransmitter receptors. The neurotransmitter diffuses across the synaptic cleft and binds to existing specialized receptors in the post-synaptic membrane.
Then it occurs the beginning of the post-junctional activity. Receptors change their conformation by binding to the transmitter and, then, the post-synaptic membrane becomes permeable to ions. Thus, increasing the concentration of sodium ions in the cytoplasm of the receiving neuron causes a potential difference, leading to a nerve impulse that will propagate in the neuron.
The last step is the dissipation of the transmitter. Because the pulses can be transmitted through the synaptic clefts, often at up to several hundred per second, there must be a mechanism to eliminate the transmitter for each pulse [4]. For this, there are enzymes in synaptic clefts which have the ability to hydrolyze the neurotransmitters, such as AChE (Figure 2), an enzyme belonging to the family of cholinesterase, whose function is to hydrolyze the neurotransmitter acetylcholine (ACh), as shown in Figure 3.
AChE and ACh are present in the CNS and peripheral nervous system (PNS). In the PNS AChE is connected to the control of heart rate, dilation of blood vessels and smooth muscle contraction, whereas in the CNS it is involved in motor control, cognition and memory.
AChE plays an important role, because the accumulation of ACh in the synaptic cleft leads to overstimulation of the innervated structures, generating a cholinergic crisis, which has various effects such as seizures, cardiac arrhythmia and death [6].
In the active site of human AChE (HssAChE) there are three amino acid residues (known as catalytic triad) directly involved in the ACh hydrolysis process, as shown in Figure 3. These amino acids are Ser203, Glu334 and His447. In the anionic active site there is a region, which serves to interact with the cationic portion of ACh by directing this substrate to the position necessary for hydrolysis [8].
The first step of the ACh hydrolysis process occurs through a nucleophilic attack by the hydroxyl group of Ser203 on the ester-carbonyl group of the substrate, promoting breakage of the ester bond [8]. During the enzymatic attack on the ester, it is formed an intermediate between the enzyme and the ester named acetylated Ser203 (acetyl-AChE complex). The acetyl-enzyme complex is easily hydrolyzed and this action, performed by water molecules, results in the formation of the fully regenerated acetate and the free enzyme.
The acetyl-AChE complex has a short life, making AChE one of the most efficient enzymes capable of hydrolyzing ACh on the order of 6 x 105 Ach molecules per molecule of enzyme per minute [8].
Figure 2. Hydrolysis of acetylcholine.
Figure 3. Simplified scheme of the HssAChE active site.
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