engleski

Comparison of pyridinium aldoximes in their reaction with the cholinesterase substrate acetylthiocholine

Oximes are a class of compounds known for their nucleophilic potency in reactions with acyl and phosphorus(V) esters. One of the major research interests in oximes is their use as effective reactivators of phosphorylated cholinesterases (ChEs). Two monopyridinium aldoximes, HI-6 (1-(((4-carbamoylpyridinium-1-yl)methoxy)methyl)-2-((hydroxyimino)methyl)pyridinium chloride) and K027 (4-carbamoyl-1-(3-(4-((hydroxyimino)methyl)pyridinium-1-yl)propyl)pyridinium bromide), and two bispyridinium aldoximes, K033 (1, 1'-(butane-1, 4-diyl)bis(2-((hydroxy-imino)methyl)pyridinium) bromide) and TMB-4 (1, 1'-(propane-1, 3-diyl)bis(4-((hydroxy-imino)methyl)pyridinium) bromide), were studied for their reactions with acetylthiocholine (ATCh). Since oximes change their ionization status depending on medium acidity, we determined the oxime dissociation constants by titration. The dissociation constants (pKa) were 7.47 for HI-6, 7.48 and 8.46 for K033, 8.42 for K027, and 8.39 and 9.51 for TMB-4. The mechanism of oxime reaction with ATCh is analogous to the oxime’ s nucleophilic attack on phosphorylated ChEs in reactivation. The rate of oximate reaction with ATCh was measured at different pH values and hence different oximate ion concentrations. The second order rate constants for the oximolysis of acetylthiocholine at 25 °C were 22 mol-1dm3 min-1 for HI-6 and K033, 231 mol-1dm3 min-1 for TMB-4 and 306 mol-1dm3 min-1 for K027. The results of kinetic measurements and conformational analysis showed that oximes K024 and TMB-4 reacted more than ten times faster with ATCh than HI-6 and K033 due to the para- position of the oxime group at the pyridinium ring, resulting in a less steric hindrance by the rest of the molecule. Therefore one who measures ChE activity assay with oxime must be aware of the oximolysis reaction that interferes with enzyme ATCh hydrolysis giving a higher activity then it is. We wish to thank Dr. Kamil Kuča (Department of Toxicology, Faculty of Military Health Sciences, Hradec Králové, Czech Republic) for preparing and supplying the oximes K027 and K033.

Pyridinium aldoxime; Oximolysis; Spectrophotometry; Titration; Dissociation constants; Conformational analysis

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