Eppley Institute, University of Nebraska Medical Center, Omaha, Nebraska 68198-5950, USA.
Toxicol Sci. 2013 Apr;132(2):390-8. doi: 10.1093/toxsci/kft011. Epub 2013 Jan 23.
The phosphoramidate nerve agent tabun inhibits butyrylcholinesterase (BChE) and acetylcholinesterase by making a covalent bond on the active site serine. The adduct loses an alkyl group in a process called aging. The mechanism of aging of the tabun adduct is controversial. Some studies claim that aging proceeds through deamination, whereas crystal structure studies show aging by O-dealkylation. Our goal was to develop a method that clearly distinguishes between deamination and O-dealkylation. We began by studying the tetraisopropyl pyrophosphoramide adduct of BChE because this adduct has two P-N bonds. Mass spectra showed that the P-N bonds were stable during trypsin digestion at pH 8 but were cleaved during pepsin digestion at pH 2. The P-N bond in tabun was also acid labile, whereas the P-O bond was stable. A scheme to distinguish aging by deamination from aging by O-dealkylation was based on the acid labile P-N bond. BChE was inhibited with Sp- and Rp-tabun thiocholine nerve agent model compounds to make adducts identical to those of tabun with known stereochemistry. After aging and digestion with pepsin at pH 2, peptide FGES198AGAAS from Sp-tabun thiocholine had a mass of 902.2 m/z in negative mode, indicating that it had aged by deamination, whereas peptide FGES198AGAAS from Rp-tabun thiocholine had a mass of 874.2 m/z in negative mode, indicating that it had aged by O-dealkylation. BChE inhibited by authentic, racemic tabun yielded both 902.2 and 874.2 m/z peptides, indicating that both stereoisomers reacted with BChE and aged either by deamination or dealkylation.
沙林膦酸酯神经毒剂塔朋通过在活性部位丝氨酸上形成共价键来抑制丁酰胆碱酯酶（BChE）和乙酰胆碱酯酶。加合物通过称为老化的过程失去一个烷基基团。塔朋加合物老化的机制存在争议。一些研究声称老化通过脱氨进行，而晶体结构研究表明老化通过 O-脱烷基进行。我们的目标是开发一种能够清楚地区分脱氨和 O-脱烷基的方法。我们首先研究了 BChE 的四异丙基焦磷酸酰胺加合物，因为该加合物具有两个 P-N 键。质谱表明，在 pH8 下用胰蛋白酶消化时，P-N 键稳定，但在 pH2 下用胃蛋白酶消化时被切断。塔朋的 P-N 键也酸不稳定，而 P-O 键稳定。区分脱氨老化和 O-脱烷基老化的方案基于酸不稳定的 P-N 键。用 Sp-和 Rp-塔朋硫代胆碱神经毒剂模型化合物抑制 BChE，以生成与已知立体化学的塔朋加合物相同的加合物。老化后，在 pH2 下用胃蛋白酶消化后，Sp-塔朋硫代胆碱的肽 FGES198AGAAS 在负离子模式下的质量为 902.2m/z，表明其已通过脱氨作用老化，而 Rp-塔朋硫代胆碱的肽 FGES198AGAAS 在负离子模式下的质量为 874.2m/z，表明其已通过 O-脱烷基作用老化。用纯、外消旋塔朋抑制的 BChE 产生 902.2 和 874.2m/z 的肽，表明两种立体异构体都与 BChE 反应，并且通过脱氨或脱烷基作用老化。
