Human BioMolecular Research Institute, San Diego, California 92121, USA.
Chem Res Toxicol. 2009 Oct;22(10):1680-8. doi: 10.1021/tx900090m.
The goal was to test 14 nerve agent model compounds of soman, sarin, tabun, and cyclohexyl methylphosphonofluoridate (GF) for their suitability as substitutes for true nerve agents. We wanted to know whether the model compounds would form the identical covalent adduct with human butyrylcholinesterase that is produced by reaction with true nerve agents. Nerve agent model compounds containing thiocholine or thiomethyl in place of fluorine or cyanide were synthesized as Sp and Rp stereoisomers. Purified human butyrylcholinesterase was treated with a 45-fold molar excess of nerve agent analogue at pH 7.4 for 17 h at 21 degrees C. The protein was denatured by boiling and was digested with trypsin. Aged and nonaged active site peptide adducts were quantified by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry of the tryptic digest mixture. The active site peptides were isolated by HPLC and analyzed by MALDI-TOF-TOF mass spectrometry. Serine 198 of butyrylcholinesterase was covalently modified by all 14 compounds. Thiocholine was the leaving group in all compounds that had thiocholine in place of fluorine or cyanide. Thiomethyl was the leaving group in the GF thiomethyl compounds. However, sarin thiomethyl compounds released either thiomethyl or isopropyl, while soman thiomethyl compounds released either thiomethyl or pinacolyl. Thiocholine compounds reacted more rapidly with butyrylcholinesterase than thiomethyl compounds. Labeling with the model compounds resulted in aged adducts that had lost the O-alkyl group (O-ethyl for tabun, O-cyclohexyl for GF, isopropyl for sarin, and pinacolyl for soman) in addition to the thiocholine or thiomethyl group. The nerve agent model compounds containing thiocholine and the GF thiomethyl analogue were found to be suitable substitutes for true soman, sarin, tabun, and GF in terms of the adduct that they produced with human butyrylcholinesterase. However, the soman and sarin thiomethyl compounds yielded two types of adducts, one of which was thiomethyl phosphonate, a modification not found after treatment with authentic soman and sarin.
目的是测试沙林、梭曼、塔崩和环己基甲基膦酸氟化物（GF）的 14 种神经毒剂模型化合物，以确定它们是否适合作为真正神经毒剂的替代品。我们想知道模型化合物是否会与真正的神经毒剂与人类丁酰胆碱酯酶反应生成相同的共价加合物。用硫代胆碱或硫甲基代替氟或氰化物的神经毒剂模型化合物被合成 Sp 和 Rp 对映异构体。用 45 倍摩尔过量的神经毒剂类似物在 pH7.4 下于 21°C 处理 17 小时，处理后将蛋白质煮沸变性并用胰蛋白酶消化。用基质辅助激光解吸/电离飞行时间（MALDI-TOF）质谱法对胰蛋白酶消化混合物中的老化和非老化活性部位肽加合物进行定量分析。用 HPLC 分离活性部位肽并用 MALDI-TOF-TOF 质谱法分析。丁酰胆碱酯酶的丝氨酸 198 被所有 14 种化合物共价修饰。所有含有硫代胆碱代替氟或氰化物的化合物的离去基团都是硫代胆碱。GF 硫甲基化合物的离去基团是硫甲基。然而，沙林硫甲基化合物释放硫甲基或异丙基，而梭曼硫甲基化合物释放硫甲基或频哪醇基。硫代胆碱化合物与丁酰胆碱酯酶的反应速度比硫甲基化合物快。用模型化合物标记导致生成除硫代胆碱或硫甲基基团外还失去 O-烷基（塔崩为 O-乙基，GF 为 O-环己基，沙林为异丙基，梭曼为频哪醇基）的老化加合物。含有硫代胆碱和 GF 硫甲基类似物的神经毒剂模型化合物被发现是真正的梭曼、沙林、塔崩和 GF 的合适替代品，因为它们与人类丁酰胆碱酯酶生成的加合物。然而，梭曼和沙林的硫甲基化合物产生了两种类型的加合物，其中一种是硫甲基膦酸酯，这是用真实的梭曼和沙林处理后未发现的修饰。
