Apart from the well-known sulfur mustard (SM), additional sulfur-containing blistering chemical warfare agents exist. Sesquimustard (Q) is one of them and five times more blistering than SM. It is a common impurity in mustard mixtures and regularly found in old munitions but can also be used in pure form. Compared to the extensive literature on SM, very little experimental data is available on Q and no protein biomarkers of exposure have been reported. We herein report for the first time the adduct of Q with the nucleophilic Cys³⁴ residue of human serum albumin (HSA) formed in vitro and introduce two novel bioanalytical procedures for detection. After proteolysis of this HSA adduct catalyzed either by pronase or by proteinase K, two biomarkers were identified by high-resolution tandem mass spectrometry (MS/HR MS), namely a dipeptide and a tripeptide, both alkylated at their Cys residue, which we refer to as HETETE-CP and HETETE-CPF. HETETE represents the Q-derived thio-alkyl moiety bearing a terminal hydroxyl group: “hydroxyethylthioethylthioethyl.” Targeting both peptide markers from plasma, a micro liquid chromatography–electrospray ionization tandem mass spectrometry method working in the selected reaction monitoring mode (μLC-ESI MS/MS SRM) was developed and validated as well suited for the verification of exposure to Q. Fulfilling the quality criteria defined by the Organisation for the Prohibition of Chemical Weapons, the novel methods enable the detection of exposure to Q alone or in mixtures with SM. We further report on the relative reactivity of Q compared to SM. Based on experiments making use of partially deuterated Q as the alkylating agent, we rule out a major role for six-membered ring sulfonium ions as relevant reactive species in the alkylation of Cys³⁴. Furthermore, the results of molecular dynamics simulations are indicative that the protein environment around Cys³⁴ allows adduct formation with elongated but not bulky molecules such as Q, and identify important hydrogen bonding interactions and hydrophobic contacts.

除了众所周知的硫芥末（SM），还存在其他含硫起泡化学战剂。Sesquimustard（Q）是其中之一，且起泡程度是SM的五倍。它是芥末混合物中的常见杂质，通常在旧弹药中发现，但也可以纯净形式使用。与有关SM的大量文献相比，关于Q的实验数据很少，也没有关于暴露的蛋白质生物标志物的报道。我们在此首次报道Q与亲核Cys ³⁴的加合物人血清白蛋白（HSA）的残留物在体外形成，并引入了两种新颖的生物分析程序进行检测。在通过链霉蛋白酶或蛋白酶K催化的这种HSA加合物发生蛋白水解后，通过高分辨率串联质谱（MS / HR MS）鉴定了两个生物标记，即二肽和三肽，两者均在其Cys残基处烷基化，我们将其称为改为HETETE-CP和HETETE-CPF。HETETE代表带有末端羟基的Q衍生的硫代烷基部分：“羟乙基硫代乙基硫代乙基”。针对血浆中的两种肽标记物，开发了一种以选定的反应监测模式（μLC-ESIMS / MS SRM）运行的微型液相色谱-电喷雾电离串联质谱法，该方法已验证并非常适合验证暴露于Q的情况。这些新方法满足了禁止化学武器组织定义的质量标准，能够检测单独暴露于Q或与SM混合的Q。我们进一步报告了与SM相比Q的相对反应性。基于使用部分氘代Q作为烷基化剂的实验，我们排除了六元环sulf离子作为Cys烷基化中的相关反应物种的主要作用³⁴。此外，分子动力学模拟的结果表明，Cys ³⁴周围的蛋白质环境允许与细长但不庞大的分子（例如Q）形成加合物，并鉴定出重要的氢键相互作用和疏水性接触。