Synthesis,Identification And Analysis Of Albumin-Adducts Of Sulfur Mustard And Its Oxidized Metabolites Divinyl Sulfone

Sulfur mustard(SM,2,2-dichloroethyl sulfide)has been used as chemical warfare agent in many wars more than 100 years.The use of chemical weapons during Iran-Iraq war in the 1980s resulted in more than 60,000 casualties,and many victims of SM exposure still suffering from injuries.Despite the development,production and use of SM were banned by the Chemical Weapons Convention for a long time,it continues pose a substantial threat to public safety used for terrorist attack or war because it is high toxicity and easy to produce.As a bifunctional alkylating agent,SM can rapidly attack and alkylate various biological molecules by forming an extremely high reactive episulfonium ion intermediate,which produce multiple adducts.Our pervious studies observed that most of SM was oxidized to mustard sulfoxide(SMO),which can be further oxidized to the mustard sulfone(SMO₂)after entering body.Due to the strong electron donating ability of sulfonyl group in SMO2,chloroethyl groups prefer to eliminated and SMO₂ is converted into DVS spontaneously in physiological weak alkaline environment.DVS contains a pair of vinyl groups with high activity which could adducted to electron-rich biological molecule via Michael addition reaction including DNA,amino acid,glutathione(GSH),and that cause oxidative stress,inflammatory responses,even cell apoptosis.DVS has a similar vesicant property to SM,whereas DVS appears to exhibit higher biotoxicity than DVS and SMO₂ reported in previous literature.The IC50 value(the concentration at which the cell growth is inhibited by 50%)for Ha Ca T cell of DVS is 34?M while that of SM is 100?M.And the dermal LD50(median lethal dose)in rabbit of DVS is 26 mg/kg,which is a quarter of that of SM(ca.100mg.kg).Recently,our research group found both DVS and DVS-GSH(secondary metabolite of DVS and GSH)could electrophilic adducted to DNA in Ha Ca T cell to generate DVS-DNA adducts including N3-HESVA,N7-HESVG,N3-AHESEHG-N7,N7-GHESEHG-N7,N3-GSH-DVS and N7-GSH-DVS,and that revealed the molecular basis of DNA damage induced by DVS and DVS-GSH.In addition,as a protein cross-linker,DVS can also react rapidly with functional groups such as sulfhydryl,carboxyl or amino groups in hemoglobin or thioredoxin,which induces aggregation effect and causes damage to the body.This cross-link property of DVS is also used for modification and labeling of proteins.Albumin is the most abundant protein in plasma,and the only free sulfhydryl group located on Cys34 which could be easily attacked by nucleophilic alkylating agents and form a variety of albumin adducts.The proteolysis of SM-exposed blood samples with pronase or proteinase K generates sulfur-hydroxyethylthioethyl-Cys-Pro-Phe([HETE]-CPF)tripeptide adduct.The tripeptide adduct with a long half-life and high abundance is used as a preferred biomarker to retrospective detection of SM exposure.However,there is still no report on the research on the reaction of DVS with albumin and the biomarker of DVS exposure.Hence,there is a significant need to investigate the addition characteristics of DVS with albumin,especially DVS albumin adducts.In this dissertation,we designed and synthesized DVS-albumin adducts using DVS,GSH and synthetic cysteine-proline-phenylalanine(CPF)tripeptide as raw materials.UPLC-HRMS/MS was used for the identification and characterization of the adducts.In addition,a sensitive,simple and reliable method has been developed for the examination of DVS-albumin adducts in human plasma specimens by optimizing the digestion conditions,SPE method and LC-MS analysis conditions.Finally,a series of new DVS albumin adducts in in DVS-exposed human plasma were identified and the addition characteristics were meticulous examined.The results indicated that DVS can covalently bind to the Cys34 of albumin,forming the monoadduct DVS-Cys-Pro-Phe(DVS-CPF)and diadduct Phe-Pro-Cys-DVS-Cys-Pro-Phe(DVS-[bi-CPF]),and producing a crosslinked adduct GSH-DVS-Cys-Pro-Phe(GSH-DVS-CPF)after addition of GSH.As an oxidative metabolite of SM in vivo,DVS plays an important role in the damage mechanism of SM.The identification of new DVS-albumin adducts provides potential tools for retrospective detection and new evidence for further clarifying the damage mechanism of SM and DVS.The main research contents are as follows:Firstly,the synthetic route of divinyl sulfone and related derivatives is optimized,and then reference materials are prepared.On this basis,UPLC-MS/MS was used to detect the mustard gas albumin adduct.The addition characteristics of DVS and DVS-GSH and albumin were studied,and the formed adducts were identified.To provide an important reference for further improving the damage mechanism in the body after mustard gas exposure.The paper is divided into seven chapters:The first chapter is the foreword,which introduces the physicochemical properties,toxicological mechanism and various metabolic pathways in the body.Focusing on the retrospective detection after sulfur mustard exposure,the toxicity and damage mechanism of its oxidative metabolite divinyl sulfone,and clarifying the albumin adduct as an important biomarker for SM exposure,establishing a sensitive and reliable detection method for traceability Testing plays an important role.The research objectives and research contents are proposed around the combination of SM and DVS with albumin.The second chapter is the synthetic preparation and identification of HETE-CPF adduct.The by-products were reduced and the yield was increased by the new synthetic route,and the identification of products were performed on GC-MS and LC-MS.The third chapter is the establishment of the detection method of SM and albumin addition products in vitro and in vivo.Based on ultra-high performance liquid chromatography tandem orbitrap high-resolution mass spectrometry,with reference to the methods reported in the literature,the resulting[S-HETE]-CPF tripeptide adduct was analyzed.We simplified the sample pretreatment steps by investigating the effect of proteinase K hydrolysis temperature,optimizing the liquid chromatography and mass spectrometry conditions.The analysis revealed that unlike human albumin,SM adducted to the Cys34 of rat albumin formed[S-HETE]-cysteine-valine-tyrosine(HETE-CPY)tripeptide adduct.The fourth chapter is the synthetic preparation and identification DVS-related references.The synthetic routes and conditions of DVS were optimized.The SM-related oxidation products(SMO,SMO₂,DVS)were prepared,and DVS-related adducts was prepared including monoadduct DVS-GSH and DVS-CPF,diadduct DVS-[bi-CPF],cross-linked adduct GSH-DVS-CPF,and the identificatio of these products were performed on GC-MS and LC-MS.The fifth chapter studies the reaction characteristics of DVS and albumin,and identifies the adducts.DVS exposed to HSA and blank plasma in vitro,then DVS adducted to Cys-34 of albumin residues can not only form monoadduct DVS-CPF,but also diadduct DVS-[bi-CPF]after enzymatic hydrolysis.In addition,based on high-resolution Q-Exactive orbitrap mass spectrometry,the identification of newly adducts of divinyl sulfone and albumin including monoadduct DVS-CPF and diadduct DVS-[bi-CPF]were performed.The sixth chapter further studied the reaction characteristics of DVS-GSH and albumin.The HSA solution and blank plasma were exposed to the previously prepared DVS-GSH reference under simulated physiological conditions,and analyzed using UPLC-MS/MS.It was confirmed that whether DVS formed DVS-GSH prior to reacting with GSH or first with cysteine 34 residue on albumin,GSH-DVS-CPF cross-linked adduct was finally formed.In contrast,the SM with stronger alkylation ability participates in the alkylation reaction via the form of sulfonium intermediate.After one-side addition,the other side is hydrolyzed to form a hydroxyethylthioethyl group.The seventh chapter briefly discussed the chronic toxicity and carcinogenic mechanism of SM,and analyzed the demographic,pathological characteristics and prognosis between current smoking and drinking(CSCD)and nonsmoking and nondrinking(NSND)oral squamous cell carcinoma(SCC)patients.Compared with CSCD goup,there were differences in sex ratio,age distribution and education level in the NSND group,but no significant differences in family history.The pathological classifications and survival rate in the CSCD group were significantly worse.Further,a Cox model confirmed the independence of smoking and drinking status for affecting LRC and DSS.This paper indicates that similar to SM,DVS can react with Cys34 of albumin via Michael addition reaction and forms new adducts including monoadduct DVS-CPF and diadduct DVS-[bi-CPF].With addition of GSH in the reaction system,DVS can produce a new crosslinked adduct GSH-DVS-CPF.Significant difference characteristics between DVS and SM were investigated in many ways,and the results showed that SM has stronger binding to Cys34 than DVS forming a stable adduct HETE-CPF.Whereas,clearly different from SM,DVS adducted to albumin produced various adduct products because the bare vinyl group in DVS-GSH or DVS-CPF with high reactively is hard to hydrolyze and could be further adduct with albumin to form cross-linked adducts DVS-[bi-CPF]and GSH-DVS-CPF.Collectively,the new albumin adducts formed by DVS is first reported for the first time,and the identification and investigation of DVS albumin adducts provide new important evidences for elucidating the damage mechanism and and provide potential biomarkers retrospective detection of DVS or SM exposure.The case-control study between CSCD and NSND of SCC can provide ideas and methods for ffurther research on the carcinogenic mechanism of SM,which offers a certain reference value and clinical significance.