Synthesis of different glutathione–sulfur mustard adducts of verified and potential biomarkers

Sulfur Mustard (SM) is a blistering agent used as a chemical weapon. Glutathione (GSH) is involved in the β-lyase degradation pathway of SM and recently, bioadducts between SM and GSH were observed in vitro. While these bioadducts have never been isolated from in vivo tests or real poisoning with SM, they could be of interest as potential future biomarkers for the retrospective validation of exposure. We herein report the synthesis of different observed and new potential GSH–SM bioadducts as reference materials for analytical investigation. Two distinct approaches were investigated: a building-block pathway and the direct reaction with GSH. The availability of these references will aid future studies and may lead to the discovery of new GSH–SM biomarkers.


Introduction
Sulfur Mustard (SM) or 2,2 0 -dichloroethyl sulde was synthesized for the rst time in 1822 by Despretz and its harmful properties were described 38 years later by Guthrie. 1 SM was used as a chemical weapon for the rst time in 1917 during the battle of Ypres, Belgium, which led to the name Yperite. During the 20 th century, SM was used on several occasions, most prominently during the Iraq-Iran War and in 2015 and 2016 during the Syrian Civil War. 2 In 1997, the development, production, stockpile and use of SM and other chemical weapons were prohibited under the Chemical Weapons Convention (CWC), which is enforced by the Organisation for the Prohibition of Chemical Weapons (OPCW). 3 SM is hydrophobic and can therefore easily pass through the skin and lipid cell membranes. Upon contact, SM acts as an irritant and aer a latency period of 2 to 24 hours, blisters occur on the skin, which can turn into skin necrosis. Heavy injuries can occur with eye contact. The most dangerous form of contact however, is by inhalation. Respiratory tracts and lungs are damaged, which can lead to pulmonary edemas, the main cause of death aer SM exposure. Treatment is purely symptomatic. Antibiotics are given to support the weakened immune system. The mortality rate aer SM exposure is low, however, already 0.01 mg cm À2 of liquid contact on the skin leads to cutaneous redness and 0.5 mg cm À2 leads to the formation of huge vesicles. 4 The reactive alkylating species is generated by intramolecular cyclisation of SM and the formation of an episulfonium ion (Scheme 1). It alkylates cellular DNA and can cross-link DNA strands (intrastrand and interstrand), which inhibits DNA replication and leads to cell death. This is also believed to be the source of the latency period, which corresponds to the time the cell needs for a division. Alkylation of DNA through SM is believed to have long term adverse effects like cancers, chronic respiratory diseases and neurological disorders. 4a,5 SM does not only form adducts with DNA, but also with other biomolecules like proteins or phospholipids. These adducts can be used as biomarkers to retrospectively give evidence for exposure. 7 Bioadducts of DNA are the most studied. They either contain a 2-(2-(hydroxy)ethylthio)ethyl (HETE) moiety aer alkylation and hydrolysis or are cross-linked with a 2-(ethylthio) ethyl (ETE) linker. Experiments with DNA incubated with 35 Slabelled SM showed that DNA adducts are mainly formed with guanine (60%) and adenine (8%). Also, cross-linked guanine was observed (16%). These adducts have been synthesized to develop analytical methods and were analyzed by LC-MS/MS. 8 Recently, an adduct between guanine, SM and glutathione (GSH) was discovered in mice exposed to SM, which could be observed by HPLC-MS/MS up to two weeks aer exposure. 9 Bioadducts are also formed with proteins like hemoglobin, albumin, globin and keratin, which can be detected aer isolation and digestion with proteases. Adduct formation happens on the nucleophilic sites of the proteins. Alkylation of Val, His, Asp, Cys and Glu has been observed. 10 GSH is a tripeptide with the formula g-Glu-Cys-Gly. It plays a major role as a redox buffer in cells and is involved in determine their potential as biomarkers. Further, we present the synthesis of the potential bioadducts bis-O-HETE-GSH and O-HETE-GSH which so far have never been observed. Having these compounds available as references might support further analytical work to establish new biomarkers for the intoxication with SM.

Results and discussion
Synthesis of S-HETE-GSH 4 The initial strategy investigated to synthesize S-HETE-GSH 4 was to alkylate GSH with the protected half mustard tBuOETECl 2. The alkylating agent was prepared by protecting one of the hydroxyl groups of thiodiglycol (TDG) with a tBu-protecting group following a modied procedure of Noort. 15 Isobutylene was bubbled through a solution of TDG and H 2 SO 4 in DCM to yield tBuOETEOH 1. While the yield remained modest, yield and purity increased from 9% to 15% and from >90% to >98% respectively.
The OH group of 1 was then replaced by Cl by slow addition of thionyl chloride, which yielded tBuOETECl 2 in high yield (99%) and purity. 15 GSH was alkylated with 2 under slightly basic conditions. 15 The resulting S-tBuOETE-GSH 3 was puried by reversed phase ash chromatography and obtained in good yield and purity.
Several conditions were tested to cleave the tert-butyl ether 3. Hydrolysis with phosphoric acid led to acceptable yield, however, the acid showed to be inseparable from the product. 16 The use of Amberlyst 15 gave low yield and purity. 17 The best result was obtained by using 90% TFA in aqueous solution. Some epimerization occurred, but the diastereomers could be separated, and S-HETE-GSH 4 was obtained in 38% yield and 95% purity (Scheme 2).
As an alternative, a building-block approach was investigated in which cysteine was rst alkylated and then used in solidphase-peptide-synthesis (SPPS). L-Cysteine was alkylated with 2 using the same procedure which was previously used to alkylate GSH. S-tBuOETE-Cys 5 was obtained in 63% yield and very high purity. In the subsequent step the amine function of 5 was protected with Fmoc-OSu to give Fmoc-Cys(ETEOtBu)-OH 6. 18 SPPS was performed on preloaded Gly-2-CT polystyrene resin and DIC/Oxyma were used as coupling agents. In the Fmoc-deprotection steps 20% piperidine in DMF was used. Coupling of 6 proved to be slow and its reaction time needed to be increased to 3 h. Cleavage from the resin and deprotection were achieved with a cocktail consisting of TFA : TIS : H 2 O (95 : 2.5 : 2.5). Aer purication by precipitation in cold ether and subsequent ash chromatography 4 was obtained in 65% yield and >97% purity (Scheme 3).
With the direct alkylation approach 4 was obtained in two steps from GSH with 28% yield and >95% purity. The buildingblock approach achieved 36% yield (>97% purity) in three steps from L-cysteine. The building-block pathway is recommended since it provides the reference material 4 in better overall yield and purity.

Synthesis of GSH-ETE-GSH 7
Another compound, which has been observed in in vitro biological assays but has never been isolated, is the adduct Scheme 3 Synthesis of S-HETE-GSH 4 by SPPS. consisting of two GSH linked by a sulfur mustard moiety via their thiol functions. Indeed, when a twofold excess of GSH reacted under slightly basic conditions with one equivalent of sulfur mustard, GSH-ETE-GSH 7 was formed. The reaction proceeded slowly, but aer ve days 7 could be isolated in 65% yield in 85% purity. The impurity was identied as residual acetonitrile (Scheme 4).

Synthesis of bis-O-HETE-GSH 8
The bioadducts bis-O-HETE-GSH 8, O-HETE-GSH 9 and GSH-O-HETE 10 have never been observed. However, SM adducts of glutamic acid and aspartic acid have been observed in vitro, aer SM reacted with their side chain acid groups. 10a Therefore it could be envisageable that the adducts 8, 9 and 10 would be formed as potential biomarkers. Both amine and thiol functionalities of GSH were protected in the presence of Boc 2 O to increase its solubility in organic solvents. 19 Protected 11 still contained S-Boc and N-Boc monoprotected GSH as minor byproducts. 11 was observed as two diastereomers in a 1 : 1 ratio. Epimerization occurred at the cysteine moiety due to the basic conditions of the coupling. Several conditions to esterify 11 with excess of TDG were screened (Table 1). Best results for the coupling were obtained using EDC, HOBt and DMAP under inert atmosphere (Table 1, entry 10). However, the bis-O-HETE-N,S-Boc-GSH adduct 12 could only be obtained in moderate purity. It was engaged without further purication in the subsequent deprotection step using a solution of TFA, TFE and water. While high yield was achieved, the nal product 8 could not be separated from a side product which was identied as the tert-butyl thioether of 8. Neither could the diastereoisomers be separated. This resulted in reduced purity of >75% (Scheme 5).

Synthesis O-HETE-GSH
In the attempt to synthesize the mono-O-HETE-GSH adducts 9 and 10 ( Fig. 1), 11 was subjected to the coupling conditions of Table 1, entry 4, but only one equivalent of TDG was used. LC-HRMS analysis of the resulting product mixture revealed that ca. 70% corresponded to either one of the two possible mono-O-HETE-N,S-Boc-GSH compounds, while ca. 11% corresponded to the bis-O-HETE-N,S-Boc-GSH 12 (data not shown). The mixture could not be separated. The experiment showed that, as expected, there was no selectivity between the two C-termini of 11. Therefore, a building-block approach to synthesize 9 was investigated instead. Commercially available Boc-Glu(OFm)-Scheme 4 Dimerization of two glutathione molecules with sulfur mustard as linker.  OH 13 was esteried with 1 using HCTU/DIPEA which gave Boc-Glu(OFm)-OETEOtBu 14. The Fm group was selectively cleaved with 20% piperidine in DMF giving Boc-Glu(OH)-O-ETEOtBu 15 in 68% yield and good purity. 15 was used in SPPS to give 9 in 77% yield and >90% purity (Scheme 6). The synthesis of 10 was not attempted.

Conclusion
Several synthesis strategies were explored to produce sulfur mustard-glutathione adducts as potential biomarkers for retrospective validation of exposure to this blistering agent. S-HETE-GSH 4 was successfully synthesized. Two pathways towards 4 were investigated. The direct alkylation of GSH with 2 and subsequent deprotection led to 28% overall yield from GSH and >95% purity. However, better yield and purity were obtained when using a building-block approach and SPPS to construct the nal peptide. While requiring an additional step, this route yielded 36% overall yield from Fmoc-Cys-OH and >97% purity. It is recommended to use the building-block pathway to obtain 4, since the reference material can be obtained in better overall yield and purity.
The dimer GSH-ETE-GSH 7 was obtained in one step by condensing GSH with sulfur mustard.
The potential biomarker bis-O-HETE-GSH 8 was synthesized directly from the reaction of SM with GSH. While 8 was only obtained in moderate purity, the characterization data will help to investigate its presence in future in vitro and in vivo screenings.
Using the protected precursor Boc-Glu(OH)-O-ETEOtBu 15 in SPPS allowed the synthesis of the mono-O-HETE-GSH derivative 9 with 77% yield and >90% purity in three steps.

Experimental part
Unless otherwise stated, all reagents were purchased from Sigma-Aldrich and were used without further purication. Glutathione was obtained from Iris Biotech. Deuterated solvents were purchased from Armar AG and Cambridge Isotope Laboratories, Inc. Boc-Glu(OFm)-OH was purchased from Bachem. Sulfur mustard was synthesized and provided by Spiez Laboratory. CAUTION: sulfur mustard is a schedule 1 chemical and is highly toxic. Adequate protection is needed and appropriate safety measures have to be taken when handling this compound.
Thin layer chromatography (TLC) was performed on silica gel 60 F-254 pre-coated aluminum sheets thin layer chromatography plates and silica gel RP-18 F-254S pre-coated aluminum sheets TLC plates from Merck.
Reaction monitoring by mass analysis was done by direct injection into a Dalton Mass Detector (ESI) from Biotage.
Flash column chromatography was carried out with an Isolera One system coupled with a Dalton Mass Detector from Biotage. Biotage SNAP Ultra cartridges (10 g, 25 g, 50 g and 100 g) and Biotage SNAP Ultra C18 cartridges (12 g, 30 g and 60 g) were used.
SPPS was performed on an Initiator+ Alstra automated peptide-synthesizer from Biotage.
LC-HRMS (ESI) analyses were done on an Agilent Technologies 1290 Innity LC System instrument with a Bruker Daltonics maXis UHR QTof 4G MS. As a column the Sigma-Aldrich Discovery HS C18 (150 mm Â 2.1 mm, particle size 5 mm) was used. As eluents H 2 O with 5 mM NH 4 Ac and MeOH with 5 mM NH 4 Ac were used with a ow-rate of 0.6 mL min À1 .
GC-MS (EI) analyses were performed on an Agilent Technologies 7890A instrument coupled with an Agilent Technologies 5975C inert MSD. The measurements were performed with the HP-1701 (30 m Â 0.25 mm Â 0.25 mm, 14% cyanopropylphenyl/86% PDMS) high resolution gas chromatography column using a temperature program (40 C for 3 min, 13 C min À1 until 280 C and 280 C for 3.54 min). The injector and the detector temperatures were 220 C and 250 C, respectively. The splitless injection mode was used to inject volumes of 1 mL (c ¼ 0.5 mg mL À1 ). Helium was used as carrier gas (1 mL min À1 ).
NMR spectra were recorded on a Bruker Avance III HD 400 MHz Nano Bay spectrometer in CDCl 3 , DMSO-d 6  IR spectra were measured on a Bruker Tensor 27 and a Jasco FT/IR-4100 spectrometer.
Purities were assessed by NMR.