Glutathione Adduct Patterns of Michael-Acceptor Carbonyls

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Glutathione Adduct Patterns of Michael-Acceptor Carbonyls Christian Slawik, Christiane Rickmeyer, Martin Brehm, Alexander Böhme, and Gerrit Schüürmann Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.6b04981 • Publication Date (Web): 22 Feb 2017 Downloaded from http://pubs.acs.org on February 24, 2017

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Environmental Science & Technology

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1 2

Glutathione Adduct Patterns of Michael-Acceptor Carbonyls

3

Christian Slawik,a,b Christiane Rickmeyer,a Martin Brehm,a Alexander Böhme,a Gerrit

4

Schüürmanna,b,*

5 6

a

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search, Permoserstraße 15, 04318 Leipzig, Germany

UFZ Department of Ecological Chemistry, Helmholtz Centre for Environmental Re-

8 9 10

b

Institute for Organic Chemistry, Technical University Bergakademie Freiberg, Leipziger

Straße 29, 09596 Freiberg, Germany

11 12 13 14 15

Corresponding Author:

16

Tel +49-341-235-1262, Fax +49-341-235-45-1262, E-mail [email protected]

17 18

ACS Paragon Plus Environment


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TOC GRAPHIC HS O

O HO

NH NH2

O

O NH

OH +

Adductome

O

GSH

E

terminal and non-terminal

GS E H

N 1

20

H

terminal only

GS E E

N

H

GS E E

2

N

E

3

tR

21


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ABSTRACT

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Glutathione (GSH) has so far been considered to facilitate detoxification of soft organic

25

electrophiles through covalent binding at its cysteine (Cys) thiol group, followed by step-

26

wise catalyzed degradation and eventual elimination along the mercapturic acid path-

27

way. Here we show that in contrast to expectation from HSAB theory, Michael-acceptor

28

ketones, aldehydes and esters may form also single, double and triple adducts with

29

GSH involving β-carbon attack at the much harder N-terminus of the γ-glutamyl (Glu)

30

unit of GSH. In particular, formation of the GSH-N single adduct contradicts the tradi-

31

tional view that S alkylation always forms the initial reaction of GSH with Michael-accep-

32

tor carbonyls. To this end, chemoassay analyses of the adduct formation of GSH with

33

nine α,β-unsaturated carbonyls employing high performance liquid chromatography and

34

tandem mass spectrometry have been performed. Besides enriching the GSH adduc-

35

tome and potential biomarker applications, electrophilic N-terminus functionalization is

36

likely to impair GSH homeostasis substantially through blocking the γ-glutamyl transfer-

37

ase catalysis of the first breakdown step of modified GSH, and thus its timely reconstitu-

38

tion. The discussion includes a comparison with cyclic adducts of GSH and furan me-

39

tabolites as reported in literature, and quantum chemically calculated thermodynamics of

40

hard-hard, hard-soft and soft-soft adducts.

41 42

KEYWORDS

43

Adductome, glutathione, Michael-acceptor, electrophile, HSAB



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44

INTRODUCTION

45 46

The exposome describes the cumulative totality of chemical exposure in the organism in

47

response to environmental factors.1,2 One possible way of providing access to this endo-

48

genous systems chemistry is the analysis of adducts formed with tissue nucleophiles.3-5

49

Depending on the subject of interest, the focus may be on proteins,5-12 lipids,12-16 the

50

DNA,17-19 or on critical peptides such as glutathione (GSH).20-22 Electrophilic natural pro-

51

ducts as part of the metabolome have led to developing activity-based protein profiling to

52

label and characterize nucleophilic functional sites of the proteome,23-25 and there is in-

53

creasing evidence that reversible binding at cysteine (Cys) thiol may represent a mecha-

54

nism of electrophile signaling.10,26,27 From the toxicological viewpoint, the Pearson HSAB

55

theory of hard and soft acids and bases appears attractive to identify preferred nucleo-

56

philic targets of electrophilic toxicants,28,29 and respective reactivities can be screened

57

quantitatively in chemico or in silico.30-33 However, it has been demonstrated that the

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HSAB concept lacks a proper account of the kinetic vs thermodynamic control of organic

59

reactions, and indeed fails to predict the reactivity of prominent ambident nu-

60

cleophiles.34,35

61

Nevertheless, α,β-unsaturated carbonyls that occur in food36-39 but also in motor

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vehicle exhaust40 are often considered as soft electrophiles, attacking preferably the soft

63

thiol group of GSH and protein Cys through Michael addition. In vivo, GSH may trap the-

64

se and other electrophiles without or with catalysis through glutathione S-transferase

65

(GST), followed by controlled degradation and eventual reconstitution through the γ-glu-

66

tamyl cycle as discussed below. Here, GSH adduct formation without GST is understood


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to concern primarily soft electrophiles41 such as Michael acceptors. This has led to the

68

perception that the α-amino group of the γ-glutamyl (Glu) residue would not participate

69

in conjugation reactions.

70

Only recently, double adducts of phenyl isocyanate with GSH involving both its

71

Cys SH and Glu α-NH2 functions were reported.21 To the best of our knowledge, the only

72

GSH adducts with a fully substituted N-terminus observed so far concern its cyclization

73

to pyrrole and pyrrolin-2-one derivatives through bifunctional cis-endiones (2-butene-1,4-

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dial, BDA) as cytochrome P450 metabolites from pro-electrophilic precursors.20,22,42-44

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Whereas thiol adducts of GSH may be readily degraded and eventually elimina-

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ted through the mercapturic pathway, a respective enzyme-catalyzed degradation of N-

77

functionalized GSH has not yet been demonstrated. In the present investigation, GSH

78

adducts formed in chemico with nine α,β-unsaturated ketones, aldehydes and esters are

79

analyzed employing UHPLC-MS/MS analysis, and evaluated from the viewpoint of

80

HSAB theory. The latter includes quantum chemical calculations of the reaction thermo-

81

dynamics, and a comparative analysis of literature findings42-44 regarding cyclic GSH

82

adducts with the furan metabolite BDA.

83

For electrophiles in excess to GSH as can be found in case of acetaminophen

84

toxification,45 our chemoassay analyses demonstrate for the first time both GSH-N sin-

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gle adducts and the formation of triple GSH adducts involving both its Cys thiol and Glu

86

N-terminus without intramolecular cyclization. Regarding the opposite setup with excess

87

GSH that is expected as normal biological condition, N alkylation as single GSH adduct

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has been observed besides single thiol (S) adducts and double (S,N) adducts through

89

bond formation to both the thiol site and the N terminus. Moreover, the associated N-Cβ-



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bond is shown to be thermodynamically favorable despite its HSAB characterization as

91

hard-soft interaction. Comparative analysis of the product patterns yields a mechanistic

92

rationale for the structural type of Michael acceptors where double and triple GSH ad-

93

ducts can be expected, and thus may contribute to downstream toxicological processes.

94 95 96

MATERIALS AND METHODS

97 98

Stock and buffer solutions. As reaction medium a buffer solution containing 0.0648

99

mol/L disodium hydrogen phosphate and 0.0153 mol/L potassium dihydrogen phosphate

100

was used and adjusted to pH 7.4. For all experiments with electrophiles in excess, the

101

GSH stock solution was prepared freshly by dissolving 0.0107 g (0.034 mmol) reduced

102

GSH in 25 mL buffer solution (cGSH = 1.4 mmol/L). For the stock solution of test com-

103

pounds, 3.3 mL DMSO was added into a 50 mL volumetric flask. Subsequently, 0.680

104

mmoL of the test compound was added gravimetrically, and the flask was filled to vol-

105

ume with buffer solution.

106

For the experiments with GSH in excess, the GSH stock solution was prepared

107

freshly by dissolving 0.659 g (2.15 mmol) in 15 mL buffer solution. The electrophile stock

108

solution was prepared through adding 1 mL DMSO into a 2 mL volumetric flask, and ad-

109

ding 15.27 mg electrophile (1-hexen-3-one (A2) or 4-hexen-3-one (B2)) gravimetrically.

110

Subsequently, the volumetric flask was filled to volume with DMSO to obtain a concen-

111

tration of 0.156 mM. All solutions were stored at 25 °C in a climate chamber prior to use.


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Adduct formation. For the adduct formation experiments 1.5-mL glass vials

113

equipped with screw caps and PTFE seals were used as batch reactors. To start the re-

114

action between GSH and the electrophile in excess, 600 µL buffer, 100 µL GSH stock

115

solution and 300 µL of test compound stock solution were added to a 1.5 mL vial, result-

116

ing in reaction mixture concentrations of 0.14 mM for GSH and 14 mM for the electro-

117

philes, respectively (electrophile-to-nucleophile ratio = 100). The excess ratio of 100 has

118

been selected to ensure that changes in concentration of the electrophile during the re-

119

action with GSH can be neglected. For the GSH excess experiments, 1470 µL of the

120

GSH stock solution was added into a 1.5-mL vial. Subsequently, 30 µL of the electro-

121

phile stock solution (A2 or B2) was added, yielding a final reaction mixture with 140 mM

122

GSH and 1.4 mM electrophile (electrophile-to-nucleophile ratio = 0.01). All reaction mix-

123

tures contained 2% (v/v) DMSO. After 24 h, GSH-electrophile adducts were analyzed

124

using HPLC-MS/MS.

125

Adduct analysis using HPLC and tandem mass spectrometry. Formed adducts

126

were analyzed with a 1290 series HPLC system from Agilent (Santa Clare, CA, USA)

127

consisting of a 1290 Infinity HPLC pump, column, oven, and thermostatic autosampler.

128

The HPLC system was equipped with a Poroshell© 120 EC-C18 column (3.0 mm i.d. x

129

50 mm length, 2.7 µm). Column temperature was set to 25°C, and the flow rate was 1

130

mL/min. The eluent consisted of doubly distilled water (solvent A) and acetonitrile (sol-

131

vent B), both containing 0.1% (ν/ν) formic acid. The following gradient has been used to

132

analyze the electrophile excess experiments: 0-0.5 min: 1% B; 0.5-1.5 min: linear in-

133

crease to 100% B; 1.5-2 min 100% B; at 2.01 min: immediate switch to 1% B. The total

134

run time was 3 min. For the experiments with GSH in excess, the linear increase to



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100% B was extended by two minutes (0.5-3.5 min) to optimize separation of GSH sin-

136

gle adducts featuring single N or S alkylation. In these cases, the total run time was 5

137

min.

138

Adduct detection proceeded with an Agilent QQQ mass spectrometer G6460 (Agi-

139

lent, Santa Clara, CA, USA) equipped with an Agilent JetStream ESI Source. GSH and

140

its adducts were detected through their protonated and positively charged molecular

141

ions ([M+H]+) using the MS2-scan mode and the adduct structures were analyzed using

142

product ion scan mode (collision energy CE 10-25 eV). Capillary and nozzle voltage

143

were set to 6000 V and 2000 V, respectively. The source temperature was 350 °C and

144

the sheath gas temperature was 380 °C. Data were recorded and analyzed using the

145

MassHunter B06.00TM software (Agilent, Santa Clara, CA, USA).

146

Quantum chemical calculations. Density functional theory using the M06-2X

147

functional46 (optimized for thermodynamics) with the basis set def2-tzvpp47 as imple-

148

mented in Orca48 has been employed to calculate enthalpies and free energies, ∆H and

149

∆G, of model reactions representing cyclic and non-cyclic GSH adduct formation at 298

150

K. The cyclic adducts42-44 were mimicked through the products of 2-butene-1,4-dial

151

(BDA, O=CH–CH=CH–CH=O) with the S- and N-nucleophiles methylsulfide (H3C–SH)

152

and methylamine (H3C–NH2), and the thermodynamics underlying the presently ob-

153

served non-cyclic Michael adducts with one, two and three GSH molecules was evaluat-

154

ed with acrolein (O=CH–CH=CH2) and 2-aminoethanethiol (H2N–CH2–CH2–SH) that

155

covers both the thiol function and the N-terminus of GSH. Besides gas-phase calcula-

156

tions including geometry optimization, aqueous solution was simulated through COS-

157

MO49 in the Gaussian CPCM50,51 implementation using UAKS radii52 to estimate the re-


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spective solution-phase thermodynamics (∆Hw and ∆Gw) through taking into account the

159

calculated solvation energies (without additional geometry optimization).

160 161 162

RESULTS and DISCUSSION

163 164

The nine α,β-unsaturated carbonyls tested include five compounds with a terminal (un-

165

substituted) β-carbon, and four non-terminal (β-substituted) Michael acceptors. Their se-

166

cond-order rate constants of reaction with GSH, kGSH [L mol–1 min–1] have been meas-

167

ured earlier employing the photometric GSH chemoassay30,53 and range from 1261 (1-

168

penten-3-one, A1 in Scheme 1) to 0.161 (ethyl crotonate, C3), thus covering almost four

169

orders of magnitude.

170 171

SCHEME 1

172 173

Terminal vs non-terminal α,β-unsaturated carbonyls. Since the chemical iden-

174

tities of GSH and the α,β-unsaturated carbonyls (Scheme 1) are well known,30,53 struc-

175

tural analysis can focus on expected GSH-electrophile adducts (see Supporting Infor-

176

mation, Schemes S1-S3). Combination of product ion spectra from triple quadrupole

177

mass spectrometry with knowledge of adduct fragmentation patterns of GSH54 and re-

178

spective NMR-confirmed information for GSH-electrophile-adducts21 enables a targeted-

179

analysis-like structure identification55 if the fragments of the most abundant mass peaks

180

can be allocated to sufficiently plausible fragmentation pathways for the GSH part of the

181

adducts.21,54,56 Typically, fragmentation of GSH occurs along the peptide chain57,58 inde-



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182

pendently of whether or not an electrophile is bound to GSH.54 Thus, S and N adducts

183

can be discriminated by the mass differences between fragment ions corresponding to

184

electrophile attachment at GSH thiol and N-terminus, respectively.54 Analytical details

185

are provided in the Supporting Information. These cover the general adduct structures

186

(Schemes S3-S4), the product ion spectra of the single, double and triple adduct of GSH

187

with 1-hexen-3-one (A2, Figures S1–S4) including fragment allocation tables for the

188

most abundant peaks (Tables S1-S4), and associated fragmentation patterns. This in-

189

formation enables one to discriminate even between those adducts that show identical

190

m/z ratios such as the single S (Table S1, fragments S-1b, S-1d, and S-1e) and single N

191

(Table S4, fragments N-1a, N-1c, and N-1f) adducts with m/z = 406.

192

Figure 1 shows a typical ion chromatogram for an electrophile excess experiment

193

indicating three different adducts of GSH (peaks 1, 2 and 3 in the figure) of terminal Mi-

194

chael acceptors, which in this case concerns the 24-h reaction with 1-hexen-3-one (A2).

195

Here, the first adduct eluted at 1.16 min (1, m/z 406), the second adduct at 1.24 min (2,

196

m/z 504), and the third one at 1.31 min (3, m/z 602). The m/z difference between 1 and

197

2 as well as between 2 and 3 is 98 Da and corresponds to the molar mass of

198

1-hexen-3-one.

199 200

FIGURE 1

201 202

For adducts 1 and 2, the proposed structures and some of the allocated frag-

203

ments are comparable to the GSH single and double adducts formed with phenyl isocy-

204

anate described previously,21 which provides further support for our proposed structure.


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These fragmentation pathways demonstrate that covalent binding always concerns the

206

Michael-acceptor β-carbon, regarding both the thiol residue and the N-terminus of GSH.

207

Whereas double adducts featuring a covalent linkage to GSH thiol and mono-

208

substitution at the N-terminus are found for all nine compounds, triple adducts involving

209

a doubly substituted N-terminus are observed only for the terminal α,β-unsaturated car-

210

bonyls (Table S5). The latter cover the three ketones 1-penten-3-one, 1-hexen-3-one

211

and 1-octen-3-one (A1-A3), the aldehyde 2-ethyl acrolein (C1), and the ester ethyl acry-

212

late (C2).

213

Interestingly, the GSH chemoassay reactivity of the three non-terminal ketones

214

(3-penten-2-one, 4-hexen-3-one, 2-octen-4-one, B1-B3) is ca. 2.5-fold larger than that of

215

ethyl acrylate (kGSH: 26.7, 26.1, 24.2 vs 10.6 L mol–1 min–1),53 demonstrating that kGSH

216

alone cannot explain whether or not a triple adduct is formed. By contrast, covalent at-

217

tack at an already monosubstituted N-terminus appears to be particularly sensitive to

218

steric hindrance, thus taking place only for terminal Michael acceptors with no substitu-

219

ent at the β-carbon.

220

Electrophile excess conditions are often used for the chemoassay screening of

221

electrophilic reactivity as related to toxicity.30,53,59 In this context, GSH is typically con-

222

sidered to mimic protein Cys30,53,59-61 without addressing the impact of the N-terminus on

223

GSH reactivity. Although the in vivo concentration of GSH is normally much higher than

224

that of electrophiles, the latter may supersede the GSH level significantly in case of pa-

225

racetamol toxication45 or agents exerting both electrophilic and oxidative stress.60

226

To demonstrate the biological relevance of GSH N-terminus adducts, GSH ex-

227

cess experiments (GSH-to-electrophile ratio = 100) have been carried out for the termi-



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nal Michael acceptor 1-hexen-3-one (A2) and the non-terminal structural isomer 4-hex-

229

en-3-one (B2). Besides GSH S and GSH S,N adducts, the respective single GSH N ad-

230

duct was observed with both electrophiles (see Supporting Information, Figure S4 and

231

Tables S4 & S6). This contrasts with the traditional expectation that the thiol group of

232

GSH would always be alkylated at first, supporting the potential biological relevance of

233

GSH N-terminus adducts.

234

Overall, the present findings suggest that Michael addition at GSH goes beyond

235

attacking its thiol group that so far has been in the focus of adductome investigations.

236

Double adducts with a singly substituted N-terminus appear to be possible for a wide

237

range of reactivity down to at least a kGSH of 0.16 L mol–1 min–1 as for ethyl crotonate,53

238

and terminal α,β-unsaturated carbonyls may even form triple adducts with a doubly sub-

239

stituted N-terminus of GSH.

240

HSAB theory vs N-terminus reactivity. According to the original Pearson classi-

241

fication, primary amino groups are hard nucleophiles,62 which would apply also for the

242

N-terminus of GSH. By contrast, more recent work classified amino acid side-chain NH2

243

as soft nucleophile,28,29 supported by quantum chemically calculated molecular parame-

244

ters based on global frontier orbital energies.29 The latter, however, do not necessarily

245

reflect the site-specific hardness or softness,32 which may also be seen by the some-

246

what surprising result that both Cys thiol and Lys amine were allocated almost identical

247

hardness and softness values (calculated for their neutral forms).29 In this context, a fur-

248

ther confounding factor is the prevalence of the protonated form, RNH3+, at physiological

249

pH (pKa: Lys ε-NH2 10.5, Glu α-NH2 9.5, GSH α-NH2 9.6), making the amino-N still har-


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250

der. Nevertheless, the HSAB theory is still considered as a valuable tool to explain toxi-

251

cant-target interactions with respect to various adverse health effects.28,29

252

In any case, the present findings demonstrate that the soft-electrophilic Michael-

253

acceptor β-carbon reacts also (without catalytic support) with the at least semi-hard

254

GSH N-terminus, which supports the recent criticism of the HSAB theory regarding its

255

application to organic chemistry.34,35 In this context, the lack of double GSH N-terminus

256

substitution with β-C-substituted Michael acceptors provides further evidence that the

257

triple (and thus also the double) adducts are confined to β-C-bonded electrophiles and

258

do not involve carbonyl-C linkages, although the latter are considered as harder electro-

259

philic sites.

260

Comparison with cyclic BDA-GSH adducts. Interestingly, the furan metabolite

261

BDA was shown to form N-alkyl-3-S- and -2-S-substituted pyrroles in adduct ratios of 4:1

262

to 10:1 upon reaction with one or more GSH molecules,42 and N-alkyl-3- and -4-pyrrolin-

263

2-ones (2,3-dihydro-2-oxo and 2,5-dihydro-2-oxo pyrroles) as adducts with Nα-acetyl-L-

264

lysine as ε-amino-N nucleophile.42,43

265

From the HSAB viewpoint, these N-heterocycle formations correspond to hard-

266

hard interactions (amino-N attack at carbonyl C). Moreover, the 3-S-substituted pyrrole

267

as prevalent GSH adduct could be interpreted as indicating that the soft-soft interaction

268

(thiol-S attack at Michael-acceptor β-carbon) would be preferred over a soft-hard reac-

269

tion (thiol-S attack at carbonyl-C yielding a 2-S-substituted pyrrole). In contrast to our

270

present findings, however, no GSH-BDA adduct resulting from an N attack at the Mi-

271

chael-acceptor β-carbon has been reported.42-44



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272

Thermodynamics of cyclic BDA-GSH adducts. To elucidate the thermody-

273

namics underlying the observed product patterns, quantum chemical calculations of the

274

enthalpies and free energies of reaction at 298 K have been performed with respective

275

model reactants (methylsulfide and methylamine as S- and N-nucleophiles) in the gas

276

phase and in simulated aqueous solution. Despite the structural simplicity of MeSH and

277

MeNH2 as compared to GSH, it is assumed that adduct formation energies involving

278

their nucleophilic sites (SH and NH2) broadly parallel the ones with respective GSH sites

279

as in previous computational models of thiol peptide reactivity,32,63,33 keeping in mind

280

that at this level of approximation GSH-specific intramolecular interactions are ignored.

281

Because previous computational analyses of aldehydes had been successful

282

without including their hydrated form,33,63 the latter has been omitted from the present

283

study, but may be of interest for a more detailed future investigation. Moreover, the Mi-

284

chael acceptor reactivity of aldehydes is confined to their α,β-unsaturated form that

285

would be regenerated from the respective germinal diol upon reaction consumption.

286

The results for all reactions shown in Scheme 2 are summarized in the top part of

287

Table 1 (normalized to the sum of all reactants to enable direct energy comparisons; the

288

calculated molecular geometries are listed in the Supporting Information).

289 290

SCHEME 2

291

TABLE 1

292 293

First, both S-substituted pyrroles – represented by their methyl analogs as prod-

294

ucts P1 (3-S-substituted pyrrole, soft-soft thiol-Cβ attack) and P2 (2-S-substituted pyr-


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295

role, soft-hard thiol-Ccarbonyl attack) of reactions (a) and (b) in Scheme 2 – are thermody-

296

namically preferred over the not-observed 3-N-substituted counterpart (P7, hard-soft

297

amine-Cβ attack). Second, the increasing prevalence of P1 over P2 analogs with in-

298

creasing reaction time42 – although conforming with the HSAB expectation (see above)

299

– contrasts with opposite trends for the P1 and P2 solution-phase reaction enthalpies

300

∆Hw (–173 vs –176 kJ/mol) and reaction free energies ∆Gw (–163 vs –165 kJ/mol),

301

which is even more pronounced in the gas phase (∆H: –158 vs –167 kJ/mol, ∆G: –148

302

vs –156 kJ/mol). Note further that the alternative epoxide pathway of the P450-catalysed

303

furan oxidation is expected to preferentially yield P2-type pyrroles.44 Accordingly, the

304

experimental time dependence of the regioselectivity suggests a more complex reaction

305

mechanism that may be subject to future investigations.

306

Third, our calculations indicate almost identical gas-phase enthalpies and free

307

energies of formation for the enamine N-methyl-4-pyrrolin-2-one P3 (2,3-dihydro-2-oxo-

308

1H-pyrrole, hard-hard amine-Ccarbonyl attack) and its Michael-acceptor isomer N-methyl-

309

3-pyrrolin-2-one P4 (2,5-dihydro-2-oxo-1H-pyrrole). Table 1 reveals further a much larg-

310

er aqueous solvation free energy for P4 than for P3 (∆Gw : –190.9 vs –171.7 kJ/mol, ∆G:

311

–156.5 vs –157.0 kJ/mol), shedding new light on the often discussed energetic prefer-

312

ence of isomers enabling π-electron conjugation (in this case as α,β-unsaturated car-

313

bonyl).

314

Fourth, formation of P5 (4-S-methyl-pyrrolidin-2-one, a γ-butyrolactam derivative)

315

as soft-soft adduct of P4 through methylthiol attacking the Michael-acceptor β-carbon

316

would be accompanied by a very large enthalpic stabilization (∆H: –233.1 vs –166.3

317

kJ/mol) but an only small free energy gain in aqueous solution (∆Gw: –196.7 vs –190.9



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318

kJ/mol). Interestingly, the possible conversion of P4 to P1 or P2 via P5 as likely interme-

319

diate was not observed for BDA exposed firstly to Nα-acetyl-L-lysine, and then to N-

320

acetyl-L-cysteine.43 The latter could now be explained by the fact that P6 as enol tauto-

321

mer of P5, possibly facilitating (at least under acidic conditions) H2O elimination to yield

322

P1 (or P2), is highly unfavorable regarding gas-phase and solution-phase thermodyna-

323

mics (∆Gw: P6 –88.4 vs P5 –196.7 kJ/mol). Overall, the experimentally observed cyclic

324

GSH-BDA adduct pattern42-44 appears to confirm the HSAB expectation. However, the

325

increase of P1 over P2 regioselectivity with increasing reaction time42 is puzzling in view

326

of the presently calculated thermodynamics, for which we have no explanation except

327

that (besides a possibly insufficient computational accuracy) the underlying reaction me-

328

chanism may be more complex. According to Table 1, the pyrrolidin-2-one P5 is the

329

thermodynamically most stable product and its precursor P4 the second-stable aqueous-

330

phase adduct, making the corresponding GSH-BDA adducts promising candidates as

331

BDA biomarkers.

332 333

SCHEME 3

334 335

Thermodynamics of non-cyclic GSH adducts with Michael acceptors. For the

336

quantum chemical calculation of the thermodynamics underlying the GSH mono, bis and

337

tris adducts with the presently analyzed set of nine α,β-unsaturated carbonyls, GSH has

338

been replaced by the bi-functional nucleophile 2-aminoethanethiol (see Scheme 3; see

339

the Supporting Information for the calculated molecular geometries). The respective gas-


Page 17 of 38


17 340

phase and solution-phase reaction enthalpies (∆H, ∆Hw) and free energies (∆G, ∆Gw)

341

are listed in the bottom part of Table 1 (again normalized to the sum of all reactants).

342

Interestingly, the HSAB-preferred mono S-Cβ-adduct (P8) is thermodynamically

343

less favorable than the corresponding N-Cβ-adduct (P11; ∆Gw: –17.3 vs –25.5 kJ/mol).

344

Moreover, the bis and tris adducts P9 and P10 involving one and two N-Cβ-bonds in ad-

345

dition to the P8 S-Cβ-bond are thermodynamically preferred over P8 (∆Gw: –29.7, –30.3

346

vs –17.3 kJ/mol). By contrast, the free energy of formation of P12 (two N-Cβ-bonds and

347

free SH) is less favorable than the one of P11 with only one N-Cβ-bond (∆Gw: –18.3 vs –

348

25.5 kJ/mol), indicating that (at least for the model nucleophile employed) multiple N-al-

349

kylation is supported by previous S substitution. Finally, N attack at the carbonyl carbon

350

yields Schiff bases P13 and P14 that are thermodynamically inferior to the correspond-

351

ing Michael adducts P8 and P9, despite support of the former through the HSAB theory

352

(hard-hard preferred over hard-soft). The surprisingly stable hard-soft mono adduct P11

353

may serve as additional biomarker for Michael-acceptor electrophiles, and the thermody-

354

namically most stable bis and tris adducts P9 and P10 appear attractive as biomarkers

355

for high-concentration electrophiles.

356

Overall the computational analysis demonstrates that the presently found non-cy-

357

clic bis and tris adducts of GSH with α,β-unsaturated carbonyls are thermodynamically

358

favorable, although the respective N-Cβ-bond formation as hard-soft interaction (with ex-

359

perimental reaction times potentially relevant for pathobiochemical events) does not con-

360

form with HSAB theory.

361

Toxicological impact. The N-terminus functionalization of GSH may impair the

362

GSH-mediated elimination of electrophilic toxicants, and as such could increase the tox-



Page 18 of 38

18

363

icological impact as compared to thiol adducts. This reasoning is based on considering

364

the likely effect of N-terminus substitution on GSH reconstitution, the latter of which

365

counteracts the otherwise occurring GSH depletion upon GSH-electrophile adduct for-

366

mation.

367 368

SCHEME 4

369 370

Scheme 4 outlines an abbreviated version of the γ-glutamyl cycle that takes care

371

of the GSH homeostasis.64 GSH catabolism is initiated through the extracellular γ-gluta-

372

myl transferase (GGT) that splits off the Glu unit from the remaining dipeptide Cys-Gly

373

(Gly = glycine). Subsequently, re-import into the cell enables the dipeptide cleavage into

374

separate Cys and Gly (catalyzed through some dipeptidase) as well as the liberation of

375

Glu through γ-glutamyl cyclotransferase followed by 5-oxoprolinase, eventually reconsti-

376

tuting GSH in two further steps catalyzed by glutamate cysteine ligase (forming the di-

377

peptide γ-Glu-Cys) and glutathione synthetase (final covalent addition of Gly), respect-

378

ively.

379 380

SCHEME 5

381 382

In case of a GS-electrophile adduct (GSH thiol linkage), breakdown to Glu, Gly

383

and Cys-electrophile is still efficient, eventually eliminating the latter through the mercap-

384

turic acid pathway (Scheme 5, left to middle). If, however, the electrophile binds at the

385

GSH N-terminus, the resultant adduct is no good GGT substrate (because GGT does


Page 19 of 38


19

386

not tolerate N-terminus substitution),65 thus impairing the cleavage of N-modified γ-Glu

387

from Cys-Gly as first catalytic step toward reconstitution of GSH (Scheme 5, right to

388

middle).

389

Because of the γ-peptide bond linking Glu and Cys, GSH is resistant to normal

390

proteases, and thus requires specialized enzymes such as GGT for liberating Glu. In

391

principle, however, proteases could cleave Gly from N-terminus adducts of GSH, possi-

392

bly followed by N-acetylation at Cys of the remaining electrophile-Glu-Cys dipeptide to

393

initiate the mercapturic acid pathway. Whether this or another degradation mechanism

394

might operate is currently not known.

395

The unavailability of the GGT-mediated transpeptidase pathway for N-terminus-

396

substituted GSH, however, suggests an increased persistence of N-bonded single, dou-

397

ble and triple adducts as compared to simple thiol adducts, possibly reinforced through

398

an increase in hydrophobicity associated with the additional non-peptide side chains.

399

The latter is supported by our chromatographic results employing a C18 column with

400

hydrophobic selectivity, which shows considerably increased retention times of the

401

formed double and triple adducts as compared to the simple GSH-electrophile adducts.

402

Since N-functionalized γ-glutamyl groups cannot be used for the recovery of GSH

403

during GSH homeostasis, N-terminus adducts may be significantly more resistant to de-

404

toxification through the mercapturic acid pathway. Moreover, non-detoxified GSH ad-

405

ducts may undergo retro-Michael addition and re-activate the electrophile for further co-

406

valent attacks. In addition, alkylation of the γ-glutamyl (Glu) unit of GSH may impair its

407

role for binding GSH to S transferases,66 and oxidized glutathione (GSSG) to reductas-



Page 20 of 38

20

408

es, respectively,67 promoting a GSH-to-GSSG ratio imbalance that in turn could increase

409

the susceptibility for oxidative stress.

410

Regarding electrophile-induced protein damage, Michael addition at non-thiol

411

sites such as Lys ε-NH2 or heterocyclic N of histidine or tryptophan would also increase

412

the hydrophobicity and correspondingly decrease the water solubility. Possible conse-

413

quences include an increased tendency for abnormal aggregation as one cause of pro-

414

tein dysfunction and associated pathophysiology, as is well known for the sickle-cell dis-

415

ease with a genetically caused exchange of Glu by the more hydrophobic valine at ami-

416

no acid position 6 of β-hemoglobin.68

417

Although both the GSH excess and the electrophile excess setup demonstrated

418

adduct formation without involving the thiol group, the in vivo relevance of single and

419

multiple N-adducts of GSH remains to be verified. To this end, the present results could

420

provide guidance for future adductome analyses of samples from human blood or urine,

421

eventually confirming non-HSAB in vivo attacks of electrophiles at GSH and proteins

422

that may shed light on their potential role as distinct molecular initiating events of down-

423

stream toxicological events. In this context, the recently introduced concept of

424

chemoavailability59,69 as trade-off between electrophilic reactivity and hydrophobicity

425

may contribute to a mechanistic understanding and predictive assessment of different

426

modes of reactive toxicity.

427 428 429


Page 21 of 38


21

430

SUPPORTING INFORMATION

431

Specification of the chemicals and reagents with purities and providers; a scheme show-

432

ing the general structures of GSH adducts formed by the reaction with α,β-unsaturated

433

carbonyls; three figures showing product ion chromatograms with the single, double and

434

triple adduct of GSH with 1-hexen-3-one (A2) and associated fragmentation patterns; six

435

figures showing extracted (normalized and overlapped) ion chromatograms of the GSH

436

adducts formed with electrophiles A1, A3, B1-B3, and C1-C3 (for A2 see Figure 1); three

437

fragment allocation tables for the most abundant peaks (Tables S1-S3) of the single,

438

double and triple adducts of GSH with 1-hexen-3-one; calculated molecular geometries

439

for the model products P1-P14 and their reactants (Schemes 2 and 3).

440 441

FUNDING SOURCES

442

Financial support through the BMBF-funded project ProHapTox (Development of a Re-

443

activity-Based Non-Animal Testing Strategy for Identifying the Skin Sensitization Poten-

444

tial of Electrophilic and Pro-Electrophilic Chemicals within the Framework of REACH,

445

FKZ 031A422A and 031A422B) is gratefully acknowledged.



Page 22 of 38

22

446

TABLE

447 448

Table 1. Calculated enthalpies and Gibbs free energies of reactions (a)-(g) of Schemes

449

2 and 3 with major products P1-P14 at 298 K in the gas phase (∆H, ∆G) and in simulat-

450

ed aqueous solution (∆Hw, ∆Gw) employing the quantum chemical density functional

451

M06-2X with basis set def2-tzvpp46-48 and the COSMO CPCM model49-51 with UAKS ra-

452

dii.52 Reaction and major product

∆H

∆G

∆Hw

∆Gw

(kJ/mol)

Cyclic adducts of 2-butene-1,4-dial with methylsulfide and methylamine (Scheme 2) (a), P1

–158.0

–148.2

–172.7

–163.0

(b), P2

–167.0

–156.1

–175.8

–165.0

(c), P3

–163.1

–157.0

–177.8

–171.7

(d), P4

–166.3

–156.5

–200.7

–190.9

(e), P5

–233.1

–176.3

–253.5

–196.7

(f), P6

–111.3

–53.2

–146.5

–88.4

(g), P7

–155.0

–142.8

–169.5

–157.3

Non-cyclic adducts of 2-aminoethanethiol with acrolein (Scheme 3) (a), P8

–64.2

–9.0

–72.5

–17.3

(b), P9

–127.4

–16.4

–140.6

–29.7

(c), P10

–191.1

–27.3

–194.1

–30.3

(d), P11

–65.2

–16.1

–74.6

–25.5

(e), P12

–123.0

–10.9

–130.4

–18.3

(f), P13

–18.3

–6.0

–24.2

–11.9

(g), P14

–77.2

–11.6

–89.8

–24.2

453


Page 23 of 38


23

454

FIGURE

455

456 457

Figure 1. Extracted and overlapped ion chromatograms of three GSH adducts formed

458

by the reaction between GSH (with the α-amino group of its γ-glutamyl moiety shown

459

explicitly) and 1-hexen-3-one (A2). The data were obtained after 24 h with an electro-

460

phile-to-GSH ratio of 100. Peak 1 (black) = single adduct, m/z 406, Peak 2 (red) = dou-

461

ble adduct, m/z 504, Peak 3 (green) triple adduct, m/z 602, E = electrophile A2 (the ge-

462

neral structure of the three GSH adducts is shown in Scheme S1 of the Supporting Infor-

463

mation).

464



Page 24 of 38

24

465

SCHEMES

466 1

2

3

O

O

O

O

O

O

O

O

O

A

B

C

O

O

467 468 469

Scheme 1. Chemical structures of the tested α,β-unsaturated carbonyls. A1 = 1- pent-

470

en-3-one, A2 = 1-hexen-3-one, A3 = 1-octen-3-one, B1 = 3-penten-2-one, B2 = 4-hex-

471

en- 3-one, B3 = 2-octen-4-one, C1 = 2-ethyl-acrolein, C2 = ethyl acrylate, C3 = ethyl cro-

472

tonate.

473 474


Page 25 of 38


25

O

O + H3C SH + 2 H3C NH2

S CH3

(a)

N CH3

+ H3C NH2 + 2 H2O

(b) S CH3

+ H3C NH2 + H3C SH

N

(d) O

CH3

N CH3

P3

P4

S CH3

(e) O

+ H3C NH2 + 2 H2O

P2

P1

(c) O

N CH3

N CH3

+ H3C NH2 + H2O

+ H3C NH2 + H3C SH

S CH3

(f) HO

N CH3

+ H3C NH2 + H2O

P6

P5 H N CH3

(g)

475 476

N CH3

+ H3C SH + 2 H2O

P7

477

Scheme 2. Possible cyclic adducts of the furan metabolite 2-butene-1,4-dial (BDA) with

478

methylsulfide and methylamine as surrogates of amino acid S- and N-nucleophiles; the

479

associated calculated enthalpies and free energies of reaction are summarized in Table

480

1. Reactions (a)-(d) with products P1-P4 mimic pyrrole and pyrrolin-2-one adducts with

481

Nα-acetyl-L-lysine, N-acetyl-L-cysteine and GSH.42-44 P5 is a hypothetical Michael thiol

482

adduct with P4, P6 its tautomer, and P7 a hypothetical bis-N-adduct (see text).

483



Page 26 of 38

26 SH

O

+ 3

H2N

O

O O O

S

(a)

O

S

(b)

+ 2

+ N H

H2N

P8

P9 O

O

O S

SH

(c)

(d) N O

O

+ 2 N H

P10

P11

O O

SH

SH

+

(e) N O

+ 2

(f)

O

+

H2O

N

P12

P13 O

O

S

+

(g)

+

H2O

N

484 485

P14

486

Scheme 3. Possible non-cyclic adducts of the GSH surrogate 2-aminoethanethiol with

487

the Michael acceptor acrolein. P8 and P11 represent soft-soft and hard-soft mono ad-

488

ducts, P9 and P10 mimic GSH bis- and tris-adducts featuring hard-soft and soft-soft

489

bonds, P12 is a bis-N-adduct (hard-soft) with a free thiol function, and P13 and P14 are

490

Schiff bases (resulting from hard-hard attack) without and with additional S-substitution.

491

Besides P8 as well known HSAB-compliant GSH-electrophile product type, the following

492

non-HSAB product types have been presently observed with GSH for the first time: P9

493

for all nine electrophiles shown in Scheme 1, P10 for the five reagents A1-A3 and C1-

494

C2, and P11 for A2 and B2.


Page 27 of 38


27

495 496

497 498 499 500

Scheme 4. GSH homeostasis through the γ-glutamyl cycle in simplified form. AA = ami-

501

no acid, GGT = γ-glutamyl transferase (γ-glutamyl transpeptidase), DPEP = dipeptidase,

502

GGCT = γ-glutamyl cyclotransferase, 5-OP = 5-oxoproline, 5-OPase = 5-oxoprolinase,

503

GCL = glutamate cysteine ligase (γ-glutamylcysteine synthetase), GSS = glutathione

504

synthetase.

505



Page 28 of 38

28

506

1 — . GG G T lu (? -N ) H -E

2 — . DP G EP ly

507

508 509 510 511

Scheme 5. GSH pathobiochemistry upon covalent attack by an electrophile E. To speci-

512

fy the different adduct types, the α-amino group of the γ-glutamyl moiety of GSH is indi-

513

cated explicitly (H2N-GSH). Besides simple thiol adduct formation (H2N-GS-E), subse-

514

quent reaction of E with α-NH2 can lead to double and triple adducts (E-NH-GS-E, E-N(-

515

E)-GS-E), impairing the GGT-mediated cleavage of γ-Glu-E or γ-Glu(E)2, and thus also

516

the eventual reconstitution of GSH. GGT = γ-glutamyl transferase (γ-glutamyl transpepti-

517

dase), DPEP= dipeptidase, NAT= N-acetyltransferase, N-Ac-Cys = N-acetylcysteine.

518 519


Page 29 of 38


29

520

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