examination of michael addition reactivity towards glutathione by transition-state calculations

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Examination of Michael additionreactivity towards glutathione bytransition-state calculationsJ.A.H. Schwöbel a , J.C. Madden a & M.T.D. Cronin aa School of Pharmacy and Chemistry, Liverpool John MooresUniversity, Liverpool, UKVersion of record first published: 29 Nov 2010.

To cite this article: J.A.H. Schwöbel , J.C. Madden & M.T.D. Cronin (2010): Examination ofMichael addition reactivity towards glutathione by transition-state calculations, SAR and QSAR inEnvironmental Research, 21:7-8, 693-710

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SAR and QSAR in Environmental ResearchVol. 21, Nos. 7–8, October–December 2010, 693–710

Examination of Michael addition reactivity towards glutathione by

transition-state calculationsy

J.A.H. Schwobel, J.C. Madden and M.T.D. Cronin*

School of Pharmacy and Chemistry, Liverpool John Moores University, Liverpool, UK

(Received 25 May 2010; in final form 1 August 2010)

Kinetic rate constants (kGSH) for the reaction of compounds acting as Michaelacceptors with glutathione (GSH) were modelled by quantum chemicaltransition-state calculations at the B3LYP/6-31G** and B3LYP/TZVP level.The data set included �,�-unsaturated aldehydes, ketones and esters, with doublebonds and triple bonds, linear and cyclic systems, both with and withoutsubstituents in the �-position. Predicted values for kGSH were found to be in goodagreement with experimental kGSH values. Factors affecting rate constants havebeen elucidated, especially solvent effects and the influence of steric hindrance.Solvent effects were examined by adding explicit solvent molecules to the systemand by using a polarizable continuum solvent model. Detailed analysis oftransition-state energies shows that the reaction is reversible. The reactive enolicintermediate plays an important role in Michael addition to GSH, while thesubsequent keto-enol-tautomerism is not rate limiting.

Keywords: Michael addition; glutathione; transition state; kinetics; peptideadduct formation; steric accessibility; solvent effect; quantum chemistry

1. Introduction

Knowledge of electrophilic reactivity is important to understand and describe interactionsbetween xenobiotic toxicants and biochemical macromolecules [1]. It has long beenunderstood that compounds capable of covalent interactions with biological macromol-ecules are liable to elicit toxic effects [2]. For instance, there have been recentimprovements in our understanding of the reactions involved between xenobiotics andimmunoproteins that result in allergic contact dermatitis (skin sensitization) [3,4] as well asfor the irreversible reaction with fish gill membranes [5] which will promote acute toxicityin excess of narcosis. Many of these mechanisms of action are electrophilic in nature.There are many further examples of electrophilic toxicity, e.g. respiratory sensitization,liver toxicity, skin irritation, etc. [6,7].

Compounds with an �,�-unsaturated carbonyl or carboxyl fragment are veryimportant industrial substances, for example in the manufacture of polymers, textiles,or auxiliary materials in medicine [8,9]. However, �,�-unsaturated compounds acting viaMichael-type reactions are considered to be particularly reactive [10]. They are capable offorming irreversible bonds with biological macromolecules, such as proteins or DNA

*Corresponding author. Email: [email protected] at the 14th International Workshop on Quantitative Structure–Activity Relationships inEnvironmental and Health Sciences (QSAR2010), 24–28 May 2010, Montreal, Canada.

ISSN 1062–936X print/ISSN 1029–046X online

� 2010 Taylor & Francis

DOI: 10.1080/1062936X.2010.528943

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molecules [11]. The types of chemical fragments associated with this mechanism are usedto define the Michael acceptor domain. Michael acceptors are characterized as havingcarbon-carbon double (C¼C) or triple (C�C) bonds with electron withdrawing substit-uents (see Figure 1). This results in a polarizable electron density at the �-bond, where the�-carbon atom is positively polarized and becomes the preferred site of an attack with softnucleophiles, for example glutathione (GSH) [10].

The tripeptide glutathione (GSH, L-�-glutamyl-L-cysteinyl-glycine) is one of the mostwidely used nucleophilic reference molecules in reactivity assays; direct GSH depletion canbe measured by a pure chemical reactivity (in chemico) assay [12]. GSH is the mostprevalent cellular thiol and the most abundant low molecular weight peptide in cells [13].It is reactive towards soft electrophiles, for example polarized �,�-unsaturated com-pounds, which act predominantly as Michael-type acceptors. Soft electrophiles arereadily polarizable and have a low electronegativity [14]. GSH protects cells bydetoxifying electrophilic compounds and acts as an antioxidant. The concentration ofGSH is depleted during attack by electrophilic compounds, commonly by alkylationmechanisms [15].

Various studies have shown good qualitative relationships between GSH reactivity ofchemicals acting as Michael-type acceptors and toxicological endpoints, e.g. acute toxicityto Tetrahymena pyriformis [16]. Therefore, chemical reactivity (in chemico) assays have thepotential to provide information to assist in the grouping and identification of toxicants.However, there is current interest to move away from reliance on experimentation to usecomputational (in silico) techniques to predict potential chemical reactivity [17]. This isan attractive approach, especially where experimental measurements are not possible orrestricted, for example because of low solubility or high volatility, and if required forprioritization of chemical inventories. Their use is foreseen in integrated testing strategies(ITS) [17], for instance to comply with the needs of the EU REACH legislation [18]. In thiscontext, a better understanding of the underlying reaction mechanisms could provideuseful information.

Recently, a quantitative structure–activity relationship (QSAR) model has beendeveloped for Michael addition reactivity based on the ground-state properties of a singlemolecule [19]. As experimental reference values, kinetic rate constants kGSH for thereaction of GSH with �,�-unsaturated xenobiotics in aqueous phosphate buffer solutionwere measured [16,20–22].

Figure 1. Michael addition domain: �, �-unsaturated carbonyl or carboxyl groups with double ortriple bonds, including cyclic systems (in addition, electron-withdrawing groups for ‘Michael-type’electrophiles might include pyridino heterocyclic groups and, although not considered in this paper,–CN, –SO2R, –NO2) [4].

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In order to resolve the issue of calculating reactivity, transition-state calculations canalso be applied. A transition state is defined as the configuration with the highest energyalong the reaction coordinate of a particular reaction. The general background behindtransition-state theory can be found in physical chemistry textbooks and a selection ofreview papers [23–26]. Several aspects of transition-state calculations for �,�-unsaturatedcompounds have been known for more than two decades. For instance, Osman et al.explained the reactivities of acrylic acid and methacrylic acid by using fluoride as areference nucleophile [27]. In addition, from a synthesis point of view, the reactionpathway including catalytic systems is an important issue, e.g. lithium organocuprate[28,29], zirconium oxychloride [30], and binaphthols [31]. Recently, Um et al. analysed theaddition of �,�-unsaturated compounds to amines [32,33]. The authors suggested threepotential transition-state structures, equivalent to Figure 2. However, such calculationshave not been applied to a large dataset of GSH reactivity values using high-levelcalculation methods.

The scope of this paper was to examine the underlying biochemical reaction pathwayof Michael addition, using base catalysed GSH reactivity data, in a systematic manner.The approach in this study was to calculate transition-state energies and the influence offactors that may affect rate constants. It has also investigated the effect of the stericaccessibility of the reaction sites and solvent effects on the transition-state structure andenergies.

2. Material and methods

2.1 Data set

The 22 compounds selected for transition-state calculations are shown in Table 1. Thisdata set is a subset of a recently developed QSAR model for Michael addition reactivity[19], and should represent the variety of �,�-unsaturated compounds acting as Michaelacceptors with a double bond or a triple bond, cyclic systems and compounds with orwithout substituents at the �-carbon. The compound classes include aldehydes, ketonesand esters.

2.2 GSH reactivity data

Experimental kinetic rate constants for the reaction between �,�-unsaturated compoundsand GSH were taken from a previous publication [19], covering four orders of magnitude(log kGSH¼�0.68��3.10 (in M�1min�1)). The standard GSH assay to measure rateconstants for electrophilic Michael acceptor-type compounds was performed in aqueous

Figure 2. Suggested transition-state structures for Michael addition: 1 simultaneous mechanism; 2stepwise mechanism (2a S–C� bond formation; 2b H–C� bond formation).

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phosphate buffer solution at T¼ 25�C and pH¼ 7.4 [16]. After a determined reaction time,the concentration of free thiol groups was measured. Free thiol groups were quantified byUV/VIS spectroscopy at 412 nm after reaction with the chromophore 5,50-dithiobis-(2-nitrobenzoic acid) (DTNB). For some compounds in the data set, slightly differentexperimental conditions were used; those values were corrected to be concordant with thestandard assay, as described by Schwobel et al. [19].

2.3 Computational details

The ground-state geometries of all molecules and the transition-state geometries of theirrespective thiol adducts were optimized at the density functional theory B3LYP/6-31G**and B3LYP/TZVP level employing Gaussian 03 [34]. Methylthiol and cysteine were usedas reference thiols. Products and reactive intermediates were optimized at the B3LYP/6-31G** level. Frequency analysis was carried out for all systems to confirm that correctground-state and transition-state geometries had been obtained. The continuum solventmodel C-PCM was applied, using Pauling radii [35]. Parameters for steric accessibilitywere calculated using the freely available MSMS program [36], using published Connolly

Table 1. Experimental rate constants (kGSH in M�1min�1; 25�C; pH 7.4) for adductformation with GSH.

# Name CAS log kGSHa

1 3-Hexyn-2-one 1679-36-3 1.902 1-Penten-3-one 1629-58-9 3.103 3-Penten-2-one 625-33-2 1.434 1-Hexen-3-one 1629-60-3 3.075 Ethyl acrylate 140-88-5 1.036 4-Hexen-3-one 2497-21-4 1.387 1-Octen-3-one 4312-99-6 3.038 3-Octen-2-one 1669-44-9 1.069 Acrolein 107-02-8 2.12b

10 (E)-Crotonaldehyde 123-73-9 1.68b

11 trans-2-Pentenal 1576-87-0 1.43b

12 trans-2-Hexenal 6728-26-3 1.21b

13 trans-2-Octenal 2548-87-0 1.12b

14 Methyl vinyl ketone 78-94-4 2.14c

15 2-Cyclohexen-1-one 930-68-7 1.24c

16 4-Methyl-2-pentenal 5362-56-1 0.74c

17 2,4-Hexadienal 142-83-6 0.72c

18 2,4-Heptadienal 5910-85-0 0.54c

19 Cinnamic aldehyde 104-55-2 0.78c

20 Mesityl oxide 141-79-7 �0.6821 3-Methyl-3-penten-2-one 565-62-8 �0.1122 2-Hydroxypropyl methacrylate 923-26-2 �0.03b

aValues taken from Schwobel et al. [19]. blog kGSH recalculated to pH¼ 7.4 (standardcondition), as reported by Schwobel et al. [19]. crate constant log kGSH based on RC50

value, as reported by Schwobel et al. [19]. Therein adopted rate constants did not havea significant impact on the model outcome (compared to measured kGSH values atpH¼ 7.4), as long as the compounds were fully soluble in the RC50 assay (which is thecase here).


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radii with explicit hydrogen atoms [37]. The solvent-accessible surface area (SAS) uses asolvent sphere of a particular radius to probe the surface of the molecule (the surface radiirsur for explicit hydrogens and aqueous systems are: 1.74 A [C]; 1.20 A [H]; 1.54 A [N];1.40 A [O]; 1.80 A [S]). The percentage of the accessible atomic surface area in theoptimized molecular structure, compared with the free atom, was obtained.

2.4 Statistical analysis

Multiple linear regression analysis was performed using the Minitab (version 15.1)statistical software to fit the coefficients of Equation (17) in Section 3.7 to experimentalkGSH values. The method uses the least squares method deriving the equation byminimizing the sum of the squared residuals. The statistical parameters are Pearson’ssquared correlation coefficient (r2), including correction for the degrees of freedom ðr2adjÞ,for a leave-one-out cross-validation r2CV

� �, the standard error (se), the F-test value (F ), and

the p value, which evaluates the statistical significance of the association between theresponse and the descriptors.

3. Results and discussion

The importance of several factors affecting the kinetic rate constant kGSH was examined bytransition-state calculations, using methylthiol and cysteine as reference molecules insteadof GSH.

3.1 Methylthiol reference system

The ground-state geometries of methylthiol and 22 Michael acceptor reactant molecules,together with their respective products, were initially optimized. Ground states wereconfirmed by obtaining real frequencies only. The transition-state geometry was searchedfor by placing the methylthiol sulphur atom close to the �-carbon atom of the Michaelacceptor molecule (initial distance rS � � �C¼ 2.35 A) and a proton close to the �-carbonatom (rH � � �C¼ 1.48 A) with the sulphur–hydrogen distance being rS � � �H¼ 1.65 A. Theactual reaction takes place in water, where the reactive nucleophile is presumed to be thethiolate ion. Therefore, it is assumed that the proton does not originate from the thiolsystem (the rS–H distance is usually 1.31 A), but from surrounding water molecules of theaqueous buffer solution. The influence of solvation will discussed later on. All transition-state structures were validated by having one imaginary frequency only. Correspondingto Um et al. (who applied the considerations for amine adducts), two different reactionpathways are suggested [32], shown in Figure 2: a concerted transition state, where bothS–C� and H–C� bond formation takes place at the same time (1), or a two-step process,with sulphur-�-carbon bond formation (2a) followed by the addition of a proton (2b).Attempts to find a negatively charged transition state of the two-step process failed, so theresults indicated simultaneous sulphur–carbon bond formation and hydrogen addition.An example of the transition-state structure is shown in Figure 3.

In order to determine the quantum chemical level needed for calculations, transition-state energies from a standard Pople basis set, 6-31G**, were compared with an advancedAhlrichs basis set, TZVP. Geometry optimizations of ground and transition states wereperformed for both basis sets. In theory, the advanced TZVP basis set should lead to


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slightly better results; however, the computational effort for TZVP is significantly greaterthan for 6-31G**. Transition-state energies were calculated by the difference, DGz, of theadduct transition-state Gz and all ground-state G0 Gibbs energies:

DGzðMeSHÞ ¼ ½GzðMeSH adductÞ� � ½G0ðMeSHÞ þ G0

ðRC¼CHXÞ�: ð1Þ

Note that the desired kinetic rate constant k can be related to the transition-state

energy DGz by the Eyring equation:

k ¼ Q �kBT

h

� �� exp �

DGz

RT

� �: ð2Þ

Here, h is the Plank constant, kB the Boltzmann constant, R the molar gas constant,

and T the temperature. Q is the partition function, derived from statistical mechanics,which will not be calculated explicitly throughout this publication, but in principle could

be fitted by experimentally known rate constants. Cespedes-Camacho et al. have used anapproach similar to this in order to calculate the alkylating potential of acrylamide [38].

The comparison of the transition-state energies is shown in Figure 4. In general, TZVPenergies are slightly higher than 6-31G** energies. Nevertheless, the general reactivity

trend remains the same for all molecules and they are strongly correlated (r2¼ 0.91). Thisindicated that neither the principal trends nor the geometrical structures are essentially

different, and that any error introduced by taking a lower level basis set can be accountedfor by a simple linear correction. Therefore, 6-31G** was applied in this investigation as a

good compromise between accuracy and computational effort, and the linear correctionmentioned above is (intrinsically) included in the coefficients of Equation (17).

It should be noted that this simple linear correction, based on 22 molecules, cannot begeneralized. TZVP calculations are preferred over 6-31G**, if more accurate results are

required, especially as energies are plugged into an exponential function and thepropagation of the error could lead to incorrect conclusions. Nevertheless, the lower basis

set level allows a toxicologically relevant classification into low, moderate and highlyreactive compounds, respectively.

Figure 3. Solvent-mediated (water; C-PCM) transition-state structure of crotonaldehyde withmethylthiol.


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3.2 Cysteine reference system

The purpose of this investigation was to model GSH reactivity. In the previous section,

the reference molecule methylthiol was chosen as the reactive part of the GSH molecule.Within the tripeptide GSH, the amino acid cysteine is responsible for toxicant–peptide

binding. Taking the whole GSH molecule as a reference system would not be practical orwarranted, because of the extraordinary increase in computational demand without any

expected increase in accuracy, compared with the cysteine system. The validity of the useof the simple methylthiol molecule has been checked by comparing transition-state

energies of the methylthiol systems to the more laborious cysteine molecule:

DGzðCysÞ ¼ ½GzðCysadductÞ� � ½G0ðCysÞ þ G0ðRC¼CHXÞ�: ð3Þ

In principle, kinetic rate constants could be calculated for both systems by

Equation (2). The comparison is shown in Figure 5, which shows a clear linear trendbetween both reference systems (r2¼ 0.91). Based on this outcome, a similar linear

correlation is expected between transition-state energies of methylthiol and of GSH (whichwill be included in the coefficients of Equation (17)). It is therefore justified to proceed

with methylthiol as a reference system, instead of the time-consuming cysteine molecule.Moreover, as observed for GSH, no potential intermolecular hydrogen bonding interferes

with the toxicant–methylthiol interaction, in contrast to cysteine where intermolecularhydrogen bonds formed by the remaining functional groups may have an effect on

reactivity [20,39].

3.3 Kinetics of the forward and backward reaction

The reaction between soft electrophiles and GSH is considered to be reversible, in contrast,

for example, to the reaction with hard lysine residues [20,40]. For this reason, thecalculation of the rate constant for the backward reaction k�1 is as important as for the

Figure 4. Comparison of transition-state free energies DGz (methylthiol adduct formation to�,�-unsaturated compounds) for the quantum chemical levels B3LYP/TZVP and B3LYP/6-31G**.


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forward reaction k1. The pseudo-first-order reaction rate constant k1st can be calculated

as follows, with KGSHa being the acid dissociation constant of GSH [41]:

k1st ¼ cðGS�Þ½ � � k1 � k�1 ¼KGSH

a

KGSHa þ cðHþÞ

� c0ðGSHÞ

� � k1 � k�1: ð4Þ

A large excess of GSH was used in the experiments, so the thiolate concentration

c(GS�) remains constant throughout the reaction time and is proportional to the initial

GSH concentration c0(GSH), the factor being a1. Therefore, the factor in parenthesis in

the first term of Equation (4) can be considered to be constant:

k1st ¼ a1 � c0ðGSHÞ½ � � k1 � k�1: ð5Þ

Second-order rate constants, kGSH, given in Table 1, are calculated by dividing the

first-order rate constants k1st by an initial GSH concentration. The actual initial GSH

concentration is appended into a new constant a�1, assuming that all experimental rate

constants were taken from one (hypothetical) standard assay:

kGSH ¼k1st

c0ðGSHÞ¼ a1 � k1 � a�1 � k�1: ð6Þ

The rate constants of the forward k1 and the backward reaction k�1 could be, in

principle, calculated by Equation (2), with transition-state energies DGz and DGzback,respectively. In this context, we prefer to relate the forward reaction rate constant k1 to the

energy of activation DEzGSH, based on the collision theory:

k1 ¼ Z1 � � � exp �DEzGSH

RT

( )ð7Þ

which is dependent on the number of molecular events per time Z1 (constant for a specific

reaction and temperature) and dependent on the steric accessibility � of a reaction site.

Figure 5. Transition-state free energies DG6¼ of methylthiol adducts and cysteine adducts with�,�-unsaturated compounds (B3LYP/6-31G**).


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The advantage of collision theory over the Eyring model (Equation (2)) will be explainedin a following section.

As stated above, methylthiol transition-state energies DEz are linearly related to GSHtransition-state energies DEzGSH (provided by a conversion factor b1 and a constant c1).In its logarithmic form, Equation (7) can now be written:

log k1 ¼ logZ1 þ log � �b1 � DEz þ c1� �

ln 10ð Þ � RT: ð8Þ

The backward reaction is calculated by the Arrhenius equation, which relates rateconstants k�1 to the activation energy DEzback, of the backward reaction:

k�1 ¼ Z�1 � exp �b�1 � DE

z

back þ c�1

�RT

8<:

9=; ð9Þ

or in its logarithmic form

log k�1 ¼ logZ�1 �b�1 � DE

z

back þ c�1

�ln 10ð Þ � RT

: ð10Þ

Z�1 is the pre-exponential factor, which is dependent on the number of molecularevents per time (again, constant for a specific reaction and temperature); the constants b�1and c�1 convert calculated transition-state energies DEzback of the methylthiol systembackward reaction into the GSH system.

3.4 Enol form as reactive intermediate

The enol forms of the respective adduct of the Michael acceptor molecule and methylthiolwere calculated for all compounds. The enolic form, shown in Figure 6, is the most likelyreactive intermediate. As will be shown, it is very likely that the backward reaction takesplace from a reactive intermediate, not the final product.

It is important to note that the hydrogen atom creating the enol form at the carbonyloxygen is not equal to the proton at the �-carbon atom. It is highly probable that bothhydrogens originate from different water molecules under physiological conditions.

Figure 6. Michael-type addition. Reaction mechanism of (a) an �,�-unsaturated compound in thereaction with glutathione (GSH) in aqueous phosphate buffer solution (PBS) by formation of (b) anenolic intermediate and (c) the final peptide adduct complex.


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The combination of the backward reaction from the enol form and the forward reaction

explains 90% of the variation in overall kGSH reactivity (see Table 2; model I), whereas the

backward reaction from the final product only explains 53% of the variation (model IV).

Therefore, the enol form is very likely to be a reactive intermediate in the experiments.

Table 2. Coefficients for the Michael acceptor model (Equation (17); all energies in kJ/mol), fittedwith 22 experimental rate constants (log kexp) of Table 1; statistical parameters are squaredcorrelation coefficients with (r2) and without adjustment to the degrees of freedom ðr2adjÞ, andstandard errors (se).

Model A B C D r2 r2adj se

Continuum solvent model (C-PCM; water)½DEzPCM� [log�SAS] ½DEzPCM,back� [const]

I. �0.0290 þ1.42 �0.0307 �2.14 0.90 0.88 0.34II. �0.0366 �0.0362 þ1.40 0.65 0.61 0.60III. �0.0134 þ1.66 þ0.00 0.51 0.46 0.81IV. �0.00619 þ1.89 �0.00082 a

�4.08 0.53 0.46 0.73

Two explicit water solvent molecules (see Figure 7)b

[DGz] [log�SAS] ½DGzback� [const]V. �0.0238 þ1.44 �0.0264 �0.98 0.87 0.84 0.38VI. �0.0310 �0.0317 þ3.04 0.61 0.56 0.63VII. �0.0063 þ1.83 �4.29 0.53 0.49 0.70

No solvent moleculesb

[DGz] [log�SAS] ½DGzback� [const]VIII. �0.0327 þ1.35 �0.0285 þ0.88 0.84 0.82 0.41

aBackward reaction from final product, not from enolic intermediate. bUsing DGz and DGzback,instead of DEzPCM and DEzPCM,back in Equation (17), respectively.

Figure 7. Transition-state structure of crotonaldehyde with methylthiol, stabilised by two explicitwater molecules. The first (lower) water molecule at the carbonyl oxygen is relatively fixed; thesecond water molecule can easily switch its position between the neighbouring group of the � carbon,the methyl group of the methylthiol molecule, and the first water molecule without significantchange in the total energy of the system.


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Furthermore, considering the forward reaction alone explains only 51% of the variation(model III). It should be noted that this proportion of variance explained is similar to thelevel of 53% mentioned above, endorsing the hypothesis that a backward reaction fromthe final product is very unlikely and rationalizes the relative stability of GSH adducts,while the backward reaction from the reactive enol form plays an important role in overallreactivity.

In this context, the question arises, how important is the subsequent keto-enol-tautomerism of the reaction? It is expected that keto-enol-tautomerism is fast and not ratelimiting. This is because the reaction energy of keto-enol-tautomerism is not statisticallysignificant, and addition of the reaction energy to Equation (17) does not improve theresults. This holds for both continuum solvent models and up to two explicit solventmolecules in the system where transition states are stabilized by a surrounding solvent, aswill be explained below.

3.5 Influence of steric hindrance on the forward reaction

It can be observed that reactivity is partially dependent on the steric accessibility of theparticular reaction site. Returning to collision theory again, Equation (8) was dependenton the steric accessibility � of a reaction site R: the greater the accessibility, the higher thereaction rate. As a good approximation, steric accessibility can be calculated in terms ofthe atomic solvent-accessible surface area (SAS), with a linear factor s:

�GSH ¼ s � SASðRÞ: ð11Þ

Ideally, there is no intercept for the conversion of SAS into � because ‘0% solvent-accessible surface area’ in fact implies ‘not accessible’. The �-carbon atom has beenselected as reaction site R. In addition, as steric requirements could be slightly different forGSH and methylthiol, additional linear correction factors d1, d2, and e1 (¼ d2þ log s) havebeen introduced to convert log(�GSH) into log(�MeSH).

log �MeSH ¼ d1 log s � SASðRÞð Þ þ d2 ¼ d1 logSASðRÞ þ e1: ð12Þ

Steric hindrance is shown to play a very important role to explain the reactivity of theforward reaction fully: without this term, only 65% of the variation of the overall reactionrate kGSH could be explained, as shown in Table 2 (model II). In other words, at least 25%of the overall reaction rate constant is influenced by steric hindrance. In a recentpublication, steric accessibility was added as a term into a GSH reactivity model, basedon ground-state properties of �,�-unsaturated molecules [19]. This outcome confirms thevalidity of using some kind of steric factor.

3.6 Solvent effects

Transition states can be stabilized by a solvent: for example, the transition-state energy DEz

for acrolein is in vacuo 196 kJmol�1, but in water only DEzPCM¼ 162 kJmol�1 (calculated bythe continuum solvation model, described below). Analogously for the backward reaction:DEzback¼ 223 kJmol�1 in vacuo, but DEzPCM,back¼ 192 kJmol�1 only in water.

Therefore, in vacuo calculations do not fully reflect the experimental assay in a testassay or biological system: while 84% of the variation in kGSH can be explained withoutthe application of any solvent models (Table 2; model VIII), the inclusion of two explicit


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water molecules led to a slight, but statistically significant, improvement, so at least 87%of the variation can now be explained (model V). The most important water moleculeis located at the carbonyl or carboxyl oxygen, which plays a role in the formation of theenolic intermediate. Any additional explicit water molecules surround the particularadduct system arbitrarily, as shown in Figure 7.

In addition, a solvent can be modelled by a conductor-like polarizable continuummodel (C-PCM) instead of considering explicit solvent molecules (in this study water).The solvent water is treated as a dielectric continuum with a permittivity " (¼ 78.39)surrounding the solute molecules outside of a molecular cavity, constructed as an assemblyof atom-centred spheres with Pauling radii. Using C-PCM in Equations (8) and (10), aneven stronger relationship is formed (model I), with one remaining explicit water moleculeat the carbonyl oxygen position.

The transition-state energy DEzforward of the forward reaction and DEzbackward of thebackward reaction is therefore calculated by the C-PCM model:

DEzforward ¼ DEzPCM: ð13Þ

DEzbackward ¼ DEzPCM,back: ð14Þ

The improvement of the overall model by the C-PCM model is relatively small butnevertheless statistically significant, meaning that 90% of the variation in of kGSH can beexplained.

3.7 kGSH reactivity model

An overall model for GSH reactivity can be now constructed by the following equationfor the forward and backward reactions (using Equations (8), (10), and (12–14)):

log k1 ¼ logZ1 þ d1 logSASðRÞ þ e1ð Þ �b1 � DE

z

PCM þ c1


, ð15Þ

log k�1 ¼ logZ�1 �b�1 � DE

z

PCM,back þ c�1


: ð16Þ

The observed rate constant kGSH can now be predicted by substituting Equations (15)and (16) into Equation (6):

log kGSH ¼ A � DEzPCM þ B � logSASðRÞn o

� C � DEzPCM,back

n oþD: ð17Þ

The coefficients A (¼�a1 � b1/[RT � ln 10]), B (¼ d1), C (¼�a�1 � b�1/[RT � ln 10]), andthe constant D (¼ e1þ [c1–c�1]/[RT � ln 10]þ log[Z1/Z�1]) were obtained by multiple linearregression analysis, as explained in Section 2.4, and are shown in Table 2. The final modeland the standard errors of the regression coefficients are as follows (model I):

log kGSH ¼ �0:0290ð�0:005Þ � DEz

PCM þ 1:42ð�0:23Þ � logSASðRÞ

þ 0:0307ð�0:004Þ � DEzPCM,back � 2:14ð�0:61Þð18Þ

r2 ¼ 0:90, r2adj ¼ 0:88, r2CV ¼ 0:83, se ¼ 0:34, F ¼ 52:61, p ¼ 0:000:


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Predicted rate constants and parameter values (energies and steric accessibility) are

listed in Table 3. There is a strong correlation between the predicted and experimental

values (r2¼ 0.90). The standard error (se) is 0.34, which is expected to be very close to the

experimental error [19]. The results are shown in Figure 8.The applicability domain of Equation (18) includes linear and cyclic �,�-unsaturated

carbonyl or carboxyl compounds, with double or triple bonds between the � carbon and

the � carbon atom, with or without �-carbon alkylation, and a kGSH value range between

10�1 and 103Mmin�1. The applicability of related Michael acceptor systems (e.g.

heterocyclic systems) is still under investigation.The signs of the coefficients in Equation (18) are in concordance with general physical

chemical perceptions: the higher the rate constant, the lower the energy barrier for the

forward reaction (negative sign for the DEzPCM term), the higher the steric accessibility of

the reaction site (positive sign for the logSAS term), and the higher the energy barrier for

the backward reaction (positive sign for the DEzPCM,back term).The intercept of Equation (18) is a consequence of many aspects (maybe even in a non-

linear manner): it accounts for different reactivity between methylthiol and GSH, the

logarithmic quotient of the pre-exponential factors for the forward and the backward

reaction, viscosity of water (which slows the reaction rate down, but is not reflected by the

transition-state calculations), the correction of the SAS term into units of the steric

Table 3. Predicted rate constants kGSH for 22 compounds (numbers according to Table 1),transition state energies and steric accessibility values (according to Equation (18); free energiesDGz and DGzback for systems including two explicit solvent molecules).

# log kpred DEzPCM DEzPCM,back log�SAS DGz DGzback[M�1 min�1] kJ/mol kJ/mol [%] kJ/ mol kJ/mol

1 þ1.98 147.7 160.9 2.44 236.9 190.12 þ2.76 160.0 186.2 2.69 239.9 214.23 þ1.35 165.1 170.7 2.14 235.1 173.54 þ2.77 162.2 188.7 2.69 243.2 218.75 þ0.85 205.5 167.6 2.68 258.4 162.56 þ1.22 164.0 168.2 2.08 248.9 190.37 þ2.76 166.2 192.1 2.69 245.8 219.28 þ0.94 171.7 172.3 1.95 245.3 194.69 þ2.85 162.3 191.6 2.69 260.5 225.410 þ1.36 171.4 176.7 2.14 260.8 202.711 þ1.19 171.7 180.6 1.95 266.3 213.712 þ1.43 172.5 179.4 2.16 256.7 206.613 þ1.04 172.3 176.8 1.94 261.5 202.714 þ2.64 165.6 187.8 2.69 245.6 217.115 þ1.44 161.5 168.2 2.18 248.4 198.916 þ0.67 206.1 209.1 1.66 236.1 184.717 þ0.19 139.1 106.3 2.19 231.4 146.018 þ0.65 141.0 123.7 2.17 230.0 158.219 þ0.60 149.5 129.6 2.18 45.6 20.320 �0.17 152.1 134.5 1.58 249.5 163.921 þ0.33 175.0 148.6 2.10 254.0 181.222 þ0.17 176.5 117.1 2.70 26.1 78.4


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accessibility �, neglected ionic effects of the aqueous buffer system, and implicitcorrections as a consequence of the moderate quantum chemical level.

In order to prove the predictivity of the model, we applied a leave-one-out cross-validation, with the r2CV value (0.83) for Equation (18) being close to the r2 value of the fulldata set (0.90). Even though an explicit external validation has not been applied so far, theprinciple outcome of the transition-state model was encoded into a local molecularparameter model with similar quality and predictivity (r2¼ 0.91; rms¼ 0.34; q2CV¼ 0.87),but using a much larger data set of 66 compounds [19]. Choosing, for example, 2-hydroxyethylacrylate from this larger data set and applying transition-state calculations in thesame way as described above, the calculated rate constant is log kGSH¼ 1.36, which isclose to the experimental value (log kGSH¼ 1.43).

All essential factors of reactivity have been reflected by model I. The reactivity of theMichael-type addition to GSH is well predicted by Equation (18). To summarize, themodel is based on the following assumptions:

(a) Michael-type reactions are reversible, and both the forward and the backwardreactions are responsible for reactivity.

(b) The enol form is a reactive intermediate.(c) A subsequent keto-enol-tautomerism is not rate determining.(d) S–C bond formation at the � carbon and H addition at the � carbon take place at

the same time.(e) Steric accessibility has an impact on reactivity and can be modelled by the solvent-

accessible surface area.(f) The solvent stabilizes the transition state.

A consequence of solvent stabilization is that reactivity might slightly differ fordifferent protein microenvironments. Interestingly, explicit water molecules are not needed

Figure 8. Predicted (Equation (18)) versus experimental (Table 2) values of kinetic rate constantskGSH (in M�1min�1) for glutathione adduct formation of �,�-unsaturated compounds at 25�C andpH¼ 7.4. Dashed lines encircle the area of twice the standard deviation, indicating that there areno systematic outliers.


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for the principle mechanism, and the transition-state structure does not change essentially

between calculations in vacuo and in water. A scheme for the reaction pathway is shown in

Figure 9. This scheme can be confirmed by quantum chemical intrinsic reaction coordinate

(IRC) calculations (IRC calculations were applied for acrolein; results not shown).

4. Conclusion

A model has been described to predict the reactivity, in terms of GSH adduct formation,

of �,�-unsaturated compounds. The transition-state calculations model peptide adduct

formation successfully. The transition-state structure implies a simultaneous S–C� and

H–C� bond formation. Essential factors of Michael-type addition are the following: steric

hindrance has a crucial impact on the reactivity of the forward reaction; the reaction is

reversible from a reactive enol intermediate; and the subsequent keto-enol tautomerism

is not rate determining. Knowledge of the energy profile can extend the knowledge of GSH

adduct formation by valuable mechanistic information. This knowledge might help in the

reactivity or toxicity profiling of electrophilic compounds. It can support mechanistic

grouping to order compounds according to reactivity and also confirm that a compound

is reactive. It should be noted in this context, that while there is no global model likely for

reactivity, the use of local models will increase confidence into the prediction of reactive

peptide binding or DNA binding for mechanistically developed groupings and categories.

Acknowledgement

This study was financially supported by the European Union through the FP6 Marie CurieInSilicoTox project (contract no. MTKD-CT-2006-42328).

Figure 9. Scheme of the reaction path for glutathione adduct formation of �,�-unsaturatedcompounds. The range of energies shown were calculated at the B3LYP/6-31G** level with theC-PCM water solvation model for 22 compounds listed in Table 2. The optimized structures ofacrolein and acrolein–methylthiol adducts are shown in the diagram.


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References

[1] A.O. Aptula and D.W. Roberts, Mechanistic applicability domains for non-animal-based

prediction of toxicological end points: General principles and application to reactive toxicity,

Chem. Res. Toxicol. 19 (2006), pp. 1097–1105.

[2] T.W. Schultz, R.E. Carlson, M.T.D. Cronin, J.L.M. Hermens, R. Johnson, P.J. O’Brien,

D.W. Roberts, A. Siraki, K.D. Wallace, and G.D. Veith, A conceptual framework for predicting

toxicity of reactive chemicals: Models for soft electrophilicity, SAR QSAR Environ. Res. 17

(2006), pp. 413–428.

[3] D.W. Roberts, A.O. Aptula, and G. Patlewicz, Electrophilic chemistry related to skin

sensitization. Reaction mechanistic applicability domain classification for a published data set

of 106 chemicals tested in the mouse local lymph node assay, Chem. Res. Toxicol. 20 (2007),

pp. 40–60.[4] D.W. Roberts, G. Patlewicz, P.S. Kern, F. Gerberick, I. Kimber, R.J. Dearman, C.A. Ryan,

D.A. Basketter, and A.O. Aptula, Mechanistic applicability domain classification of a

local lymph node assay dataset for skin sensitization, Chem. Res. Toxicol. 20 (2007),

pp. 1019–1030.[5] P.C. von der Ohe, R. Kuhne, R.-U. Ebert, R. Altenburger, M. Liess, and G. Schuurmann,

Structural alerts – A new classification model to discriminate excess toxicity from narcotic effect

levels of organic compounds in the acute daphnid assay, Chem. Res. Toxicol. 18 (2005),

pp. 536–555.[6] M.T.D. Cronin, J.C. Dearden, J.C. Duffy, R. Edwards, N. Manga, A.P. Worth, and

A.D.P. Worgan, The importance of hydrophobicity and electrophilicity descriptors in mechan-

istically-based QSARs for toxicological endpoints, SAR QSAR Environ. Res. 13 (2002),

pp. 167–176.[7] S.J. Enoch, D.W. Roberts, and M.T.D. Cronin, Electrophilic reaction chemistry of low molecular

weight respiratory sensitizers, Chem. Res. Toxicol. 22 (2009), pp. 1447–1453.

[8] H. Tanii and K. Hashimoto, Structure–toxicity relationship of acrylates and methacrylates,

Toxicol. Lett. 11 (1982), pp. 125–129.[9] D.W. Potter and T.-B. Tran, Rates of ethyl acrylate binding to glutathione and protein, Toxicol.

Lett. 62 (1992), pp. 275–285.

[10] T.W. Schultz, J.W. Yarbrough, R.S. Hunter, and A.O. Aptula, Verification of the structural

alerts for Michael acceptors, Chem. Res. Toxicol. 20 (2007), pp. 1359–1363.[11] Y.K. Koleva, J.C. Madden, and M.T.D. Cronin, Formation of categories from structure-activity

relationships to allow read-across for risk assessment: Toxicity of �,�-unsaturated carbonyl

compounds, Chem. Res. Toxicol. 21 (2008), pp. 2300–2313.

[12] J.A.H. Schwobel, Y.K. Koleva, S.J. Enoch, F. Bajot, M. Hewitt, J.C. Madden, D.W. Roberts,

T.W. Schultz, and M.T.D. Cronin, Measurement and estimation of electrophilic reactivity for

predictive toxicology, Chem. Rev. (2010), in press.[13] A.O. Aptula, G. Patlewicz, D.W. Roberts, and T.W. Schultz, Non-enzymatic glutathione reac-

tivity and in vitro toxicity: A non-animal approach to skin sensitization, Toxicol. Vitro 20 (2006),

pp. 239–247.[14] I.A. Blair, Endogenous glutathione adducts, Curr. Drug Metab. 7 (2006), pp. 853–872.[15] A.P. Freidig, H.J.M. Verhaar, and J.L.M. Hermens, Comparing the potency of chemicals with

multiple modes of action in aquatic toxicology: Acute toxicity due to narcosis versus reactive

toxicity of acrylic compounds, Environ. Sci. Technol. 33 (1999), pp. 3038–3043.[16] A. Bohme, D. Thaens, A. Paschke, and G. Schuurmann, Kinetic glutathione chemoassay to

quantify thiol reactivity of organic electrophiles – Application to �,�-unsaturated ketones,

acrylates, and propiolates, Chem. Res. Toxicol. 22 (2009), pp. 742–750.[17] M.T.D. Cronin, F. Bajot, S.J. Enoch, J.C. Madden, D.W. Roberts, and J. Schwobel, The in

chemico–in silico interface: Challenges for integrating experimental and computational chemistry

to identify toxicity, ATLA 37 (2009), pp. 513–521.


Dow

nloa

ded

by [

Purd

ue U

nive

rsity

] at

13:

09 1

8 M

arch

201

3

[18] G. Schaafsma, E.D. Kroese, E.L.J.P. Tielemans, J.J.M. van de Sandt, and C.J. van Leeuwen,

REACH, non-testing approaches and the urgent need for a change in mind set, Regul. Toxicol.

Pharmacol. 53 (2009), pp. 70–80.[19] J. Schwobel, D. Wondrousch, Y.K. Koleva, J.C. Madden, M.T.D. Cronin, and

G. Schuurmann, Prediction of Michael type acceptor reactivity toward glutathione, Chem. Res.

Toxicol. 23 (2010), pp. 1576–1585.[20] H. Esterbauer, H. Zollner, and N. Scholz, Reaction of glutathione with conjugated carbonyls,

Z. Naturforsch. 30c (1975), pp. 466–473.[21] T.J. McCarthy and G. Witz, Structure–activity relationships in the hydrolysis of acrylate and

methacrylate esters by carboxylesterase in vitro, Toxicology 116 (1997), pp. 153–158.

[22] A.P. Freidig, H.J.M. Verhaar, and J.L.M. Hermens, Quantitative structure–property relation-

ships for the chemical reactivity of acrylates and methacrylates, Environ. Toxicol. Chem. 18

(1999), pp. 1133–1139.

[23] K.J. Laidler and M.C. King, Development of transition-state theory, J. Phys. Chem. 87 (1983),

pp. 2657–2664.[24] D.G. Truhlar, B.C. Garrett, and S.J. Klippenstein, Current status of transition-state theory,

J. Phys. Chem. 100 (1996), pp. 12771–12800.

[25] M.D. Kostin, Transition state theory and grand canonical partition functions, J. Chem. Phys. 107

(1997), pp. 5811–5814.[26] L.-P. Ju, K.-L. Han, and J.Z.H. Zhang, Global dynamics and transition state theories:

Comparative study of reaction rate constants for gas-phase chemical reactions, J. Comput. Chem.

30 (2009), pp. 305–316.[27] R. Osman, K. Namboodiri, H. Weinstein, and J.R. Rabinowitz, Reactivities of acrylic and

methacrylic acids in a nucleophilic addition model of their biological activity, J. Am. Chem. Soc.

110 (1988), pp. 1701–1707.[28] E. Nakamura, S. Mori, and K. Morokuma, Reaction pathway of the conjugate addition of lithium

organocuprate clusters to acrolein, J. Am. Chem. Soc. 119 (1997), pp. 4900–4910.[29] S. Mori and E. Nakamura, Density functional studies on conjugate addition of (Me2CuLi)2

to cyclohexenone: Stereoselectivity and rate-determining step, Chem. Eur. J. 5 (1999),

pp. 1534–1543.

[30] B. Hemmateenejad, M. Sanchooli, and A. Mehdipour, Quantitative structure-reactivity

relationship studies on the catalyzed Michael addition reactions, J. Phys. Org. Chem. 22 (2009),

pp. 613–618.

[31] R.S. Paton, J.M. Goodman, and S.C. Pellegrinet, Theoretical study of the asymmetric conjugate

alkenylation of enones catalyzed by binaphthols, J. Org. Chem. 73 (2008), pp. 5078–5089.[32] I.-H. Um, S.-J. Hwang, and E.-J. Lee, A kinetic study on Michael-type reactions of 1-(X-

substituted phenyl)-2-propyn-1-ones with amines: Effect of amine nature on reactivity and

mechanism, Bull. Korean Chem. Soc. 29 (2008), pp. 767–771.[33] I.-H. Um, E.-J. Lee, J.-A. Seok, and K.-H. Kim, The a-effect in reactions of sp-hybridized carbon

atom: Michael-type reactions of 1-aryl-2-propyn-1-ones with primary amines, J. Org. Chem. 70

(2005), pp. 7530–7536.[34] M.J. Frisch, G.W. Trucks, H.B. Schlegel, G.E. Scuseria, M.A. Robb, J.R. Cheeseman,

J.A. Montgomery, T. Vreven, K.N. Kudin, J.C. Burant, J.M. Millam, S.S. Iyengar, J. Tomasi,

V. Barone, B. Mennucci, M. Cossi, G. Scalmani, N. Rega, G.A. Petersson, H. Nakatsuji,

M. Hada, M. Ehara, K. Toyota, R. Fukuda, J. Hasegawa, M. Ishida, T. Nakajima, Y. Honda,

O. Kitao, H. Nakai, M. Klene, X. Li, J.E. Knox, H.P. Hratchian, J.B. Cross, C. Adamo,

J. Jaramillo, R. Gomperts, R.E. Stratmann, O. Yazyev, A.J. Austin, R. Cammi, C. Pompelli,

J.W. Ochterski, P.Y. Ayala, K. Morokuma, G.A. Voth, P. Salvador, J.J. Dannenberg,

V.G. Zakrzewski, S. Dapprich, A.D. Daniels, M.C. Strain, O. Farkas, D.K. Malick,

A.D. Rabuck, K. Raghavavachari, J.B. Foresman, J.V. Ortiz, Q. Cui, A.G. Baboul,

S. Clifford, J. Cioslowski, B.B. Stefanov, G. Liu, A. Liashenko, P. Piskorz, I. Komaromi,

R.L. Martin, D.J. Fox, T. Keith, M.A. Al-Laham, C.Y. Peng, A. Nanayakkara,


Dow

nloa

ded

by [

Purd

ue U

nive

rsity

] at

13:

09 1

8 M

arch

201

3

M. Challacombe, P.M.W. Gill, B. Johnson, W. Chen, M.W. Wong, C. Gonzalez, andJ.A. Pople, Gaussian 03, Revision C.02, Gaussian, Inc., Pittsburgh, PA, USA, 2003.

[35] M. Cossi, N. Rega, G. Scalmani, and V. Barone, Energies, structures, and electronic properties ofmolecules in solution with the C-PCM solvation model, J. Comput. Chem. 24 (2003), pp. 669–681.

[36] M. Sanner, MSMS, Molecular Graphics Laboratory, La Jolla, CA, USA, 1996.[37] M.L. Connolly, The molecular surface package, J. Mol. Graphics 11 (1992), pp. 139–141.[38] I.F. Cespedes-Camacho, J.A. Manso, M.T. Perez-Prior, R. Gomez-Bombarelli, M. Gonzalez-

Perez, E. Calle, and J. Casado, Reactivity of acrylamide as an alkylating agent: A kineticapproach, J. Phys. Org. Chem. 23 (2009), pp. 171–175.

[39] X. Wang and M.T. Ashby, Reactive sulfur species: Kinetics and mechanism of the reaction of

thiocarbamate-S-oxide with cysteine, Chem. Res. Toxicol. 21 (2008), pp. 2120–2126.[40] J. Cai, A. Bhatnagar, and W.M. Pierce Jr, Protein modification by acrolein: Formation and

stability of cysteine adducts, Chem. Res. Toxicol. 22 (2009), pp. 708–716.

[41] C.F. Bernasconi and R.B. Killion Jr, Nucleophilic addition to olefins. Part 23. High intrinsicrate constant and large imbalances in the thiolate ion addition to substituted a-nitrostilbenes,J. Am. Chem. Soc. 110 (1988), pp. 7506–7512.


Dow

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examination of michael addition reactivity towards glutathione by transition-state calculations

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