Substituent Effects on the Reactivity of Benzoquinone Derivatives with Thiols

Abstract

Benzoquinone (BQ) is an extremely potent electrophilic contact allergen that haptenates endogenous proteins through Michael addition (MA). It is also hypothesized that BQ may haptenate proteins via free radical formation. The objective of this study was to assess the inductive effects (activating and deactivating) of substituents on BQ reactivity and mechanistic pathway of covalent binding to a nucleophilic thiol. The BQ binding of Cys34 on human serum albumin was studied and for reactivity studies, nitrobenzenethiol (NBT) was used as a surrogate for protein binding of the BQ and benzoquinone derivatives (BQD). Stopped flow techniques were used to determine pseudo-first order rate constants (k) of methyl, t-butyl and chlorine substituted BQD reactions with NBT whereas electron pair resonance (EPR) studies were performed to investigate the presence a free radical mediated binding mechanism of BQD. Characterization of adducts was performed using mass spectrometry and nuclear magnetic resonance spectroscopy (NMR). The rate constant values demonstrated the chlorine substituted (activated) BQD to be more reactive toward NBT than the methyl and t-butyl substituted (deactivated) BQD, and this correlated with the respective EPR intensities. The EPR signal, however, was quenched in the presence of NBT suggesting MA as the dominant reaction pathway. MS and NMR results confirmed adduct formation to be a result of MA onto the BQ ring with vinylic substitution also occurring for chlorine substituted derivatives. The binding positions on BQ and NBT:BQ(D) stoichiometric ratios were affected by whether the inductive effects of the substituents on the ring were positive or negative. The reactivity of BQ and BQD is discussed in terms of the potential relationship to potential allergenic potency.

Reactivity measurement in estimation of benzoquinone and benzoquinone derivatives’ allergenicity

“…Reaction rate constants of BQ and BQD toward NBT that were previously determined (Mbiya et al, 2012), along with predicted EC3 values derived from equations correlating electrophilic allergen reactivity (to NBT) with pEC3, are shown in Table 1. Reactivity of BQ and BQD toward NBT was used as a surrogate for protein binding of the electrophiles in phosphate buffer at pH 7.4.…”

“…The k a values reported in Mbiya et al, (2012), where BQ and BQD were reacted with NBT, were adopted. The structures of test compounds are given in Fig.…”

“…BQ is an extreme skin sensitizer EC3 = 0.013% (EC3 is the estimated concentration that produces a 3 fold increase in lymph node cell proliferation over the vehicle control) (Roberts and Aptula, 2009) but the effects of inserting substituents on the BQ ring have not been studied in animal assays. BQ and BQD are known to covalently modify proteins via the Michael addition (MA) reaction and/or through nucleophilic vinylic substitution (SNV), depending on whether it is attached to either electron donating groups (EDGs) or electron withdrawing groups (EWGs) that are good leaving groups (Mbiya et al, 2012). …”

“…The reported (Mbiya et al, 2012) reactivity constants ( k a ) for BQ and BQD were used to predict the LLNA EC3 values for the BQD after which LLNA studies were then performed for BQ and BQD to determine the EC3 values. LLNA studies also served to test the predictive power of the reactivity data thus evaluating the potential utility of the reactivity approach as an alternative method for skin sensitization testing.…”

A comparison of the C‒H bond dissociation enthalpies of sulfur-containing fused heterocyclic compounds to the C‒H bond dissociation enthalpies in other heterocycles

“…[44][45][46] Therefore, five sulfur heterocyclic compounds were selected to systematically investigate the substituent effect on C−H BDEs (see Table 5 for their structures). The substituents are classified into three types such as electronwithdrawing groups (EWGs) including − NO 2 , − CF 3 , electron-donating groups (EDGs) including − NH 2 , − N(CH 3 ) 2 , and conjugation effect groups (CEGs) including − CH=CH 2 , − Ph.…”