First Advisor

Reuben H. Simoyi

Date of Publication

Summer 8-13-2013

Document Type

Dissertation

Degree Name

Doctor of Philosophy (Ph.D.) in Chemistry

Department

Chemistry

Language

English

Subjects

Quinone -- Reactivity, Quinone -- Allergenicity, Protein binding

DOI

10.15760/etd.1406

Physical Description

1 online resource (xiv, 109 pages)

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 the mechanistic pathway of covalent binding to nucleophilic thiols. The BQ binding by 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 possible free radical mediated binding mechanism of BQD. Characterization of adducts was performed using mass spectrometry (MS) 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 of NBT onto the BQ ring with vinylic substitution also occurring for chlorine-substituted derivatives. The binding positions on BQ and NBT/BQD 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 allergenic potency. Hammett and Taft (HT) constants were then used to estimate the influence of these substituents on chemical reactivity. HT values demonstrated chlorine substituted BQD to be more reactive than methyl substituted BQD. BQ and BQD dermal allergenicity, as evaluated in the murine local lymph node assay, (LLNA) was consistent with that predicted by reactivity and HT parameters. These results demonstrate the effect of substituents on BQ reactivity and dermal allergic sensitization, and suggest the potential utility of chemical reactivity data and HT values for electrophilic allergen identification and potency ranking.

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Persistent Identifier

http://archives.pdx.edu/ds/psu/10029

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