Disentangling the Puzzling Regiochemistry of Thiol Addition to o-Quinones

Abstract

The addition of thiol compounds to o-quinones, as exemplified by the biologically relevant conjugation of cysteine to dopaquinone, displays an anomalous 1,6-type regiochemistry compared to the usual 1,4-nucleophilic addition, for example, by amines, which has so far eluded intensive investigations. By means of an integrated experimental and computational approach, herein, we provide evidence that the addition of glutathione, cysteine, or benzenethiol to 4-methyl-o-benzoquinone, modeling dopaquinone, proceeds by a free radical chain mechanism triggered by the addition of thiyl radicals to the o-quinone. In support of this conclusion, DFT calculations consistently predicted the correct regiochemistry only for the proposed thiyl radical-quinone addition pathway. These results would prompt a revision of the commonly accepted mechanisms for thiol-o-quinone conjugation and stimulate further work aimed at assessing the impact of the free radical processes in biologically relevant thiol-quinone interactions.

Scheme 1. Oxidative Coupling Reactions of Catechols with Thiol or Amine Compounds Leading to C-5 or C-6 Conjugates, Respectively

Scheme 2. Schematic Reaction of 4-Methyl-o-benzoquinone with Thioacetic Acid under Different Reaction Conditions

Figure 1

(A) Yields of the 5-S-cysteinyl adduct of the reaction of cysteine with 4-MBQ (10:1 M ratio) as a function of pH in the presence and in the absence of air. (B) Effect of Trolox on the yields of the 5-S-cysteinyl adduct at a 1:5 catechol-cysteine/Trolox ratio as a function of pH in the absence of air.

Scheme 3. Thiyl Radical-Mediated Isomerization of Olefins

Figure 2

EPR spectra (dotted lines) recorded in spin-trapping experiments with (A) 30 mM PBN, 15 mM PhSH, 15 mM 4-MBQ in a 1:1 MeCN/acetate buffer (pH = 5.0) or (B) 30 mM DEPMPO, 15 mM PhSH, 15 mM 4-MBQ in a 1:1 MeCN/acetate buffer (pH = 5.0). Corresponding computer simulations (full lines) have been obtained with the following parameters: (A) a_N = 15.1 G, a_H = 3.9 G (g = 2.0067); (B) a_N = 14.6 G, a_H = 20.9 G, a_P = 47.4 G (g = 2.0064).

Figure 3

EPR spectrum (dotted lines) and corresponding computer simulation (full line) recorded in spin-trapping experiments with 50 mM DEPMPO, 8.5 mM cysteine, 5 mM 4-MBQ in 1:1 MeCN/acetate buffer (pH = 5.0), showing the trapping of the CyS^• radical.

Figure 4

Spin trapping of the thiyl radical GS^• upon photolyzing a solution of GS-NO (1.7 mM) in the presence of PBN (9 mM) in MeCN/acetate buffer (pH = 5) at 30 °C (dashed line; the asterisk indicates the persistent signal of the EPR cavity Dewar). Simulated spectrum (full line): a_N = 15.1 G, a_H = 3.1 G, g-factor = 2.0070.

Scheme 4. Generation of 4-MBQ/GS Adduct under Radical Conditions

Scheme 5. Proposed Pathways of the 4-MBQ Reactions with Thiols

Scheme 6. Proposed Mechanism for the Formation of the 5-S-Adduct