Chlorination increases the persistence of semiquinone free radicals derived from polychlorinated biphenyl hydroquinones and quinones

Abstract

Polychlorinated biphenyls (PCBs) comprise a group of persistent organic pollutants that differ significantly in their physicochemical properties, their persistence, and their biological activities. They can be metabolized via hydroxylated and dihydroxylated metabolites to PCB quinone intermediates. We have recently demonstrated that both dihydroxy PCBs and PCB quinones can form semiquinone radicals (SQ(*-)) in vitro. These semiquinone radicals are reactive intermediates that have been implicated in the toxicity of lower chlorinated PCB congeners. Here we describe the synthesis of selected PCB metabolites with differing degrees of chlorination on the oxygenated phenyl ring, e.g., 4,4'-dichloro-biphenyl-2,5-diol, 3,6,4'-trichloro-biphenyl-2,5-diol, 3,4,6,-trichloro-biphenyl-2,5-diol, and their corresponding quinones. In addition, two chlorinated o-hydroquinones were prepared, 6-chloro-biphenyl-3,4-diol and 6,4'-dichloro-biphenyl-3,4-diol. These PCB (hydro-)quinones readily react with oxygen or via comproportionation to yield the corresponding semiquinone free radicals, as detected by electron paramagnetic resonance spectroscopy (EPR alias ESR). The greater the number of chlorines on the (hydro-)quinone (oxygenated) ring, the higher the steady-state level of the resulting semiquinone radical at near neutral pH.

Figure 1. Molecular structures derived from the crystal structure of 13 and 20

(a) Molecular structure of 4-chloro-biphenyl-2,5-diol (13) showing the atom-labeling scheme and (b) view of 4-chloro-biphenyl-2,5-diol (13) along the C1–C1′ axis illustrating the non-planar conformation of the molecule; (c) molecular structure of 3,6,4′-trichloro-biphenyl-2,5-diol (20) showing the atom-labeling scheme and (d) view of 3,6,4′-trichloro-biphenyl-2,5-diol (20) along the C1–C1′ axis illustrating the non-planar conformation of the molecule. Displacement ellipsoids are drawn at the 50% probability level.

Figure 2. EPR spectra of semiquinones generated from PCB hydroquinones with differing numbers and positions of chlorine on the phenyl rings

(Final concentrations of the original hydroquinones were 100 μM in phosphate buffer, pH = 7.4). (a) SQ^•− generated from 4′-chloro-biphenyl-2,5-diol (28); (b) SQ^•− generated from 4-chloro-biphenyl-2,5-diol (13); (c) SQ^•− generated from 4,4′-dichloro-biphenyl-2,5-diol (14); (d) SQ^•− generated from 4′-chloro-biphenyl-3,4-diol (30); (e) SQ^•− generated from 6-chloro-biphenyl-3,4-diol (9); (f) SQ^•− generated from 6,4′-dichloro-biphenyl-3,4-diol (10).

Figure 3. EPR spectra of semiquinones generated from PCB hydroquinones and quinones with differing numbers of chlorine on the oxygenated phenyl rings

(a) SQ^•− generated from 1 mM of 4′-chloro-biphenyl-2,5-diol (28) (yield: 0.000 092); (b) SQ^•− generated from 1 mM of 2-(4-chloro-phenyl)-[1,4]benzoquinone (29) (yield: 0.000 12); (c) computer simulation of SQ^•− with three hydrogens; (d) SQ^•− generated from 100 μM of 4,4′-dichloro-biphenyl-2,5-diol (14) (yield: 0.004 5); (e) SQ^•− generated from 100 μM of 2-chloro-5-(4-chloro-phenyl)-[1,4]benzoquinone (15) (yield: 0.003 5); (f) computer simulation of SQ^•− with two hydrogens; (g) SQ^•− generated from 10 μM of 3,6,4′-trichloro-biphenyl-2,5-diol (20) (yield: 0.026); (h) SQ^•− generated from 10 μM of 2,5-dichloro-3-(4-chloro-phenyl)-[1,4]benzoquinone (21) (yield: 0.025); (i) computer simulation of SQ^•− with one hydrogen; (j) SQ^•− generated from 1 μM of 3,4,6-trichloro-biphenyl-2,5-diol (25) (yield: 0.23); (k) SQ^•− generated from 1 μM of 2,3,5-trichloro-6-phenyl-[1,4]benzoquinone (26) (yield: 0.23); (l) computer simulation of SQ^•− with no hydrogen. These spectra were collected using a modulation amplitude of 1.0 Gauss. Lowering the modulation amplitude to 0.1 Gauss (for spectra d and e) or 0.01 Gauss (for spectra g, h, j, and k) did not reveal any possible hyperfine splittings from the chlorines on the semiquinone moiety.

Figure 4. The equilibrium constant for the comproportionation reaction to form SQ^•− increases with the number of chlorines on the oxygenated ring

Assay conditions: quinone and hydroquinone mixture in 100 mM PBS buffer, pH 7.4. (a) Zero chlorine, 50 μM of 28 and 50 μM of 29 yields [SQ^•−]_ss = 63 nM; (b) one chlorine, 5 μM of 14 and 5 μM of 15 yields [SQ^•−]_ss = 70 nM; (c) two chlorines, 500 nM of 20 and 500 nM of 21 yields [SQ^•−]_ss = 87 nM; (d) three chlorines, 50 nM of 25 and 50 nM of 26 yields [SQ^•−]ss = 65 nM. Where K = [SQ^•−]² / [hydroquinone][quinone]. This equilibrium constant will actually be dependent on pH because the pK_as for the hydroquinones will be above pH or near 7, while the pK_a of the semiquinone radicals will be somewhat acidic; for example, the pK_a of the semiquinone radical of unsubstituted benzoquinone/hydroquinone is 4.25.

Scheme 1. Synthesis of PCB hydroquinones/quinones with one chlorine on the oxygenated ring

Scheme 2. Synthesis of PCB hydroquinones/quinones with two chlorines on the oxygenated ring

Scheme 3. Synthesis of PCB hydroquinones/quinones with three chlorines on the oxygenated ring