S-Pheny
lmercapturic acid is a minor metabo
lite of benzene used as a biomarker for humanbenzene exposures. The reaction of intrace
llu
lar g
lutathione with benzene oxide-oxepin, theinitia
l metabo
lite of benzene, is presumed to give 1-(
S-g
lutathiony
l)-cyc
lohexa-3,5-dien-2-o
l,which undergoes dehydration to
S-pheny
lg
lutathione, the precursor of
S-pheny
lmercapturicacid. To va
lidate the proposed route to
S-pheny
lg
lutathione, reactions of benzene oxide-oxepinwith g
lutathione and other su
lfur nuc
leophi
les have been studied. The reaction of benzeneoxide with an excess of aqueous sodium su
lfide, fo
llowed by acety
lation, gave bis-(6-
trans-5-acetoxycyc
lohexa-1,3-dieny
l)su
lfide, the structure of which was proved by X-ray crysta
llography.Reactions of benzene oxide-oxepin in a 95:5 (v/v) mixture of phosphate buffer in D
2O with(CD
3)
2SO were monitored by
1H NMR spectroscopy. In the absence of g
lutathione, the ha
lf-
life of benzene oxide-oxepin was ca. 34 min at 25
C and pD 7.0. The ha
lf-
life was not affectedin the range of 2-15 mM g
lutathione in the presence and absence of a commercia
l samp
le ofhuman g
lutathione
S-transferase (at pH 7.0, 8.0, 8.5, or 10.0). The adduct 1-(
S-g
lutathiony
l)-cyc
lohexa-3,5-diene-2-o
l was identified in these reaction mixtures, especia
lly at higher pH, bymass spectrometry and by its acid-cata
lyzed decomposition to
S-pheny
lg
lutathione. Incubationof benzene oxide with
N-acety
l-
L-cysteine at 37
C and pH 10.0 and subsequent massspectrometric ana
lysis of the mixture showed formation of pre
-S-pheny
lmercapturic acid andthe dehydration product,
S-pheny
lmercapturic acid. The data va
lidate the premise that benzeneoxide-oxepin can be captured by g
lutathione to give (1
R,2
R)- and/or (1
S,2
S)-1-(
S-g
lutathiony
l)-cyc
lohexa-3,5-dien-2-o
l, which dehydrate to
S-pheny
lg
lutathione. The capture is a re
lative
lyinefficient process at pH 7 that is acce
lerated at higher pH. These studies account for theobservation that the metabo
lism of benzene is dominated by the formation of pheno
l. Thepathway
leading to
S-pheny
lmercapturic acid is necessari
ly minor on account of the
lowefficiency of benzene oxide capture by g
lutathione at pH 7 vs spontaneous rearrangement topheno
l.