We recently communicated that the
free-radical-mediated oxidation
(autoxidation) of cholesterol yields a more complex mixture of hydroperoxide
products than previously appreciated. In addition to the epimers of
the major product, cholesterol 7-hydroperoxide, the epimers of each
of the regioisomeric 4- and 6-hydroperoxides are formed as is the
5α-hydroperoxide in the presence of a good H-atom donor. Herein,
we complete the story by reporting the products resulting from competing
peroxyl radical addition to cholesterol, the stereoisomeric cholesterol-5,6-epoxides,
which account for 12% of the oxidation products, as well as electrophilic
dehydration products of the cholesterol hydroperoxides, 4-, 6-, and
7-ketocholesterol. Moreover, we interrogate how their distributionand
abundance relative to the H-atom abstraction productschanges
in the presence of good H-atom donors, which has serious implications
for how these oxysterols are used as biomarkers. The resolution and
quantification of all autoxidation products by LC-MS/MS was greatly
enabled by the synthesis of a new isotopically labeled cholesterol
standard and corresponding selected autoxidation products. The autoxidation
of cholesteryl acetate was also investigated as a model for the cholesterol
esters which abound in vivo. Although esterification of cholesterol
imparts measurable stereoelectronic effects, most importantly reflected
in the fact that it autoxidizes at 4 times the rate of unesterified
cholesterol, the product distribution is largely similar to that of
cholesterol. Deuteration of the allylic positions in cholesterol suppresses
autoxidation by H-atom transfer (HAT) in favor of addition, such that
the epoxides are the major products. The corresponding kinetic isotope
effect (k
H/k
D ∼ 20) indicates that tunneling underlies the preference for
the HAT pathway.