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H-Atom Abstraction vs Addition: Accounting for the Diverse Product Distribution in the Autoxidation of Cholesterol and Its Esters

Zielinski, Zosia A. M., Pratt, Derek A.
Journal of the American Chemical Society 2019 v.141 no.7 pp. 3037-3051
Lewis acids, acetates, autoxidation, biomarkers, cholesteryl esters, epoxides, esterification, hydroperoxides, isotopes, liquid chromatography, models, oxysterols, tandem mass spectrometry
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 distribution—and abundance relative to the H-atom abstraction products—changes 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 (kH/kD ∼ 20) indicates that tunneling underlies the preference for the HAT pathway.