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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.