Department of Chemistry and Biomolecular Sciences , University of Ottawa , Ottawa , Ontario K1N 6N5 , Canada.
J Am Chem Soc. 2019 Feb 20;141(7):3037-3051. doi: 10.1021/jacs.8b11524. Epub 2019 Feb 8.
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 ( k/ k ∼ 20) indicates that tunneling underlies the preference for the HAT pathway.
我们最近曾交流过，胆固醇的自由基介导氧化（自动氧化）会产生比以前认为更为复杂的过氧化物产物混合物。除了主要产物胆固醇 7-过氧化物的差向异构体之外，还形成了每个区域异构体 4-和 6-过氧化物的差向异构体，以及在良好的 H-原子供体存在下形成的 5α-过氧化物。在此，我们通过报告竞争过氧自由基与胆固醇加成的产物、占氧化产物 12%的立体异构胆固醇-5,6-环氧化物，以及胆固醇过氧化物的亲电脱水产物 4-、6-和 7-酮胆固醇，来完成这个故事。此外，我们还探讨了在良好的 H-原子供体存在下，这些产物的分布和丰度（相对于 H-原子抽取产物）如何变化，这对这些氧化甾醇如何被用作生物标志物有严重影响。通过合成一种新的同位素标记胆固醇标准品和相应的选定自动氧化产物，大大促进了所有自动氧化产物通过 LC-MS/MS 的分离和定量。还研究了胆固醇乙酸酯的自动氧化，作为体内大量存在的胆固醇酯的模型。尽管胆固醇的酯化赋予了可测量的立体电子效应，最重要的是反映了它的自动氧化速率是未酯化胆固醇的 4 倍，但产物分布在很大程度上与胆固醇相似。胆固醇的烯丙位氘化抑制了通过 H-原子转移（HAT）的自动氧化，有利于加成，使得环氧化物成为主要产物。相应的动力学同位素效应（ k/ k ∼ 20）表明，隧道效应是 HAT 途径偏好的基础。
