Toy P H, Newcomb M, Hager L P
Department of Chemistry, Wayne State University, 5101 Cass Avenue, Detroit, Michigan 48202, USA.
Chem Res Toxicol. 1998 Jul;11(7):816-23. doi: 10.1021/tx9800295.
The oxidation of hypersensitive radical probes by chloroperoxidase from Caldariomyces fumago (CPO) was studied in an attempt to "time" a putative radical intermediate. Oxidation of (trans-2-phenylcyclopropyl)methane, previously studied by Zaks and Dodds [Zaks, A., and Dodds, D. R. (1995) J. Am. Chem. Soc. 115, 10419-10424] was reinvestigated. Unrearranged oxidation products were found as previously reported, and control experiments demonstrated that the cyclic alcohol from oxidation at the cyclopropylcarbinyl position, while subject to further oxidation, survives CPO oxidation as detectable species. However, in contrast to the report by Zaks and Dodds, the rearranged alcohol product expected from ring opening of a cyclopropylcarbinyl radical intermediate was shown to be unstable toward the enzyme oxidation reaction. Because of this instability, two new hypersensitive radical probes, (trans-2-phenylcyclopropyl)ethane and 2-(trans-2-phenylcyclopropyl)propane, and their potential cyclic and acyclic products from oxidation at the cyclopropylcarbinyl position were synthesized and tested. Oxidation of both of these probes at the cyclopropylcarbinyl position by CPO gave unrearranged alcohol products only, but control experiments again demonstrated that the rearranged alcohol products were unstable toward CPO oxidation conditions. From the combination of the probe and control studies, the lifetime of a putative radical intermediate must be less than 3 ps. Whereas the results are consistent with an insertion mechanism for production of alcohol product, they do not exclude a very short-lived intermediate.
为了对假定的自由基中间体进行“计时”，研究了烟曲霉氯过氧化物酶（CPO）对超敏自由基探针的氧化作用。重新研究了（反式-2-苯基环丙基）甲烷的氧化反应，该反应先前已由扎克斯和多兹进行过研究[扎克斯，A.，和多兹，D. R.（1995年）《美国化学会志》115，10419 - 10424]。如先前报道的那样，发现了未重排的氧化产物，对照实验表明，环丙基甲基位置氧化产生的环醇虽然会进一步氧化，但在CPO氧化过程中作为可检测物种存活下来。然而，与扎克斯和多兹的报告不同，由环丙基甲基自由基中间体开环预期得到的重排醇产物对酶氧化反应不稳定。由于这种不稳定性，合成并测试了两种新的超敏自由基探针，即（反式-2-苯基环丙基）乙烷和2-（反式-2-苯基环丙基）丙烷，以及它们在环丙基甲基位置氧化可能产生的环状和非环状产物。CPO对这两种探针在环丙基甲基位置的氧化仅产生未重排的醇产物，但对照实验再次表明，重排醇产物对CPO氧化条件不稳定。综合探针研究和对照研究结果，假定的自由基中间体的寿命必须小于3皮秒。虽然结果与醇产物生成的插入机制一致，但并不排除存在一个寿命极短的中间体。
