Ay a key role in P450-catalyzed nitration [16 ]. P450 catalytic diversification in nature is thus enabled by the generation of multiple potentially reactive species during the P450 catalytic cycle, as well as the potency of P450-derived oxidants, which can react with substrates in different ways. Though many potential oxidants occur during the cycle, natural P450s are often quite specific in the reactions that they catalyze. Specificity is directed by protein sequences molded by the force and filter of natural selection to favor certain intermediates while tuning their reactivity and selectivity. Beginning with compound I-derived oxidations, one particularly interesting P450-mediated reaction occurs during biosynthesis of the natural product aureothin (Figure 2, green). The P450 enzyme AurH first catalyzes hydroxylation of the aureothin precursor, followed by intramolecular C-O bond formation to give a tetrahydrofuran ring, with both reactions presumably occurring with the intermediacy of compound I [17]. Hertweck and coworkers have exploited this unusual enzyme to accomplish a biomimetic total synthesis of aureothin, as well as the synthesis of several aureothin derivatives [18?0 ; one paper [20 describes an active site mutation that converts AurH into a six-electron oxidase, leading to the conversion of a substrate methyl group all the way to a carboxylic acid. Several natural examples of sequential hydroxylations to yield ketones or carboxylates from unactivated C-H bonds have been described recently [21,22]. For example, in xiamycin biosynthesis, the P450 enzyme XiaM was shown to catalyze sequential hydroxylation of a methyl group to a carboxylate [21]. Another example of multiple P450-catalyzed oxidations was published by H er et al. in their investigation of the first steps of the biosynthesis of bioactive alkaloids vinblastine and secologanin (Figure 2, green) [22]. Though more typical of di-iron monooxygenases and -ketoglutarate-dependent dioxygenases, desaturation has been observed with a few P450 enzymes [8]. An interesting example of P450-catalyzed desaturation was recently reported by Bell et al. [23 . CYP199A4 was previously found to catalyze demethylation of several aromatic compounds, including 4-methoxybenzoic acid and veratric acid, as well as hydroxylation (major product) and desaturation (minor product) of 4-ethylbenzoic acid. In their recent Isovaleryl-Val-Val-Sta-Ala-Sta-OH custom synthesis report, these authors found two active site mutations (F185V and F185I) that markedly increase desaturation of 4ethylbenzoic acid to yield 4-vinylbenzoic acid, with the isoleucine variant giving exclusively the desaturation product (Figure 2, green). Several examples of P450-catalyzed decarboxylation are associated with biosynthesis and drug metabolism. One biotechnologically interesting P450-catalyzed decarboxylation leads to the synthesis of terminal alkenes from fatty acids [24 . The authors propose a mechanism in which compound I abstracts the -hydrogen, followed by 1-electron oxidation of the resulting radical to yield a -carbocation, which spontaneously decarboxylates to give the product.NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author ManuscriptCurr Opin Chem Biol. Author manuscript; available in PMC 2015 April 01.McIntosh et al.buy ML390 PageThe above compound I-mediated transformations most likely proceed via hydrogen atom abstraction. Another mechanism by which compound I can mediate oxidation is through sequential 1-electron oxidations. Vancomycin and related ant.Ay a key role in P450-catalyzed nitration [16 ]. P450 catalytic diversification in nature is thus enabled by the generation of multiple potentially reactive species during the P450 catalytic cycle, as well as the potency of P450-derived oxidants, which can react with substrates in different ways. Though many potential oxidants occur during the cycle, natural P450s are often quite specific in the reactions that they catalyze. Specificity is directed by protein sequences molded by the force and filter of natural selection to favor certain intermediates while tuning their reactivity and selectivity. Beginning with compound I-derived oxidations, one particularly interesting P450-mediated reaction occurs during biosynthesis of the natural product aureothin (Figure 2, green). The P450 enzyme AurH first catalyzes hydroxylation of the aureothin precursor, followed by intramolecular C-O bond formation to give a tetrahydrofuran ring, with both reactions presumably occurring with the intermediacy of compound I [17]. Hertweck and coworkers have exploited this unusual enzyme to accomplish a biomimetic total synthesis of aureothin, as well as the synthesis of several aureothin derivatives [18?0 ; one paper [20 describes an active site mutation that converts AurH into a six-electron oxidase, leading to the conversion of a substrate methyl group all the way to a carboxylic acid. Several natural examples of sequential hydroxylations to yield ketones or carboxylates from unactivated C-H bonds have been described recently [21,22]. For example, in xiamycin biosynthesis, the P450 enzyme XiaM was shown to catalyze sequential hydroxylation of a methyl group to a carboxylate [21]. Another example of multiple P450-catalyzed oxidations was published by H er et al. in their investigation of the first steps of the biosynthesis of bioactive alkaloids vinblastine and secologanin (Figure 2, green) [22]. Though more typical of di-iron monooxygenases and -ketoglutarate-dependent dioxygenases, desaturation has been observed with a few P450 enzymes [8]. An interesting example of P450-catalyzed desaturation was recently reported by Bell et al. [23 . CYP199A4 was previously found to catalyze demethylation of several aromatic compounds, including 4-methoxybenzoic acid and veratric acid, as well as hydroxylation (major product) and desaturation (minor product) of 4-ethylbenzoic acid. In their recent report, these authors found two active site mutations (F185V and F185I) that markedly increase desaturation of 4ethylbenzoic acid to yield 4-vinylbenzoic acid, with the isoleucine variant giving exclusively the desaturation product (Figure 2, green). Several examples of P450-catalyzed decarboxylation are associated with biosynthesis and drug metabolism. One biotechnologically interesting P450-catalyzed decarboxylation leads to the synthesis of terminal alkenes from fatty acids [24 . The authors propose a mechanism in which compound I abstracts the -hydrogen, followed by 1-electron oxidation of the resulting radical to yield a -carbocation, which spontaneously decarboxylates to give the product.NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author ManuscriptCurr Opin Chem Biol. Author manuscript; available in PMC 2015 April 01.McIntosh et al.PageThe above compound I-mediated transformations most likely proceed via hydrogen atom abstraction. Another mechanism by which compound I can mediate oxidation is through sequential 1-electron oxidations. Vancomycin and related ant.