Anti-Markovnikov alkene oxidation by metal-oxo–mediated enzyme catalysis

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Science  13 Oct 2017:
Vol. 358, Issue 6360, pp. 215-218
DOI: 10.1126/science.aao1482
  • Fig. 1 Anti-Markovnikov oxidation of alkenes.

    (A) Alkene oxidation reactions. (B) Comparison of proposed catalytic cycles for P450-catalyzed alkene epoxidation and anti-Markovnikov oxidation. (i) Formation of metal-oxo complex, termed compound I (framed). (ii) Energetically preferred, highly concerted olefin epoxidation. (iii to v) Alternative stepwise anti-Markovnikov oxidation via an additional electron transfer (iv), yielding a high-energy carbocation intermediate. Subsequent 1,2-hydride migration (v) generates the anti-Markovnikov oxidation product. Steps iv and v might occur stepwise or in a concerted fashion. (C) The anti-Markovnikov oxygenase (aMOx) can be combined with various established biocatalysts in short (two-step) enzyme pathways to catalyze a variety of challenging anti-Markovnikov functionalization reactions (fig. S10). R, aryl or alkyl; M, metal.

  • Fig. 2 Directed evolution of aMOx and mechanistic insights.

    (A) aMOx was engineered in the laboratory in 10 rounds of directed evolution using styrene 1 as a substrate (tables S2 and S3). The error bars represent the standard deviation of the total turnover number (TTN) for anti-Markovnikov oxidation in reactions run in at least two independent triplicates (minimum of six reactions). (B) The aMOx-catalyzed anti-Markovnikov oxidation is a direct oxidation without an epoxide intermediate (fig. S6). (C) Isotopic labeling experiments support a 1,2-hydride migration in the catalytic cycle (fig. S8). (D) aMOx-catalyzed enantioselective anti-Markovnikov oxidation (fig. S9). (E) Enantiocontrol of aMOx derives from the enzyme’s capacity to lock the substrate in a specific conformation. Carbocation 1,2-rearrangements are best described as concerted reactions via a suprafacial 1,2-shift (25, 44). NADH, reduced form of NAD+ (nicotinamide adenine dinucleotide, oxidized form); NADPH, reduced form of NADP+ (NAD+ phosphate); D, deuterium; Me, methyl.

  • Fig. 3 Application of aMOx in anti-Markovnikov redox hydration of alkenes.

    The reaction conditions were 5 mM alkene, 0.25 μM aMOx, 10 units alcohol dehydrogenase (ADH), 1% i-PrOH, 0.1 equivalents NADP+, and 2 hours reaction time at room temperature. Red, percentage of total product that is the anti-Markovnikov oxidation product. *The major product is the epoxide, with minor allylic oxidation by-product (<8%). †The major product is the epoxide, with very minor allylic oxidation by-product (<1%). i-PrOH, isopropanol; er, enantiomeric ratio.

Supplementary Materials

  • Anti-Markovnikov alkene oxidation by metal-oxo-mediated enzyme catalysis

    Stephan C. Hammer, Grzegorz Kubik, Ella Watkins, Shan Huang, Hannah Minges, Frances H. Arnold

    Materials/Methods, Supplementary Text, Tables, Figures, and/or References

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    • Materials and Methods
    • Figs. S1 to S11
    • Tables S1 to S3
    • References

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