Non-natural anti-Markovnikov oxidation of olefins by redirecting the oxygen transfer in P450 monooxygenases
Organic Molecular Chemistry - Synthesis and Characterisation
Final Report Abstract
The selective oxidation of the end carbon of a terminal alkene to generate an aldehyde, a so called anti-Markovnikov alkene oxidation, is a great challenge in catalysis because Markovnikov oxidation and epoxidation are highly kinetically favored. To perform this reaction, chemists depend on fairly unproductive methods. We showed that anti-Markovnikov alkene oxidation can be achieved by evolving a cytochrome P450 enzyme in the laboratory. This enzyme has been used as a starting point because it applies metal-oxo species for alkene epoxidation, in which a metallinked oxygen attacks the alkene. We demonstrated that reactive intermediates in the epoxidation reaction can be re-directed towards anti-Markovnikov oxidation. It is possible to access this new chemical transformation because enzymes are macromolecular catalyst and can efficiently stabilize and control transition states and promote reactions through challenging pathways. Such a control is difficult to achieve by small-molecule catalysts due to competition with other, lower energy reaction pathways. Since it was impossible predict how an active site should look like to perform this reaction, we used directed evolution, an iterative protein-mutation and screening process, to generate an enzyme called aMOx (anti-Markovnikov oxygenase) that catalyzes this reaction with high efficiency and selectivity. We used aMOx in scale-up reactions to demonstrate that it can be applied in synthesis. We also performed first mechanistic studies which revealed that aMOx catalyzes a 1,2-hydride migration as part of the reaction mechanism. A surprising finding is that aMOx can perform this 1,2-hydride migration with high enantioselectivity. Next to enzymology studies, we have also applied aMOx in enzyme cascades. Design and evolution of new enzyme function provides access to novel metabolic pathways and we showed that formal anti- Markovnikov hydration of alkenes can be achieved by combining aMOx with an alcohol dehydrogenase. A major challenge in the project was the laboratory evolution of the new enzyme function since it needed 12 mutations to control the reaction. Overall, we have been very lucky with the research project since it has worked as proposed. We have also received good feedback. Our manuscript was highlighted in C&EN (2017, 95, page 4) and Synfacts (2018, 14, 0083). The results were also highlighted together with other research articles by ACS and C&EN as Research of the year 2017 in chemistry.
Publications
- Anti- Markovnikov alkene oxidation by metal-oxo–mediated enzyme catalysis. Science 2017, 358:215–218
Hammer SC, Kubik G, Watkins E, Huang S, Minges H, Arnold FH
(See online at https://doi.org/10.1126/science.aao1482) - Design and evolution of enzymes for nonnatural chemistry. Curr. Opin. Green Sustain. Chem. 2017, 7:23–30
Hammer SC, Knight AM, Arnold FH
(See online at https://doi.org/10.1016/j.cogsc.2017.06.002)
