Steering ene-reductases for non-natural C(sp2)-C(sp3) couplings through photoinduced single-electron-oxidation

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The Huang group at Nanjing University, China, which was newly established in April 2021, is dedicated to advancing innovative bio-catalysis by merging synthetic chemical strategies with synthetic biological techniques. Within my lab, I prioritize four key principles for our projects. These include developing catalysis that is new-to-nature, difficult or even unattainable using conventional chemo-catalysis, showcasing mechanistic interest, and yielding valuable products with practical applications. Building upon my previous experience in asymmetric photocatalysis[1] and photoenzymatic catalysis[2], I conceived a photoenzymatic system that utilizes direct excited quinone state flavin for single-electron-oxidation, thereby triggering asymmetric radical transformations.

I am pursuing this direction for several reasons. Firstly, the proposed photoenzymatic system represents a new-to-nature approach that is distinct from the ene-reductase-catalysed native two-electron hydride reductions of activated alkenes. Second, the chemo- and stereo-selectivities associated with photo-induced radical transformations inherently pose significant challenges. Lastly, and perhaps most importantly, the mechanistic pathway of the proposed photoenzymatic single-electron-oxidation triggered system differs from the previously reported ene-reductase-based photoenzymatic catalysis involving an enzymatic EDA (electron donor acceptor) intermediate, as pioneered by Hyster[3], Zhao[4], Wu[5], and Xu[6] (Fig. 1).

 

Fig. 1. An unprecedented photoenzymatic hydroarylation. a, Previous work: Reported visible-light-driven unnatural biocatalysis relies on the excitation of EDA complexes accessing to reductive biotransformations. b, This work: Direct visible-light-excitation of FMN-dependent ene-reductase enables an unnatural redox-neutral hydroarylation of alkenes with arenes. FAD(ox), (oxidation form of) flavin adenine dinucleotide; FMN(ox/hq/sq), (oxidation/hydroquinone/semiquinone form of) flavin mononucleotide; NADPH, reduced nicotinamide adenine dinucleotide phosphate; EDA, electron donor-acceptor.

 

Indeed, I believed that this system was highly feasible as supported by various findings from both nature and synthetic chemistry. For instance, Beisson and co-workers recently discovered a fatty acid photodecarboxylase from Chlorella variabilis (CvFAP). The key enzymatic mechanism involves the visible-light-excited flavin adenine dinucleotide (FAD) cofactor initiating the single electron transfer oxidation of fatty acids[7]. Furthermore, synthetic flavin derivatives have been extensively utilized as organic photoredox catalysts. These examples provided valuable insights and established a foundation for exploring the potential of the proposed photoenzymatic system.

       My doctoral student Beibei Zhao, one of the original members of my lab, eagerly embraced the task and demonstrated remarkable dedication and intelligence. He came across a very inspiring publication from the Shu group at SUSTech, which reported a radical coupling between electron-rich arenes and alkenes enabled by the photoexcitation of an acridinium-based organic dye[8]. Inspired by this work, our team decided to focus on using 3-methyoxythiophene 1a and α-methyl styrene 2a as the model substrates and finally developed this redox-neutral enantiodivergent biotransformation.

       The paper thoroughly outlines the scientific challenges involved in this reaction and provides comprehensive details on how we effectively addressed them. However, it is worth noting that the efforts invested and the technical difficulties faced during the research went beyond what can be described in the paper. This is especially significant considering the context of being in a newly established lab and being a novice mentor with a fresh Ph.D. student.

This journey of collaboration has been an incredibly pleasant one. Prof. Binju Wang from Xiamen University has been our dedicated long-term collaborator for computational calculations. He and his Ph.D. student Jianqiang Feng conducted professional computational studies to unravel the reaction mechanism and explain the origin of enantioselectivity. We have also received valuable support from Prof. Changlin Tian at the University of Science and Technology of China. Under the guidance of Prof. Tian, Dr. Lu Yu and Mr. Aokun Liu performed meticulous EPR experiments, demonstrating the key radical pathway. Thanks to the hard work of the team, we were able to complete the manuscript, allowing us to submit the paper on Jan. 22nd, which coincided with the first day of the Chinese Lunar New Year in 2023.

As my first independent research paper, I am thrilled to see the recognition from colleagues regarding the novelty of this work, leading to its publication in Nature Catalysis. This encouragement motivates me to further explore non-natural biocatalytic systems that feature novel mechanisms and are difficult to achieve by traditional catalysis.

For details of this work, please see our paper titledDirect Visible-Light-Excited Flavoproteins for Redox-Neutral Asymmetric Radical Hydroarylation” in Nature Catalysis. https://www.nature.com/articles/s41929-023-01024-0

References

[1] Huang, X. & Meggers, E. Asymmetric Photocatalysis with Bis-cyclometalated Rhodium Complexes. Acc. Chem. Res. 52, 833-847, (2019).

[2] Harrison, W., Huang, X. & Zhao, H. Photobiocatalysis for Abiological Transformations. Acc. Chem. Res. 55, 1087-1096, (2022).

[3] Biegasiewicz, K. F. et al., Photoexcitation of Flavoenzymes Enables a Stereoselective Radical Cyclization. Science 364, 1166-1169 (2019).

[4] Huang, X. et al. Photoenzymatic enantioselective intermolecular radical hydroalkylation. Nature 584, 69-74, doi:10.1038/s41586-020-2406-6 (2020).

[5] Peng, Y. et al. Photoinduced Promiscuity of Cyclohexanone Monooxygenase for the Enantioselective Synthesis of α-Fluoroketones. Angew. Chem. Int. Ed. 61, e202211199 (2022).

[6] Duan, X. et al. A Photoenzymatic Strategy for Radical-Mediated Stereoselective Hydroalkylation with Diazo Compounds. Angew. Chem. Int. Ed. Engl. 62, e202214135 (2023).

[7]     Sorigue, D. et al. An Algal Photoenzyme Converts Fatty Acids to Hydrocarbons. Science 357, 903-907 (2017).

[8] Chen, B. H., Du, Y. D. & Shu, W. Organophotocatalytic Regioselective C-H Alkylation of Electron-Rich Arenes Using Activated and Unactivated Alkenes. Angew. Chem. Int. Ed. Engl. 61, e202200773 (2022).

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