SYNLETT0936-52141437-2096 © Georg Thieme Verlag Stuttgart · New York 2020, 31, 248–254 synpacts 248 en Syn lett T. K. Hyster Synpacts Radical Biocatalysis: Using Non-Natural Single Electron Transfer Mechanisms to Access New Enzymatic Functions Todd K. Hyster* O O Department of Chemistry, Princeton University, ±e Princeton, NJ 08544, USA R R R + R [email protected] LG substrate non-natural R promiscuous electron transfer R oxidoreductases mechanisms racemic enantioenriched O R R R prochiral radical Received: 13.03.2019 Accepted after revision: 14.04.2019 Published online: 07.05.2019 DOI: 10.1055/s-0037-1611818; Art ID: st-2019-p0146-sp Abstract Exploiting non-natural reaction mechanisms within native enzymes is an emerging strategy for expanding the synthetic capabili- ties of biocatalysts. When coupled with modern protein engineering techniques, this approach holds great promise for biocatalysis to ad- dress long-standing selectivity and reactivity challenges in chemical synthesis. Controlling the stereochemical outcome of reactions involv- ing radical intermediates, for instance, could benefit from biocatalytic solutions because these reactions are often difficult to control by using existing small molecule catalysts. General strategies for catalyzing non- Todd Hyster is a native of Minnesota and obtained his BS in chemistry natural radical reactions within enzyme active sites are, however, unde- from the University of Minnesota – Twin Cities in 2008. He received his veloped. In this account, we highlight three distinct strategies devel- PhD from Colorado State University in 2013, working in the labs of oped in our group that exploit non-natural single electron transfer Tomislav Rovis. He then went on to be a NIH postdoctoral fellow in the mechanisms to unveil previously unknown radical biocatalytic func- labs of Frances Arnold at Caltech. Since the fall of 2015, he has been an tions. These strategies allow common oxidoreductases to be used to assistant professor in the chemistry department at Princeton University. address the enduring synthetic challenge of asymmetric hydrogen atom transfer. 1 Introduction 2 Photoinduced Electron Transfer from NADPH strategy for addressing this limitation is mining nature to 3 Ground State Electron Transfer from Flavin Hydroquinone discover new types of chemical reactivity.2 Although this 4 Enzymatic Redox Activation in NADPH-Dependent Oxidoreduc- tases approach has proven to be effective, it is intrinsically limit- 5 Conclusion ed to reactivity patterns evolved in nature. To realize the This document was downloaded for personal use only. Unauthorized distribution is strictly prohibited. full potential of enzymes to improve chemical synthesis, Key words asymmetric catalysis, radical reactions, biocatalysis, pho- approaches need to be developed in which reactivity pat- tochemistry, single electron transfer, catalytic promiscuity terns currently unknown to nature may be catalyzed.3 Strategies for enabling enzymes to catalyze non-natural 1 Introduction reactions have primarily relied on the catalytic promiscuity inherent to metalloenzymes and hydrolases.3c,4 In the case Enzymes are attractive catalysts for asymmetric synthe- of metalloenzymes, organometallic cofactors located within sis as they provide unmatched control over the stereochem- enzyme active sites are responsible for providing the de- ical outcome of chemical reactions. As a consequence, en- sired reactivity while the protein scaffold provides enantio- zymes are increasingly used in chemical synthesis, includ- and diastereoselectivity (Figure 1).5 Alternatively, the cata- ing in the preparation of pharmaceutically valuable lytic promiscuity of hydrolases relies on the ability of the molecules.1 A factor limiting the expanded use of enzymes oxyanion hole to catalyze other reactions that proceed via in chemical synthesis is the narrow breadth of distinct oxyanionic intermediates (Figure 1).6 Inspired by these ex- chemical reactions catalyzed by enzymes. The primary amples, we sought to develop strategies to enable enzymes © Georg Thieme Verlag Stuttgart · New York — Synlett 2020, 31, 248–254 249 Syn lett T. K. Hyster Synpacts to catalyze new reactions mediated by non-natural free- 2 Photoinduced Electron Transfer from radical intermediates. These represented a family of reac- NADPH tivity for which robust enzymatic catalysts had not been developed. Moreover, as controlling the stereochemical out- Nicotinamide adenine dinucleotide (NADH) is a biologi- come of radical reactions remains a challenge for small cal reductant that functions as a hydride donor for many molecule catalysts, we anticipated that a biocatalytic solu- synthetically valuable oxidoreductases.10 In an effort to un- tion would be of high synthetic value (Figure 1).7 derstand its biological function, a variety of NADH ana- Central to our goal is the development of general strate- logues have been prepared, including N-benzyl nicotin- gies for forming radical species in enzyme active sites. We amide (BNAH) and Hantzsch esters.11 While these ana- ruled out intercepting radical intermediates formed by logues typically function as hydride donors in chemical monooxygenases, such as P450s, because of their exceed- synthesis, Fukuzumi found that when irradiated with visi- ingly short lifetimes.8 Alternatively, the radical SAM family ble light, BNAH was promoted to is excited triplet state in of enzymes, while capable of generating synthetically ver- which it would function as a potent single electron reduc- 12 satile radical intermediates, are typically plagued by poor tant (Eox* = –2.60 V). This function was demonstrated in a substrate scope and sensitivity to oxygen, thus limiting series of dehalogenation reactions in which BNAH* can re- their application in chemical synthesis.9 Instead, we sought ductively cleave C–Br bonds to afford alkyl radicals that to develop a biocatalytic method that would adopt the char- readily abstract a hydrogen atom from BNAH to afford the acteristics of the most ubiquitous enzymes in chemical syn- reduced product (Scheme 1). We recognized that if NADH thesis: namely stability and substrate promiscuity. We displayed a similar reactivity profile, it would be possible to identified commonly used oxidoreductases as attractive render radical dehalogenations asymmetric using nicotin- scaffolds for their broad use in organic synthesis (Figure 1). If a non-natural electron transfer event could be induced to Fukuzumi (1983) occur within their active sites, it would enable existing col- O O Br H lections of these enzymes to be quickly applied to non-nat- ν NH2 > 360 nm h NH2 ural reactions. This approach would also significantly lower pyridine (5 mol%) N N the barrier for reaction adoption because libraries of oxidore- MeCN Br Bn Bn ductases can be purchased from many chemical vendors. 80% yield Scheme 1 Fukuzumi’s photodehalogenation of benzyl bromide with BNAH This document was downloaded for personal use only. Unauthorized distribution is strictly prohibited. Figure 1 Strategies for revealing non-natural enzyme functions © Georg Thieme Verlag Stuttgart · New York — Synlett 2020, 31, 248–254 250 Syn lett T. K. Hyster Synpacts amide-dependent ketoreductases (KREDs). In this scenario, spectrum was subtracted from the NADPH/KRED/substrate the protein scaffold would be responsible for controlling spectrum, a new broad absorption feature at 395 nm was the delivery of a hydrogen atom to an intermediary prochi- observed. We attributed this new absorption feature to the ral radical. Asymmetric delivery of hydrogen atom rep- formation of an electron donor–acceptor (EDA) complex resents a long-standing challenge in chemical synthesis for formed between NADPH and the substrate within the en- which small molecule reagents have proven largely ineffec- zyme active site. Control experiments confirm that this tive at delivering products with synthetically useful levels complex does not form in the absence of enzyme, providing of enantioselectivity.13 a mechanism for gating electron transfer to occur only in We began by testing a collection of engineered KREDs the enzyme. Although this mechanism for electron transfer from Codexis.14 As a model reaction we explored the deha- has experienced renewed interest in the preceding decade, logenation of -bromo--aryl lactones because of their it has never before been observed for biocatalytic reac- modest reduction potentials and the presence of a carbonyl tions.17 functional group permitting substrate binding. By using A survey of the substrate scope of the Codexis KRED standard reaction conditions for KREDs and irradiating P2D03 established the generality of this approach (Scheme with blue light, the desired dehalogenation product was ob- 4). A variety of substituted -aryl--valerolactones can be served, with Codexis P2D03 providing product in 81% yield effectively dehalogenated under the standard reaction con- with 98:2 er favoring the R enantiomer (Scheme 2). Struc- ditions. Moreover, -butyrolactones can also be dehaloge- tural information, generously provided by Gjalt Huisman at nated, albeit with slightly diminished enantioselectivities. Codexis, revealed the most active KREDs to be variants of Finally, substrates with -benzylic substituents can also be the short-chain dehydrogenase from L. kefir which possess- dehalogenated in modest yield but good levels of enantiose- es an expanded active site.15 With this knowledge in hand, lectivity. The short chain dehydrogenase from Cupriavidus we engineered a KRED from