Ketoreductase Catalyzed (Dynamic) Kinetic Resolution for Biomanufacturing of Chiral Chemicals

Biocatalyzed asymmetric reduction of ketones is an environmentally friendly approach and one of the most cost-effective routes for producing chiral alcohols. In comparison with the well-studied reduction of prochiral ketones to generate chiral alcohols with one chiral center, resolution of racemates by ketoreductases (KREDs) to produce chiral compounds with at least two chiral centers is also an important strategy in asymmetric synthesis. The development of protein engineering and the combination with chemo-catalysts further enhanced the application of KREDs in the efficient production of chiral alcohols with high stereoselectivity. This review discusses the advances in the research area of KRED catalyzed asymmetric synthesis for biomanufacturing of chiral chemicals with at least two chiral centers through the kinetic resolution (KR) approach and the dynamic kinetic resolution (DKR) approach.


INTRODUCTION
Since the discovery of microbial resolution of tartaric acid in 1858 by Louis Pasteur (Pasteur, 1858), the kinetic resolution (KR) of stereoisomers has become as one of the most important methods to obtain optical pure compounds, especially in the pharmaceutical industry due to the regulatory requirements for single stereoisomer of chiral drugs. In the kinetic resolution, the enantiomers of a racemic substrate react by chiral catalysts or enzymes at different rates; one of the enantiomers with a much faster rate completes the reaction first while the other enantiomer with a slower reaction rate is left (Figure 1). In theory, the maximum yield of the formed product is 50% (Keith et al., 2001). There are some common kinetic resolution reactions, such as 1) acylative/deacylative resolution (Sheldon, 1996;Somfai, 1997); 2) oxidative kinetic resolution (Komatsu et al., 1992;Hashiguchi et al., 1995); 3) reductive kinetic resolution (Harada et al., 1990;Kurosu et al., 1998); 4) organometallic kinetic resolution (Hayashi et al., 1986;Morken et al., 1994); 5) kinetic resolution of epoxides (Martin et al., 1981;Hodgson et al., 1996).
Owing to the limitation of a maximum 50% theoretical yield, it is urgently needed to increase the yields of KR methods. The first clear dynamic kinetic resolution (DKR) was achieved by Weygand and coworkers in 1966, in which the synthesized dipeptides from racemic azlactones were unequal (Weygand et al., 1966). As shown in Figure 1, DKR also relies on enantiomers of a racemic substrate that react (by catalysts) at different rates, but the great difference of DKR is the rapidly dynamic equilibrium (occurred spontaneously, or by catalysis) between the enantiomers of a racemic substrate. Under this circumstance, the certain production of the resolution reaction can achieve 100% theoretically.
Ketoreductases catalyzed reductions of ketones into chiral alcohols are the widely used and important biocatalytic reactions (Kaluzna et al., 2005a;Brenna, 2013;Hollmann et al., 2021;Li et al., 2021;Musa, 2022). The increasing studies on protein engineering or directed evolution (the Nobel prize in chemistry 2018) (Arnold, 2019) of KREDs, and combining KREDs with other catalysts, such as biocatalysts, metal catalysts (the Nobel prize in chemistry 2001) (Noyori, 2002), organocatalysts (the Nobel prize in chemistry 2021) (Hargittai, 2022), and photocatalysts (DeHovitz et al., 2020), further encourage the application of KREDs in asymmetric synthesis. For instance, several blockbuster drugs, such as atorvastatin, montelukast, and crizotinib, could be synthesized by the KREDs catalyzed process in the pharmaceutical industry (Bornscheuer et al., 2012;Devine et al., 2018;Campos et al., 2019). In comparison with the wellknown reduction of prochiral ketones to chiral alcohols with one chiral center, (dynamic) kinetic resolution of racemates by KREDs to produce chiral compounds with at least two chiral centers is also an attractive approach in asymmetric synthesis (Stewart, 2001;Kalaitzakis and Smonou, 2013;Applegate and Berkowitz, 2015;Yang et al., 2022). In this review, we describe the advances in the research area of KREDs catalyzed asymmetric synthesis of chiral compounds with at least two stereocenters through KR and DKR approaches. Of the DKR, the dynamic equilibrium between the enantiomers of substrates could be archived by spontaneous reaction, organocatalysis, and photoredox reactions. For a clearer overview, the ketoreductases catalyzed oxidative KR of chiral alcohols, and the desymmetrization of achiral materials (Chen et al., 2019;Li J. et al., 2020) were not involved in this review.
In 1985 and 1986, the KR of bicyclic ketones with [3.2.0]-ring system were also reported (Butt et al., 1985;Davies et al., 1986). As shown in Figure 3A, reduction of the rac-5a with an alcohol dehydrogenase from Thermoanaerobium brockii (TabADH) gave alcohol (1S,5R,6S)-6a in high optical purity (>95% ee) (Butt et al., 1985). However, another enzyme 3α,20β-hydroxysteroid dehydrogenase (3α,20β-HSDH) from Streptomyces hydrogenans FIGURE 1 | Kinetic resolution (KR) and dynamic kinetic resolution (DKR) based on ketoreductases catalyzed reductions. For both KR and DKR, the racemic substrate was used as the start material, and only one of the enantiomers with a faster rate was highlighted in KR process for a clear explanation.
In addition to the kinetic resolution of the aforementioned cyclic ketones, some studies about the KR of aliphatic ketones by ketoreductases have also been reported. For example, Murry and coworkers reported that the baker's yeast-mediated reduction could resolve the β-keto esters 19a-e by conversion of (6S)ketones to corresponding β-hydroxy esters (6S,3′S)-20a-e (Hudlicky et al., 1991) ( Figure 5A). Interestingly, the selective reduction indicated that the microbial reaction could recognize the remote chiral centers beyond the reactive groups. Similar KR reactions were done by other groups, who also used baker's yeast for kinetic resolution of β-keto esters (Eh and Kalesse, 1996) ( Figure 5B).

KETOREDUCTASES INVOLVED IN DYNAMIC KINETIC RESOLUTION PROCESSES
For kinetic resolution, one of the most unfavorable disadvantages is the yield limitation of <50%. In addition to that, the low selectivity of ketoreductases might also produce the undesired stereoisomers. In addition, the need for the separation of resultant products and unreacted substrates is also an issue. To overcome the disadvantages in KR, especially the yield limitation, one of the most important strategies that have been developed is dynamic kinetic resolution. In recent decades, ketoreductases based on DKR of racemic substrates to obtain products with two chiral centers have appeared a lot. Racemic functional compounds such as ketoesters, ketoamines, ketothioesters, keto arylphosphonates, and cyclic ketones could be efficiently resolved with excellent yields and stereoselectivities.
As mentioned in the introduction section, the DKR relies on the rapid interconversion (occurring spontaneously or by catalysis) of substrate enantiomers. For racemic ketones capable of spontaneous racemization or base-catalyzed racemization, their common feature is that there is an acidic proton at the α-position of ketones. The stereochemistry can often be established on the adjacent carbon, due to keto-enol tautomerism and selective reduction one of the ketone enantiomers by enzymes .

Ketoreductases Involved in DKR of α-Heteroatom-Substituted β-Keto Esters
The C-F bond of α-fluoro-β-keto esters can affect the physical and chemical properties of the compound tremendously (O'Hagan, 2008), and fluorine also has a great influence on the biological activity of pharmacologically active compounds and drugs (Meanwell, 2018). Accordingly, Green and coworkers focus on selective catalytic reduction of α-fluoro-β-keto esters (23a-i) by DKR (Green et al., 2019) ( Figure 6A). Using commercially available ketoreductases, most of the substrates could be reduced to corresponding α-fluoro-β-hydroxy esters in high optical purities and yields. Importantly, either anti-or syndiastereomers could be produced with high diastereomeric and enantiomeric excess ( Figure 6A), depending on the ketoreductases used. The authors also utilized in situ 19 F NMR spectroscopy to monitor the enzyme reaction in real-time, which is convenient for analyses. The DKR of α-chloro-β-keto ester (23k) was also reported by Stewart and coworkers (Kaluzna et al., 2005b). In their study, syn-(2R,3S)-24k and syn-(2S,3R)-24k were obtained by recombinant reductases from Saccharomyces cerevisiae, and a chemoenzymatic route for synthesis of the side chain of Taxol (25) was developed. In addition, Berkowitz and coworkers also succeeded in gaining optical pure (2S,3R)-24k (95% de, 99% ee) by DKR of 23k using an alcohol dehydrogenase from Clostridium acetobutylicum (CaADH) (Applegate et al., 2011).
The biocatalytic reduction of racemic α-alkoxy-β-keto esters via DKR to prepare 1,2-diols was first reported by Li C. et al. (2020). The structure of chiral 1,2-diols consists widely of natural products and also serves as building blocks for several useful compounds.
The γ-lactones are usually found in natural products and methods for selective construction of these compounds are highly desired. Recently, Scheidt and coworkers developed a chemoenzymatic strategy for the synthesis of γ-lactones based on enzymatic DKR and intramolecular cyclization (Maskeri et al., 2020). Commercially available KRED-P2-C02 (Codexis) was chosen to catalyze the reduction of substrates (43) and gave the γ-lactones (44) with high enantio-and diastereoselectivity. They also established that the optimal solvent condition for catalytic reduction was 1:3:6 isopropanol:1-chlorododecane: 0.1 M tris buffer at pH = 9.
As is well-known, ketoreductase domains (KR domains) were involved in modular polyketide synthases (PKS). Ludeke and coworkers demonstrated that Tyl-KR1, an isolated PKS ketoreductase domain in the biosynthesis of tylactone, has reductive activity toward α-alkyl-β-keto thioesters 45a and 45b ( Figure 7B), but with medium selectivity (Häckh et al., 2013). In addition, through structure-activity relationship study and rational engineering, the Keatinge-Clay group investigated the stereocontrols of ketoreductase domains of modular PKS. The reverse stereoselectivity of EryKR1, from (2S,3R)-to (2S,3S)-, was found in the reduction of 45b-d by mutations (Bailey et al., 2016). Bailey and coworkers also reported the engineering of modular PKS ketoreductase domains for altering their stereochemical controls (reduction of 45h for an example) (Drufva et al., 2020).

Ketoreductase Involved in DKR of α-Alkyl-β-Keto Amides and α-Substituted β-Arylphosphonates
Chiral α-substituted-β-hydroxy amides are important building blocks of some biologically active compounds . The stereoselectivity synthesis of these compounds is still a challenge although various synthetic strategies have been developed. Lavandera and coworkers reported that these compounds can be acquired by ADHs catalyzed DKR of αalkyl-β-ketone amides (Méndez-Sánchez et al., 2019) (47a-g in Figure 8A). Through screening a set of ADHs, syn-α-alkyl-βhydroxy amides could be obtained with high diastereo-and enantioselectivities ( Figure 8A). In addition, the ADH-A from Rhodococcus ruber can get access to the diastereoisomers (2R,3S)-48a-f with 59-95% de and >99% ee at 25 mM scales.
Vicinal amino alcohols are useful building blocks in the synthesis of natural products and pharmaceuticals (Orcel and Waser, 2015). For example, the (1R,2S)-55a is an important intermediate for the synthesis of compound 56, a mGluR5 allosteric modulator developed for the treatment of neurological disorders. DKR of N-protected α-amino ketones is an interesting strategy for preparation of the chiral amino alcohols, however, only a few studies on DKR of N-protected α- amino β-keto esters have been reported (Zou et al., 2018;Giannopoulos et al., 2020). To prepare optically pure (1R,2R)-55a, the scientists at Bristol-Myers Squibb recently developed a DKR process of N-protected α-amino ketones . Through screening, hundreds of commercial ketoreductases and microbial strains, the desired syn-(1R,2S)-55a and the other three diastereoisomers could be obtained with excellent enantiomeric excess and diastereoisomeric excess through DKR. The obtained chiral alcohols (1R,2S)-55a-b with >99% de and >99% ee by KRED-145 catalyzed DKR has been Similarly, the scientist at Merck and Codexis also reported the DKR of N-protected α-amino ketone 57 (Xu et al., 2018). Unlike the compounds 54a-b, the racemization of which occurs at pH 7, the fast racemization of 57 could only be realized at elevated temperature (≥45°C) and high pH (≥ pH 10), while these conditions were challenges for ketoreductases. Thus, through Frontiers in Bioengineering and Biotechnology | www.frontiersin.org June 2022 | Volume 10 | Article 929784 10 the incorporation of structurally guided design and directed enzyme evolution, a ketoreductase suitable for high-pH and elevated-temperature conditions was developed. The DKR of 57 by evolved KRED-p301 gave the desired (1R,2R)-58 with >99% ee and >100:1 dr. Finally, a chemoenzymatic route for the synthesis of Vibegron (59), an approved drug for the treatment of overactive bladder, was developed based (1R,2R)-58 from DKR.
The chiral cyclic alcohols are also building blocks in the synthesis of pharmaceuticals. In 2016, the scientists at Bristol-Myers Squibb and Merck reported the synthesis of GPR40 modulator (71)  and GPR40 partial agonist MK-8666 (74) (Hyde et al., 2016) using chiral cyclic alcohols obtained from DKR of 69 and 72, respectively ( Figure 10). In the first study, a series of ketoreductases were screened for DKR of 69. Surprisingly, the glucose dehydrogenase (GDH) displayed the highest selectivity among the tested enzymes and gave the desired (3R,4R)-70 with 37.5:1 dr and >99% ee . In the next study, the scientists at Merck screened several commercial KREDs to produce 73, a key intermediate for MK-8666, by DKR of 72 (Hyde et al., 2016). Stereodivergent synthesis of all four diastereoisomers of 73 was achieved by KREDs with different stereo preferences and the desired (6S,7S)-73 was obtained with >30:1 dr and >99% ee by optimized KRED 264. And a chemoenzymatic route for the synthesis of MK-8666 was developed based on DKR.
At the same time, the couple of ketoreductases catalyzed DKR and nitrile hydratases/amidases catalyzed hydrolysis was reported to provide enantiopure β-hydroxy carboxylic acids by Rebolledo and coworkers . For the first step, the authors utilized commercially available KREDs to reduce 2oxocycloalkanecarbonitriles (75a-c in Figure 10). The diastereoisomers (1R,2R)-76a-c and (1S,2S)-76a-c could be obtained with high selectivity (88:12 to >99:1 dr and 95% to >99% ee) by using different KREDs. For the next step, wellstudied Rhodococcus rhodochrous were used for hydrolysis of 2oxocycloalkanecarbonitriles. Finally, the bio-cascade process gave the β-hydroxy carboxylic acids with excellent overall yield and optical purity.
As shown in Figure 10, another type of substrate for DKR was α, β-unsaturated cyclic ketones (Kosjek et al., 2006). Transition metal-based hydrogenation of 77a-b gave the allylic alcohol in low diastereomeric excess. In contrast, the enzymatic reduction of 77a-b resulted in both high enantio-and diastereoselectivity. The obtained chiral allylic alcohols are important building blocks in the synthesis of natural compounds.
In contrast to the aforementioned and well-studied DKR of αsubstituted ketones, DKR of β-substituted ketones is still challenging since the lack of racemization methods for static β-keto stereocenters under mild conditions. In 2020, the Macmillan and Hyster groups describe a general method for the racemization of traditionally static β-keto stereocenters by a combination of photoredox catalysis and organocatalysis (DeHovitz et al., 2020). The method was then subjected to ketoreductases catalyzed reduction to achieve the DKR of βsubstituted ketones 79. Related alcohols (1S,3R)-80 could be obtained with high yields (68%-82%) and high selectivities (up to >20:1 dr, >99:1 er) by a combination of photoredox-and enzyme-catalysis. The stereodivergent synthesis of all four stereoisomers of 80 could also be achieved by using ketoreductases with different stereopreferences. In addition, other enzymes, such as transaminases, could be incorporated Frontiers in Bioengineering and Biotechnology | www.frontiersin.org June 2022 | Volume 10 | Article 929784 into this DKR process instead of ketoreductases, indicating the power of this strategy toward static stereocenters.

DISCUSSION AND CONCLUSION
Biocatalyzed (dynamic) kinetic resolution is an environmentally friendly strategy for the preparation of chiral compounds. In this review, we summarized a series of examples of KREDs catalyzed kinetic resolution (KR) or dynamic kinetic resolution (DKR).
With the development of enzyme mining and protein engineering, KREDs have been successfully applied to KR or DKR of several ketone compounds, including but not limited to α-substituted β-keto esters, α-alkyl-β-keto amides, α-substituted β-arylphosphonates, α-substituted β-ketones, and cyclic ketones. All the summarized KR or DKR reactions could produce corresponding chiral products with at least two chiral centers.
In addition, the KRED-based KR and DKR also show potential for industrial applications. Despite this, challenges or limitations still remain, especially in DKR. As mentioned, the KREDs catalyzed DKR relies on efficient racemization between two enantiomers and selective reduction of one of them by KREDs. First, racemization in most reviewed DKR is spontaneous, a general racemization method for substrates that cannot be racemized spontaneously is lacking. Although Macmillan and Hyster groups showed a remarkable example for racemization of static stereocenters (DeHovitz et al., 2020), other studies are rarely reported, especially in comparison with the successful application of metal catalysts for racemization of secondary alcohols in lipase-mediated DKR (Zhu, 2021). Second, as reviewed, a lot of screening efforts were performed to get the desired alcohols in KRED catalyzed reduction reactions. However, lower yields or wrong stereoisomers were obtained in some cases.
Thus, the ketoreductases with higher selectivity, activity, stability, and tolerance to adverse reaction conditions are still needed. Nevertheless, the increasing studies on protein engineering or directed evolution of KREDs, and combining KREDs with other catalysts, such as biocatalysts, metal catalysts (Burda et al., 2008;Ríos-Lombardía et al., 2016), organocatalysts (Baer et al., 2009;Rulli et al., 2011;Rudroff et al., 2018), photocatalysts (DeHovitz et al., 2020), and electrocatalysts (Wan et al., 2021), in one-pot or by consecutive reactions, further encourage the application of KREDs in asymmetric synthesis and also (dynamic) kinetic resolution. We finally believe that the novel methods of racemization, the further engineering of KREDs, and the combination of KREDs with other catalytic systems will continue to encourage the applications of KREDs catalyzed (dynamic) kinetic resolutions for biomanufacturing of chiral chemicals.

AUTHOR CONTRIBUTIONS
CH, JL, JF, and XJ conducted the literature search and wrote the original manuscript. ZZ, SY, and BQ conceived the idea and provided financial support. CH, JL, and BQ polished the final manuscript. All authors contributed to the article and approved the submitted version.