Enantioselective Reduction of α,β-Unsaturated Ketones and Aryl

Letter Cite This: Org. Lett. XXXX, XXX, XXX−XXX

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Enantioselective Reduction of α,β-Unsaturated Ketones and Aryl Ketones by Perakine Reductase Sheng Cai,†,¶ Nana Shao,‡,¶ Yuanyuan Chen,†,¶ Anbang Li,§ Jie Pan,† Huajian Zhu,⊥ Hongbin Zou,‡ Su Zeng,† Lianli Sun,*,† and Jinhao Zhao*,§

Org. Lett. Downloaded from pubs.acs.org by BETHEL UNIV on 05/02/19. For personal use only.

†

Institute of Drug Metabolism and Pharmaceutical Analysis, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, China ‡ Institute of Drug Discovery and Design, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, China § Institute of Pesticide and Environmental Toxicology, Ministry of Agriculture Key Lab of Molecular Biology of Crop Pathogens and Insects, Zhejiang University, Hangzhou 310029, China ⊥ School of Medicine, Zhejiang University City College, Hangzhou 310015, China S Supporting Information *

ABSTRACT: This report describes the enantioselective reduction of structurally diverse α,β-unsaturated ketones and aryl ketones by perakine reductase (PR) from Rauvolf ia. This enzymatic reduction produces α-chiral allylic and aryl alcohols with excellent enantioselectivity and most of the products in satisfactory yields. Furthermore, the work demonstrates 1 mmol scale reactions for product delivery without any detrimental effect on yield and enantioselectivity. The catalytic mechanism, determined by 3D-structure-based modeling of PR and ligand complexes, is also described.

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unsatisfactory enantioselectivity, and low yield.4 An especially challenging process is competitive 1,2- and 1,4-reduction, used for preparing chiral allylic alcohols through asymmetric 1,2reduction of α,β-unsaturated ketones.5 Recently, the research groups of Lipshutz and Zhu greatly advanced enantioselective reduction of enones by exploiting CuH and NiH, respectively.6 Despite this progress, the reactions still had to be carried out at a low temperature (−25 °C), and the toxic metal issue remained unsolved. Compared to chemical methods, enzymatic methods are a green and efficient alternative for the preparation of chiral alcohols.7 Currently, the two major approaches are enzymatic kinetic resolution of racemic alcohols and enzyme-catalyzed asymmetric reduction of prochiral ketones. Theoretically, the maximum yield of enzymatic resolution is 50%, which greatly restricts its application.8 Through great efforts, various enzymes have been developed for catalyzing the asymmetric reduction of prochiral aryl ketones.9 However, enzymatic 1,2-reduction of α,β-unsaturated ketones has rarely been reported. To date, few studies have attempted this approach, using a very limited number of enones.10 Moreover, existing ketone reductases have some intrinsic limitations, including narrow substrate spectrum, low catalytic efficiency toward certain substrates, and thermal instability. There is, therefore, a requirement for new enzymes with a robust capacity for synthesis of chiral allylic alcohols and aryl alcohols.11

mong the chiral alcohols, chiral allylic and aryl alcohols are groups of specific interest for chemists. Chiral allylic alcohols are structural subunits frequently found in natural products and pharmaceuticals,1 for instance, epothilone A, bervastatin, and calcipotriol (Figure 1A). Chiral aryl alcohols are key intermediates in the synthesis of several pharmaceuticals,2 including mirabegron (β3 receptor agonist), ezetimibe, and crizotinib (Figure 1B). Asymmetric reduction of prochiral ketones is the most efficient approach to obtain these chiral alcohols. Transition metals are the major catalysts used for the production of a series of chiral alcohols by chemical means.3 However, these approaches suffer from several drawbacks; for example, toxicity to the environment, strong chemical reducing conditions,

Figure 1. Typical chiral allylic alcohol (A) and aryl alcohol (B) structural elements in pharmaceuticals. © XXXX American Chemical Society

Received: March 17, 2019

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DOI: 10.1021/acs.orglett.9b00950 Org. Lett. XXXX, XXX, XXX−XXX

Letter

Organic Letters Perakine reductase (PR) converts the aldehyde alkaloid perakine to its corresponding alcohol raucaffrinoline in the presence of the cofactor NADPH through a side branch of the main ajmaline pathway of the plant Rauvolfia serpentina.12 Based on sequence alignment, PR has been identified as the first member of aldo-ketone reductase (AKR) 13D family.13 The enzyme’s aldehyde substrate spectrum and kinetic behavior have been comprehensively studied. PR accepts relatively small nitrobenzaldehydes over medium-sized cinnamic aldehydes and structurally bulky alkaloids.12 The demonstrated catalytic promiscuity toward aldehydes suggested to us that PR might also have activity toward ketones, which would grant it the potential to be of use in the synthesis of more valuable chiral alcohols. Because of the notable value of the chiral aryl and allylic alcohols, in this work, we focused on PR-catalyzed conversion of prochiral α,β-unsaturated and aromatic ketones to the desired chiral alcohols (Scheme 1).

Scheme 2. PR Catalyzed Asymmetric Reduction of Ketonesa,b,c

Scheme 1. PR Catalyzed Asymmetric Reduction of Enones and Aromatic Ketones, Leading to α-Allylic Alcohols and αAromatic Alcohols

Recombinant His6-PR was expressed, purified, and analyzed as previously reported.12 A collection of structurally diverse enones and aromatic ketones was used to explore the recombinant PR’s ketone catalytic capability. Glucose dehydrogenase from Bacillus megaterium (BmGDH) was used as the cofactor regeneration enzyme. Surprisingly, all the evaluated substances, which included eight β-substituted enones (S1−S8) and ten acetophenone derivates (S9−S18), were converted by PR (Scheme 2, Table S1), which exhibited a robust capacity to reduce ketones with excellent enantiomeric excesses (ee’s) on the order of 99%. Particularly, when compared to the reported samples that are active toward enones,10 PR exhibits a relatively boarder substrate scope and higher stereoselectivity. Moreover, PR displayed excellent activity within a relatively wide temperature range (30−50 °C) and maintained >80% of its activity under pH 6.5−8.5 (Figure S1). The high thermal stability and pH tolerance indicate the potential for industrial application. Among the evaluated substances, 4′-nitroacetophenone (S12) was found to be the best ketone substrate, with yield and ee values exceeding 99%. Furthermore, it had a Km value of 3.22 mM and catalytic efficiency (kcat/Km) of 0.08 mM−1 s−1 (Figure S2, Table S2). However, kcat/Km of PR toward 4′nitrobenzaldehyde is 29.5 mM−1 s−1,12 which is ∼369-fold higher than that of 4′-nitroacetophenone. This significantly higher activity of aldehydes compared to that of ketones may largely be attributed to the more active aldehyde group. Varying the substituents on the aryl ring highly impacted product yields. With the exception of allylic alcohol (2), the electronwithdrawing groups consistently gave a higher yield, while the electron-donating groups led to lower yields. Compared with (S)-phenylethanol (9), derivates with electron-withdrawing groups (10−12, 14, 16) on the aromatic ring all achieved yields exceeding 92%. In contrast, inclusion of

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Reactions were carried out using a concentration of 0.8 mM in 5 mL volume. bYield is the analytical yield of product in the enzymatic reaction system (aqueous phase), determined by reverse phase HPLC. c Optical purities were determined by chiral HPLC analysis using purified product. dAbsolute configuration was assigned by comparing the retention time with that of the standard chiral compound as determined by chiral HPLC.

electron-donating groups (13, 15) sharply decreased yields, which were as low as 21.7 and 14%. Both of the additional αhalohydrins (17, 18) were found achieving 99% yield. It is possible that as the CC bond disrupted direct conjugation between the aromatic ring and the carbonyl group, the influence on the yield exerted by the substitution on β-aryl ring of enones is milder than that exerted by acetophenone derivates. Thus, with the exception of (7), where the bulky strong electrondonating substitution led to a relatively low yield (18%), most of the secondary allylic alcohols gave intermediate yields ranging from 50 to 89%. Moreover, changing the position of the substituent did not alter the outcome of the reaction. In addition, we carried out two sets of 1 mmol scale reactions using allylic ketone (S1) and aryl ketone (S12) as the two typical substrates (Supporting Information). Both of the desired products (1) and (12) were produced without any detrimental effects on yield and enantioselectivity. As illustrated in Scheme 2, all the desired products displayed excellent ee values, revealing the exclusive enantioselectivity of PR. The products were assigned as α-configuration alcohols by comparing standard chiral and racemic compounds by chiral B

DOI: 10.1021/acs.orglett.9b00950 Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters

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HPLC (Supporting Information). Thus, it may be deduced that PR follows the Prelog rule.14 PR, along with some anti-Prelog reductases, whose catalyzed reductions lead to β-chiral alcohols,15 could serve as stereocomplementary enzymes for the synthesis of chiral alcohols. In Prelog enzymes, large and small pockets are formed for substrate binding.14 Lacking the crystal structure of the PR−substrate complex, we predicted the substrate-binding mode by means of molecular modeling. In terms of the catalytic mechanism of the AKR superfamily, cofactors and substrates bind separately in two different pockets, and the two pockets are conjugated by the reaction site.16 Based on these clues, two typical products (7) and (12) were modeled in PR as examples. In the model, the oxygen atom of the product’s hydroxy group approached the carboxamide group of NADPH, located within a hydrogen-bond distance of the γ-N of the imidazole ring of His126 (2.34 Å) (Figure 2, Figure S3).

Letter

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Hongbin Zou: 0000-0001-6784-2737 Lianli Sun: 0000-0002-8640-4780 Jinhao Zhao: 0000-0002-9198-5399 Author Contributions ¶

S.C., N.S., and Y.C. contributed equally.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Research reported in this publication was supported by NSF of China (81302653), Public Welfare Research Project of Zhejiang Province (LGG19B020003), and Scientific Foundation of Zhejiang Educational Committee (Y201328460).

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Figure 2. Prediction of product binding in PR by molecular modeling. Panels A and C present the accommodation of allylic alcohol (7) in the binding site, while panels B and D present the accommodation of aryl alcohol (12) in the binding site.

Furthermore, the large pocket is formed by the surface of residues Ile56, Ile87, Ile 90, His126, and Arg127. The three clustered Ile residues contributing to the hydrophobic property of the pocket may facilitate substrate binding. The small pocket comprises the surface of the Met21, Tyr57, and Lys84 residues, together with the nicotinamide riboside component of NADPH. It was also observed that PR converted one additional compound, 4-phenyl-2-butanone (S19), which does not contain a conjugated moiety to the carbonyl group (Supporting Information). The yield and ee value of the corresponding product, (S)-4-phenylbutan-2-ol (19), were 73 and 95%, respectively, which are comparable to those of the substrates with conjugated rings. In conclusion, perakine reductase demonstrates a robust ability to asymmetrically reduce structurally diverse enones and aromatic ketones. It is therefore a powerful biocatalyst for the synthesis of various chiral allylic alcohols and aryl alcohols.

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REFERENCES

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.orglett.9b00950. Experimental procedures, enzyme assay, optical purity analyses of products, molecular modeling, and NMR data (PDF) C

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DOI: 10.1021/acs.orglett.9b00950 Org. Lett. XXXX, XXX, XXX−XXX