Catalytic Asymmetric Reduction of Difficult-to-Reduce Ketones: Triple

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Catalytic Asymmetric Reduction of Difficult-to-Reduce Ketones: Triple Code Saturation Mutagenesis of an Alcohol Dehydrogenase Zhoutong Sun, Richard Lonsdale, Adriana Ilie, Guangyue Li, Jiahai Zhou, and Manfred T. Reetz ACS Catal., Just Accepted Manuscript • DOI: 10.1021/acscatal.5b02752 • Publication Date (Web): 22 Jan 2016 Downloaded from http://pubs.acs.org on January 28, 2016

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Catalytic Asymmetric Reduction of Difficult-to-Reduce Ketones: Triple Code Saturation Mutagenesis of an Alcohol Dehydrogenase Zhoutong Sun1,2, Richard Lonsdale1,2, Adriana Ilie1,2, Guangyue Li1,2, Jiahai Zhou3 and Manfred T. Reetz1,2,* 1

Max-Planck-Institut für Kohlenforschung, Kaiser-Wilhelm-Platz 1, 45470 Mülheim an der Ruhr, Germany Fachbereich Chemie der Philipps-Universität, Hans-Meerwein-Strasse, 35032 Marburg, Germany 3 State Key Laboratory of Bio-organic and Natural Products Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, Shanghai 200032, China 2

ABSTRACT: Catalytic asymmetric reduction of prochiral ketones with formation of enantio-pure secondary alcohols is of fundamental importance in organic chemistry, chiral man-made transition metal catalysts or organocatalysts and enzymes of the type alcohol dehydrogenase (ADH) being the options. A distinct limitation is the traditional requirement that the α- and α’-moieties flanking the carbonyl function differ sterically and/or electronically. Difficult-to-reduce ketones such as tetrahydrofuran-3-one and tetrahydrothiofuran-3-one and related substrates are particularly challenging, irrespective of the catalyst type. The ADH from Thermoethanolicus brockii (TbSADH) is an attractive industrial biocatalyst due to its high thermostability, but also fails in the reduction of such ketones. We have successfully applied directed evolution using the previously developed concept of triple code saturation mutagenesis at sites lining the TbSADH binding pocket with tetrahydrofuran-3-one serving as the model compound. Highly (R)- and (S)-selective variants were evolved (95-99% ee) with minimal screening. These robust catalysts also proved to be effective in the asymmetric reduction of tetrahydrothiofuran-3-one and other challenging prochiral ketones as well. The chiral products, generally prepared by multi-step routes, serve as synthons in the preparation of several important therapeutic drugs. KEYWORDS: directed evolution, saturation mutagenesis, triple code, enantioselectivity, alcohol dehydrogenase.

INTRODUCTION The catalytic asymmetric reduction of prochiral ketones with formation of enantiomerically pure or enriched alcohols is a fundamentally important transformation in synthetic organic and medicinal chemistry.1 Noyori-type Ru-complexes2 and Corey-type oxazaborolidines3 have been applied successfully in literally thousands of studies. More recently certain chiral organocatalysts were shown to catalyze ketone reductions, although the scope of this method appears to be limited thus far.4 In all of these approaches, high enantioselectivity is achieved only when the α- and α’-substituents flanking the carbonyl function are sterically very different. The challenging problem of asymmetric reduction of prochiral benzophenones can be solved by the chiral oxazaborolidine method, provided the two substituted phenyl groups are sterically different and/or electronically biased.3 As an alternative to man-made catalysts, enzymes of the type alcohol dehydrogenases (ADHs) have been used for a long time.5 Here again, for high enantioselectivity the nature of the α- and α’-substituents needs to be sufficiently different. We were interested in the biocatalytic asymmetric reaction of the difficult-to-reduce ketones such as 1 with formation of (R)and (S)-2 on an optional basis (Scheme 1). The (S)-enantiomer is a precursor of the HIV-inhibitor amprenavir® and fosamprenavir® used currently in AIDS treatment, but prepared by different routes.6 In our hands a standard chiral Rucatalyst7 led to an enantioselectivity of only 70%. Two commercial ADH kits totaling 47 enzymes were also tested, the

best (R)-selective enzyme resulting in 22% ee, and the best (S)-selective enzyme leading to 91% ee, although both at suboptimal conversions under the chosen conditions (see Table S1). The ADH from Thermoethanolicus brockii (TbSADH),8 which is an attractive ADH in biotechnology5 due to its high thermostability, proved to be slightly (R)-selective (23% ee) at full conversion. Our plan was to perform directed evolution on TbSADH as the catalyst in the reduction of 1, and to use the best evolved mutants as catalysts in the reduction of the likewise challenging substrates 3, 5 and 7 without performing any additional mutagenesis experiments. As can be seen in Scheme 1, the chiral alcohols with the appropriate absolute configuration, (S)-2,6 (R)-4,9 (R)-6,10 and (S)-8,11 have been used as precursors in the production of important therapeutic drugs. In the case of (S)-2, the only enzymatic route reported thus far involves the hydrolytic kinetic resolution of the acetate of rac-1 by an evolved lipase leading to a selectivity factor of E = 10,6b which hardly suffices for practical applications. The equally industrially important intermediate (R)-4 was prepared by Pfizer in several steps starting from L-aspartic acid.9a Codexis reported the directed evolution of a non-identified ADH as the catalyst in the reduction of ketone 3, requiring 8 cycles of mutagenesis, expression and screening using error-prone polymerase chain reaction (epPCR) and DNA shuffling, but the details of this multi-step protein engineering process were not revealed, nor was the sequence data of the best mutant reported.9b

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O O

HO

HO ADH NAD(P)H

1

O +

O

(R)-2

O

(S)-2

HO

S

ADH NAD(P)H

3

S +

HO

N Boc

ADH NAD(P)H

5 O 7

(S)-4 HO N Boc +

(R)-6 Boc ADH HO N NAD(P)H

ketone 1 into its binding pocket. Figure 1 reveals five prominent contact points at CAST residues A85, I86, W110, L294 and C295 which are therefore candidates for potential SM.

S

(R)-4

O

Amprenavir (HIV inhibitor)

HO

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Boc HO N + (R)-8

Sulopenem (antibiotic) N Boc (S)-6 N (S)-8

Boc

Several chiral pharmaceuticals

Imbruvica (lymphoma treatment)

Scheme 1. Asymmetric transformations of difficult-to-reduce ketones. Directed evolution of enzymes constitutes a powerful technique for engineering the stereoselectivity of enzymes as catalysts in synthetic organic chemistry and biotechnology.12 The most commonly used mutagenesis methods are epPCR, saturation mutagenesis (SM) and DNA shuffling, and all of them have been applied in enhancing and inverting enantioselectivity of a variety of different enzymes. The real challenge is to devise mutagenesis strategies that reliably generate highquality mutant libraries requiring a minimum of screening (bottleneck of directed evolution). We and others have previously used reduced amino acid alphabets in SM at sites lining the binding pocket (CASTing), employing iterative saturation mutagenesis (ISM) if necessary, but it was not routinely clear which and how many amino acid building blocks should be chosen.13,14 In the preceding study,15 we showed that triple code saturation mutagenesis (TCSM) is particularly efficient for manipulating the stereoselectivity of limonene epoxide hydrolase (LEH). This approach utilizes three properly chosen amino acids (in addition to WT) as building blocks in SM at a large randomization site lining the enzyme’s binding pocket. In order to reduce screening to a minimum for 95% library coverage,16 the site is split into smaller segments which are then individually targeted by SM. We now report that TCSM is also well suited for the directed evolution of TbSADH as a catalyst in the asymmetric reduction of the challenging ketones 1, 3, 5, and 7. RESULTS AND DISCUSSION Directed evolution of TbSADH using ketone 1 as the model substrate. The crystal structure of TbSADH has been solved at 2.5 Å resolution,8e which allowed us to dock the model

Figure 1. Five CAST residues A85, I86, W110, L294 and C295 identified by docking ketone 1 into the crystal structure8e of TbSADH. In contrast to other protein engineering studies of ADHs in which the α- and α’ substituents flanking the carbonyl function differ considerably, allowing for reasonably rational choices of potential amino acid substitutions,17 the docking pose shown in Figure 1 does not really help in making such a decision and underscores the difficulty in asymmetric reduction of substrates of this type. Therefore, in order to choose an appropriate triple code, the five residues were subjected individually to NNK-based SM, requiring in each case the screening of 96 transformants for 95% library coverage. At positions 85, 86, and 294 both improved (R)-selective and inverted (S)-selective single mutants were identified, whereas the libraries generated at positions 110 and 295 harbored only (S)- and (R)-selective variants, respectively (Table 1). This information served as a guide in the actual TCSM experiments. Since TCSM of a 5-residue would entail formidable screening for 95% library coverage (3072 transformants), the residues were grouped into two SM sites, and two different triple codes were designed on the basis of the NNK-based SM experiments: Site A (A85/I86/L294/C295) using a valineasparagine-leucine (V-N-L) triple code, and site B (A85/I86/W110/L294) employing a valine-glutamine-leucine (V-Q-L) triple code.

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Table 1. Saturation mutagenesis of single residue using NNK codon degeneracy for ADH. Bold mutations were chosen as candidates for triple code amino acid alphabets rational design. Code

SZ2109 SZ2139 SZ2141 SZ2142 SZ2019 SZ2036 SZ2066 SZ2067 SZ2108 SZ2114 SZ2173 SZ2174 SZ2176 SZ2201 SZ2205

Mutation

A85V A85G A85W A85N I86R I86D I86M I86E I86T I86A I86L I86G I86Q I86V I86N

ee%

29 32 13 54 29 51 32 44 39 18 40 68 85 60 89

c%

93 87 72 19 >99 >99 >99 >99 >99 >99 >99 >99 >99 99 87

Favored enantiomer

(S) (R) (S) (R) (R) (R) (R) (R) (R) (S) (S) (R) (R) (S) (R)

For 95% library coverage, only 576 transformants had to be screened in each case. We expected the best (R)-selective variants to occur in library A, and the best (S)-selective variants to be in library B. Indeed, highly (R)-and (S)-selective

Code

SZ2006 SZ2007 SZ2012 SZ2110 SZ2111 SZ2147 SZ2011 SZ2116 SZ2119 SZ2124 SZ2015 SZ2130 SZ2132 SZ2136 SZ2239

Mutation

W110A W110M W110E W110S W110L W110W L294V L294A L294H L294Q C295E C295V C295D C295N C295H

ee%

27 4 11 47 37 45 51 73 57 41 42 77 85 95 56

c%

>99 >99 >99 97 94 77 85 95 91 93 >99 99 98 >99 28

Favored enantiomer

(S) (S) (S) (S) (S) (S) (R) (R) (S) (S) (R) (R) (R) (R) (R)

variants were obtained in the two libraries, respectively, with 95-99% ee and full conversion (Fig. 2 and Table S2). Thus, further optimization using ISM was not necessary.

Figure 2. Results of screening ADH library A (a) and B (b) for substrate 1. The triple code V-N-L was used for library A at site A (A85/I86/L294/C295) and V-Q-L was used for library B at site B (A85/I86/W110/L294).

Table 2. Thermostability and kinetic parameters of the WT and best mutants for substrate 1. 1 ThermostabilKm kcat kcat/Km Km Mutant ity (T5015) (mM) (s−1) (s−1 M−1) (mM) WT 86℃ 2.21 ± 0.22 1.71 ± 0.06 773.75 0.06 ± 0.006 SZ2074 70℃ 1.68 ± 0.14 1.43 ± 0.04 851.19 0.024 ± 0.001 SZ2172 75℃ 22.79 ± 2.10 0.92 ± 0.04 40.37 0.036 ± 0.002

Kinetic parameters, thermostability and up-scaled reactions. WT TbSADH and the best (R)- and (S)-selective mu-

NADPH kcat (s−1) 8.12 ± 0.56 6.76 ± 0.12 7.96 ± 0.17

kcat/Km (s−1 mM−1) 135.33 281.67 221.11

tants SZ2074 and SZ2172, respectively, were characterized by enzyme kinetics and thermostability experiments (Table 2).

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Both mutants show lower Km and consequently higher catalytic efficiency (kcat/Km) toward cofactor NADPH relative to WT, but the kinetic results are different when focusing on the turnover of the substrate 1. The Km of SZ2074 is decreased contributing slightly to an increase in catalytic efficiency. In the case of SZ2172, Km is increased 10-fold compared to WT, which results in notably lower kcat/Km. However, kcat/Km is not always a reliable parameter for efficiency in applications.18 In industrially relevant scale-up processes in which high substrate loading needs to be tolerated, a reasonably high Km value may actually be beneficial. Concerning the question of enzyme robustness of the two variants, the thermostablility as measured by T5015 values is reduced (Table 2). Since WT is unusually robust, such a tradeoff is of no practical concern, both variants showing T5015 values which exceed 70℃. We also tested the best mutants SZ2074 and SZ2172 for up-scaled reactions using 100 mM of substrate 1 in 50 ml of reaction volume. In both cases full conversion within 4h was achieved, while maintaining the originally evolved high enantioselectivity of 99% and 94% ee, respectively.

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Shedding light on the origin of enhanced and reversed enantioselectivity. In order to explain the effect of mutation at the molecular level, docking calculations were performed. The reduction of 1 by ADHs of the type TbSADH is believed to occur via the following mechanism: the carbonyl oxygen of the substrate coordinates to the Zn2+ and a hydride is supplied from the NADPH cofactor to the carbonyl carbon atom of the substrate.5,8,17 The binding orientation of the substrate with respect to the NADPH determines which face of the C=O bond undergoes nucleophilic attack and consequently the stereochemistry of the product. Docking of 1 to WT ADH results in several binding poses in which the reduction of the C=O bond can occur from either face, therefore indicating that the stereoselectivity of product formation will be low. This is indeed found to be the case experimentally, where the formation of the R-enantiomer of the product is preferred with an ee of 23%. Figure 3 shows two docking poses found for 1 in the WT: the binding poses shown in Figures 3a and 3b would result in formation of (S)and (R)-2, respectively. The binding scores for these docking poses are within 0.1 kcal/mol and can therefore be assumed to be equally favorable.

Figure 3. Docking poses for 1 in WT TbSADH: (a) (S)-selective docking pose; (b) (R)-selective docking pose. (c) SZ2074 and (d) SZ2172 mutants of TbSADH. Interactions are highlighted by yellow dashed lines. For induced fit docking of 1 to the SZ2074 mutant, 18 docking poses were found. Two of these place the substrate in a position such that hydrogen-bonding interactions are observed

between the substrate and the mutated residues. Both of these poses favor the formation of (R)-2, which is consistent with the experimental selectivity observed for this mutant. The pose

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with the most favorable docking score is displayed in Figure 3c. The dihydrofuranyl ring oxygen forms a hydrogen-bonding interaction with the side chain NH2-group of N295. This interaction anchors the substrate in an orientation such that formation of (R)-2 is favored. The distance corresponding to the hydride transfer reaction from NADPH to the substrate is 2.703 Å for this docking pose. The N295 NH2 group forms an additional hydrogen bond with the carbonyl group of the NADPH nicotinamide moiety. N86 is also found to interact with the N295 side chain via hydrogen bonding from the former’s NH2 group to the latter’s C=O. In the case of the SZ2172 mutant, 8 docking poses were found. The docking pose with the most favorable score places the substrate in an orientation favoring the formation of (S)-2, also consistent with experiment. Here the ring oxygen atom forms a hydrogen bond to the Q294 NH2 group (Figure 3d). The distance corresponding to the hydride transfer reaction in this case is 2.774 Å. The docking calculations presented here indicate that mutations at sites 295 and 294 in ADH can be used as a selectivity switch for the reduction of 1, by anchoring the substrate via hydrogen bonding to the ring oxygen and boosting the degree of stereoselectivity. Testing the best variants as catalysts in the asymmetric reduction of other substrates. Some of the best TbSADH variants evolved for substrate 1 were tested as catalysts in the asymmetric reduction of ketones 3, 5 and 7, particular attention being paid on enantioselective variants which lead to the chiral intermediates of use in pharmaceutical applications (Scheme 1). The data is summarized in Tables S3 and S4, the most important results being highlighted here. Variants SZ2055, SZ2057, SZ2063 and SZ2074 ensure quantitative conversion of ketone 3 with excellent (R)-selectivity (95-99% ee). Reduction using a standard chiral Ru-catalyst led to poor results (72% ee; see SI). The BOC-protected N-analog (S)-6 is a useful intermediate in the synthesis of several pharmaceuticals, the best previous preparation in this case being the reduction of ketone 5 by phenylacetaldehyde reductase.10 Variants SZ2172 and SZ2257 are excellent catalysts for this transformation (>99% ee at conversions of 98% and 74%, respectively). Finally, in the reduction of ketone 7, variants SZ2172 (99% ee (S); 95% conversion) and SZ2257 (99% ee (S); 92% conversion) are effective, similar to WT TbSADH (99%ee (S); 95% conversion). This finding suggests that this substrate is somewhat less difficult than the other ketones. Alcohol (S)-8 is a useful synthon in the synthesis of imbruvica® for the treatment of lymphoma.11 CONCLUSIONS AND PERSPECTIVES This study supports the previous conclusion that triple code saturation mutagenesis (TCSM)16 is a viable approach to directed evolution of stereoselective enzymes as catalysts in organic chemistry and biotechnology. TCSM utilizes a 3membered reduced amino acid alphabet (in addition to WT) at individual segments of large randomization sites, which dramatically reduces the degree of oversampling in the screening step relative to the use of conventional NNK codon degeneracy encoding all 20 canonical amino acids. Consequently, TCSM constitutes an effective compromise between structural

diversity and screening effort. In the present study, a new twist to TCSM was implemented by employing a different triple code at two randomization sites. In this way the initial SM libraries harbored highly enantioselective and active hits, which made ISM not necessary. Step-economy, a term borrowed from synthetic organic chemistry,19 needs to be considered in directed evolution more so than in the past.12,16 We also suggest that a different triple code can be applied at every individual residue of a given randomization site, provided convincing data is available for making such precise decisions. The choice of a triple code depends upon the specific enzyme under study. A universal triple code for all enzyme types is unrealistic. In a curiosity-driven experiment, we nevertheless tested the triple code Val-Phe-Tyr previously designed for the SM-based directed evolution of the epoxide hydrolase LEH,16 and indeed observed the expected failure when applying it to TbSADH (see Table S5). In addition to mechanistic considerations, three guidelines help in making rational decisions regarding the choice of a triple code: X-ray structure (or homology model), consensus sequence data, and, if needed, information derived from NNK-based SM at individual residues of a large randomization site. We expect TCSM to be successful in other types of enzymes as well, including P450 monooxygenases. MATERIALS AND METHODS Materials KOD Hot Start DNA Polymerase was obtained from Novagen. Restriction enzyme Dpn I was bought from NEB. The oligonucleotides were synthesized by Life Technologies. Plasmid preparation kit was ordered from Zymo Research, and PCR gel extraction kit was bought from QIAGEN. DNA sequencing was conducted by GATC Biotech. All commercial chemicals were purchased from Sigma-Aldrich, Tokyo Chemical Industry (TCI) or Alfa Aesar. Lysozyme and DNase I were purchased from AppliChem. SM-based methods for library construction Libraries were constructed using the Over-lap PCR and megaprimer approach with KOD Hot Start polymerase. 50 µL reaction mixtures typically contained 30 µL water, 5 µL KOD hot start polymerase buffer (10×), 3 µL 25 mM MgSO4, 5 µL 2 mM dNTPs, 2.5 µL DMSO, 0.5 µL (50~100 ng) template DNA, 100 µM primers mix 0.5 µL each and 1 µL KOD hot start polymerase. The PCR conditions for short fragment: 95 °C 3 min, (95 °C 30 sec, 56 °C 30 sec, 68 °C 40 sec) × 32 cycles, 68 °C 120 sec, 16 °C 30 min. For mega-PCR: 95 °C 3 min, (95 °C 30 sec, 60 °C 30 sec, 68 °C 5 min 30 sec) × 24 cycles, 68 °C 10 min, 16 °C 30 min. The PCR products were analyzed on agarose gel by electrophoresis and purified using a Qiagen PCR gel extraction kit. 2 µL NEB CutSmart™ Buffer and 2 µL Dpn I were added in 50 µL PCR reaction mixture and the digestion was carried out at 37 °C for more than 3 h. After Dpn I digestion, the PCR products (1 µL) were directly transformed into electro-competent E. coliBL21(DE3) to create the final library for Quick Quality Control20 and screening.

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Primer design and library creation Library A was created by mixed primers F1/R1, see primer design in Figure S1 and primers list in Table S6. Library B was created using a different strategy according to the following steps (Fig. S2): 1) Primers F2/R2 and F3/R3 were used to amplify WT of ADH separately. The PCR products were purified by a gel extraction kit to remove the original plasmid; 2) Step 1 products were used as a template with primers F2/R3 to do overlap PCR. Then, the PCR fragments were purified to remove any remaining primers; 3) Step2 products were used as megaprimers to amplify plasmid pRSFduetADHwt.17f The PCR products were digested by Dpn I to remove the template plasmid and 1 µL aliquot to transform E. coliBL21(DE3) to create the variants plasmid library for screening. The list of primers is shown in Table S7.

taining 500 mM NaCl and 20 mM sodium phosphate (pH 7.4). The target protein fraction was collected and desalted through HiPrep 26/10 Desalting colomn(GE Healthcare) against 20 mM phosphate buffer containing 100 mM NaCl (pH7.4). Protein concentration was measured by Bradford method. Determination of kinetic parameters The kinetic parameters were obtained by measuring the initial velocities of the enzymic reaction and curve-fitting according to the Michaelis–Menten equation using GraphPad Prism 5 software (GraphPad Software Inc). For substrate 1, the activity assay was performed in a mixture containing a varying concentration of substrate 1 (0.075–52.5mM) and 0.7 mM of NADPH. For NADPH, 40 mM of substrate 1 and NADPH in the range of 0.01–0.2 mM were used for the activity assay. All experiments were conducted in triplicate.

Screening Procedures Colonies were picked and transferred into deep-well plates containing 300 µL LB medium with 50 µg/mL kanamycin and cultured overnight at 37 °C with shaking. An aliquot of 120 µL was transferred to glycerol stock plate and stored at -80 °C. Then, 800 µL TB medium with 0.2 mM IPTG and 50 µg/mL kanamycin was added directly to the culture plate for 8 h at 30 °C with shaking for protein expression. The cell pellets were harvested and washed with 400 µL 50 mM pH 7.4 potassium phosphate buffer and centrifuged for 10 min 4000 rpm at 4 °C. Then, the cell pellets were resuspended in 400 µL of the same buffer with 6 U DNase I and 1 mg/mL lysozyme for breaking the cell at 30 °C for 1 h with shaking. The crude lysate was centrifuged for 30 min 4000 rpm at 4 °C. 300 µL supernatant was transferred into new deep-well plates for reaction with 5 mM substrate 3 and 50 µM NADP+ and 10% isopropanol for 14~16 h at 30 °C 800 rpm, the final volume was 400 µL. The product and remaining substrate were extracted using equal volumes of EtOAc for GC analysis by chiral column (Table S8).

Activity assay and thermostability Enzyme activity was determined in a 0.2 ml volume of phosphate buffer (50 mM, pH 7.4) containing substrate 1 (10 mM), NADPH (0.5 mM), and 50 uL (0.5 mg/ml) of the purified enzyme. The substrate was dissolved in acetonitrile, which did not exceed 1% of the total volume. The reaction was initiated by the addition of the enzyme, and monitored for 1.5 min with SPECTRAMAX M5 (MD, USA) at 30 ◦C. The activity was determined by measuring NADPH oxidation from the decrease in absorbance at 340 nm using a molar absorption coefficient of 6.22 mM−1 cm−1. One unit of enzyme activity was defined as the amount of enzyme catalyzing the conversion of 1 umol NADPH per minute. For thermostability, the enzyme was incubated at different temperatures (50–90°C) for 30 min followed by measuring the residual activity by following the standard assay procedure. All experiments were conducted in triplicate.

Protein expression and purification E. coli BL21 (DE3) cells carrying the recombinant plasmid were cultivated in 5 ml LB medium containing Kanamycin (50 ug/ml) overnight at 37 ◦C. The overnight culture was inoculated into 500mL TB medium containing Kanamycin (50 ug/ml) and grown at 37°C. The culture was induced by addition of isopropyl ß-D-1-thiogalactopyranoside (IPTG) with a final concentration of 0.2 mM when OD600 reached 0.6, and then allowed to grow for additional 12 h at 30 ◦C. After centrifugation at 6000×g for 15 min at 4 ◦C, the bacterial pellet was washed with phosphate buffer (50 mM, pH 7.4), and resuspended in a phosphate buffer (20 mM pH 7.4) containing 0.5 M of NaCl and 20 mM of imidazole. The cells were lysed by sonication and the supernatant was collected by centrifugation at 10,000×g for 60 min at 4 ◦C. Protein purification was performed at 23 ◦C using an AKTA purifier 10 system with UNICORN 5 software (GE Healthcare). The WT and mutants was purified using affinity chromatography with a HisTrapTM FF crude column (GE, USA). The column was preequilibrated with 50 ml equilibrium buffer (20 mM phosphate buffer, 0.5 M NaCl, 20 mM imidazole, pH 7.4). The sample (30 ml) was loaded with a flow rate of 2.0 ml/min. After washing with 50 ml of the equilibrium buffer, the bounded target protein was washed with 10–500 mM imidazole solution con-

Docking analysis The X-ray structure of ADH from Thermoanaerobium brockii was used as the basis for docking calculations.21 The 1YKF structure has NADP present, but not substrate. However, the NADP is positioned too close to the zinc to allow for the substrate to bind, and preliminary docking calculations were unsuccessful. Superposition of the horse liver ADH crystal structure (6ADH22) with the 1YKF structure shows a better positioning of the NADP that allows for the substrate to bind. Hence the coordinates of the NADP were obtained from the 6ADH crystal structure to replace those in the 1YKF structure. Homology models of the SZ2074 and SZ2172 mutants were constructed using the Structure Prediction Wizard in Prime.23 The WT model was used as a template and the Knowledgebased approach was used. The resulting structures were prepared for docking using the Protein Preparation Wizard.24 Hydrogen atoms were added according to the protonation states indicated by PROPKA.25 The positions of all hydrogen atoms were energy minimized using the Impact program26 and the OPLS2005 forcefield. The substrate 1 was prepared for docking using the LigPrep program.27 Docking to the WT enzyme was performed using Glide28 with standard precision (SP) settings. Twenty docking poses were requested and a constraint was applied such that only the docking poses in which the substrate coordinates to the active site zinc ion were saved. For the SZ2074 and SZ2172 mutants, induced fit docking (IFD)29 was performed, in order to allow

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the mutated residues to adapt to the presence of the substrate. For the IFD calculations, the same docking constraint and maximum number of saved docking poses was used as for the Glide SP docking to the WT model. Semi-preparative scale reactions using ADH (alcohol dehydrogenase) best mutants An Erlenmeyer flask (100 mL) containing LB (20 mL) and kan (50µg/mL) was inoculated with ADH mutant (SZ 2074 or SZ 2172) and incubated 6h (37°C, 220 rpm). This pre-culture was then inoculated into TB (400 mL) containing kanamycin (50µg/mL) and allowed to grow at 37°C until O.D. was 0.80.9. IPTG was then added to a final concentration of 0.2 mM and the culture was grown 16 h (30°C, 150 rpm). Cells were pelleted by centrifugation, washed once with pH=7.4 potassium phosphate buffer (100 mM) and then resuspended in pH=7.4 potassium phosphate buffer (50 mL, 100 mM). Cells were then transferred to a 50 mL Erlenmeyer flask and 1.4 mg/mL lysozyme (70 mg), 6U DNase I (50 µL), NADP+ 100mM (100 µL) were added. In continuation, substrate (100 mM) dissolved in 10% isopropanol (5 mL) were added. Bioconversion was carried out at 30°C, 220 rpm. Reaction completion was monitored by GC and full conversion was reached within 4h. The organic phase was extracted with ethyl acetate (500 mL) and crude reaction products were purified using column chromatography. Chemistry General remarks Tetrahydrofuran-3-one, tetrahydrothiophen-3-one, RuCl(pcymene)[(R,R)-Ts-DPEN], [DPEN=1,2bis(diphenylethylenediamine)] and dry 1,2-dichloroethane were purchased from TCI chemicals and Sigma-Aldrich and used without further purification. NMR spectra were recorded on a Bruker Avance 300 spectrometer (1H: 300 MHz, 13C: 75 MHz) using TMS as internal standard (d=0), chemical shifts δ are given in ppm. Conversion and enantiomeric excess was determined by chiral GC and HPLC. Analytical thin layer chromatography was performed on Merck silica 60 F254q and for column chromatography on Merck silica 60 (230-400 mesh ATM). Optical rotation measurements were performed on a Krüss Optronic P8000 Digital Automatic High-Speed Polarimeter at 25°C. Absolute configuration was assigned after comparison with commercial available enantiomeric pure samples. Synthesis of alcohols Synthesis (R)-3-Hydroxytetrahydrofuran (2) (R)-3-Hydroxytetrahydrofuran (2) with SZ2074 catalyzed bioconversion was upscaled as described above. Purification on column chromatography (EA:PE 3:1, rf=0.5) afforded (R)-2 as a ligh yellow liquid. The absolute configuration was assigned by GC profile comparison with an authentic enantiomeric pure (R) sample. 1H NMR (300 MHz, CDCl3) δ 4.41 (ddt, 3J = 5.8, 3.9, 1.9 Hz, 1H), 3.94–3.86 (m, 1H), 3.80– 3.69 (m, 2H), 3.68–3.64 (m, 1H), 3.15 (s, 1H, OH), 2.07–1.95 (m, 1H), 1.88–1.79 (m, 1H). 13C NMR (75 MHz, CDCl3) δ 75.56 (s), 71.80 (s), 66.79 (s), 35.55 (s).

Synthesis (S)-3-Hydroxytetrahydrofuran (2) (S)-3-Hydroxytetrahydrofuran (2) with SZ2172 catalyzed bioconversion was upscaled as described above. Purification on column chromatography (EA:PE 3:1, rf=0.5) afforded (S)-2 as a ligh yellow liquid. The absolute configuration was assigned by GC profile comparison with an authentic enantiomeric pure (S) sample. 1H NMR (300 MHz, CDCl3) δ 4.40 (ddt, 3J = 5.8, 3.9, 1.9 Hz, 1H), 3.93–3.85 (m, 1H), 3.79– 3.68 (m, 2H), 3.67–3.63 (m, 1H), 3.32 (s, 1H, OH), 2.06–1.94 (m, 1H), 1.87–1.78 (m, 1H). 13C NMR (75 MHz, CDCl3) δ 75.52 (s), 71.74 (s), 66.76 (s), 35.52 (s). Synthesis (R)-3-Hydroxytetrahydrothiophen (4) (R)-3-Hydroxytetrahydrothiophen (4) with SZ2074 catalyzed bioconversion was upscaled as described above. Purification on column chromatography (EA:PE 3:1, rf=0.47) afforded (R)4 as an orange oil (292 mg, yield=94%). Absolute configuration of (R)-4 was assigned after comparison the optical rotation sign and GC profile with sample (R)-4 obtained by enantioselective Noyori asymmetric hydrogenation of substrate 3. 1 H NMR (300 MHz, CDCl3) δ 4.59–4.55 (m, 1H), 2.98–2.81 (m, 3H), 2.80–2.75 (m, 1H), 2.51 (s, 1H, OH), 2.15–2.06 (m, 1H), 1.88–1.76 (m, 1H). 13C NMR (75 MHz, CDCl3) δ 74.65 (s), 39.82 (s), 38.09 (s), 28.18 (s). HRMS (EI) calcd for C4H8OS [M]+: 104.0296; [α]D= 0.257, (c=1, MeOH), lit.30

ASSOCIATED CONTENT Supporting Information: including supplementary tables and figures for commercial ADH kits screening, primers list, library design, creation and screening with details of the result. Also 1 including GC and HPLC analytic conditions with GC profiles, H NMR profiles with chemistry reactions to characterize the best mutants. This material is available free of charge via the Internet at http://pubs.acs.org.”

AUTHOR INFORMATION Corresponding Author * Correspondence should be addressed to (M.T.R.) [email protected]

ACKNOWLEDGMENT Support from the Max-Planck-Society and the LOEWE Research cluster SynChemBio is gratefully acknowledged in addition to grants from Science and Technology Commission of Shanghai Municipality (15JC1400403). REFERENCES (1) (a) Morris, R. H. Chem. Soc. Rev. 2009, 38, 2282-2291; (b) Gladiali, S.; Alberico, E. Chem. Soc. Rev. 2006, 35, 226-236. (2) (a) Noyori, R. Angew. Chem. Int. Ed. 2002, 41, 2008-2022; (b) Sandoval, C. A.; Li, Y.; Ding, K.; Noyori, R. Chem. Asian J. 2008, 3, 1801-1810. (3) Corey, E. J.; Helal, C. J. Angew. Chem. Int. Ed. 1998, 37, 19862012. (4) (a) Li, D. R.; He, A.; Falck, J. R. Org. Lett. 2010, 12, 1756-1759; (b) Rossi, S.; Benaglia, M.; Massolo, E.; Raimondi, L. Catal. Sci. Technol. 2014, 4, 2708-2723; (c) Denizalti, S.; Mercan, D.; Sen, B.; Gokce, A. G.; Cetinkaya, B. J. Organomet. Chem. 2015, 779, 62-66.

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