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Synthesis of enantiopure glycidol derivatives via a one-pot two-step enzymatic cascade† Yu-Chang Liu,a,b,c Yan Liua,b,c and Zhong-Liu Wu*a,b Styrene monooxygenase (SMO) can catalyze the kinetic resolution of secondary allylic alcohols to provide enantiopure glycidol derivatives. To overcome the low theoretical yield of kinetic resolution, we designed a one-pot two-step enzymatic cascade using prochiral α,β-unsaturated ketones as the substrates. An

Received 15th October 2014, Accepted 8th December 2014

S-specific ketoreductase ChKRED03 was screened for the efficient bioreduction of the substrates to provide (S)-allylic alcohols, which underwent SMO-catalyzed epoxidation to achieve glycidol derivatives

DOI: 10.1039/c4ob02186j

with contiguous stereogenic centers. Excellent enantioselectivity (ee > 99%) and diastereoselectivity (de >

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99%) were achieved for the majority of the substrates, and product yields reached up to >99%.

Introduction Enantiomerically enriched glycidol derivatives are important building blocks in the synthesis of natural products and biologically active compounds.1–6 A series of chiral compounds can be derived by opening the reactive oxirane moiety with diverse nucleophiles.7,8 The Sharpless–Katsuki asymmetric epoxidation represents a classic catalytic approach to prepare enantiopure glycidol derivatives. A wide range of primary allylic alcohols can be epoxidized with high stereoselectivity.9,10 The reaction is also suitable for the kinetic resolution of secondary allylic alcohols, but is sometimes sensitive to steric hindrance.1,11,12 For the aromatic substrate 1-phenylprop-2-en-1-ol (1b), it required several days at−20 °C to reach 50% conversion, and yielded an epoxide with 90% ee.11 Styrene monooxygenase (SMO) is an enzyme involved in the upper catabolic pathway of styrene degradation.13,14 It shows excellent enantioselectivity in the epoxidation of styrene and its derivatives.15–19 In our previous work on the SMO from Pseudomonas sp. LQ26, we have found that the racemic secondary allylic alcohol 1-phenylprop-2-en-1-ol (1b) could be epoxidized within 2 h with excellent enantioselectivity and diastereoselectivity (>99% ee, >99% de) using recombinant Escherichia coli expressing the SMO,20 and the unreacted (R)-1b could be recovered with >99% ee. The enzymatic method

a Key Laboratory of Environmental and Applied Microbiology, Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu 610041, China. E-mail: [email protected]; Fax: +86-28-82890434; Tel: +86-28-82890434 b Environmental Microbiology Key Laboratory of Sichuan Province, Chengdu 610041, China c University of the Chinese Academy of Sciences, Beijing 100049, China † Electronic supplementary information (ESI) available. See DOI: 10.1039/ c4ob02186j

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shows a clear advantage over previously established chemistry methods, and provides a valuable eco-friendly approach that employs molecular oxygen as the oxidant. However, kinetic resolution only provides the desired glycidol product with a maximum theoretical yield of 50%. To overcome such a limitation, in this study, we employed a two-step enzymatic cascade reaction involving an S-specific ketoreductase and SMO, and using an α,β-unsaturated ketone as the starting material, which allowed us to achieve high conversions as well as enantiomeric and diastereomeric excesses.

Results and discussion Identification of S-specific ketoreductase ChKRED03 SMO specifically catalyzes the epoxidation of (S)-1-phenylprop2-en-1-ol (1b), which could be produced by the bioreduction of 1a catalyzed with ketoreductases following the Prelog’s rule. Therefore, six recombinant ketoreductases in our inventory that follow the Prelog’s rule21 were assayed against substrate 1a. Three of them, namely ChKRED03, ChKRED11 and ChKRED15, displayed excellent enantioselectivity (>99% ee), yielding a single enantiomer of (S)-1b without any by-product. ChKRED03 performed the best in terms of activity (Fig. 1). ChKRED03 shared the maximal similarity of 43% with a known alcohol dehydrogenase RasADH from Ralstonia sp. DSM642822 (PDB: 4BMS). It also shared 90% similarity with one predicted protein from Chryseobacterium hispalense (NCBI accession no. WP_029296933). Protein sequence alignment of ChKRED03 with the confirmed ketoreductases is shown in Fig. 2. ChKRED03 contains the cofactor binding motif (G12xxxG16xG18) and the essential segment (S137, Y150, K154) for the catalytic activity of SDRs (Fig. 2), which are highly

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Fig. 1 Bioreduction of 1-phenylprop-2-en-1-one (1a) with S-specific ketoreductases. Shown are the conversion rate (white) and enantiomeric excess of the product (black).

conserved residues in the reductase (SDR) superfamily.23

short-chain

dehydrogenase/

due to the generation of gluconic acid. Considering the reaction conditions of the subsequent SMO-catalyzed epoxidation (optimal pH 6.5), the bioreduction was performed at pH 7.0, which yielded the same reaction efficiency. ChKRED03 could as well catalyze the bioreduction of a series of α,β-unsaturated ketones, analogues of 1a (Table 1). The reaction proceeded smoothly without any by-product observed. All the tested substrates were converted to the corresponding (S)-alcohols (b) with excellent activity and enantioselectivity. In the majority of cases, complete conversion was observed, and products with >99% ee were achieved (Table 1). Substrates 6a and 14a showed slightly lower reactivity, but still yielded >80% conversion within 5 h with good to excellent enantioselectivity (Table 1, entries 6 & 14). The results indicated that ChKRED03 could serve as a suitable ketoreductase

Reaction conditions and substrate adaptability of ChKRED03 The purified ChKRED03 catalyzed the reduction of 1a to enantiopure (S)-1b over the pH range of 4.0–9.0 and displayed the maximum activity at pH 7.5 in either Tris-HCl or potassium phosphate buffer (Fig. 3A). No significant change of activity occurred over the pH range of 5.5–8.0 (>90% of the maximum activity). In addition, the optimum reaction temperature of ChKRED03 was determined to be 30 °C (Fig. 3B). Hence, the pH- and temperature-dependence of activities was compatible with that of the bioepoxidation reaction using whole cells of recombinant E. coli producing SMO, which displayed the maximum activity at 30 °C and pH 6.5 in potassium phosphate buffer.24 ChKRED03 was an NADPH-dependent ketoreductase and did not accept NADH as a cofactor. Therefore, a cofactor regeneration system based on GDH-catalyzed glucose oxidation was applied to ensure a sufficient supply of NADPH. Complete conversion was achieved within 1 h with excellent enantioselectivity (ee > 99%), which was higher than the 80% conversion without using the NADPH-regeneration system. After the reaction, the resulting mixture had a pH drop of 0.5

Fig. 3 Effect of (A) pH and (B) temperature on the activity of ChKRED03. Buffers used: sodium citrate–citric acid (●), potassium phosphate (○), Tris-HCl (▲).

Fig. 2 Sequence alignment of ChKRED03 with members of the short chain dehydrogenase/reductase (SDR) family. ChKRED03, predicted ketoreductase in this work (KC342003); RasADH, short chain alcohol dehydrogenases from Ralstonia sp. DSM6428 (4MBS); LBADH, alcohol dehydrogenases from Lactobacillus brevis, (1NXQ); BKR, β-keto acyl carrier protein reductase from Brassica napus (1EDO). Residues that are highly conserved in the SDR family are shaded in grey. The cofactor-binding motif (GxxxGxG sequence) is underlined. The catalytic triad, Ser-Tyr-Lys, is highlighted with asterisks.

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Table 1 Asymmetric bioreduction of α,β-unsaturated ketones to secondary allylic alcohols

Entry

R

t (h)

Conv.a (%)

ee (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14

Ph (1a) m-FC6H4 (2a) p-FC6H4 (3a) m-MeC6H4 (4a) p-MeC6H4 (5a) 2-Thienyl (6a) 3-Thienyl (7a) m-ClC6H4 (8a) p-ClC6H4 (9a) m-BrC6H4 (10a) p-BrC6H4 (11a) m-OMeC6H4 (12a) p-OMeC6H4 (13a) Benzyl (14a)

1 1 1 5 5 5 5 5 5 1 1 5 5 5

>99 >99 >99 >99 >99 82 >99 >99 >99 >99 >99 >99 >99 86

>99 >99 >99 >99 >99 94 >99 >99 >99 >99 >99 >99 >99 >99

a Conversions were determined by HPLC. The unreacted substrates were detected for substrates 6a and 14a.

to generate the corresponding (S)-alcohols for the sequential epoxidation reaction. One-pot two-step biotransformation of α,β-unsaturated ketones ChKRED03 and SMO could be coexpressed in E. coli, but efficient reduction of the CvC double bond of 1a was observed

Table 2

when using the recombinant whole cells as the catalyst, resulting in 1-phenyl-1-propanol. E. coli is known to contain several ene-reductases,25–27 which apparently could take 1a as a substrate, and competed with SMO for the CvC double bond. To avoid the side reaction, a one-pot enzymatic cascade was designed that used ChKRED03 and SMO in a step-wise fashion. In the first step, the substrate 1a was reduced to (S)-1b using ChKRED03 as a free enzyme in the presence of a GDH-catalyzed cofactor recycling system. After the reaction reached completion, the whole cells of E. coli expressing SMO were added into the reaction mixture. As expected, SMO efficiently catalyzed the epoxidation of (S)-1b, yielding the product (1R, 2R)-phenyl glycidol (1c) with excellent stereoselectivity (ee > 99%, de > 99%) at 100% conversion in 2 h (Table 2, entry 1). Encouraged by this result, we then extended the one-pot two-step cascade to the panel of α,β-unsaturated ketones listed in Table 1 that were accepted as substrates by ChKRED03. Because substituents at the ortho-positions of the benzene ring are known to strongly prevent the bioepoxidation reaction,20 only meta- and para-substituted substrates were tested (Table 2). After the two-step cascade, all the substrates (2a– 14a) were transformed to the corresponding glycidol derivatives with excellent enantioselectivity (ee > 99%) and diastereoselectivity (de 86–99%). The conversions of substrates a to the final product c were generally over 50%, which was the maximum theoretical yield of the kinetic resolution. A single (1R,2R)-enantiomer was achieved in the majority of cases. Compared with previous results using SMO as the only catalyst,20 the two-enzyme cascade delivered much improved substrate conversion and product yield. The isolated yields reached up to 93% (for 1c) in a 50 ml reaction system.

One-pot two-step cascade for the synthesis of enantiopure glycidol derivatives

Entry

R

ta (h)

Conv.b (%)

eec (%)

de1 d (%)

de2 e (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14

Ph (1a) m-FC6H4 (2a) p-FC6H4 (3a) m-MeC6H4 (4a) p-MeC6H4 (5a) 2-Thienyl (6a) 3-Thienyl (7a) m-ClC6H4 (8a) p-ClC6H4 (9a) m-BrC6H4 (10a) p-BrC6H4 (11a) m-OMeC6H4 (12a) p-OMeC6H4 (13a) Benzyl (14a)

2 15 15 15 15 15 15 24 24 36 36 36 36 36

>99 97 98 87 86 79 98 83 84 54 64 37 75 55

>99 >99 >99 >99 >99 >99 >99 >99 >99 >99 >99 >99 >99 >99

>99 >99 >99 >99 >99 91 >99 >99 >99 >99 >99 >99 >99 >99

>99 >99 >99 >99 >99 >99 >99 >99 >99 86 >99 >99 >99 >99

a

The reaction time for the epoxidation step. b Conversions of a to c were determined by HPLC. c The enantiomeric excess of the major product: ee = (|[1R,2R] − [1S,2S]|)/([1R,2R] + [1S,2S]). d The diastereomeric excess for the hydroxyl group: de1 = (|[1R,2R] − [1S,2R]|)/([1R,2R] + [1S,2R]). e The diastereomeric excess for the oxiranyl group de2 = (|[1R,2R] − [1R,2S]|)/([1R,2R] + [1R,2S]).

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In the two-step cascade, the first step was generally more efficient than the second one. For substrates 2a–5a and 7a– 13a, which had a lower conversion than 1a, no by-product formation or unreacted substrate was observed. The HPLC trace showed a portion of intermediate b, indicating incomplete epoxidation of b to c. For substrates 6a and 14a, due to the incomplete bioreduction, the remaining substrate underwent CvC double bond reduction catalyzed with the ene-reductases in the E. coli cells, yielding by-products 1-(thiophen-2-yl)propan-1-one (6d) and 1-phenylbutan-2-one (14d), respectively.

Conclusions In summary, an enzymatic cascade reaction employing an S-specific ketoreductase ChKRED03 and a styrene monooxygenase has been accomplished for the stereoselective synthesis of glycidol derivatives with excellent diastereomeric and enantiomeric excess. This convenient one-pot two-step approach shows significant improvement over our previously established process based on SMO alone, and provides a valuable alternative to classic chemo-catalyzed kinetic resolution for the epoxidation of secondary allylic alcohols.

Experimental Materials and methods Materials and general methods. Substrate 1a was synthesized from 3-chloro-1-phenylpropan-1-one (Alfa-Aesar, Tianjin, China).28 Other substrates were synthesized using Jones oxidation from alcohols.29 Racemic 1b was purchased from Sigma-Aldrich (St. Louis, MO, USA), and other α-substituted secondary allylic alcohols (b) were synthesized using Grignard reaction at −10 °C.30 Racemic epoxides (c) were synthesized according to a previous method.20 Glucose dehydrogenase (GDH) was purchased from Sigma-Aldrich (St. Louis, USA). Restriction enzymes and T4 DNA ligase were purchased from New England Biolabs (Beverly, MA, USA). 1 H NMR spectra were recorded on a Bruker-600 (600/ 150 MHz) spectrometer in CDCl3. All signals are expressed in ppm downfield from tetramethylsilane. Optical rotations were measured with a Perkin Elmer 341 polarimeter. Chiral HPLC was conducted with a Shimadzu Prominence LC-20AD system connected to a PDA-detector using Daicel OJ-H (2a–13a, 2b– 13b), Chiralcel OD-H (14a, 14b) and Chiralpak AS-H (1c–14c) columns. The conversion rate and enantiomeric excess of (S)1-phenylprop-2-en-1-ol (1b) were determined by chiral GC analysis using a CP-Chirasil-DEX column (25 m × 0.25 mm, Varian, USA), and nitrogen as the carrier gas on a Fuli 9790 II GC system connected to a flame ionization detector. Heterologous expression of SMO Recombinant Escherichia coli BL21 harboring the plasmid pET28a(+) encoding the styAB gene from Pseudomonas sp. LQ26 (NCBI accession no. GU593979) was cultivated overnight

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at 37 °C in LB media containing kanamycin (50 μg ml−1). Two ml of overnight culture was then inoculated into 200 ml of terrific broth containing kanamycin (50 μg ml−1) in a 500 ml flask, and incubated at 37 °C for 3 h, followed by incubation at 20 °C for another 20 h with gyratory shaking at 220 rpm. Cells were harvested by centrifugation, washed twice with potassium phosphate buffer (0.1 M, pH 7.0) and stored at 4 °C. Cloning, expression and purification of ChKRED03 The DNA fragment encoding ChKRED03 (NCBI accession no. KC342002) was amplified from the genomic DNA of Chryseobacterium sp. CA49 with primers 5′-CATATGATGAATTT CACAGATAAAAATGTAATC-3′ (forward) and 5′-CTCGAGTTA TCTGCGGATCGTTACTC-3′ (reverse), and cloned into the pMD-19 T vector (Novagen, Madison, WI, USA). The resulting product was digested with EcoRI and HindIII, and subcloned into the pET28a(+) plasmid (Novagen) digested with the same restriction enzymes. The resulting plasmid encoding ChKRED03 was transformed into E. coli BL21(DE3) for protein expression. Single colonies were grown overnight at 37 °C in Luria– Bertani (LB) medium containing 50 μg kanamycin ml−1. Then 2 ml of the starter culture was transferred into 200 ml of terrific broth containing 50 μg kanamycin ml−1, and incubated at 37 °C. The induction of protein expression was initiated with the addition of 0.5 mM IPTG when OD600 reached 0.6–0.8, and the incubation was continued at 30 °C for 16 h. Cell pellets were collected, resuspended in buffer A (100 mM potassium phosphate, 300 mM NaCl and 10 mM imidazole, pH 8.0), and disrupted using a high-pressure homogenizer (ATS-AH100B, ATS Engineering Inc., Canada) at 60 MPa. After removing the cell debris, the resulting supernatant was loaded onto a Ni2+-nitrilotriacetic acid column (Qiagen, Valencia, CA, USA). The His-tagged enzyme was eluted with buffer A containing 250 mM imidazole, and dialyzed against potassium phosphate buffer (20 mM, pH 7.5). Protein analysis was done using SDS-PAGE and the BCA Protein Assay Kit (Beyotime, China) with bovine serum albumin as the standard. Measurement of the enzyme activity of ChKRED03 The assay was performed by adding 0.2 mg of the enzyme to 1 ml potassium phosphate buffer (100 mM, pH 7.5) containing 7.5 mM 1a and 1 mM NADPH. Continuous spectrophotometric measurements were then performed to monitor the oxidation of NADPH at 340 nm for 2 min at 25 °C on a Shimadzu UV-1800 spectrophotometer. One unit of activity was defined as the amount of the enzyme catalyzing the oxidation of 1 µmol of NADPH per min. To determine the pH-dependence of the activity of ChKRED03, sodium citrate ( pH 4.0–6.0), potassium phosphate ( pH 6.0–8.0) and Tris-HCl ( pH 7.0–9.0) were used in different pH ranges. To determine the temperature optima, the reactions were performed under the standard conditions for 20 min, and terminated by extraction with ethyl acetate. The conversion rate was measured using GC.

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General procedure for biotransformation The bioreduction of ketones (a) was performed in 10 ml potassium phosphate buffer (100 mM, pH 7.0) containing 7.5 mM substrate, 50 mM glucose, 25 µM NADP+, 3U ChKRED03 and 3U GDH. The reaction was conducted at 30 °C, and the process was monitored using HPLC or GC. For the one-pot two-step biotransformation, the second catalyst was added when the bioreduction of the ketone reached an end. Freshly harvested whole cells of recombinant E. coli expressing SMO (0.4 g dry cell weight) were added into the reaction mixture, and then the incubation was continued at 30 °C for 2–36 h with shaking at 240 rpm. The reaction was terminated by extraction with diethyl ether (3 × 20 ml). The organic layers were dried over anhydrous Na2SO4 and concentrated under reduced pressure. All the samples were analyzed by chiral HPLC to determine the yield and enantiomeric purity of products. Preparative biotransformations were performed at 30 °C in 50 ml potassium phosphate buffer (100 mM, pH 7.0) following the same procedure. The extracted and concentrated products were purified by silica gel column chromatography, and identified by NMR analysis. Spectral data for biotransformation products (S)-1-Phenylprop-2-en-1-ol (1b). Elute: petroleum ether–ethyl acetate 15 : 1, Rf: 0.4, 93% yield; [α]20 D –2.5 (c 1.0 in CHCl3); 1 {lit.31 [α]25 D −5.9 (c 1.73 in C6H6)}; H NMR(600 MHz, CDCl3): δ 7.25–7.36 (m, 5H, Ar–H), 6.04–6.06 (m, 1H, vCH), 5.32–5.35 (d, J = 18 Hz, 1H, –CH), 5.17–5.19 (m, 1H, vCH2), 2.09 (br, 1H, OH). Retention times: tR (R) 12.5 min, tR (S) 13.3 min; 1a, tR 5.9 min. (1R,2R)-Phenyl-2,3-glycidol (1c). Elute: petroleum ether– ethyl acetate 7 : 1, Rf: 0.3, 93% yield; 1H NMR(600 MHz, CDCl3): δ 7.37–7.43 (m, 5H, Ar–H), 4.94 (s, 1H, –CH), 3.22–3.25 (m, 1H, –CH), 2.85–2.87 (m, 1H, –CH2), 2.76–2.77 (m, 1H, –CH2), 2.23 (br, 1H, OH). Retention times: tR1 12.5 min, tR2 14.8 min, tR3 15.7 min, tR4 17.7 min; 1b, tR min. (S)-1-(3-Fluorophenyl)prop-2-en-1-ol (2b). Elute: petroleum ether–ethyl acetate 15 : 1, Rf: 0.4, 90% yield; [α]20 D + 7.6 (c 0.83 in CHCl3);{lit.31 [α]35 + 12.1 (c 0.56, CHCl3)}; 1H NMR D (600 MHz, CDCl3): δ 6.96–7.34 (m, 4H, Ar–H), 5.99–6.03 (m, 1H, vCH), 5.38(d, J = 18 Hz, 1H, –CH), 5.24 (d, J = 12 Hz, 1H, vCH2), 5.20 (d, J = 6 Hz, 1H, vCH2), 1.98(br, 1H, OH). Retention times: tR (S) 8.9 min, tR (R) 9.3 min; 2a, tR 7.1 min. (1R,2R)-(3-Fluorophenyl)-2,3-glycidol (2c). Elute: petroleum ether–ethyl acetate 7 : 1, Rf: 0.3, 85% yield; 1H NMR(600 MHz, CDCl3): δ 7.01–7.37 (m, 4H, Ar–H), 4.94 (s, 1H, –CH), 3.19–3.21 (m, 1H, –CH), 2.87–2.88 (m, 1H, –CH2), 2.76–2.77 (m, 1H, –CH2), 2.28 (br, 1H, OH). Retention times: tR1 12.1 min, tR2 14.4 min, tR3 14.9 min, tR4 15.8 min; 2b, tR 6.5 min. (S)-1-(4-Fluorophenyl)prop-2-en-1-ol (3b). Elute: petroleum ether–ethyl acetate 15 : 1, Rf: 0.3, 92% yield; [α]20 D +11.3 (c 0.81 in CHCl3); 1H NMR(600 MHz, CDCl3): δ 7.25–7.37 (m, 2H, Ar– H), 7.04–7.06 (m, 2H, Ar–H), 5.98–6.03 (m, 1H, vCH), 5.36 (d, J = 18 Hz, 1H, –CH), 5.21–5.23 (m, 2H, vCH2), 2.03 (br, 1H,

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OH). Retention times: tR (S) 9.7 min, tR (R) 10.3 min; 3a, tR 8.1 min. (1R,2R)-(4-Fluorophenyl)-2,3-glycidol (3c). Elute: petroleum ether–ethyl acetate 7 : 1, Rf: 0.25, 88% yield; 1H NMR(600 MHz, CDCl3): δ 7.36–7.41 (m, 2H, Ar–H), 7.05–7.09 (m, 2H, Ar–H), 4.48 (d, J = 6 Hz, 1H, –CH), 3.19–3.21 (m, 1H, –CH), 2.87–2.88 (m, 1H, –CH2), 2.77–2.78 (m, 1H, –CH2), 2.24 (br, 1H, OH). Retention times: tR1 13.6 min, tR2 18.1 min, tR3 21.4 min; 3b, tR 7.2 min. (S)-1-m-Tolylprop-2-en-1-ol (4b). Elute: petroleum ether– ethyl acetate 15 : 1, Rf: 0.35, 91% yield; [α]20 D −8.4 (c 0.57 in CHCl3); 1H NMR(600 MHz, CDCl3): δ 7.23–7.25 (m, 1H, Ar–H), 7.19 (s, 1H, Ar–H), 7.16 (d, J = 12 Hz, 1H, Ar–H), 7.10 (d, J = 12 Hz, 1H, Ar–H), 6.02–6.08 (m, 1H, vCH), 5.35 (d, J = 12 Hz, 1H, –CH), 5.16–5.20 (m, 2H, vCH2), 2.36 (m, 3H, –CH3). Retention times: tR (S) 8.9 min, tR (R) 9.6 min; 4a, tR 7.4 min. (1R,2R)-(m-Tolylprop)-2,3-glycidol (4c). Elute: petroleum ether–ethyl acetate 5 : 1, Rf: 0.35, 76% yield; 1H NMR(600 MHz, CDCl3): δ 7.14–7.29 (m, 4H, Ar–H), 4.45 (d, J = 6 Hz, 1H, –CH), 3.21–3.23 (m, 1H, –CH), 2.83–2.86 (m, 1H, –CH2), 2.76–2.78 (m, 1H, –CH2), 2.37 (s, 3H, –CH3), 2.34 (br, 1H, OH). Retention times: tR1 11.4 min, tR2 12.8 min, tR3 13.4 min, tR4 14.1 min; 4b, tR 6.3 min. (S)-1-p-Tolylprop-2-en-1-ol (5b). Elute: petroleum ether–ethyl acetate 15 : 1, Rf: 0.3, 91% yield; [α]20 D −5.7 (c 0.63 in CHCl3); 1 {lit.32 [α]20 D −3.8 (c 0.8 in CHCl3)}; H NMR(600 MHz, CDCl3): δ 7.26 (d, J = 12 Hz, 2H, Ar–H), 7.17 (d, J = 6 Hz, 2H, Ar–H), 6.02–6.07 (m, 1H, vCH), 5.24 (d, J = 18 Hz, 1H, –CH), 5.16–5.19 (m, 2H, vCH2), 2.35 (m, 3H, –CH3), 1.98 (br, 1H, OH). Retention times: tR (S) 10.5 min, tR (R) 12.3 min; 5a, tR 12.3 min. (1R,2R)-( p-Tolylprop)-2,3-glycidol (5c). Elute: petroleum ether–ethyl acetate 5 : 1, Rf: 0.3, 78% yield; 1H NMR(600 MHz, CDCl3): δ 7.28–7.32 (m, 2H, Ar–H), 7.19–7.20 (m, 2H, Ar–H), 4.45 (d, J = 6 Hz, 1H, –CH), 3.21–3.23 (m, 1H, –CH), 2.84–2.86 (m, 1H, –CH2), 2.76–2.77 (m, 1H, –CH2), 2.36 (s, 3H, –CH3), 2.25 (br, 1H, OH). Retention times: tR1 12.5 min, tR2 15.1 min, tR3 16.8 min, tR4 19.6 min; 5b, tR 7.1 min. (S)-1-(Thiophen-2-yl)prop-2-en-1-ol (6b). Elute: petroleum ether–ethyl acetate 15 : 1, Rf: 0.3, 74% yield; [α]20 D +13.1 (c 0.42 1 in CHCl3); {lit.31 [α]26 D +18.4 (c 0.5, CHCl3)}; H NMR(600 MHz, CDCl3): δ 7.25–7.27 (m,1H, Ar–H), 6.97–6.99 (m, 2H, Ar–H), 6.09–6.15 (m, 1H, vCH), 5.39–5.42 (br, 2H, vCH2, –CH), 5.25 (d, J = 6 Hz, 1H, vCH2), 2.03 (br, 1H, OH). Retention times: tR (S) 11.6 min, tR (R) 14.7 min; 6a, tR 10.7 min. (1R,2R)-(Thiophen-2-yl)-2,3-glycidol (6c). Elute: petroleum ether–ethyl acetate 5 : 1, Rf: 0.3, 70% yield; 1H NMR(600 MHz, CDCl3): δ 7.32–7.33 (m, 1H, Ar–H), 7.10–7.11 (m, 1H, Ar–H), 7.02–7.03 (m, 1H, Ar–H), 4.79 (d, J = 6 Hz, 1H, –CH), 3.31–3.32 (m, 1H, –CH), 2.90–2.92 (m, 1H, –CH2), 2.84–2.87 (m, 1H, –CH2). Retention times: tR1 15.9 min, tR2 18.5 min, tR3 22.5 min, tR4 27.7 min; 6b, tR 7.6 min. (S)-1-(Thiophen-3-yl)prop-2-en-1-ol (7b). Elute: petroleum ether–ethyl acetate 15 : 1, Rf: 0.25, 89% yield; [α]20 D +9.8 (c 0.33, 1 CHCl3; {lit.31 [α]35 D +13.4 (c 0.5, CHCl3)}; H NMR(600 MHz, CDCl3): δ 7.25–7.26 (m, 1H, Ar–H), 7.15–7.16 (m, 1H, Ar–H),

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7.03 (d, J = 6 Hz, 1H, Ar–H), 6.00–6.05 (m, 1H, vCH), 5.30 (d, J = 18 Hz, 1H, –CH), 5.16–5.19 (m, 2H, vCH2). Retention times: tR (S) 11.3 min, tR (R) 14.2 min; 7a, tR 10.6 min. (1R,2R)-(Thiophen-3-yl)-2,3-glycidol (7c). Elute: petroleum ether–ethyl acetate 2 : 1, Rf: 0.2, 85% yield; 1H NMR(600 MHz, CDCl3): δ 7.35–7.36 (m, 2H, Ar–H), 7.14–7.15 (m, 1H, Ar–H), 4.61 (br, 1H, –CH), 3.26–3.28 (m, 1H, –CH), 2.88–2.90 (m, 1H, –CH2), 2.84–2.86 (m, 1H, –CH2). Retention times: tR1 17.3 min, tR2 19.7 min, tR3 27.2 min, tR4 38.90 min; 7b, tR 8.1 min. (S)-1-(3-Chlorophenyl)prop-2-en-1-ol (8b). Elute: petroleum ether–ethyl acetate 15 : 1, Rf: 0.35, 92% yield; [α]20 D +10.4 (c 0.65 in CHCl3); 1H NMR(600 MHz, CDCl3): δ 7.36 (s, 1H, Ar–H), 7.22–7.28 (m, 3H, Ar–H), 5.96–6.01 (m, 1H, vCH), 5.34(d, J = 18 Hz, 1H, –CH), 5.21 (d, J = 12 Hz, 1H, vCH2), 5.15 (d, J = 6 Hz, 1H, vCH2). Retention times: tR (S) 8.9 min, tR (R) 9.3 min; 1a, tR 7.0 min. (1R,2R)-(3-Chlorophenyl)-2,3-glycidol (8c). Elute: petroleum ether–ethyl acetate 5 : 1, Rf: 0.25, 71% yield; 1H NMR(600 MHz, CDCl3): δ 7.28–7.43 (m, 4H, Ar–H), 4.48 (s, 1H, –CH), 3.20–3.23 (m, 1H, –CH), 2.88–2.89 (m, 1H, –CH2), 2.76–2.78 (m, 1H, –CH2), 2.38 (br, 1H, OH). Retention times: tR1 13.3 min, tR2 14.5 min, tR3 14.9 min, tR4 15.8 min; 8b, tR 6.7 min. (S)-1-(4-Chlorophenyl)prop-2-en-1-ol (9b). Elute: petroleum ether–ethyl acetate 15 : 1, Rf: 0.3, 90% yield; [α]20 D +1.2 (c 0.81 in 1 CHCl3); {lit.31 [α]26 D +15.3 (c 1.0, CHCl3)}; H NMR(600 MHz, CDCl3): δ 7.29–7.33 (m, 4H, Ar–H), 5.97–6.02 (m, 1H, vCH), 5.34 (d, J = 12 Hz, 1H, –CH), 5.21 (d, J = 6 Hz, 1H, vCH2), 5.17 (br, 1H, vCH2), 2.03 (br, 1H, –OH). Retention times: tR (S) 9.1 min, tR (R) 9.8 min; 1a, tR 7.9 min. (1R,2R)-(4-Chlorophenyl)-2,3-glycidol (9c). Elute: petroleum ether–ethyl acetate 5 : 1, Rf: 0.3, 72% yield; 1H NMR(600 MHz, CDCl3): δ 7.31–7.35 (m, 2H, Ar–H), 4.45 (d, J = 6 Hz, 1H, –CH), 3.19–3.21 (m, 1H, –CH), 2.85–2.86 (m, 1H, –CH2), 2.74–2.76 (m, 1H, –CH2), 2.51 (br, 1H, OH). Retention times: tR1 13.5 min, tR2 18.3 min, tR3 20.7 min; 9b, tR 7.4 min. (S)-1-(3-Bromophenyl)prop-2-en-1-ol (10b). Elute: petroleum ether–ethyl acetate 15 : 1, Rf: 0.35, 93% yield; [α]20 D +7.6 (c 0.48 in CHCl3); 1H NMR(600 MHz, CDCl3): δ 7.52 (s, 1H, Ar–H), 7.40 (d, J = 12 Hz,1H, Ar–H), 7.25–7.28 (m, 1H, Ar–H), 7.19–7.22 (m, 1H, Ar–H), 5.95–6.00 (m, 1H, vCH), 5.33(d, J = 12 Hz, 1H, –CH), 5.21 (d, J = 12 Hz, 1H, vCH2), 5.13 (d, J = 6 Hz, 1H, vCH2). Retention times: tR (S) 9.2 min, tR (R) 9.7 min; 1a, tR 7.4 min. (1R,2R)-(3-Bromophenyl)-2,3-glycidol (10c). Elute: petroleum ether–ethyl acetate 5 : 1, Rf: 0.25, 40% yield; 1H NMR (600 MHz, CDCl3): δ 7.46–7.47 (d, J = 6 Hz, 2H, Ar–H), 7.24–7.27 (m, 2H, Ar–H), 4.47 (br, 1H, –CH), 3.19–3.22 (m, 1H, –CH), 2.87–2.89 (m, 1H, –CH2), 2.76–2.77 (m, 1H, –CH2), 2.46 (br, 1H, OH). Retention times: tR1 14.2 min, tR2 15.3 min, tR3 15.9 min, tR4 16.6 min; 10b, tR 7.1 min. (S)-1-(4-Bromophenyl)prop-2-en-1-ol (11b). Elute: petroleum ether–ethyl acetate 15 : 1, Rf: 0.3, 92% yield; [α]20 D +8.4 (c 0.78 in CHCl3); {lit.33 [α]D +11.59 (c 3.28, PhH) }; 1H NMR(600 MHz, CDCl3): δ 7.45 (d, J = 12 Hz, 2H, Ar–H), 7.20 (d, J = 12 Hz, 2H, Ar–H), 5.93–5.99 (m, 1H, vCH), 5.30 (d, J = 18 Hz, 1H, –CH), 5.18 (d, J = 12 Hz, 1H, vCH2), 5.10 (br, 1H, vCH2), 2.01 (br,

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1H, OH). Retention times: tR (S) 9.6 min, tR (R) 10.4 min; 1a, tR 8.5 min. (1R,2R)-(4-Bromophenyl)-2,3-glycidol (11c). Elute: petroleum ether–ethyl acetate 5 : 1, Rf: 0.3, 52% yield; 1H NMR (600 MHz, CDCl3): δ 7.52–7.54 (m, 2H, Ar–H), 7.29–7.32 (m, 2H, Ar–H), 4.49 (d, J = 6 Hz, 1H, –CH), 3.18–3.22 (m, 1H, –CH), 2.87–2.89 (m, 1H, –CH2), 2.76–2.78 (m, 1H, –CH2), 2.24 (br, 1H, OH). Retention times: tR1 14.4 min, tR2 19.4 min, tR3 22.5 min; 11b, tR 7.8 min. (S)-1-(3-Methoxyphenyl)prop-2-en-1-ol (12b). Elute: petroleum ether–ethyl acetate 10 : 1, Rf: 0.3, 88% yield; [α]20 D +20.7 1 (c 0.9 in CHCl3); {lit.34 [α]20 D +23.1 (c 0.75, CHCl3)}; H NMR (600 MHz, CDCl3): δ 7.25–7.28 (m, 1H, Ar–H), 6.93–6.95 (m, 2H, Ar–H), 6.81–6.83 (m, 1H, Ar–H), 6.01–6.06 (m, 1H, vCH), 5.35 (d, J = 18 Hz, 2H, –CH), 5.16–5.20 (m, 2H, vCH2), 3.80 (s, 3H, –CH3). Retention times: tR (S) 14.5 min, tR (R) 15.7 min; 1a, tR 10.0 min. (1R,2R)-(3-Methoxyphenyl)-2,3-glycidol (12c). Elute: petroleum ether–ethyl acetate 4 : 1, Rf: 0.25, 30% yield; 1H NMR (600 MHz, CDCl3): δ 7.27–7.30 (m, 1H, Ar–H), 6.95–6.98 (m, 2H, Ar–H), 6.85–6.86 (m, 1H, Ar–H), 4.41 (d, J = 6 Hz, 1H, –CH), 3.81 (s, 3H, –OCH3), 3.18–3.20 (m, 1H, –CH), 2.80–2.84 (m, 2H, –CH). Retention times: tR1 20.6 min, tR2 21.9 min, tR3 26.8 min, tR4 28.3 min; 12b, tR 9.5 min. (S)-1-(4-Methoxyphenyl)prop-2-en-1-ol (13b). Elute: petroleum ether–ethyl acetate 10 : 1, Rf: 0.2, 90% yield; [α]20 D −3.9 1 (c 0.4 in CHCl3); {lit.31 [α]26 D −6.04 (c 1.0, CHCl3)}; H NMR (600 MHz, CDCl3): δ 7.28 (d, J = 12 Hz, 2H, Ar–H), 6.88 (d, J = 6 Hz, 2H, Ar–H), 6.01–6.06 (m, 1H, vCH), 5.32 (d, J = 18 Hz, 2H, –CH), 5.14–5.18 (m, 2H, vCH2), 3.79 (s, 3H, –CH3), 2.03 (br, 1H, OH). Retention times: tR (S) 21.4 min, tR (R) 23.0 min; 1a, tR 17.9 min. (1R,2R)-(4-Methoxyphenyl)-2,3-glycidol (13c). Elute: petroleum ether–ethyl acetate 3 : 1, Rf: 0.2, 65% yield; 1H NMR (600 MHz, CDCl3): δ 7.31–7.36 (m, 2H, Ar–H), 6.91–6.92 (m, 2H, Ar–H), 4.44 (d, J = 6 Hz, 1H, –CH), 3.83 (s, 3H, –OCH3), 3.20–3.22 (m, 1H, –CH), 2.84–2.85 (m, 1H, –CH2), 2.77–2.78 (m, 1H, –CH2), 2.29 (br, 1H, OH). Retention times: tR1 25.9 min, tR2 30.2 min, tR3 33.0 min; 13b, tR 15.7 min. (S)-1-Phenylbut-3-en-2-ol (14b). Elute: petroleum ether–ethyl acetate 20 : 1, Rf: 0.2, 78% yield; [α]20 D +7.1 (c 0.38 in CHCl3) 1 {lit.35 [α]25 D +12.7 (c 1, CHCl3)}; H NMR(600 MHz, CDCl3): δ 7.22–7.32 (m, 5H, Ar–H), 5.90–59.6 (m, 1H, vCH), 5.24 (d, J = 18 Hz, 1H, vCH2), 5.13 (d, J = 6 Hz, 1H, vCH2), 4.33–4.36 (m, 1H, –CH), 2.88 (dd, J = 6 Hz, J = 12 Hz, 1H, –CH2−), 2.79 (dd, J = 6 Hz, J = 12 Hz, 1H, –CH2−). Retention times: tR (S) 10.1 min, tR (R) 11.4 min; 1a, tR 12.1 min. (1R,2R)-Phenylbut-2,3-glycidol (14c). Elute: petroleum ether–ethyl acetate 5 : 1, Rf: 0.4, 44% yield; 1H NMR (600 MHz, CDCl3): δ 7.22–7.33 (m, 5H, Ar–H), 3.88–3.90 (m, 1H, –CH), 3.02–3.05 (m, 1H, –CH), 2.87–2.92 (m, 1H, –CH2), 2.84–2.86(m, 1H, –CH2), 2.76–2.79 (m, 1H, –CH2), 2.72–2.73 (m, 1H, –CH2). Retention times: tR1 13.9 min, tR2 16.2 min, tR3 23.5 min, tR4 26.8 min; 14b, tR 6.4 min. 1-(Thiophen-2-yl)propan-1-one (6d). 9% yield; 1H NMR (600 MHz, CDCl3): δ 7.71 (d, J = 6 Hz, 1H, Ar–H), 7.61 (d, J =

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6 Hz, 1H, Ar–H), 7.11–7.13 (m, 1H, Ar–H), 2.92–2.96 (m, 2H, –CH2), 1.23–1.25 (m, 3H, –CH3). Phenylbutan-2-one (14d). 6% yield; 1H NMR(600 MHz, CDCl3): δ 7.20–7.34 (m, 5H, Ar–H), 3.69 (s, 2H, –CH2), 2.47 (dd, J = 6 Hz, J = 12 Hz, 2H, –CH2), 1.01–1.04 (m, 3H, –CH3).

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Acknowledgements This work was supported by the National Natural Science Foundation of China (21072183 & 21372216), and the Open Fund of Key Laboratory of Environmental and Applied Microbiology (KLCAS-2013-06) and the Key Research Program (KGZD-EW-606-14) of the Chinese Academy of Sciences.

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Synthesis of enantiopure glycidol derivatives via a one-pot two-step enzymatic cascade.

Styrene monooxygenase (SMO) can catalyze the kinetic resolution of secondary allylic alcohols to provide enantiopure glycidol derivatives. To overcome...
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