*4.9. Preparative-Scale Synthesis of Enantiomeric β-Phenylalcohols (S)-***2a***–***2i***, (S)-***2m** *and (S)-***2n** *by Resting Cells*

For isolation and characterization of the bioreduction product, the reaction was performed on a preparative scale: 300 g resting cells of *R. rubra* AS 2.2241 were resuspended in 1000 mL of Na2HPO4-KH2PO4 buffer (100 mM, pH 7.0) with 50 g glucose and 10 mM of each substrate (**1a**–**1i**, **1m** and **1n**). The reaction mixture was incubated at 25 ◦C and shaken at 220 rpm for 24 h. The cells were removed by centrifugation and the supernatant was saturated with NaCl. The supernatant was extracted with EtOAc (1000 mL × 3). The organic phases were washed with brine, dried over Na2SO4, filtered and concentrated in vacuo. The residue was purified by flash chromatography on silica gel (eluent: EtOAc/PE 1:20) to give the enantiomerically pure alcohols **2a**–**2i**, **2m**, **2n**. The isolated yield and *ee* of preparative-scale are comparable to those obtained from screening biotransformations. The spectroscopic data (1H and <sup>13</sup> C NMR, and HPLC retention times) of enantiomerically alcohols **2a**–**2i**, **2m** and **2n** are in agreement with those obtained for racemic forms, as described in the Supplementary Materials.

#### *4.10. Assay Methods*

Reaction products were analysed by chiral HPLC analysis using a Shimadzu LC-10AT VP series (Tokyo, Japan) and a Shimadzu SPD-M10Avp photo diode array detector (190–370 nm) with a Chiralcel AD-H column [eluent: *n*-hexane/*i*-PrOH (95:5, *v/v*), flow rate: 0.5 mL/min, column temperature 25 ◦C]. The yields (quantified using calibration curves) and product *ee* values of analytes were determined by chiral HPLC analyses according to the following retention time data: 1-(2-bromophenyl)ethanone (**1a**) [**1a**, 11.85 min; (*R*)-**2a**, 12.71 min (*S*)-**2a**, 13.23 min], 1-(3-bromophenyl)ethanone (**1b**) [**1b**, 10.66 min; (*S*)-**2b**, 16.39 min; (*S*)-**2b**, 17.26 min], 1-(3-bromophenyl)ethanone (**1c**) [**1c**, 11.21 min; (*S*)-**2c**, 16.83 min; (*R*)-**2c**, 17.99 min], 1-(2-nitrophenyl)ethanone (**1d**) [**1d**, 12.54; (*R*)-**2d**, 21.33 min; (*S*)-**2d**, 22.63 min], 1-(3-nitrophenyl)ethanone (**1e**) [**1e**, 21.14 min; (*R*)-**2e**, 26.25 min; (*S*)-**2e**, 27.06 min], 1-(3-nitrophenyl)ethanone (**1f**) [**1f**, 20.24 min; (*S*)-**2f**, 35.54 min; (*R*)-**2f**, 38.20 min], 1-(2-(trifluoromethyl)phenyl)ethanone (**1g**) [**1g**, 12.49 min; (*R*)-**2g**, 11.83 min; (*S*)-**2g**, 12.34 min], 1-(3-(trifluoromethyl)phenyl)ethanone (**1h**) [**1h**, 11.13 min; (*S*)-**2h**, 14.06 min; (*R*)-**2h**, 15.57 min], 1-(4-(trifluoromethyl)phenyl)ethanone (**1i**) [**1i**, 11.68 min; (*S*)-**2i**, 16.55 min; and (*R*)-**2i**, 17.79 min], 1-(2,4-dichlorophenyl)ethanone (**1m**) [**1m**, 15.09 min; (*R*)-**2m**, 19.09 min; (*S*)-**2m**, 21.26 min], 1-(3,4-dichlorophenyl)ethanone (**1n**) [**1n**, 12.39 min; (*R*)-**2n**, 12.79 min; and (*S*)-**2n**, 13.67 min].

The optical rotations of products isolated from the biocatalytic reactions were determined in a 1-dm cuvette using a Perkin-Elmer model 241 polarimeter and were referenced to the Na–D line. The experimental and reported data are listed below: (*S*)-**2a**, [α] 20 <sup>D</sup> = −62.4 (*c* 1.00, CHCl3) {(*S*)-1-(2-bromophenyl)ethanol [α] 27 <sup>D</sup> = −29.8 (*c* 0.68, CHCl3) [42]}; (*S*)-**2b**, [α] 20 <sup>D</sup> = −43.9 (*c* 1.00, CHCl3) {(*S*)-1-(3-bromophenyl)ethanol [α] 25 <sup>D</sup> = −27.6 (*c* 1.00 in CHCl3) [35]}; (*S*)-**2c**, [α] 20 <sup>D</sup> = −17.3 (*c* 1.00, MeOH, {(*S*)-1-(4-bromophenyl)ethanol [α] 21 <sup>D</sup> = −20.6 (*c* 1.07, MeOH) [39]}; (*S*)-**2d**, [α] 20 <sup>D</sup> = +64.9 (*c* 0.1, MeOH) {(*S*)-1-(2-nitrophenyl)ethanone [α] 25 <sup>D</sup> = +18.5 (*c* 0.23, MeOH) [40]}; (*S*)-**2e**, [α] 20 <sup>D</sup> = −88.5 (*c* 0.1, CHCl3) {(*S*)-1-(3-nitrophenyl)ethanone [α] 25 <sup>D</sup> = −20.5 (*c* 1.0, CHCl3) [40]}; (*S*)-**2f**, [α] 20 <sup>D</sup> = −67.2 (*c* 0.1, CHCl3) {(*S*)-1-(4-nitrophenyl)ethanone [α] 25 <sup>D</sup> = −20.5 (*c* 1.2, CHCl3) [40]}; (*S*)-**2g**, [α] 20 <sup>D</sup> = −43.1 (*c* 0.1, MeOH) {(*S*)-1-(2-Trifluoromethylphenyl) ethanol [α] 26 <sup>D</sup> = −37.7 (*c* 1.0, CH3OH) [42]}; (*S*)-**2h**, [α] 20 <sup>D</sup> = −55.1 (*c* 0.2, MeOH) {(*S*)-1-(3-(trifluoromethyl)phenyl)ethanol [α] 25 <sup>D</sup> = −21.9 (*c* 1.40, MeOH) [35]}; (*S*)-**2i**, [α] 20 <sup>D</sup> = −62.2 (*c* 0.2, CHCl3) {(*S*)-1-(4-(trifluoromethyl)phenyl)ethanol [α] 25 <sup>D</sup> = −33.7 (*c* 5.52, CHCl3) [35]}; (*S*)-**2m**, [α] 20 <sup>D</sup> = −75.05 (*c* 0.2, CHCl3) {(*S*)-1-(2,4-dichlorophenyl)ethanol [α] 25 <sup>D</sup> = −52.4 (*c* 0.55, CHCl3) [43]}; (*S*)-**2n**, [α] 20 <sup>D</sup> = −30.0 (*c* 0.3, CHCl3) {(*R*)-1-(3,4-dichlorophenyl)ethanol [α] 20 <sup>D</sup> = +35.8 (*c* 1.00, CHCl3) [41]}.

**Supplementary Materials:** The following are available online at www.mdpi.com/1660-3397/16/2/62/s1. The NMR spectra and HPLC spectra of all the compounds are shown.

**Acknowledgments:** This study was funded by the Basic Research Program of Sun Yat-Sen University (Grant No. 17lgpy58), Natural Science Foundation of Guangdong Province (Grant No. 2017A030310232), the National Natural Science Foundation of China (Grant No. 41706148), China's Marine Commonweal Research Project (Grant No. 201305017), and the special financial fund of innovative development of marine economic demonstration project (Grant No. GD2012-D01-001).

**Author Contributions:** B.-S.C. and F.Z.R.d.S. designed the study. B.-S.C. wrote the paper. H.L. performed the enzymatic experiments. B.-S.C. and L.L. supervised the study. All authors made substantial contributions to the discussion of data and approved the final manuscript.

**Conflicts of Interest:** The authors declare no conflict of interest.
