Amino Acids
Stereoselective introduction of two chiral centers by a single
diketoreductase: an efficient biocatalytic route
for the synthesis of statin side chains
Xuri Wu . Lili Wang . Shuzhen Wang . Yijun Chen
Laboratory of Chemical Biology, China Pharmaceutical University,
24 Tongjia Street, Nanjing, Jiangsu Province, 210009, People’s Republic of China
E-mail: yjchen@cpu.edu.cn
Characterization of 5
Compound 5 was confirmed by NMR spectroscopy and mass spectrometry. 1H NMR (500 MHz,
CDCl3, signals of isopropylidene are given): δ = 7.26-7.33 (m, 5H, Ph), 4.55 (dd, J = 12.7 Hz, 2H,
Ph-CH2), 4.31 (m, 1H, H-3), 4.16 (q, J = 7.1 Hz, 2H, CO2CH2), 4.11 (m, 1H, H-5), 3.51 (dd, J = 11.0,
5.6Hz, 1H, H-6), 3.41 (dd, J = 11.0, 5.6 Hz, 1H, H-6), 2.52 (dd, J = 10.0, 7.1 Hz, 1H, H-2), 2.37 (dd, J
= 10.0, 5.9 Hz, 1H, H-2), 1.62 (m, 1H, H-4), 1.47 (s, 3H, C(CH3)), 1.39 (s, 3H, C(CH3)), 1.27 (t, J =7.2,
3H, CH3), 1.25 (m, 1H, H-4); 13C NMR (125 MHz, CDCl3, 20°C): δ = 14.2, 19.7, 30.0, 33.3, 41.6, 73.4,
60.6, 65.7, 68.4, 73.5, 98.9, 127.6, 127.7, 128.4, 138.2, 170.8; MS (ESI) m/z 345.2 [M+Na].
Characterization of 6
The structure of 6 was identified by NMR spectroscopy and high resolution mass spectrometry, and
its optical rotation was determined. 1H NMR (500 MHz, CDCl3):δ = 4.34 (m, 1H, H-3), 4.15 (q, J = 7.3
Hz, 2H, CO2CH2), 4.02 (m, 1H, H-5), 3.60 (dd, J = 11.4, 5.1Hz, 1H, H-6), 3.41 (dd, J = 11.4, 5.1 Hz,
1H, H-6), 2.54 (dd, J = 15.4, 7.2 Hz, 1H, H-2), 2.37 (dd, J = 15.4, 6.0 Hz, 1H, H-2), 1.76 (brs,1H, OH),
1.53 (m, 1H, H-4), 1.47 (s, 3H, C(CH3)), 1.38 (s, 3H, C(CH3)), 1.26 (t, J =7.2, 3H, CH3),1.24 (m, 1H,
H-4). 13C NMR (125 MHz, CDCl3, 20°C): δ = 14.1, 19.8, 29.9, 31.8, 41.5, 65.5, 60.5, 65.9, 69.4, 99.0,
170.9. HRMS (ESI) calcd. for C11H20O5 [M+Na]: 255.1203, found: 255.1209; [α]20
D = + 7.4 (c = 0.86 in
CHCl3) { literature data for t-butyl ester [α]20
= + 9.9 (c = 2.0 in CHCl3) } (Sun et al. 2007).
D
Supplementary Fig. 1 Time courses of the reduction of 1 by rDKR with 2-propanol and NADP+.
Substrate 1 at 10 g/L was transformed by recombinant whole cell suspensions with 5% 2-propanol and
NADP+. All data represent averages of three experiments. Open diamond: 1; closed diamond: 2; closed
triangle: 3; open triangle: 4.
Supplementary Fig. 1 Time courses of the reduction of 1 by rDKR with glucose.
The conversion of 1 was performed by recombinant whole cells and glucose. All data represent
averages of three experiments. Open diamond: 1; closed diamond: 2; closed triangle: 3; open triangle:
4.
Supplementary Table 1.
Effects of substrate concentration on the biocayalytic conversion.
Concentration of 1 (mg/ml)
a
Conversion of 1 (%) a
de (%) b
ee (%) b
1
97.1
> 99.5
>99.5
2
96.0
>99.5
>99.5
5
95.4
> 99.5
>99.5
10
93.5
> 99.5
>99.5
15
65.1
> 99.5
>99.5
Conversion of 1 to 2 was determined by HPLC.
b
The de and ee values of 2 were determined by
Chiralcel OD-RH column according to previous reports (Wu et al. 2008; Wu et al. 2009; Liu et al.
2008).
Supplementary References
Sun FL, Xu G, Wu JP, Yang LR (2007) A new and facile preparation of tert-butyl (3R,
5S)-6-hydroxy-3, 5-O-isopropylidene-3, 5-dihydroxyhexanoate. Tetrahedron: Asymmetry 18:
2454-2461
Wu XR, Liu N, He YM, Chen YJ (2008) A bacterial enzyme catalyzing double reduction of a
β,δ-diketo ester with unprecedented stereoselectivity. Nature Proceedings <http://hdl. handle.
net/10101/npre.2008.1697.1>
Wu XR, Liu N, He YM, Chen YJ (2009) Cloning, expression and characterization of a novel
diketoreductase from Acinetobacte baylyi. Acta Biochim Biophys Sin 41: 163-170
Liu JH, Yu BY, Chen YJ (2008) Determination of enantiomeric excess of ethyl 3, 5- dihydroxy
-6-benzyloxy hexanoate by chiral reverse phase high performance liquid chromatography. Chirality
25:51-53