X.-Q. Pei et al. / Journal of Molecular Catalysis B: Enzymatic 123 (2016) 91–99
93
Fig. 1. Phylogenetic relationship of amino acid sequences of Chr-OYE1 and Chr-OYE2 to other known OYEs. The distance neighbor-joining tree was created using the Mega5.2
with Clustal W method. Bootstrap value at braches was expressed as percentages of 1000 replications.
organic phase was combined and concentrated under reduced pres-
sure. The final product was purified with column chromatography
and subjected to GC or HPLC analysis, as well as NMR analysis to
confirm the structure and purity.
data were in agreement with literature [20]; retention times: tR (R)
12.4 min, tR (S) 12.8 min.
(R)-N-phenyl-2-methylsuccinimide (12b): 1H NMR (600 MHz,
CDCl ): ı 1.45 (d, 3H, CH , J = 7.2 Hz), 2.50 (dd, 1H, CH , J = 4.80 Hz,
3
3
2
1H NMR spectra were recorded on a Bruker Advance 600 MHz
spectrometer (Bruker, Switzerland) and optical rotations were col-
lected on a PerkinElmer-341 polarimeter (PerkinElmer, USA). GC
analysis was performed on a Fuli 9790 II GC system equipped with
a flame ionization detector. The conversion of substrates 1a, 2a,
17.70 Hz), 3.09 (dd, 1H, CH , J = 9.00 Hz, 17.70 Hz), 3.06–3.01 (m, 1H,
2
CH), 7.29 (d, 2H, ArH), 7.39 (t, 1H, ArH), 7.47(t, 2H, ArH); The NMR
25
data were in agreement with literature [20]; [˛]D = +7.1 (c = 2.0,
25
CHCl ) for >99% ee (lit. [28] [˛]D = +7.3 (c = 0.5, CHCl ) for 100%
3
3
ee, (R)); retention times: tR (R) 20.3 min, tR (S) 22.1 min.
1
4
a, 5a, 8a–11a, 18a–19a to the corresponding products was deter-
(R)-1-Nitro-2-phenylpropane (13b): H NMR (600 MHz, CDCl ):
3
mined on a 30QC2/AC5 column (30 m × 0.22 mm ID, 0.25 m film
thickness; SGE Analytical Science, Australia). The substrate con-
version and enantiomeric excess of products for 3a, 6a and 7a
were determined on a CP-Chirasil-DEX column (25 m × 0.25 mm,
Varian, USA) with the temperatures of the injector and detector
ı 1.38 (d, 3H, CH , J = 7.2 Hz), 3.60–3.66 (Ar–CH, m, 1H), 4.48 (dd,
3
1H, NO CH , J = 8.4 Hz, 12.0 Hz), 4.55 (dd, 1H, NO CH , J = 7.2 Hz,
2
2
2
2
12.0 Hz), 7.22–7.35 (m, 5H, ArH); The NMR data were in agreement
2
5
with literature [23]; [˛]D = +38.0 (c = 1.0, CHCl ) for 99% ee (lit.
3
[29] [˛]D27 = +44.3 (c = 3.4, CHCl ) for 98 % ee, (R)); retention times:
3
◦
◦
set at 260 C and 280 C, respectively. HPLC analysis was per-
formed on a Shimadzu Prominence LC-20AD system equipped with
a PDA detector. The flow rate was set to be 0.8 ml/min and the
tR (S) 13.4 min, tR (R) 14.6 min.
1-Phenyl-2-nitropropane (14b): light yellow oil; 1H NMR
(600 MHz, CDCl ); ı 1.55(d, 3H, CH ), 3.01(dd, 1H, ArCH , J = 6.6 Hz,
3
3
2
◦
column temperature was 35 C. The conversion and enantiomeric
13.8 Hz), 3.33(dd, 1H, ArCH , J = 7.8 Hz, 14.1 Hz), 4.76–4.79(CH NO
2
2
,
excess of substrates 12a–17a to the corresponding product were
determined on a Chiralcel OD–H column (12a) with n-hexane/2-
propanol (90:10 v/v), or OJ–H column (4.6 mm × 250 mm, Daicel,
Japan) with n-hexane/2-propanol (90:10 v/v, 13a–14a, 16a–17a)
or with n-hexane/2-propanol (75:25 v/v, 15a).
m, 1H), 7.16–7.32(m, 5H, ArH); The NMR data were in agreement
with literature [26]; retention times: tR (R) 11.1 min, tR (S) 12.0 min.
1
(R)-Ethyl-3-nitro-2-phenylpropanoate
(15b):
H
NMR
(600 MHz, CDCl ): ı 1.22 (t, 3H, CH , J = 7.1 Hz), 4.2 (m, 2H,
3
3
CH CH ), 4.42 (dd, 1H, CH, J = 5.1 Hz, 10.0 Hz), 4.54 (dd, 1H,
2
3
CH NO , J = 5.1 Hz, J = 14.6 Hz), 5.10 (dd, 1H, CH NO , J = 14.6 Hz,
2
2
2
2
J = 10.0 Hz), 7.38–7.25 (m, 5H, ArH); The NMR data were in agree-
2
5
2
.6. Spectral data for biotransformation products
ment with literature [23]; [˛]D = +130 (c = 0.4, CHCl3) for 96% ee
(
lit. [24] [˛]D25 = −126.2 (c = 2.8, CHCl ) for 94 % ee, (S)); retention
3
Cyclohexanone (4b): 1H NMR (600 MHz, CDCl ): ı 1.71–1.75
times: tR (R) 14.3 min, tR (S) 18.0 min.
3
(
m, 2H, CH (CH CH ) ), 1.85–1.89 (m, 4H, CH CH CO), 2.34 (t, 4H,
(E)-4-Phenylbut-3-en-2-one (16a, product of 17a reduction):
H NMR (600 MHz, CDCl3): ı 2.38 (s, 3H, CH3), 6.72 (d, 1H, CHCO,
2
2
2 2
2
2
1
CH CO, J = 7.2 Hz,); The NMR data were in agreement with literature
2
[
20]; retention time: tR 5.9 min.
,2,6-Trimethylcyclohexane-1,4-dione (6b): H NMR (600 MHz,
CDCl ): ı 1.12 (s, 3H, (CH ) C), 1.15 (d, 3H, CH CH, J = 6.5 Hz), 1.21
J = 16.2 Hz), 7.39–7.40 (m, 3H, ArH), 7.51 (d, 1H, CHCH, J = 16.2 Hz),
7.54–7.55 (m, 2H, ArH); retention time: tR 11.6 min.
1
2
4-Phenylbutan-2-one (16b): 1H NMR (600 MHz, CDCl3): ı 2.13
(s, 3H, CH3), 2.75 (t, 2H, CH2CO, J = 7.2 Hz), 2.89 (t, 2H, CH2CH2,
3
3 2
3
(
(
(
s, 3H, (CH ) C), 2.34 (dd, 1H, CH CHCH , J = 13.2 Hz, 17.4 Hz), 2.52
3 2 2 3
d, 1H, CH C(CH ) , J = 15.0 Hz), 2.73–2.77 (m, 1H, CH CHCH ), 2.75
2
3 2
2
3
d, 1H,CH C(CH ) , J = 14.4 Hz), 2.98–3.03 (m, 1H, CHCH ); The NMR
2
3 2
3