Communications
3
4
) (S)-CHBN is produced in 86% yield, which breaks the bot-
tleneck of kinetic resolution, for which the yield is limited
to 50%.
temperatures were set at 2208C. The retention times of (S)-2 and
(R)-2 were 4.38 and 4.56 min, respectively.
The chiral separation of (R,S)-3 and (R,S)-4 was performed with
a GC-14C equipped with a FID detector and an Astec CHIRALDEX
G-TA column (30 m0.25 mm, 0.12 mm film thickness, Supelco,
) Extraction and separation of (S)-CHBN are simple and con-
venient, even though a biocatalyst loading of 5% is used in
this process.
USA). Helium was used as the carrier gas at a flow rate of
À1
1
2
1
1
.0 mLmin . The inlet and detector temperatures were both set at
208C. The temperature program was isothermal at 1008C for
0 min, increasing at 28Cmin to 1208C, with the final 30 min at
208C. Retention times were: R =40.0 min for (S)-3, R =41.7 min
However, several practical problems emerge from this pro-
À1
À1
cess. For example, a substrate concentration of 10 gL is not
high enough for industrial applications, and the substrate-to-
catalyst ratio is high, which affects production costs and the
generation of solid waste. In our future work, the directed evo-
lution of W249F will also be performed to improve the catalyt-
ic efficiency and enantioselectivity for higher substrate concen-
trations. On the other hand, engineering strategies, such as im-
mobilization of the enzyme and in situ product removal, will
also be employed to promote the process so that it can be ap-
plied on a large scale.
t
t
for (R)-3, R =14.0 min for (S)-4, and R =14.8 min for (R)-4.
t
t
1
13
H NMR and C NMR spectra were recorded with Bruker AVANCE III
1
13
(
H, 500 MHz) and Agilent 600 MHz DD2 ( C, 600 MHz) spectrome-
ters in CDCl , respectively. Chemical shifts are given in ppm down-
3
field from tetramethylsilane as the internal standard. GC–MS was
performed with a 6890–5975C (Agilent, CA, USA) by using the pro-
gram (7890A) mentioned above. The optical activity was deter-
mined by using an automatic polarimeter (AUTOPOL IV, RudolphIn-
strument, USA).
In conclusion, an efficient one-step method to prepare (S)-4-
chloro-3-hydroxybutyronitrile [(S)-CHBN] from prochiral 1,3-di-
chloro-2-propanol (1,3-DCP) by using HheC from A. radiobacter
AD1 was developed. The high yield and enantioselectivity of
Construction of mutants
(
S)-CHBN and the inexpensive starting material indicate that
The 16 mutants were constructed by the standard PCR-based site-
directed mutagenesis method. The reactions were performed in
a total volume of 50 mL containing 10 mL 5PS buffer, 4 mL
dNTPmix, 1 mL HheC plasmids (100–200 ng), 0.5 mL PrimerSTAR HS
DNA polymerase (Takara, Dalian, China), 0.5 mL each of the forward
and reverse primers (50 mm), and 33.5 mL deionized water. The PCR
was performed as follows: 958C for 5 min, followed by 30 cycles of
this process is interesting and promising for the production of
the atorvastatin intermediate ethyl (S)-4-chloro-3-hydroxybuta-
noate, which is currently prepared by reduction of the expen-
sive substrate ethyl 4-chloroacetoacetate by coupling with ke-
[12b,21,24]
toreductase and glucose dehydrogenase.
our knowledge, this is the first report on the biosynthesis of
S)-CHBN from prochiral 1,3-DCP.
To the best of
10 s at 988C, 15 s at 588C, 8 min at 728C, and a final incubation at
728C for 10 min. After analysis by agarose gel electrophoresis,
20 mL of the PCR products was digested with restriction enzyme
(
DpnI (1 mL, Takara) at 378C for 3 h. After incubation at 658C for
0 min, 10 mL of the digested products was transformed into
Experimental Section
3
chemically competent E. coli BL21 (DE3) and plated on the agar/ka-
namycin/LB plates. Successful construction of these mutants was
verified by sequencing.
Materials and analytic methods
(R,S)-ECH and (S)-ECH were purchased from J & K Chemical Tech-
nology (Shanghai, China). PrimerSTAR HS DNA polymerase and re-
striction enzymes were obtained from Takara (Dalian, China). 30%
(
wt/vol) NaCN solution (CAUTION: NaCN is hypertoxic) was added
by using a 902 Titrando system (Metrohm, Switzerland) equipped
with a magnetic stirrer (IkA, Japan) and a pH stat. Gene synthesis
and DNA sequencing were completed by Sangon Biotech (Shang-
hai, China). Cloning and expression of HheAAD2, HheBGP1, HheC, and
Scale-up of the process for (S)-CHBN by using mutant
W249F
The reaction was performed in a 500 mL reactor containing PBS
[25]
HHDH-PL were described in previous work.
(200 mL, 200 mm, pH 7.5). To the mixture, 50% H SO was added
2
4
to adjust the pH to 7.0Æ0.1. After heating the mixture to 408C,
Compounds 1–5 were analyzed by using a GC-7890A system
equipped with a HP-5 capillary column (30 m0.32 mm, 0.25 mm
film thickness) fitted with a flame ionization (FID) detector. Nitro-
1
,3-DCP (4.03 g, 0.0312 mol) and the W249F enzyme (10 g, wet
cell) were added, and the pH of the mixture was controlled at
7.5Æ0.1 by adding 30% NaCN solution by using a pH stat. The
whole process was monitored by taking samples from the mixture
and by analyzing them by GC. Once the conversion of 1,3-DCP
reached 99% after 1 h, the reaction was stopped. After extraction
with ethyl acetate (2200 mL), the organic extracts were separat-
ed, combined, and dried with anhydrous sodium sulfate. (S)-CHBN
À1
gen was used as the carrier gas at a flow rate of 1.0 mLmin . The
inlet and detector temperatures were 220 and 2508C respectively.
The temperature program was isothermal at 608C for 4 min, in-
À1
creasing at 208Cmin to 2008C, with the final 2 min at 2008C. Re-
tention times were: R =4.0 min for 2, R =5.8 min for 4, R =
t
t
t
6
.4 min for 1, R =8.3 min for 3, and R =9.9 min for 5.
t t
(
3.22 g, 0.0269 mol, 86%) was obtained as a brown liquid after
The chiral separation of (R,S)-2 was determined by using a GC-14C
Shimadzu, Japan) equipped with a FID detector and a chiral capil-
evaporating under vacuum. The (S)-CHBN product was character-
25
(
ized by optical rotation, GC, and NMR spectroscopy. ½a =À12.28
D
1
lary BGB-175 column (0.25 mm30 m0.25 mL film thickness).
(c=1, H O); H NMR (500 MHz, CDCl ): d=4.22 (s, 1H), 3.69–3.67
(dd, J=5.2, 3.6 Hz, 2H), 2.74–2.71 ppm (t, J=5.8 Hz, 2H). C NMR
2
3
À1
13
Helium was used as the carrier gas at a flow rate of 1.0 mLmin .
The inlet and detector temperatures were both set at 2208C. The
temperature program was 908C for 10 min. The inlet and detector
(151 MHz, CDCl ): d=119.15, 70.05, 50.17, 25.89 ppm. Purity (GC):
96.5%; 97.5%ee.
3
ChemCatChem 2015, 7, 2446 – 2450
2449
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim