Stereo-complementary bioreduction of saturated N-heterocyclic ketones

Article abstract of DOI:10.1016/j.procbio.2017.03.002

The asymmetric bioreduction of several saturated N-heterocyclic ketones is demonstrated in a stereo-complementary fashion using the ketoreductases READH and ChKRED20 for the production of (S)- and (R)-alcohols, respectively. The reaction accepts substrates with a five-, six- or seven-membered ring, and exhibits excellent stereoselectivity when using 2-propanol as both the ultimate reducing agent and cosolvent, achieve >99% ee in the majority of cases for both enantiomers.

Full text of DOI:10.1016/j.procbio.2017.03.002

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ARTICLE IN PRESS
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Contents lists available at ScienceDirect
Process Biochemistry
journal homepage: www.elsevier.com/locate/procbio
Stereo-complementary bioreduction of saturated N-heterocyclic
ketones
Chao Lia,b,c, Yan Liua,b, Xiao-Qiong Peia,b, Zhong-Liu Wua,b,∗
a
Key Laboratory of Environmental and Applied Microbiology, Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu 610041, China
Environmental Microbiology, Key Laboratory of Sichuan Province, Chengdu 610041, China
Graduate University of the Chinese Academy of Sciences, Beijing 100049, China
b
c
a r t i c l e i n f o
a b s t r a c t
Article history:
The asymmetric bioreduction of several saturated N-heterocyclic ketones is demonstrated in a stereo-
complementary fashion using the ketoreductases READH and ChKRED20 for the production of (S)- and
Received 6 January 2017
Received in revised form 27 February 2017
Accepted 1 March 2017
Available online xxx
(R)-alcohols, respectively. The reaction accepts substrates with a five-, six- or seven-membered ring,
Keywords:
Ketoreductase
Hydroxypiperidine
Hydroxypyrrolidine
Hydroxyazepane
Bioreduction
Chemical compounds studied in this article:
tert-butyl 3-oxopyrrolidine-1-carboxylate
(
(
PubChem CID: 471360)
S)-tert-butyl
3
-hydroxypyrrolidine-1-carboxylate
(
(
PubChem CID: 854055)
R)-tert-butyl
3
-hydroxypyrrolidine-1-carboxylate
(
PubChem CID: 6544479)
1
6
-benzylpyrrolidin-3-one (PubChem CID:
9890)
(
S)-1-benzylpyrrolidin-3-ol (PubChem CID:
733875) ;(R)-1-benzylpyrrolidin-3-ol
PubChem CID: 643472)
benzyl 3-oxopyrrolidine-1-carboxylate
2
(
(
(
PubChem CID: 561203)
S)-benzyl
3
-hydroxypyrrolidine-1-carboxylate
(
PubChem CID: 13438604)
(R)-benzyl
3
-hydroxypyrrolidine-1-carboxylate
PubChem CID: 11183628)
ethyl 3-oxopyrrolidine-1-carboxylate
(
(
PubChem CID: 277754)
(S)-ethyl
3
-hydroxypyrrolidine-1-carboxylate
(
PubChem CID: 66772749)
(R)-ethyl
3
-hydroxypyrrolidine-1-carboxylate
(
PubChem CID: 66291444)
∗
Corresponding author at: Chengdu Institute of Biology, Chinese Academy of Sciences, 9 South Renmin Road, 4th Section, Chengdu 610041, Sichuan, China.
Tel.: +86 28 82890434.
E-mail address: wuzhl@cib.ac.cn (Z.-L. Wu).
http://dx.doi.org/10.1016/j.procbio.2017.03.002
1
359-5113/© 2017 Elsevier Ltd. All rights reserved.
Please cite this article in press as: C. Li, et al., Stereo-complementary bioreduction of saturated N-heterocyclic ketones, Process Biochem
(
2017), http://dx.doi.org/10.1016/j.procbio.2017.03.002

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C. Li et al. / Process Biochemistry xxx (2017) xxx–xxx
tert-butyl
and exhibits excellent stereoselectivity when using 2-propanol as both the ultimate reducing agent and
cosolvent, achieve >99% ee in the majority of cases for both enantiomers.
3
-oxopiperidine-1-carboxylate
(
(
PubChem CID: 2756825)
S)-tert-butyl
3
-hydroxypiperidine-1-carboxylate
(
(
PubChem CID: 1514399)
R)-tert-butyl
3
-hydroxypiperidine-1-carboxylate
PubChem CID: 1514398)
-benzylpiperidin-3-one (PubChem
CID: 96650)
S)-1-benzylpiperidin-3-ol (PubChem
CID: 693762)
R)-1-benzylpiperidin-3-ol (PubChem
(
1
(
(
CID: 693761)
benzyl 3-oxopiperidine-1-carboxylate
(
(
PubChem CID: 1514169)
S)-benzyl
3
-hydroxypiperidine-1-carboxylatel
(
(
PubChem CID: 6932653)
R)-benzyl
3
-hydroxypiperidine-1-carboxylate
PubChem CID: 6932652)
tert-butyl 4-oxoazepane-1-carboxylate
(
(
(
PubChem CID: 1512679)
S)-tert-butyl
4
-hydroxyazepane-1-carboxylate
(
(
PubChem CID: 40481276)
R)-tert-butyl
4
-hydroxyazepane-1-carboxylate
(
PubChem CID: 40481277)
1
1
-benzylazepan-4-one (PubChem CID:
514350)
(
S)-1-benzylazepan-4-ol (PubChem
CID: 1514352)
R)-1-benzylazepan-4-ol (PubChem
(
CID: 1514351)
benzyl 4-oxoazepane-1-carboxylate
(
(
PubChem CID: 1512681)
S)-benzyl
4
-hydroxyazepane-1-carboxylate
(
(
PubChem CID: 51892891)
R)-benzyl
4
-hydroxyazepane-1-carboxylate
PubChem CID: 51892892)
tert-butyl 3-oxoazepane-1-carboxylate
(
(
(
PubChem CID: 22831870)
S)-tert-butyl
3
-hydroxyazepane-1-carboxylate
(
(
PubChem CID: 73424851)
R)-tert-butyl
3
-hydroxyazepane-1-carboxylate
(
PubChem CID: 73424814)
1
1
1
2
-benzylazepan-3-one (PubChem CID:
5152264)
-benzylazepan-3-ol (PubChem CID:
2933843).
©
2017 Elsevier Ltd. All rights reserved.
1
. Introduction
a convenient choice to provide a concise and atom economic
approach.
Optically pure saturated N-heterocyclic alcohols, such as
However, despite of the well-documented asymmetric reduc-
tion of aromatic ketones, it still lacks efficient catalytic systems
to prepare aliphatic alcohols, including N-heterocyclic alcohols,
with high enantioselectivity. Li et al. have reported a novel chi-
ral surfactant-type catalyst to achieve the asymmetric transfer
hydrogenation of aliphatic ketones, resulting in good to excellent
conversions and stereoselectivities [8]. However, for the substrate
N-Boc-3-oxopyrrolidine (1a), the enantiopurity of the product (R)-
N-Boc-3-hydroxypyrrolidine (1b) was only 92% ee even at strictly
hydroxypiperidine and hydroxypyrrolidine, are important building
blocks for the synthesis of a variety of natural compounds and phar-
maceutically relevant products including some marketed drugs
[
1–3]. They are mainly prepared from chiral pools through time-
consuming multi-step chemical conversions, or enzymatic kinetic
resolution via hydrolysis or acylation [4–6], or from chiral azido-
containing precursors that are synthesized from azido-aldehydes,
using aldolases [7]. On the other hand, the corresponding ketone
precursors of many saturated N-heterocyclic alcohols are com-
mercially available, which would make asymmetric reduction
◦
controlled reaction temperatures as low as 5 C [8].
As a potential alternative to chemical strategies, the tissue of
Daucus carota has been applied to catalyze the Prelog-reduction
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of several saturated N-heterocyclic ketones to the correspond-
ing (S)-alcohols with varied stereoselectivity. Unfortunately, the
application of the reaction system is limited by impractically low
substrate concentration (2–3 mM) as well as high catalyst loading
Then, 2 mL of the starter culture was transferred into terrific broth
medium containing 50 ␮g kanamycin/mL and incubated at 37 C.
The expression was induced by the addition of 0.5 mM IPTG, and
◦
◦
the incubation was continued at 30 C for another 18 h.
4
(
23%, w/v) [9]. Practical biocatalytic solutions have been reported
The cell pellets were collected by centrifugation at 2 × 10 × g
using recombinant ketoreductases (KRED) for the substrates 1a
and N-Boc-piperidin-3-one (5a) to produce the corresponding (S)-
alcohols by our group and others [10–13]. Substrate concentrations
of 50–200 g/L could be applied, demonstrating the great potential
of KREDs in the production of this group of chiral alcohols. How-
ever, up to now, the reported substrate spectra remains extremely
limited for saturated N-heterocyclic ketones. Moreover, the anti-
Prelog bioreduction of this group of compounds to provide the
for 10 min, resuspended in 100 mM potassium phosphate buffer
(pH 7.0), and disrupted using a high-pressure homogenizer (ATS-
AH100B, ATS Engineering Inc., Canada) at 60 MPa. Cell debris was
4
removed by centrifugation at 2 × 10 × g for 20 min to achieve the
crude enzyme extract. The quantification of the crude enzyme
extracts followed the common practice of reported industrial-scale
productions catalyzed with KREDs [20–22]. Briefly, a small fraction
of the crude enzyme extracts was lyophilized and weighed. Then
all references to enzyme loading (g/L) are based on the amount of
lyophilized powder prepared from the crude enzyme extracts.
The crude enzyme extract was loaded onto Ni2+-nitrilotriacetic
acid columns (Bio-Rad) and eluted with potassium phosphate
buffer (20 mM, pH 8.0) containing 500 mM NaCl and 250 mM imi-
dazole. The purified enzyme was dialyzed and used freshly. Protein
estimations were done with a commercial BSA Protein Assay Kit
with bovine serum albumin as a standard (Tiangen, Beijing, China).
(
7
R)-alcohols is scarcely reported. Kizaki et al have screened over
00 strains to achieve a strain of Devosia riboflavin that yields (R)-
N-benzyl-3-pyrrolidinol (2b) with 99.3% ee and 6% yield after 24-h
reaction at a substrate concentration of 1 g/L [14]. The Leifsonia
alcohol dehydrogenase (LSADH) and an immobilized ketoreductase
P1B2 have been used in the production of (R)-1b and (R)-benzyl 4-
hydroxyazepane-1-carboxylate (10b), respectively [15,16]. LSADH
displayed absolute selectivity, but the product yield was only 24%
after 24 h for 10 g/L substrate [15]. P1B2 could convert 50 g/L sub-
strate within 20 h, yielding (R)-10b with 96% ee [16].
2.3. Specific activity measurement
In this work, we re-screened an in-house inventory of recom-
binant KREDs for complementary stereoselectivity in order to
produce both enantiomers of more structurally diverse saturated
N-heterocyclic alcohols. This inventory consists of the alcohol
dehydrogenase from Rhodococcus erythropolis DSM 43297 (READH)
that follows Prelog’s rule [17,18] as well as 27 KREDs mined
from the genome of Chryseobacterium sp. CA49 with 20 of
them giving anti-Prelog stereochemistry for the substrate of 3,5-
bis(trifluoromethyl)acetophenone [19]. The use of 2-propanol as
the ultimate reducing agent was found to be essential to achieve
The reaction mixture containing 0.1 mg purified enzyme, 20 mM
substrate 1a and 20 mM NADH in 1 mL potassium phosphate buffer
◦
◦
(100 mM, pH 7.0) was incubated at 50 C for READH, or at 40 C
for ChKRED20. After 5 min, the reaction was terminated by extrac-
tion with ethyl acetate. The organic phase was then concentrated
and analyzed with HPLC. One unit of enzyme was defined as the
quantity of enzyme required to catalyze the conversion of 1 ␮mol
substrate in 1 min.
>99% ee for both enantiomers.
2.4. General procedure for biotransformation
The reaction mixture containing 200 mM substrate, 1 mM NAD+,
2
. Materials and methods
5
% (v/v) 2-propanol and 10 mg crude enzyme READH in 1 mL potas-
◦
sium phosphate buffer (100 mM, pH 7.0) was incubated at 50 C.
2.1. General
For ChKRED20, 40% (v/v) 2-propanol and a reaction temperature of
◦
4
0 C were applied instead. The reaction was monitored by TLC, and
The substrates (1a–12a) were purchased from Alfa-Aesar (Tian-
terminated by extracting with methyl tert-butyl ether (1 mL). The
organic extract was dried over anhydrous sodium sulfate and con-
centrated. The samples were subjected to chiral HPLC to determine
the conversion and enantiomeric excess. The products were puri-
fied by silica gel column chromatography, and identified by NMR
analysis, optical rotation measurements and mass spectrometry.
jin, China). The racemic alcohols were prepared by reducing the
ketones with sodium borohydride. All the other reagents were
obtained from general commercial suppliers and used without fur-
ther purification.
The GenBank accession numbers of ChKRED20 from Chryseobac-
terium sp. CA49 and READH from R. erythropolis DSM43297 are
KC3420020 and AY161280, respectively. The recombinant plas-
mids encoding the two enzymes were previously constructed in
pET28a(+) [18,19].
2
.5. Spectral data of biotransformation products
(
S)-tert-butyl
3-hydroxypyrrolidine-1-carboxylate
(S)-1b:
1H NMR spectra were recorded on a Brucker-600 (600/150 MHz)
25
25
3
+
(
5.5 mg, 98% yield; [␣]D
23.2 (c 0.9, CHCl ), 96% ee; H NMR (600 MHz, CDCl ) ı 4.67
+22.6 (c 1.13, CHCl ); lit. [9] [␣]D
3
spectrometer in CDCl . All signals are expressed as ppm down field
3
1
3
3
from tetramethylsilane. Optical rotations were measured with a
Perkin Elmer 341 polarimeter. Chiral HPLC analysis was conducted
with a Shimadzu Prominence LC-20AD system connected to a PDA-
detector using Chiralpak AS-H (1b–4b) Daicel IC-H (5b), Chiralpak
AD-H (6b, 7b, 10b), Chiralcel OB-H (8b, 9b) and Chiralcel OD-H
s, 1H, OH), 4.40 (dd, J = 4.2, 1.9 Hz, 1H, CH), 3.64–3.17 (m, 4H,
13
CH2, CH ), 2.05–1.76 (m, 2H, CH ), 1.43 (s, 9H, CH );
C
2
2
3
NMR (151 MHz, CDCl ) ı 154.88, 79.40, 70.96 and 70.11, 54.30
3
and 54.10, 43.95 and 43.48, 33.96 and 33.55, 28.49; HR–ESI–MS:
+
2
10.1100 (calc. 210.1101 for [C H NNaO ] ); retention times:
9
17
3
(
11b, 12b) columns (See Table S1 of the Supporting Information for
tR(S) 11.3 min, tR(R) 12.1 min.
details).
(
R)-tert-butyl 3-hydroxypyrrolidine-1-carboxylate (R)-1b:
25
26
3
4.4 mg, 92% yield; [␣]D −20.8 (c 1.06, CHCl ); lit. [15][␣]D
3
1
2.2. Expression and purification of proteins
−19.7 (c 0.3, CHCl ), 99%ee; H NMR (600 MHz, CDCl ) ı 4.48–4.37
3
3
(
m, 1H, CH), 3.54–3.25 (m, 4H, CH2, CH ), 2.04–1.76 (m, 3H,
2
The recombinant plasmids were
Escherichia coli BL21 (DE3) (Novagen, Madison, WI, USA) competent
transformed
into
CH , OH), 1.45 (s, 9H, CH ).
2 3
(S)-1-benzylpyrrolidin-3-ol (S)-2b: 32.5 mg, 92% yield; [␣]D
25
25
cells for protein expression. Single colonies were grown overnight
−5.7 (c 1.05, CHCl ); lit. [23] [␣]D −5.3 (c 1, CHCl ), 98.5% ee;
3
3
◦
1
at 37 C in Luria-Bertani medium containing 50 ␮g kanamycin/mL.
H NMR (600 MHz, CDCl ) ı 7.35–7.22 (m, 5H, Ar–H), 4.32 (m, 1H,
3
Please cite this article in press as: C. Li, et al., Stereo-complementary bioreduction of saturated N-heterocyclic ketones, Process Biochem
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CH), 3.63 (s, 2H, CH ), 2.98–2.76 (m, 2H, CH , OH), 2.67 (d,
ee; H NMR (600 MHz, CDCl ) ı 7.35–7.22 (m, 5H, Ar–H), 3.85–3.75
2
2
3
J = 10.1 Hz, 1H, CH ), 2.56 (dd, J = 10.2, 5.3 Hz, 1H, CH ), 2.39–2.28
(m, 1H, CH ), 3.51 (s, 2H, CH ), 3.01–1.96 (m, 5H, OH, CH2,
2
2
2
2
(
m, 1H, CH ), 2.24–2.12 (m, 1H, CH ), 1.80–1.67 (m, 1H, CH );
CH ), 1.85–1.74 (m, 1H, CH ), 1.66–1.46 (m, 3H, CH2, CH ).
2 2 2
2
2
2
13
C NMR (151 MHz, CDCl ) ı 138.44, 128.91, 128.30, 127.14, 71.23,
(S)-benzyl 3-hydroxypiperidine-1-carboxylate (S)-7b: 44.6 mg,
3
95% yield; [␣]D25 +10.5 (c 1.32, CHCl ); H NMR (600 MHz, CDCl ) ı
1
6
2.87, 60.19, 52.39, 34.93; HR–ESI–MS: 178.1218 (calc.178.1226 for
3
3
+
[
C11H16NO] ); retention times: tR(R) 10.2 min, tR(S) 10.9 min.
7.40–7.27 (m, 5H, Ar–H), 5.12 (s, 2H, CH ), 3.95–3.47 (m. 3H, CH,
2
2
5
(
R)-1-benzylpyrrolidin-3-ol (R)-2b: 31.8 mg, 90% yield; [␣]D
CH ), 3.37–2.99 (m, 2H, CH ), 2.49 (s, OH), 1.97–1.70 (m, 2H),
2
2
20
13
+
4.83 (c 0.96, CHCl ); lit. [24] [␣]D +3.9 (c 0.5, CH OH), 99.5% ee;
1.64–1.36 (m, 2H); C NMR (151 MHz, CDCl ) ı 155.71, 136.73,
3
3
3
1
H NMR (600 MHz, CDCl ) ı 7.37–7.21 (m, 5H, Ar–H), 4.38–4.27 (m,
128.49, 128.00, 127.84, 67.21, 65.93, 50.77, 44.16, 32.29, 22.34;
3
+
1
H, CH), 3.63 (s, 2H, CH ), 2.93–2.73 (m, 2H, OH, CH ), 2.68 (d,
HR–ESI–MS: 258.1098 (calc. 258.1101 for [C13H17NNaO ] ); reten-
2
2
3
J = 10.0 Hz, 1H, CH ), 2.56 (dd, J = 10.3, 5.4 Hz, 1H, CH ), 2.33 (dd,
tion times: t (S) 23.2 min, t (R) 24.7 min.
2
2
R
R
J = 15.7, 8.9 Hz, 1H, CH ), 2.26–2.11 (m, 1H, CH ), 1.79–1.68 (m,
(R)-benzyl 3-hydroxypiperidine-1-carboxylate (R)-7b: 42.7 mg,
2
2
91% yield, [␣]D25 −10.4 (c 1.19, CHCl ); lit. [28] [␣]
25
−11.9 (c
1
H, CH ).
2
3
D
1
(
S)-benzyl 3-hydroxypyrrolidine-1-carboxylate (S)-3b: 38.0 mg,
1, CHCl ), 96% ee; H NMR (600 MHz, CDCl3) ı 7.40–7.28 (m, 5H,
3
2
5
21
8
6% yield; [␣]D +19.1 (c 1.26, CHCl ); lit. [25] [␣]D +21.9 (c 1.04,
Ar–H), 5.13 (s, 2H, CH ), 3.85–3.50 (m, 3H, CH, CH ), 3.31–3.10
3
2
2
1
CH OH), 99% ee; H NMR (600 MHz, CDCl ) ı 7.38–7.26 (m, 5H,
(m, 2H, CH ), 1.97–1.70 (m, 3H, CH , OH), 1.61–1.39 (m, 2H,
2 2
3
3
Ar–H), 5.18–5.03 (m, 2H, CH ), 4.39 (s, 1H, CH), 3.61–3.34 (m,
CH ).
2
2
13
4
H, CH2, CH ), 2.97 (s, 1H, OH), 1.92 (s, 2H, CH ); C NMR
(S)-tert-butyl 4-hydroxyazepane-1-carboxylate (S)-8b: 35.2 mg,
82% yield; [␣]D25 −6.7 (c 1.01, CHCl ); H NMR (600 MHz, CDCl ) ı
2
2
1
(
151 MHz, CDCl ) ı 155.23, 136.85, 128.46, 127.94, 127.82, 70.81
3
3
3
and 69.95, 66.84, 54.63 and 54.13, 44.17 and 43.87, 33.96 and 33.45;
HR–ESI–MS: 244.0936 (calc. 244.0950 for [C12H15NNaO ] ); reten-
3.94–3.75 (m, 1H, CH), 3.57–3.15 (m, 4H, CH2, CH ), 2.14–1.52
2
+
13
3
(m, 7H, OH, CH2, CH2, CH ), 1.44 (s, 9H, CH ); C NMR
2
3
tion times: tR(S) 11.3 min, tR(R) 12.9 min.
(151 MHz, CDCl ) ı 155.59, 79.27, 70.95 and 70.70, 46.53 and 45.68,
3
(
R)-benzyl
3-hydroxypyrrolidine-1-carboxylate
(R)-3b:
41.41 and 41.01, 37.37 and 37.29, 35.48 and 35.20, 28.49, 22.56 and
2
5
20
+
3
7.1 mg, 84% yield; [␣]D −20.5 (c 1.15, CHCl ); lit. [26][␣]D
22.09; HR–ESI–MS: 238.1412 (calc. 238.1414 for [C11H21NNaO ] );
3
3
1
−
21.9 (c 1, CHCl ), 98% ee. H NMR (600 MHz, CDCl ) ı 7.41–7.27
retention times: t (S) 17.9 min, t (R) 20.0 min.
3
3
R
R
(
m, 5H, Ar–H), 5.12 (s, 2H, CH ), 4.43 (s, 1H, CH), 3.74–3.23 (m,
H, CH2, CH ), 2.53–1.81 (m, OH, 3H).
(R)-tert-butyl
4-hydroxyazepane-1-carboxylate
(R)-8b:
2
33.5 mg, 78% yield; [␣]D25 +5.7(c 1.15, CHCl ); H NMR (600 MHz,
1
4
2
3
(
S)-ethyl 3-hydroxypyrrolidine-1-carboxylate (S)-4b: 29.5 mg,
CDCl ) ı 3.90–3.79 (m, 1H, CH), 3.55–3.12 (m, 4H, CH2, CH ),
3
2
2
5
1
9
4
4
3% yield; [␣]D +15.2 (c 1.07, CHCl ); H NMR (600 MHz, CDCl ) ı
.47–4.40 (m, 1H, CH), 4.11 (q, J = 7.1 Hz, 2H, CH ), 3.59–3.28 (m,
H, CH2, CH ), 2.36 (s, 1H, OH), 2.05–1.84 (m, 2H, CH ), 1.24
t, J = 7.1 Hz, 3H, CH ); C NMR (151 MHz, CDCl ) ı 155.46, 71.01
2.07–1.51 (m, 7H, OH, CH2, CH2, CH ), 1.44 (s, 9H, CH ).
3
3
2
3
25
2
(S)-1-benzylazepan-4-ol (S)-9b: 18.4 mg, 45% yield; [␣]
D
1
2
2
−15.4 (c 1.27, CHCl ); H NMR (600 MHz, CDCl ) ı 7.34–7.22 (m,
3
3
1
3
(
5H, Ar–H), 4.15–4.01 (m, 1H, CH), 3.72–3.55 (m, 2H, CH ), 3.50
3
3
2
and 70.14, 61.07, 54.51 and 54.09, 43.91 and 43.71, 34.03 and 33.56,
(s, 1H, OH), 2.93–2.82 (m, 1H, CH ), 2.79–2.71 (m, 1H, CH ),
2
2
+
1
4.78; HR–ESI–MS: 182.0783 (calc. 182.0788 for [C7H13NNaO ] );
2.62–2.52 (m, 1H, CH ), 2.49–2.40 (m, 1H, CH ), 2.02–1.92 (m,
3
2
2
retention times: tR(S) 16.1 min, tR(R) 17.8 min.
R)-ethyl 3-hydroxypyrrolidine-1-carboxylate (R)-4b: 28.6 mg,
1H, CH ), 1.91–1.83 (m, 1H, CH ), 1.82–1.70 (m, 2H, CH ),
2
2
2
13
(
1.70–1.63 (m, 1H, CH ), 1.60–1.51 (m, 1H, CH );
C NMR
2
2
2
5
1
9
0% yield; [␣]D −15.9 (c 1.02, CHCl ); H NMR (600 MHz, CDCl3)
(151 MHz, CDCl ) ı 138.99, 128.82, 128.35, 127.12, 70.06, 63.41,
3
3
ı 4.48–4.40 (m, 1H, CH), 4.12 (q, J = 7.1 Hz, 2H, CH ), 3.57–3.25
55.16, 51.29, 35.78, 34.40, 24.18; HR–ESI–MS: 206.1531 (calc.
2
+
(
m, 4H, CH ), 2.06 (s, 1H, OH), 2.05–1.83 (m, 2H, CH ), 1.25 (t,
206.1539 for [C13H20NO] ); retention times: t (S) 11.4 min, t (R)
2
2
R
R
J = 7.1 Hz, 3H, CH ).
14.0 min.
3
2
5
(
S)-tert-butyl
3-hydroxypiperidine-1-carboxylate
(S)-5b:
(R)-1-benzylazepan-4-ol (R)-9b: 16.8 mg, 41% yield; [␣]
+12.1 (c 1.22, CHCl ); H NMR (600 MHz, CDCl ) ı 7.34–7.23 (m, 5H,
Ar–H), 4.13–4.05 (m, 1H, CH), 3.63 (dd, J = 35.3, 13.2 Hz, 2H, CH ),
D
2
0
25
1
3
8.1 mg, 95% yield; [␣]D +24.1 (c 0.63, C H5OH); lit. [9] [␣]D
2
3
3
1
+
23.8 (c 0.9, C H5OH), 95% ee; H NMR (600 MHz, CDCl ) ı
2
3
2
3
.79–3.65 (m, 2H, CH ), 3.59–3.46 (m, 1H), 3.18–2.97 (m, 2H,
2.96–2.82 (m, 1H, CH ), 2.83–2.70 (m, 1H, CH ), 2.64–2.52 (m,
2 2
2
CH ), 2.05 (s, 1H, OH), 1.94–1.81 (m, 1H, CH ), 1.79–1.69 (m,
1H, CH ), 2.46 (t, J = 10.6 Hz, 1H, CH ), 2.06–1.93 (m, 1H, CH ),
1.93–1.84 (m, 1H, CH ), 1.84–1.70 (m, 2H, CH ), 1.70–1.62 (m,
2 2
2
2
2 2 2
1
H, CH ), 1.58–1.37 (m, 11H, CH2, CH ); 13C NMR (151 MHz,
2
3
CDCl ) ı 155.25, 76.81, 66.15, 50.59, 44.25, 32.54, 28.41, 22.42;
1H, CH ), 1.62–1.51 (m, 1H, CH ).
3
2
2
+
HR–ESI–MS: 224.1254 (calc. 224.1257 for [C10H19NNaO ] );
(S)-benzyl 4-hydroxyazepane-1-carboxylate (S)-10b: 41.8 mg,
84% yield; [␣]D25 +10.8 (c 1.06, CHCl ); H NMR (600 MHz, CDCl ) ı
3
1
retention times: tR(S) 15.3 min, tR(R) 16.3 min.
3
3
(
R)-tert-butyl
3-hydroxypiperidine-1-carboxylate
(R)-5b:
7.38–7.27 (m, 5H, Ar–H), 5.13 (s, 2H, CH ), 3.93–3.82 (m, 1H, CH),
2
2
5
20
3
7.3 mg, 93% yield; [␣]D −24.4 (c 1.05, C H5OH); lit. [6][␣]D
3.61–3.46 (m, 2H, CH ), 3.46–3.29 (m, 2H, CH ), 2.04–1.85 (m,
2
2
2
1
13
−
18.92 (c 0.122, C H5OH), 99% ee; H NMR (600 MHz, CDCl ) ı
2H, CH ), 1.85–1.53 (m, 5H, OH, CH2, CH ); C NMR (151 MHz,
2
3
2
2
3
.76–3.64 (m, 2H, CH ), 3.62–3.42 (m, 1H, CH), 3.18–2.96 (m,
CDCl ) ı 156.14, 136.98, 128.47, 127.91 and 127.77, 70.68 and
2
3
2
H, CH ), 2.33 (s, 1H, OH), 1.93–1.83 (m, 1H, CH ), 1.78–1.67
70.50, 66.99, 46.39 and 46.25, 41.37 and 40.98, 37.42 and 37.20,
35.38 and 35.17, 22.28 and 21.89; HR–ESI–MS: 272.1255 (calc.
2
2
(m, 1H, CH ), 1.56–1.36 (m, 11H, CH2, CH ).
2
3
25
+
(
S)-1-benzylpiperidin-3-ol (S)-6b: 35.9 mg, 94% yield; [␣]D
272.1257 for [C14H19NNaO ] ); retention times: t (S) 22.4 min,
3
R
20
+12.3 (c 1.21, CH OH); lit. [24] [␣]D
+13.5 (c 0.5, CH OH),
tR(R) 23.8 min.
3
3
1
9
8% ee; H NMR (600 MHz, CDCl ) ı 7.33–7.23 (m, 5H, Ar–H),
(R)-benzyl 4-hydroxyazepane-1-carboxylate (R)-10b: 24.9 mg,
3
50% yield; [␣]D25 −11.7 (c 1.12, CHCl , 95%ee); lit. [16] [␣]
25
3
.84–3.78 (m, 1H, CH), 3.51 (s, 2H, CH ), 2.64–2.37 (m, 4H,
2
3
D
1
OH, CH2, CH ), 2.27 (t, 1H, CH ), 1.88–1.72 (m, 1H, CH ),
−10.1(c 1, DMF, 96% ee); H NMR (600 MHz, CDCl ) ı 7.38–7.27 (m,
2
2
2
3
1
1
2
.67–1.45 (m, 3H, CH2, CH ); 13C NMR (151 MHz, CDCl ) ı
38.13, 129.09, 128.24, 127.10, 66.32, 63.03, 60.29, 53.50, 31.81,
1.71; HR–ESI–MS: 192.1381 (calc. 192.1383 for [C12H18NO] );
5H, Ar–H), 5.14 (s, 2H, CH ), 3.96–3.82 (m, 1H, CH), 3.65–3.47
2
3
2
(m, 2H, CH ), 3.47–3.28 (m, 2H, CH ), 2.04–1.86 (m, 2H, CH ),
2
2
2
+
1.86–1.50 (m, 5H, OH, CH2, CH ).
2
retention times: tR(R) 18.0 min, tR(S) 20.5 min.
R)-1-benzylpiperidin-3-ol (R)-6b: 35.5 mg, 94% yield; [␣]D
(S)-tert-butyl
3-hydroxyazepane-1-carboxylate
(S)-11b:
25
25
25
(
30.9 mg, 72% yield; [␣]D +33.6 (c 1.06, CHCl ); lit. [29] [␣]
3
D
20
−
11.9 (c 1.08, CH OH); lit. [27][␣]D −13.3 (c 0.22, CH OH), 98%
3
3
Please cite this article in press as: C. Li, et al., Stereo-complementary bioreduction of saturated N-heterocyclic ketones, Process Biochem
2017), http://dx.doi.org/10.1016/j.procbio.2017.03.002
(

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C. Li et al. / Process Biochemistry xxx (2017) xxx–xxx
5
+
32.1 (c 1, CHCl , 98% ee); 1H NMR (600 MHz, CDCl ) ı 3.98–3.81
3
3
(
m, 1H, CH), 3.76–2.70 (m, 5H, OH, CH2, CH ), 1.89–1.79 (m,
2
1
H, CH ), 1.77–1.58 (m, 3H, CH2, CH ), 1.57–1.48 (m, 1H,
2 2
13
CH ), 1.44 (s, 9H, CH ), 1.39–1.27 (m, 1H, CH ); C NMR
2
3
2
(
151 MHz, CDCl ) ı 157.53, 155.33, 79.89, 71.01 and 70.56, 53.66
3
and 53.34, 48.73 and 47.76, 36.43 and 35.77, 28.76 and 28.39, 27.95,
+
2
0.92; HR–ESI–MS: 238.1418 (calc. 238.1414 for [C11H21NNaO ] );
3
retention times: tR(S) 13.6 min, tR(R) 16.2 min.
R)-tert-butyl 3-hydroxyazepane-1-carboxylate
(
(R)-11b:
25
25
1
−
7.6 mg, 41% yield; [␣]D −32.4 (c 1.31, CHCl ); lit. [29] [␣]D
3
1
31.6 (c 1, CHCl ), 99% ee; H NMR (600 MHz, CDCl ) ı 4.01–3.79
3
3
(
m, 1H, CH), 3.74–2.81 (m, 5H, CH2, CH , OH), 1.90–1.78 (m,
2
1
H, CH ), 1.79–1.60 (m, 3H, CH2, CH ), 1.58–1.49 (m, 1H,
2 2
CH ), 1.44 (s, 9H, CH ), 1.41–1.27 (m, 1H, CH ).
2
3
2
25
(
S)-1-benzylazepan-3-ol (S)-12b: 34.8 mg, 85% yield; [␣]D
1
+
4.3 (c 1.32, CHCl ); H NMR (600 MHz, CDCl ) ı 7.39–7.20 (m, 5H,
3
3
Ar–H), 3.83–3.77 (m, 1H, CH), 3.71 (s, 2H, CH ), 3.05–2.69 (m,
2
4
H, OH, CH2, CH ), 2.51–2.41 (m, 1H, CH ), 1.87–1.75 (m, 2H,
2
2
CH ), 1.73–1.63 (m, 1H, CH ), 1.56–1.45 (m, 3H, CH2, CH );
2
2
2
13
C NMR (151 MHz, CDCl ) ı 138.92, 129.02, 128.41, 127.27, 67.60,
3
6
3.99, 57.82, 54.91, 37.38, 28.18, 20.80; HR–ESI–MS: 206.1535
+
(
calc. 206.1539 for [C13H20NO ] ); retention times: tR(S) 16.1 min,
3
tR(R) 17.6 min.
(
R)-1-benzylazepan-3-ol (R)-12b: 35.2 mg, 86% yield; [␣]D25
1
−
3.8 (c 1.19, CHCl ); H NMR (600 MHz, CDCl ) ı 7.37–7.22 (m,
3
3
5
H Ar–H), 3.84–3.74 (m, 1H, CH), 3.71 (s, 2H, CH ), 2.88–2.69
2
(
m, 3H, CH2, CH ), 2.52–2.40 (m, 1H, CH ), 1.87–1.74 (m, 2H,
2
2
CH ), 1.74–1.62 (m, 1H, CH ), 1.58–1.43 (m, 3H, CH2, CH ).
2
2
2
Fig. 1. Reduction of N-Boc-3-oxopyrrolidine (1a) with various recombinant ketore-
ductases. Shown are (A) the reaction scheme and (B) the performance of the
ChKRED13, ChKRED15, ChKRED20 and READH (ꢀ), and other ketoreductases (᭹).
The result of the control reaction catalyzed with the crude cell extract of E. coli
harboring the empty vector is marked in red. The reaction was performed in 1 mL
3
. Results and discussion
3.1. Preliminary screening for ketoreductases
+
+
potassium phosphate buffer (100 mM, pH 7.0) containing NAD /NADP (2 mM),
To screen the inventory for complementary stereoselectivity,
GDH (3 g/L), glucose (5% w/v), crude enzyme extract of recombinant ketoreductases
◦
(
10 g/L) and substrate (10 mM), and incubated at 30 C for 12 h. (For interpretation of
the recombinant KREDs were expressed in E. coli and used as
crude cell extract in the assay for the bioreduction of N-Boc-3-
oxopyrrolidine (1a) (Fig. 1A ). A coenzyme regeneration system
consisting of a glucose dehydrogenase (GDH) and glucose was
applied to supply the NAD(P)H. All the reactions catalyzed with
KREDs went smoothly, yielding the desired alcohol product with
no detectable by-products. However, the control reaction cat-
alyzed with the crude cell extract of E. coli harboring the empty
vector showed significant catalytic activity as well, affording the
the references to color in this figure legend, the reader is referred to the web version
of this article.)
(
S)-1b with 33% HPLC yield and 90% ee (Fig. 1B). Only 16 KREDs
afforded product yields higher than the control reaction. Most of
the KREDs followed Prelog’s rule and delivered the (S)-enantiomer,
and the highest enantiomeric excess was achieved with ChKRED13
and READH (97–99% ee). Only two of the tested KREDs, namely,
ChKRED15 and ChKRED20, yielded the (R)-enantiomer, and the
enantiomeric excesses of (R)-1b was 53% ee and 76% ee, respec-
tively (Fig. 1B).
Fig. 2. Bioreduction of 1a using purified alcohol dehydrogenases/ketoreductases.
Background reaction was performed using the cell extract prepared from E. coli
BL21 carrying the empty vector. Shown are conversions (white) and enantiomeric
excesses of the S- (black) or R- enantiomer (with dashed lines). The reaction was
performed in 1 mL potassium phosphate buffer (100 mM, pH 7.0) containing NADH
3
.2. Improvement of reaction conditions
The significant yield and unsatisfactory stereoselectivity of the
(
10 mM), NADPH (10 mM), enzymes (1 g/L) and substrate (10 mM), and incubated
control reaction was apparently unfavorable to the enantiopurity
of the products. It was deteriorative to the enantiopurity of (R)-
◦
at 30 C for 12 h.
1
b with the opposite selectivity. Therefore, we further investigated
the stereoselectivity of several selected KREDs as well as GDH using
purified enzymes in the presence of stoichiometric NADH/NADPH.
As a comparison, the cell extract of E. coli harboring the empty
vector was assayed side by side as the background reaction.
The results showed that by using the purified enzymes, the
enantiomeric excesses achieved for (R)-1b increased to 86 and >99%
for ChKRED15 and ChKRED20, respectively (Fig. 2), indicating excel-
lent stereoselectivity of purified ChKRED20. For the (S)-enantiomer,
READH was confirmed to be a good catalyst, consistently produc-
ing enantiopure product of >99% ee (Fig. 2). On the other hand,
the purified GDH also displayed significant activity but with poor
stereoselectivity toward the S-enantiomer (Fig. 2), which posed
negative effect on the enantiopurity of the products. The back-
ground reaction also afforded the S-enantiomer from substrate 1a
Please cite this article in press as: C. Li, et al., Stereo-complementary bioreduction of saturated N-heterocyclic ketones, Process Biochem
2017), http://dx.doi.org/10.1016/j.procbio.2017.03.002
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Fig. 3. Effect of 2-propanol concentration to the background reduction of 1a. Shown
are conversions (᭹) and enantiomeric excesses (ꢀ) of (S)-1b. The reaction was per-
formed in 1 mL potassium phosphate buffer (100 mM, pH 7.0) containing NADH
(
10 mM), cell extract prepared from E. coli BL21 carrying the empty vector (3 g/L)
◦
and substrate (10 mM), and incubated at 40 C for 12 h.
(Fig. 2). Although the conversion was low, such an activity would
certainly affect the enantiopurity of (R)-1b if crude enzyme extract
of ChKRED15 or ChKRED20 was to be applied as a catalyst.
Hence, it was evident that the use of GDH should be avoided
in the production of either (R)- or (S)-1a. A previous report has
briefly mentioned the activity of GDH toward cyclic alkyl ketones,
and indicated using phosphite and formate dehydrogenases as
alternatives to recycle the cofactor [30]. In our case, since both
ChKRED20 and READH are known to catalyze the dehydrogenation
of 2-propanol to regenerate NADH [17,19], the co-substrate strat-
egy would certainly be preferred for a more economic and practical
process.
Based on the known properties of ChKRED20 [31–33] and
◦
READH [17], the reaction temperature was set to be 40 and 50 C
for ChKRED20 and READH, respectively. With special concern for
the (R)-enantiomer, we investigated the effect of 2-propanol to
the activity and stereoselectivity of the background transforma-
tion under the similar conditions as ChKRED20. To our satisfaction,
Fig. 4. Structures of various saturated N-heterocyclic ketones.
2
-propanol displayed increased inhibition to the background enzy-
matic activity with increased concentration, and no conversion of
a was observed after 12-h reaction at 2-propanol concentration of
30% (Fig. 3). Such an inhibition effect would facilitate the use of the
practical level of 200 mM (Fig. 4). The results are summarized in
Table 1.
1
>
It is well known that the intrinsic specificity of the enzymes
often lead to significant change of enzymatic activity and selectivity
toward substrates with different electronic and steric character-
istics. As expected, the activities of both ChKRED20 and READH
were greatly affected by both the size of the heterocyclic ring
and the substituents (Table 1). Substrates with the six-membered
ring are typically associated with higher activity (Table 1, entries
crude enzyme extract instead of purified enzyme in the production
of (R)-1b without compromising of the product enantiopurity.
Considering the robustness of the crude enzyme extract
of ChKRED20 toward 2-propanol [32], the concentration of 2-
propanol was set to be 40% (v/v) for ChKRED20-catalyzed reactions.
On the contrary, READH only tolerated low concentration of 2-
propanol [17] with its activity decreasing significantly in the
presence of 10% (v/v) 2-propanol (data not shown). Therefore, we
used 5% (v/v) 2-propanol for NADH recycling purpose. Although the
background enzymatic activity was not completely inhibited with
5
–7). In addition, the Boc substituted substrates with five- or six-
membered ring appeared to be preferred over others (Table 1,
entries 1 vs 2–4, 5 vs 6–7). It is most likely due to the favorable
steric effect and the electron withdrawing nature of the Boc sub-
stituent [33]. Although the reaction conditions were not optimized
for each substrate, the isolated yields reached >80% in the majority
of cases. For the READH system, the (S)-enantiomer was obtained
with absolute enantiopurity in all the cases, while for the ChKRED20
system, the (R)-enantiomer was occasionally produced with traces
of (S)-alcohol, especially for the seven-membered rings, but the
enantiomeric excesses remained good with the lowest being 88%
ee (Table 1).
5
% (v/v) 2-propanol, fortunately it afforded the same enantiomer as
READH did with excellent selectivity. Therefore, the product enan-
tiopurity should not be affected.
3.3. Bioreduction of various N-heterocyclic ketones
The crude enzyme extracts of ChKRED20 and READH were each
applied to the biotransformation of 1a using 2-propanol as the
ultimate reducing agent. At a low substrate loading of 10 mM,
ChKRED20 and READH afforded the (R)- and (S)-1b, respectively,
with absolute stereoselectivity and complete conversion after 5 h.
To investigate the scope and limitation of ChKRED20 and
READH, both the (R)- and (S)-systems were then extended to the
biotransformation of various N-heterocyclic ketones with 5- to 7-
membered rings, and the substrate concentration was raised to a
4. Conclusions
We have developed an efficient biocatalytic method to syn-
thesize optically pure saturated N-heterocyclic alcohols from the
corresponding ketones. Both the R- and S-enantiomers are accessi-
ble by using ketoreductases ChKRED20 and READH, respectively.
The reactions use 2-propanol as a cheap co-substrate to regen-
Please cite this article in press as: C. Li, et al., Stereo-complementary bioreduction of saturated N-heterocyclic ketones, Process Biochem
2017), http://dx.doi.org/10.1016/j.procbio.2017.03.002
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Table 1
Reduction of various heterocyclic ketones by READH and ChKRED20.
READH systema
ChKRED20 systemb
Entry
substrate
Reaction time
h)
Yieldc (%)
eed (%)
Specific activitye
(U/mg)
Reaction time
(h)
Yieldc (%)
eed (%)
Specific activitye
(U/mg)
(
1
2
3
4
5
6
7
8
9
1a
2a
3a
4a
5a
6a
7a
8a
2
95
92
86
93
95
94
95
82
45
84
72
85
>99 S
>99 S
>99 S
>99 S
>99 S
>99 S
>99 S
>99 S
>99 S
>99 S
>99 S
>99 S
24
10
24
24
10
6
14
10
24
24
24
24
24
92
90
84
90
93
93
91
78
41
50
41
89
>99 R
>99 R
98 R
>99 R
>99 R
>99 R
>99 R
>99 R
88 R
5.5
2.5
3.1
4.8
9.9
3.8
6.9
2.3
1.7
2.8
2.9
3.6
21
21
10
0.5
4
1.5
21
24
24
24
24
4.1
4.7
4.9
38
16
23
4.3
2.9
3.7
3.8
3.2
9a
1
1
1
0
1
2
10a
11a
12a
95 R
>99 R
>99 R
a
+
The reaction mixture containing 200 mM substrate, 1 mM NAD , 5% (v/v) 2-propanol and 10 mg crude enzyme in 1 mL potassium phosphate buffer (100 mM, pH 7.0) was
◦
incubated at 50 C.
b
+
The reaction mixture containing 200 mM substrate, 1 mM NAD , 40% (v/v) 2-propanol and 10 mg crude enzyme in 1 mL potassium phosphate buffer (100 mM, pH 7.0)
◦
was incubated at 40 C.
c
Isolated yield.
Determined using HPLC analysis.
d
e
One unit of activity was defined as the quantity of enzyme required to catalyze the conversion of 1 ␮mol substrate in 1 min.
erate NADH. The practical substrate loading and overall excellent
stereoselectivity of both enzymes toward the tested 12 substrates
indicates great potential of this method.
[10] X. Ju, Y.Y. Tang, X.L. Liang, M.Q. Hou, Z.H. Wan, J.H. Tao, Development of a
biocatalytic process to prepare (S)-N-Boc-3-hydroxypiperidine, Org. Process
Res. Dev. 18 (2014) 827–830.
[
[
[
11] G.-P. Xu, H.-B. Wang, Z.-L. Wu, Efficient bioreductive production of
S)-N-Boc-3-hydroxypiperidine using ketoreductase ChKRED03, Process
(
Biochem. 51 (2016) 881–885.
Acknowledgments
12] Y. Makino, T. Dairi, N. Itoh, Engineering the phenylacetaldehyde reductase
mutant for improved substrate conversion in the presence of concentrated
2-propanol, Appl. Microbiol. Biotechnol. 77 (2007) 833–843.
13] N. Itoh, M. Matsuda, M. Mabuchi, T. Dairi, J. Wang, Chiral alcohol production
by NADH-dependent phenylacetaldehyde reductase coupled with in situ
regeneration of NADH, Eur. J. Biochem. 269 (2002) 2394–2402.
This study was funded by the National Natural Science Foun-
dation of China (21372216 and 21572220), the Open Fund of Key
Laboratory of Environmental and Applied Microbiology (KLCAS-
2
016-08 and KLCAS-2015-01), and the Key Research Program
[14] N. Kizaki, Y. Yasohara, N. Nagashima, J. Hasegawa, Characterization of novel
alcohol dehydrogenase of Devosia riboflavina involved in stereoselective
reduction of 3-pyrrolidinone derivatives, J. Mol. Catal. B: Enzym. 51 (2008)
(
KGZD-EW-606-14) of the Chinese Academy of Sciences.
7
3–80.
Appendix A. Supplementary data
[15] K. Inoue, Y. Makino, N. Itoh, Production of (R)-chiral alcohols by a
hydrogen-transfer bioreduction with NADH-dependent Leifsonia alcohol
dehydrogenase (LSADH), Tetrahedron: Asymmetry 16 (2005) 2539–2549.
16] H.M. Li, J. Moncecchi, M.D. Truppo, Development of an immobilized
ketoreductase for enzymatic (R)-1-(3,5-bis(trifluoromethyl)phenyl)ethanol
production, Org. Process Res. Dev. 19 (2015) 695–700.
17] K. Abokitse, W. Hummel, Cloning, sequence analysis, and heterologous
expression of the gene encoding a (S)-specific alcohol dehydrogenase from
Rhodococcus erythropolis DSM 43297, Appl. Microbiol. Biotechnol. 62 (2003)
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.procbio.2017.03.
[
002.
[
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