Efficient Synthesis of Chiral Indolines using an Imine Reductase from Paenibacillus lactis

Article abstract of DOI:10.1002/adsc.201500160

An enzymatic process for the efficient asymmetric reduction of 3H-indoles as well as 3H-indole iodides was developed for the first time. Using a new imine reductase identified from Paenibacillus lactis (PlSIR), various chiral indolines were facilely synthesized in good yields and excellent enantiopurities (up to >99% ee) under mild reaction conditions.

Full text of DOI:10.1002/adsc.201500160

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DOI: 10.1002/adsc.201500160
Efficient Synthesis of Chiral Indolines using an Imine Reductase
from Paenibacillus lactis
Hao Li,a Zheng-Jiao Luan,a Gao-Wei Zheng,a,* and Jian-He Xua,*
a
State Key Laboratory of Bioreactor Engineering, Shanghai Collaborative Innovation Center for Biomanufacture, East
China University of Science and Technology, 130 Meilong Road, Shanghai 200237, Peopleꢀs Republic of China
Fax: (+86)-21-6425-0840; e-mail: gaoweizheng@ecust.edu.cn or jianhexu@ecust.edu.cn
Received: February 15, 2015; Revised: April 28, 2015; Published online: May 12, 2015
Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/adsc.201500160.
Abstract: An enzymatic process for the efficient
asymmetric reduction of 3H-indoles as well as 3H-
indole iodides was developed for the first time.
Using a new imine reductase identified from Paeni-
bacillus lactis (PlSIR), various chiral indolines were
facilely synthesized in good yields and excellent
enantiopurities (up to >99% ee) under mild reac-
tion conditions.
an Ir-BICP catalyst with a reaction time of 100 h.[9a]
In addition, 100% conversion and 86% ee were ob-
tained using an Ir-XYLIPHOS catalyst in ionic liquid
[C10mim][BF4] for 15 h.[9b] Therefore, this kind of
substrate remains a challenge for the transition metal-
catalyzed asymmetric hydrogenation.[9g] Alternatively,
these issues may be addressed by biocatalysis. Here
we describe a new imine reductase and a synthetic
strategy for the highly enantioselective reduction of
3H-indoles as well as 3H-indole iodides, affording
a variety of chiral indolines with up to >99% ee.
Keywords: asymmetric hydrogenation; biocatalysis;
chiral indolines; imine reductase; 3H-indoles
First, to identify a novel imine reductase (IRED)
with high activity and enantioselectivity towards these
sterically hindered substrates, we cloned and ex-
pressed 50 putative imine reductases in Escherichia
Optically active cyclic amines are present in numer- coli (Supporting Information, Table S1), and tested
ous biologically active pharmaceutical drugs and natu- their activity on 2,3,3-trimethylindolenine (1a) as
ral alkaloids.[1] Chiral indolines are important struc- a model substrate. Twelve of the tested enzymes ex-
ture motifs commonly used in the pharmaceutical hibited significant activity towards substrate 1a. The
molecules, such as D2/D4 receptor antagonists.[2] Re- 12 candidate enzymes were then further compared
cently, the asymmetric hydrogenation of imines has based on the enantiomeric excesses of the formed
attracted considerable attention because it has the ad- products. As shown in Table 1, an imine reductase
vantage of a 100% theoretical yield.[3] Various metal- from Paenibacillus lactis (PlSIR) displayed the highest
mediated and organic-molecule catalysts have been
developed for the reduction of cyclic imines with high
efficiency and selectivity.[4] As asymmetric bioreduc-
tion is environmentally friendly, the exploration of
novel reductases for the asymmetric synthesis of drug
intermediates remains an important topic of industrial
interest.[5]
To date, only a few biocatalysts have been reported
for the asymmetric reduction of cyclic imines, specifi-
cally limited to pyrrolines (Scheme 1a),[6] dihydroiso-
quinolines (Scheme 1b),[7] and b-carboline imines
(Scheme 1c).[8] However, to the best of our knowl-
edge, there has been no report on the bioreduction of
3H-indoles (Scheme 1d), another kind of cyclic imine
reported frequently in many reports on chemical re-
duction.[9] Among the previous methods, the highest
enantiomeric excess (ee) of 95% was obtained using Scheme 1. Asymmetric bioreduction of cyclic imines.
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Efficient Synthesis of Chiral Indolines using an Imine Reductase
Table 1. Enzyme screening for the reduction of 1a.
Table 2. Reduction of 3H-indoles using PlSIR.[a]
Entry
Enzyme
Activity [U/mg][a]
ee [%][b,c]
1
2
3
4
5
6
7
8
PlSIR
KfSIR
AaRIR
BbSIR
SeSIR
SvSIR
StRIR
PlRIR
SnRIR
AdRIR
SvRIR
SeRIR
3.58
3.31
0.95
0.43
0.40
0.33
0.22
0.21
0.17
0.12
0.10
0.07
99.3 (S)
95.1 (S)
35.1 (R)
96.9 (S)
97.4 (S)
95.8 (S)
59.1 (R)
96.3 (R)
91.8 (R)
69.1 (R)
97.9 (R)
45.1 (R)
Entry Substrate 1 (R1 =) Time [h] Yield [%][c] ee [%][d.e]
9
1
2
3
4
5
6
1a (H)
1b (5-F)
2.5
4.0
2.5
4.0
18
9.0
3.0
24
75 (2a)
74 (2b)
82 (2c)
80 (2d)
86 (2e)
83 (2f)
79 (2g)
68 (2h)
82 (2i)
>99 (S)
>99 (S)
>99 (S)
>99 (S)
97 (S)
89 (S)
>99 (S)
89 (S)
10
11
12
1c (5-Cl)
1d (5-Br)
1e (5-NO2)
1f (5-CF3)
1g (5-Me)
1h (5-OMe)
1i (4,6-F)
[a]
Activity was measured at 308C and pH 7.0 using cell-free
extract.
Enantioselectivity was determined by chiral HPLC.
Absolute configuration was assigned by comparing the
sign of the optical rotation with those reported in the lit-
erature.
[b]
[c]
7
8[b]
9
12
99 (S)
10[b]
24
24
n.d.[f] (2j)
–
–
specific activity of 3.58 U/mg[10] cell-free extract for
the reduction of 1a, giving the corresponding (S)-
product 2a with 99.3% ee (Table 1, entry 1).
11[b]
n.d.[f] (2k)
To investigate the intrinsic properties of PlSIR, the
N-terminal His-tagged recombinant enzyme was puri-
fied to electrophoretic homogeneity using nickel-af-
finity chromatography (Supporting Information, Fig-
ure S1). The specific activity of the pure protein was
15.1 U/mg at 308C and pH 6.0. The kinetic parame-
ters of PlSIR for substrate 1a and NADPH were also
determined at 308C and pH 6.0, indicating a catalytic
efficiency constant (kcat/Km) of 23.4 sÀ1 mMÀ1 (Sup-
porting Information, Table S2), which is 30-fold
higher than that of the best (S)-imine reductase re-
ported so far.[7a] The enzyme also exhibited excellent
thermal stability, with a half-life of 49.5 h at 508C
(Supporting Information, Table S3).
[a]
Reaction conditions: 1 mmol substrate, 100 mg PlSIR
(cell-free extract), 10 mg BmGDH (cell-free extract),
1.5 mmol glucose, 1 mmol NADP+, 0.5 mL DMSO and
9.5 mL potassium phosphate buffer (0.1M, pH 6.0) at
308C.
The PlSIR load was 500 mg.
Isolated yields.
Enantioselectivity was determined by chiral HPLC.
The absolute configurations of the products were as-
signed by comparing their CD spectra with that of 2a.
No product was detected.
[b]
[c]
[d]
[e]
[f]
Preparative asymmetric reductions of various 3H-
We also investigated the direct reduction of imini-
indoles were then performed, using a coupled system um salts to chiral tertiary amines (Table 3). Consider-
consisting of PlSIR and BmGDH (a glucose dehydro- ing the good solubility of iminium salts in water,
genase from Bacillus megaterium for NADPH regen- DMSO was not added to the reaction mixture. The
eration[11]). The reactions were performed on reductions of both N-methyl- and N-ethyl-substituted
a 1.0 mmol scale (100 mM substrate in 10 mL reaction indole iodides (Table 3, entries 1 and 2) were faster
mixture) at 308C and pH 6.0; the results are summar- than that of 1a (Table 2, entry 1), and afforded the de-
ized in Table 2. Most substrates tested (Table 2, en- sired products in 71% yield with excellent enantiose-
tries 1–7 and 9) were facilely reduced to chiral indo- lectivities (>99% ee) within 20 min. To explain the
lines in good yields (74–86%) and with excellent difference of catalytic activity between imines and
enantiopurities (89% to >99% ee). The enzyme dis- iminium salts, the kinetic parameters of PlSIR to-
played a low activity for substrate 1h (Table 2, wards representative substrates were determined. As
entry 8). It is worth noting that substrates with bulky shown in Table 4, significantly higher kcat values for
groups at the 2-position or 3-position on the indole 3a, 3d and 3e were observed compared to 1a, 1b and
ring (Table 2, entries 10 and 11) could not be convert- 1g, resulting in a faster reactivity.
ed by PlSIR; this is probably because the narrow
With the increase of chain length, the reaction rate
active pocket of the enzyme hinders the entrance of decreased rapidly (Table 3, entry 3). In light of the
these bulky substrates.
higher activity observed for the reduction of N-
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Table 3. Reduction of 3H-indole iodides using PlSIR.[a]
Entry Substrate 3
(R1 =, R2 =)
Time [h] Yield [%][c] ee [%][d,e]
1
2
3
4
5
3a (H, Me)
3b (H, Et)
3c (H, Pr)
3d (5-F, Me)
0.33
0.33
9.0
71 (4a)
71 (4b)
74 (4c)
83 (4d)
87 (4e)
>99 (S)
>99 (S)
96 (S)
>99 (S)
>99 (S)
Scheme 2. Asymmetric hydrogenation of iminium salts or
enamines to chiral tertiary amines.
0.25
3e (5-Me, Me) 0.50
6b)
6.0
76 (4f)
94 (À)[f]
direct reduction of iminium salts by PlSIR to tertiary
amines eliminated the double-bond isomerization step
involved in the asymmetric hydrogenation of enam-
ines (Scheme 2).[12]
The activities of PlSIR towards other imine sub-
strates were also measured, including 2-methyl-1-pyr-
roline (7a), 6-methyl-2,3,4,5-tetrahydropyridine (7b)
and 1-methyl-3,4-dihydroisoquinoline (7c). The PlSIR
exhibited a certain activity towards 7a (0.20 U/mg)
and 7b (0.62 U/mg), giving directly the (S)-products
2-methylpyrrolidine (8a) and 2-methylpiperidine (8b)
with 96% ee and 97% ee, respectively. Unfortunately,
no activity was detected towards 7c.
In summary, we have obtained a new imine reduc-
tase PlSIR with high activity and excellent enantiose-
lectivity. Using the imine reductase as a catalyst, an
efficient biocatalytic approach for the synthesis of
chiral indolines was developed through the asymmet-
ric reduction of 3H-indoles and iminium salts. The
corresponding N-unprotected indolines and N-alkylin-
dolines were obtained in good yields (up to 87%) and
with excellent enantioselectivities (up to >99% ee).
Further engineering of PlSIR to extend the scope of
substrates is still ongoing in our laboratory.
[a]
Reaction conditions: 1 mmol substrate, 50 mg PlSIR
(cell-free extract), 50 mg BmGDH (cell-free extract),
1.5 mmol glucose, 1 mmol NADP+ and 10 mL potassium
phosphate buffer (0.1M, pH 6.0) at 308C.
The PlSIR and BmGDH loads were 100 mg and 10 mg,
respectively.
[b]
[c]
[d]
Isolated yields.
Enantioselectivity was determined by chiral HPLC or
GC.
The absolute configurations of the products were as-
signed by comparing their CD spectra with that of 2a.
Absolute configuration is not determined; optical rota-
tion sign is given.
[e]
[f]
Table 4. Comparison of kinetic parameters of PlSIR towards
cyclic imines and corresponding iminium salts.
Entry
Substrate
Km
[mM]
kcat
kcat/Km
(R1 =)
[sÀ1]
[sÀ1 mMÀ1]
1
2
3
4
5
6
1a (H)
0.48
0.15
0.037
0.14
0.19
0.096
11.3
4.51
1.91
78.3
97.2
14.9
23.4
30.1
51.6
559
512
155
1b (5-F)
1g (5-Me)
3a (H)
3d (5-F)
3e (5-Me)
Experimental Section
Cloning and Expression of PlSIR
PlSIR gene (WP_007128231.1) was amplified by PCR, and
methyl-substituted 3H-indole iodides, other similar the gene fragment was then inserted into plasmid pET-28a.
The resultant plasmid was then transformed into E. coli
BL21 (DE3) for overexpression. The recombinant cells were
cultivated in LB medium at 378C with shaking at 180 rpm
until OD600nm reached 0.6–0.8, and then the encoding genes
were induced by addition of IPTG (final concentration of
0.2 mM) at 168C for further 24 h. The cells were harvested
by centrifugation (8800”g, 10 min), and subsequently dis-
rupted by sonication and the homogenate was centrifuged
again (13,500”g, 48C, 20 min) to remove the cells debris.
Enzyme activity was determined by monitoring the decrease
substrates (Table 3, entries 4–6) were also examined.
The reduction proceeded smoothly for substrates with
either electron-withdrawing (Table 3, entry 4) or elec-
tron-donating (Table 3, entry 5) groups, giving the cor-
responding indolines in high yields (83% and 87%, re-
spectively) and with excellent enantioselectivities (>
99% ee) within 30 min. Even the bulky substrate 3f
was well converted. Notably, the space-time yield of
4d (Table 3, entry 4) reached 1538 gLÀ1 dÀ1, demon-
strating the great potential of PlSIR. Furthermore, in the absorbance of NADPH at 340 nm.
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Efficient Synthesis of Chiral Indolines using an Imine Reductase
terson, A. Kieltyka, R. Brodbeck, R. Primus, J. W. F.
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Experimental Details for Bioreduction
The reaction mixture was composed of 1 mmol 3H-indole
1a, 1.5 mmol glucose, 1 mmol NADP+, 100 mg PlSIR (75 U),
10 mg BmGDH (100 U), 0.5 mL DMSO and 9.5 mL potassi-
um phosphate buffer (100 mM, pH 6.0). The reaction was
performed at 308C, and pH was automatically adjusted to
6.0 by titrating 1M NaOH solution. The reaction was termi-
nated by adding 0.5 mL NaOH (2M), and the mixture was
extracted with ethyl acetate, dried over Na2SO4, filtered and
concentrated under reduced pressure. The crude product
was purified by column chromatography eluting with petro-
leum ether and ethyl acetate (20:1 to 10:1) to afford indo-
line 2a as yellow oil; yield: 120 mg (75%).
The reaction mixture was composed of 1 mmol 3H-indole
iodide 3a, 1.5 mmol glucose, 1 mmol NADP+, 50 mg PlSIR,
50 mg BmGDH and 10 mL potassium phosphate buffer
(100 m, pH 6.0). The reaction was performed at 308C, and
pH was automatically adjusted to 6.0 by titrating 1M NaOH
solution. The reaction was terminated by adding 0.5 mL
NaOH (2M), and the mixture was extracted with ethyl ace-
tate, dried over Na2SO4, filtered and concentrated under re-
duced pressure. The crude product was purified by column
chromatography eluting with petroleum ether and ethyl ace-
tate (40:1 to 20:1) to afford indoline 4a as yellow oil; yield:
125 mg (71%).
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This work was financially supported by the National Natural
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