A biocatalytic redox cascade approach for one-pot deracemization of carboxyl-substituted tetrahydroisoquinolines by stereoinversion

Article abstract of DOI:10.1039/c9gc02795e

Optically pure 1,2,3,4-tetrahydroisoquinoline carboxylic acids are important chiral building blocks in the pharmaceutical and fine chemical industries. However, the existing chemo-enzymatic deracemization method employing d-amino acid oxidase from Fusarium solani M-0718 (FsDAAO) suffers from the requirement for a large excess of a nonselective chemical reducing agent. To explore an alternative method, we envisaged a concurrent biocatalytic oxidation and reduction cascade in one pot. Herein, we report a novel biocatalytic route for the asymmetric reduction of 3,4-dihydroisoquinoline-1-carboxylic acids employing Δ¹-piperidine-2-carboxylate/Δ¹-pyrrolidine-2-carboxylate reductase from Pseudomonas putida KT2440 (PpDpkA) as a biocatalyst, yielding the corresponding (S)-1-carboxyl-substituted tetrahydroisoquinolines with high conversions and enantiomeric excess (>99% ee). By combining FsDAAO and PpDpkA in one pot, a fully biocatalytic method was demonstrated for the deracemization of a range of racemic 1-carboxyl substituted tetrahydroisoquinolines to produce the corresponding (S)-enantiomers with >99% conversions and >99% ee. Furthermore, preparative-scale biotransformation of racemic 1,2,3,4-tetrahydroisoquinoline-1-carboxylic acid gave the (S)-enantiomer with 89% isolated yield and >99% ee. Taken together, we provide an enantioselective biocatalytic redox cascade method for the one-pot synthesis of enantiopure 1,2,3,4-tetrahydroisoquinoline carboxylic acids.

Full text of DOI:10.1039/c9gc02795e

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DOI: 10.1039/C9GC02795E
ARTICLE
A biocatalytic redox cascade approach for one-pot deracemization
of carboxyl-substituted tetrahydroisoquinolines by
stereoinversion
Received 00th January 20xx,
Accepted 00th January 20xx
a
b
a
a
a
a
Shuyun Ju, Mingxin Qian, Jing Li, Gang Xu, Lirong Yang, and Jianping Wu*
DOI: 10.1039/x0xx00000x
Optically pure 1,2,3,4-tetrahydroisoquinolines carboxylic acids are important chiral building blocks in the pharmaceutical
and fine chemical industries. However, the existing chemo-enzymatic deracemization method employing D-amino acid
oxidase from Fusarium solani M-0718 (FsDAAO) suffers from the requirement for a large excess of nonselective chemical
reducing agent. To explore an alternative method, we envisaged a concurrent biocatalytic oxidation and reduction cascade
in one pot. Herein, we report a novel biocatalytic route for the asymmetric reduction of 3,4-dihydroisoquinoline-1-carboxylic
1
1
acids employing Δ -piperidine-2-carboxylate/Δ -pyrrolidine-2-carboxylate reductase from Pseudomonas putida KT2440
PpDpkA) as biocatalyst, yielding the corresponding (S)-1-carboxyl-substituted tetrahydroisoquinolines with high
(
conversions and enantiomeric excess (>99% ee). By combining FsDAAO and PpDpkA in one pot, a fully biocatalytic method
was demonstrated for the deracemization of a range of racemic 1-carboxyl substituted tetrahydroisoquinolines to produce
the corresponding (S)-enantiomers with >99% conversions and >99% ee. Furthermore, preparative-scale biotransformation
of racemic 1,2,3,4-tetrahydroisoquinoline-1-carboxylic acid gave the the (S)-enantiomer with 89% isolated yield and >99%
ee. Taken together, we provide an enantioselective biocatalytic redox cascade method for the one-pot synthesis of
enantiopure 1,2,3,4-tetrahydroisoquinoline carboxylic acids.
R1
R2
R1
R2
R1
R2
FsDAAO
+
Introduction
Optically pure 1,2,3,4-tetrahydroisoquinolines (THIQs)
constitute a class of privileged structural moieties that exist in a
variety of drugs and natural isoquinoline alkaloids with diverse
biological activities.1,2 Compared with chemical synthetic
NH
N
NH
O2 H2O2
COOH
COOH
COOH
rac-1a-4a
Catalase
1b-4b
(S)-1a-4a
(
A) Previous work
(B) This work
NH3·BH3
PpDpkA
methodologies generally involving toxic organic reagents or
transition metal catalysts,3-9 elegant and environmentally
NADP+
NADPH
TbADH
acetone
1
2-propanol
friendly enzymatic methods for the preparation of chiral THIQs,
especially 1-substituted THIQs, have aroused widespread
attention in recent years. Several oxidoreductase-mediated
biocatalytic approaches have been investigated for the
preparation of chiral 1-alkyl-, 1-benzyl-, or 1-aryl-substituted
THIQs.10-15 For those carboxyl-substituted THIQs, we recently
developed a chemo-enzymatic approach to (S)-enantiomers
employing D-amino acid oxidase from Fusarium solani M-0718
1
2
2
1
a: R =H, R =H
3a: R =OH, R =H
1
2
1
2
2
a: R =Cl, R =H
4a: R =OMe, R =OMe
Scheme 1 One-pot enantioselective synthesis of (S)-Carboxyl
substituted THIQs via (A) previously reported chemo-enzymatic
deracemization
demonstrated in this work.
or
(B)
multi-enzymatic
deracemization
3 3
reducing agent such as ammonia-borane (NH ·BH ). We
envisaged that we might be able to couple the FsDAAO-
catalyzed oxidation of racemic substrates with a (S)-selective
FsDAAO) (Scheme 1A).16
(
Although this system provided an effective route to (S)-
reductase-mediated
asymmetric
reduction
of
1
,2,3,4-tetrahydroisoquinoline carboxylic acids, it suffered from
dihydroisoquinoline carboxylic acids in one pot, thus providing
a “greener” alternative method for the production of (S)-
enantiomers (Scheme 1B). Theoretically, the use of an (S)-
the requirement for a large excess of nonselective chemical
a. Institute of Bioengineering, College of Chemical and Biological Engineering,
Zhejiang University, Hangzhou, 310027, People’s Republic of China. Fax: (+86)-
selective reductase would lead to
a more efficient
5
71-8795-2363; E-mail: wjp@zju.edu.cn
deracemization process than the reported chemo-enzymatic
approach, because no reduction to the (R)-enantiomer would
b.
Tongli Biomedical Co., Ltd, 1# Guotai North Road, Zhangjiagang Economic
Development Zone, Zhangjiagang, 215600, Jiangsu, People’s Republic of China
Footnotes relating to the title and/or authors should appear here.
†
occur. Compared with the FsDAAO-NH
3 3
·BH cascade, the
Electronic Supplementary Information (ESI) available: [details of any supplementary
information available should be included here]. See DOI: 10.1039/x0xx00000x
addition of NH ·BH and the time-consuming and yield-reducing
3
3
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ARTICLE
Journal Name
removal of this chemical reducing agent would be avoided in considering the structural characteristics of the moVdieewlAsruticblestOrnalitnee
this multi-enzyme system. Deracemization of racemic mixtures 1b, which also bears a carboxylic acid sub
D
s
O
t
I
i
:
t
1
u
0
e
.1
n
0
t
39
o
/
n
C9
t
G
h
C
e
0
i
2
m
79in
5E
e
through such concurrent biocatalytic oxidation and reduction carbon atom, we speculated that DpkAs might be potential
cascade is an attractive approach and has been widely applied in the enzyme toolbox for our reduction process.
synthesis of chiral sec-alcohols,17,18, 2‑hydroxy acids,19-21 amino
To search for an ideal biocatalyst, a genome-mining strategy
acids,22-25 and amines.26-30 Recently, the deracemization of 1- was adopted. A mini-library of DpkAs was constructed using the
methyl-1,2,3,4-tetrahydroisoquinoline by combining an protein-protein basic local alignment search tool (BLAST) with
engineered
amine
oxidase
(AO)
with
artificial the amino acid sequence of PpDpkA as the template. According
87%), three candidate enzymes
transferhydrogenase (ATHase) has been studied.31,32 Despite to the sequence identity (43%
–
the advantages and attractiveness, this system was not suitable were selected (Supporting Information, Table S1). These four
for the preparation of chiral carboxyl-substituted THIQs enzyme genes were cloned and overexpressed in E. coli BL21
because of the limitation of substrate specificities of these two (DE3). To investigate the expression patterns, sodium dodecyl
enzymes. To date, no general comparable biocatalytic approach sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was
for the synthesis of chiral carboxyl-substituted THIQs has been performed. As shown in Figure S2, these four enzymes were
reported.
successfully expressed in a soluble form. The imine reduction
In this study, we successfully construct a multi-enzymatic activity of these enzymes toward substrate 1b was evaluated at
cascade for the deracemization of racemic 1-carboxyl- 30 °C and pH 8.0 using crude extract. As shown in Table 1, all of
substituted THIQs in one pot (Scheme 1B). To achieve this, a the tested enzymes exhibited significant activities toward the
panel of diverse imine-reducing enzymes for the reduction of substrate and produced the corresponding (S)-1,2,3,4-
model substrate 3,4-dihydroisoquinoline-1-carboxylic acid (1b) tetrahydroisoquinoline-1-carboxylic acid (1a) with excellent
1
was cloned and evaluated. The Δ -piperidine-2-carboxylate enantioselectivity (>99% enantiomeric excess (ee)). Among
1
reductase/Δ -pyrrolidine-2-carboxylate
reductase
from them, PpDpkA, which displayed the highest activity of
Pseudomonas putida KT2440 (termed PpDpkA) was achieved 31.07±0.91 U/mL, was selected for further research.
with highest activity and excellent enantioselectivity. By Table 1 Evaluation of enzymatic imine reduction activity toward
combining FsDAAO with PpDpkA in one pot, a range of racemic 1b
Entry Enzyme
Activity (U/mL)a
31.07±0.91
17.65±0.21
16.09±0.65
0.36±0.01
ee (%) /Config.c
b
1
-carboxyl substituted THIQs were transformed to the
corresponding (S)-enantiomers. Therefore, this system provides
a novel and sustainable method for the efficient synthesis of
optically pure carboxyl-substituted THIQs.
1
2
3
4
PpDpkA
PeDpkA
PfDpkA
PaDpkA
>99% (S)
>99% (S)
>99% (S)
>99% (S)
a
Enzyme activity was determined by monitoring the decrease
in the absorbance of NADPH at 340 nm.
The product ee was determined by chiral HPLC.
Configuration was determined by comparison with literature
data or by analogue.
Results and discussion
Enzyme exploitation for the asymmetric reduction of 3,4-
dihydroisoquinoline-1-carboxylic acid
b
c
The asymmetric reduction of these sterically hindered
dihydroisoquinoline carboxylic acids to the corresponding (S)-
enantiomers with excellent enantioselectivity is critical to the
proposed biocatalytic cascade. Unfortunately, no applicable
enzyme has been reported for this process to date. Although
various microbial imine reductases (IREDs) have been
successfully employed for the reduction of analogues, such as
Purification and characterization of PpDpkA
The recombinant PpDpkA with a His-tag at the N-terminal was
purified using nickel-affinity chromatography, and analyzed by
SDS-PAGE (Supporting Information, Figure S3A), indicating a
subunit size of approximately 40 kDa. The enzymatic properties
of PpDpkA were then investigated using compound 1b as the
substrate. PpDpkA exhibited maximal activity at pH 6.5 and 42%
relative activity at pH 8.0 (Supporting Information, Figure S3B).
The enzyme was stable from pH 6.5 to 8.5 (retained more than
1
-methyl-3,4-dihydroisoquinoline,12,33-36 previous applications
of these enzymes mainly focused on the analogous non-
carboxylate imines and iminium ions.37,38 Interestingly, another
class of natural imine-reducing enzymes such as bacterial Δ1-
9
0% activity) for 24 h at 4 °C (Supporting Information, Figure
piperidine-2-carboxylate (Pip2C) reductase (EC 1.5.1.21) and Δ1- S3C). The specific activities from 20 to 55 °C were then
pyrrolidine-2-carboxylate (Pyr2C) reductases (EC 1.5.1.1) are measured. The enzyme showed maximal activity at 40 °C and
known to catalyze the reduction of Pip2C or Pyr2C to L-pipecolic 80% relative activity at 30 °C (Supporting Information, Figure
acid or L-proline.39 The most prominent and best characterized S3D). The thermostability of PpDpkA was assessed at 30, 40, and
members are those from Pseudomonas spp. (also known as 50 °C (Supporting Information, Figure S3E). The half-lives of the
enzyme were calculated to be 52 h at 30 °C, 21 h at 40 °C and
DpkAs or N-methyl-L-amino acid dehydrogenases), which are
0
.4 h at 50 °C, indicating relative good thermal stability under
biologically involved in the catabolism of D-lysine and D-
proline.40,41 DpkAs as imine-reducing biocatalysts have been
reported to exhibit a narrow substrate scope.38 Nevertheless,
mild reaction conditions. The kinetic parameters of purified
PpDpkA toward substrate 1b were determined at pH 8.0 and 30
2
| J. Name., 2012, 00, 1-3
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ARTICLE
°
C, indicating a catalytic efficiency constant (kcat/Km) of 759.84 dehydrogenase
from
Thermoanaerobacter
brockii
View Article Online
DOI: 10.1039/C9GC02795E
-1
-1
44
s mM (Table 2).
(TbADH) /isopropanol regeneration system was selected. To
access the potential effect of this cofactor regeneration system
on FsDAAO and PpDpkA, the specific activities of these two
enzymes were separately examined in the presence of different
concentrations (0–100 mM) of isopropanol and co-product
acetone. As shown in Fig. S3 and S4, the activities of these two
enzymes were not affected.
Table 2 Kinetic parameters of PpDpkAa
-
1
-1
-1
Sub.
Km (mM)
0.77±0.02
k
cat (s )
k
cat/Km (s mM )
1
b
587.52±5.88
759.84
a
The assay was performed using the HPLC method, and each value
represents mean±standard deviation of three measurements.
Enantioselective synthesis of (S)-1a employing the PpDpkA-TbADH
system
Evaluation of the feasibility and compatibility of the proposed
biocatalytic cascade
Based on these results, the asymmetric reduction of substrate
Constructing an efficient concurrent multi-enzymatic cascade in 1b was then performed using purified PpDpkA. A control
one pot was considered to be rather challenging due to the experiment without cofactor regeneration system was initially
different reaction conditions suitable for individual conducted. The cofactor NADPH was supplied at a sufficient
biotransformation. Furthermore, the combination of reactions concentration (8 mM). Substrate 1b (5 mM) was transformed
may lead to the enzyme activity inhibition by reaction into (S)-1a at a high conversion (>99%) and >99% ee within 1 h
intermediates.4
2,43
Hence, it is extremely important and (Table 3, entry 1). Then, we applied PpDpkA for the reduction of
necessary to take each reaction into full consideration and substrate 1b in combination with the TbADH/isopropanol
+
identify compatible conditions.
regeneration system. A relatively low concentration of NADP
To investigate the feasibility of the proposed (5 μM) was used. The chiral HPLC analysis showed that (S)-1a
deracemization, we initially performed the FsDAAO-PpDpkA was obtained with excellent enantioselectivity (>99% ee).
cascade reaction in one pot without cofactor regeneration However, the conversion of substrate 1b only reached 93% at 1
system. Considering the effect of pH and temperature on h (Table 3, entry 2). To further improve the efficiency, we
enzymatic activity and stability, a compromised mild reaction increased the amount of TbADH. As a result, PpDpkA (2 U/mL)
condition (pH 8.0 and 30 °C) was adopted. Biotransformation and TbADH (5 U/mL) were combined and afforded high
reaction was performed in 1 mL of Na
mM, pH 8.0) that contained rac-1a (10 mM), purified FsDAAO enantioselectivity (>99% ee) within 1 h (Table 3, entry 4).
0.1 mg/mL, 2 U/mL), flavine adenine dinucleotide (FAD) (100
2 4 2 4
HPO -NaH PO
buffer (50 conversion (>99%) of substrate 1b to (S)-1a with excellent
(
Table 3 Asymmetric reduction of 1b to (S)-1a employing PpDpkAa
μM), catalase (200 U/mL), an excess of purified PpDpkA (10
U/mL) and reduced nicotinamide adenine dinucleotide
phosphate (NADPH) (8 mM). After incubation for 1 h, chiral
HPLC results revealed that the concentration of (S)-1a was
indeed improved, which is the prerequisite for the one-pot
simultaneous cascade. However, 60% of (R)-1a still existed in
the solution. This result was inconsistent with the previously
described kinetic resolution, in which (R)-1a (5 mM) could be
completely oxidized by purified FsDAAO (2 U/mL) in 1 h. We
speculated that the activity of FsDAAO may have been inhibited
by the reduction process, which subsequently resulted in low
efficiency in the oxidation process.
e
Entry PpDpkA
TbADH
(U/mL)
/
Cofactor Conv.
ee (%) /
b
c
d
e
(U/mL)
(μM)
(c) (%)
Config.
1
2
3
4
2
NADPH
(8000)
NADP
>99%
>99 (S)
>99 (S)
>99 (S)
>99 (S)
+
2
2
2
1
3
5
93%
96%
>99%
(
5)
NADP
5)
NADP
5)
Reaction conditions (1ml): substrate 1b (5 mM), purified PpDpkA
+
1
6
(
+
(
a
(
2 U/mL), purified TbADH (1
–
5 U/mL), cofactor NADPH (8000 μM)
To identify the problem, the specific activity of FsDAAO was
separately determined with different concentrations (0–10
mM) of NADPH and NADP . As shown in Figure S4C, no
+
or NADP (5 μM), and isopropanol (6 mM) in Na
2
HPO -NaH PO
4
2
4
buffer (50 mM, pH 8.0). Reactions were incubated at 30 °C with
00 rpm shaking for 1 h.
+
6
+
significant activity inhibition was observed with NADP .
b
c
One unit (U) was defined as the amount of enzyme that converted
However, only 38% of relative activity was retained in the
presence of 8 mM NADPH (Figure S4B), suggesting that the
relative high concentration of NADPH was the predominant
reason for the low efficiency of the FsDAAO-catalyzed
oxidation. Fortunately, the presence of low concentrations of
NADPH (0–0.5 mM) had no negative effect on the specific
activity of FsDAAO.
Given the high cost of NADPH and the negative effect of this
cofactor at relatively high concentrations on FsDAAO, cofactor
regeneration was essential for our deracemization reaction.
Considering the product separation and the activity of the
enzyme used for NADPH regeneration, an alcohol
+
1
8
μmol of NADPH to NADP per minute using 1b as substrate at pH
.0 and 30 °C.
One unit (U) was defined as the amount of enzyme that converted
+
1
μmol of NADP to NADPH per minute using isopropanol as
substrate at pH 8.0 and 30 °C.
d
e
Conversion (c) of 1b to (S)-1a was determined by HPLC analysis.
The ee was determined by chiral HPLC analysis, and configuration
was determined by comparison with literature data or by
analogue.
One-pot synthesis of (S)-carboxyl-substituted THIQs via a multi-
enzymatic cascade
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 3
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ARTICLE
Journal Name
Since the kinetic resolution of rac-1a employing FsDAAO and system
the asymmetric synthesis of (S)-1a using the PpDpkA-TbADH
View Article Online
DOI: 10.1039/C9GC02795E
Table 4 Deracemization of rac-1a–
4a employing FsDAAO-PpDpkA cascade a
Entry
Sub.
mM)
FsDAAO
(U/mL)
2
10
20
2
2
2
PpDpkA
(U/mL)
2
10
20
40
40
40
TbADH
(U/mL)
5
25
50
5
5
5
NADP+
(μM)
5
25
50
5
5
5
Time
(h)
1
1
6
1
1
5
Conv.e
(C) (%)
>99
>99
>95
>99
>99
>99
ee (%)f/
Config.
b
c
d
f
(
1
2
3
4
5
6
1a (10)
1a (50)
1a (100)
2a (10)
3a (10)
4a (5)
>99 (S)
>99 (S)
>99 (S)
>99 (S)
>99 (S)
>99 (S)
a
Reaction conditions (1ml): racemic substrates (5-100 mM), purified FsDAAO (2
–
–
20 U/mL), FAD (100 μM), catalase (100 times the activity
+
of FsDAAO), purified PpDpkA (2
Na HPO -NaH PO
–
40 U/mL), NADP (5
–
50 μM), purified TbADH (5
50 U/mL), and isopropanol (1.2 equivalents of 1b 4b) in
–
2
4
2
4
buffer (50 mM, pH 8.0). Reactions were shaken at 30 °C, 600 rpm.
b
c
One unit (U) was defined as the amount of enzyme that catalyzed the oxidation of 1 μmol (R)-1a per minute at pH 8.0 and 30 °C.
One unit (U) was defined as the amount of enzyme that converted 1 μmol of NADPH to NADP per minute using 1b as substrate at pH 8.0
+
and 30 °C.
d
e
f
+
One unit (U) was defined as the amount of enzyme that converted 1 μmol of NADP to NADPH per minute using isopropanol as substrate
at pH 8.0 and 30 °C.
Conversion (C) of (R)-enantiomer to (S)-enantiomer was determined by chiral HPLC analysis. C = [(the residual (S)-a - the initial (S)-a)/the
initial (R)-a] × 100%.
The ee was determined by chiral HPLC analysis, and configuration was determined by comparison with literature data or by analogue.
have been demonstrated, we sought to develop a novel method catalyzed kinetic resolution was carried out separately at
for the synthesis of (S)-1a by combining these two reactions in analytical scale. Upon completion of the reaction, the solution
one pot.
was incubated at 99 °C for 10 min to denature the enzymes. The
To realize this process, we began our synthesis with a low purified PpDpkA and NADPH were then added to the reaction
concentration of substrate rac-1a (10 mM). Biotransformation mixture at 30 °C. After incubation for 1 h, chiral HPLC analysis
was performed by combining purified FsDAAO, PpDpkA, and revealed that the concentrations of (S)-2a–4a were significantly
TbADH in one pot. Based on the feasibility and compatibility increased, and no (R)-enantiomers were observed. Our
+
analysis, NADP (5 μM) and isopropanol (6 mM) were used for preliminary results demonstrated that compounds 2b–4b could
the cofactor regeneration, which had no negative effect on the be reduced by PpDpkA with excellent enantioselectivity (>99%
activity of FsDAAO or PpDpkA. Catalase was utilized to ee), which indicated a broad and remarkable complementary
4
5
decompose the H
analysis revealed that (S)-1a was obtained with high conversion
(
(
2
O
2
generated by FsDAAO. Chiral HPLC substrate scope to IREDs and ATHase.
Subsequently, the two reactions were performed
>99%) and excellent enantioselectivity (>99% ee) within 1 h simultaneously in one pot. As shown in Table 4, the racemic
Table 4, entry 1). substrates 2a–4a were successfully transformed into the
Encouraged by this result, we further investigated the corresponding (S)-enantiomers with high conversions (up to
application of this cascade with higher substrate >99%) and excellent enantioselectivity (>99% ee) in 1 or 5 h.
concentrations. As expected, 50 mM of rac-1a was completely Therefore, the presented cascade successfully extended the
deracemized within 1 h (Table 4, entry 2). When the substrate biocatalytic toolbox for the synthesis of chiral THIQs.
concentration was 100 mM, the reaction efficiency decreased, Preparative-scale deracemization of rac-1a
which was due to the slight insolubility of 1a at this high
concentration. Prolonging the reaction time to 6 h, chiral HPLC
analysis revealed that 95% of (R)-1a was transformed to (S)-1a
with >99% ee (Table 4, entry 3).
To further access the synthetic potential of the FsDAAO-PpDpkA
cascade, the one-pot deracemization of substrate 1a (50 mM)
was performed on a preparative scale. The reaction process was
monitored by chiral HPLC. As shown in Fig. 1, the amount of (R)-
Having established the FsDAAO-PpDpkA cascade in one pot,
1
1
a decreased rapidly in the first 2 h, and the conversion of (R)-
a to (S)-1a reached 98% at 2.5 h. In the next 2.5 h, complete
we were curious to explore whether this system would be
applicable to derivatives of 1a. The FsDAAO-catalyzed kinetic
resolution of racemic substrates 2a–4a with different
substituent groups (Cl, OH, OMe) on phenyl ring has been
demonstrated.16 However, the feasibility of the asymmetric
reduction procedure was not certain. To investigate the
substrate tolerance of PpDpkA for phenyl substituted
dihydroisoquinoline-1-carboxylic acids (2b–4b), step-by-step
sequential reactions were performed. The purified FsDAAO-
deracemization was achieved with >99% conversion and >99%
ee. Finally, (S)-1a was obtained with good isolated yield (89%)
and excellent enantioselectivity (>99% ee). In comparison with
the previously described chemo-enzymatic deracemization, this
multi-enzymatic cascade was more efficient owing to the direct
reduction of 1b to (S)-1a. Noteworthily, the removal of large
excesses of the chemical reducing agent was circumvented,
4
| J. Name., 2012, 00, 1-3
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ARTICLE
which not only saves resources and operation time but also Substrates racemic 6-chloro-1,2,3,4-tetrahydroisoquinoline-1-
View Article Online
DOI: 10.1039/C9GC02795E
carboxylic acid hydrochloride (rac-2a) and racemic 6,7-
enables significant improvement of the yield.
dimethoxy-1,2,3,4-tetrahydroisoquinoline-1-carboxylic
hydrochloride (rac-4a) were purchased from BOC sciences
Shirley, NY). Substrate racemic 6-hydroxy-1,2,3,4-
tetrahydroisoquinoline-1-carboxylic acid (rac-3a) was
purchased from J&K scientific Ltd (Beijing, China). Substrate
racemic 1-phenyl-1,2,3,4-tetrahydroisoquinoline was
acid
(
purchased from TCI (Shanghai) Development co., Ltd (China).
Methanol (MeOH) and methyl cyanide (MeCN), diethyl amine
(DEA) for HPLC, catalase from bovine liver, isopropyl-β-D-
thiogalactopyranoside (IPTG), and kanamycin were purchased
from Sigma-Aldrich (Munich, Germany). Genome DNA
extraction kit, plasmid purification kit, DNA gel extraction kit,
restriction endonucleases, T4 DNA ligase, and PrimerSTAR MAX
DNA polymerase were purchased from Takara (Beijing, China).
All other reagents and compounds used were of analytical grade
and commercially available.
Cloning, expression, purification and characterization of enzymes
Fig. 1 The time-course study of multi-enzymatic deracemization of
rac-1a.
FsDAAO, DpkAs and TbADH genes were cloned and
overexpressed in E. coli BL21 (DE3). Their activities were
detected using HPLC method or monitoring the changes in the
absorbance of NADPH at 340 nm. Recombinant enzymes were
purified by His-tag affinity chromatography. The enzymatic
properties were characterized. All these methods were
described in the Supporting Information in detail.
Conclusions
In summary, we have shown the first application of DpkAs for
the asymmetric reduction of 3,4-dihydroisoquinoline-1-
carboxylic acids with high activity and excellent
enantioselectivity, yielding the synthetically challenging (S)-1-
carboxyl-substituted THIQs. By combining FsDAAO and PpDpkA
in one pot, we developed a novel fully biocatalytic cascade
approach for the deracemization of a range of racemic carboxyl-
substituted THIQs to the corresponding (S)-enantiomers with
high conversions (up to >99%) and excellent enantioselectivity
Analytical-scale asymmetric reduction of 1b and deracemization of
rac-1a–4a
For the asymmetric reduction of substrate 1b, a typical 1 mL
reaction mixture contained substrate 1b (5 mM), purified
PpDpkA (2 U/mL), purified TbADH (1–5 U/mL), cofactor NADPH
+
(
8 mM) or NADP (5 μM), and isopropanol (6 mM) in Na
2
HPO
4
-
NaH
0 °C with 600 rpm shaking for 1 h.
The analytical-scale deracemization of racemic substrates
2 4
PO buffer (50 mM, pH 8.0). Reactions were incubated at
(>99% ee). The cofactor NADPH regeneration system was
3
essential for this process. Preparative-scale deracemization of
substrate 1a was demonstrated with good isolated yield (89%)
and excellent enantioselectivity (>99% ee). Compared to the
chemo-enzymatic method, the non-selective chemical reducing
agent was replaced with environmentally benign biocatalysts in
this multi-enzymatic cascade, thereby providing an alternative
promising and sustainable route to the synthesis of optically
pure carboxyl-substituted THIQs. Owing to the broad substrate
scope of FsDAAO and PpDpkA, we anticipate that this approach
might be further applied for the preparation of other
pharmaceutically important chiral building blocks.
were performed at pH 8.0 and 30 °C. Unless stated otherwise, a
typical 1 mL reaction mixture contained substrate rac-1a–4a (5–
1
00 mM), purified FsDAAO (2–20 U/mL), FAD (100 μM), catalase
(
100 times the activity of FsDAAO), purified PpDpkA (2–40
+
U/mL), NADP (5–50 μM), purified TbADH (5–50 U/mL), and
2 4 2 4
isopropanol (1.2 equivalents of 1b-4b) in Na HPO -NaH PO
buffer (50 mM, pH 8.0).
Preparative-scale deracemization of rac-1a
The reaction was carried out in a 50 mL three-necked flask
equipped with a magnetic stirrer. The rac-1a hydrochloride salt
(
214 mg, 1 mmol) were dissolved in 8 mL water. The pH was
Experimental
Chemicals
carefully adjusted to a value of pH 8.0 with ammonium
hydroxide. Isopropanol (0.6 mmol) and NADP (0.5 μmol) were
+
added to the substrate solution. The lyophilized E.coli cells
containing FsDAAO or PpDpkA or TbADH from 200 or 50 or 200
mL ferments was resuspended in 12.5 or 5 or 10 mL water, and
the crude lysate was prepared by sonication and centrifugation.
Cofactor FAD, formaic acid (FA), trifluoroacetic acid (TFA) were
purchased from Aladdin (Shanghai, China). Cofactor NADP and
NADPH were purchased from Bontac Bio-engineering Co., Ltd.
+
(Shenzhen, China). Tryptone and yeast extract were obtained
5
or 1 or 6 mL crude lysate and 50 mg catalase were added to
from Oxoid (Basingstoke, England). Substrates racemic 1,2,3,4-
tetrahydroisoquinoline-1-carboxylic acid hydrochloride (rac-1a)
and (R)-1a were provided by Tongli Biomedical Co., Ltd.
the substrate solution. The pH was kept at 8.0. The reactor was
kept at 30 °C through a thermostatic water bath. The reaction
was monitored by chiral HPLC. Upon the completion of reaction,
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Journal Name
the proteins were removed through ultrafiltration. The solution 21.
were then evaporated off under vacuum, and the residue was
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2
2
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washed with warm water. The solution was concentrated so
that compound (S)-1a was recrystallized as white solid.
9
Conflicts of interest
There are no conflicts to declare.
25.
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Acknowledgements
This work was financially supported by the Natural Science
Foundation of China (No. 21676240).
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DOI: 10.1039/C9GC02795E
ARTICLE
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Green Chemistry
Page 8 of 8
View Article Online
DOI: 10.1039/C9GC02795E
One-pot Multi-enzyme Cascade
NADP+ + isopropanol
R1
R2
(for NADPH regeneration)
R1
NH
NH
R2






COOH
  
COOH
(S)


rac
1
2
R , R = H, Cl, OH, OMe
Mild conditions
Metal-free
Up to >99% conv.
Facile product isolation
Highly atom-economic
Up to >99% ee
We describe a biocatalytic redox cascade reaction for one-pot enantioselective
synthesis of (S)-carboxyl substituted tetrahydroisoquinolines.