Application of Imine Reductases (IREDs) in Micro-Aqueous Reaction Systems

Article abstract of DOI:10.1002/adsc.201501154

Here we present the applicability of different imine reductases (IREDs) in micro-aqueous reaction systems. Subjects of the study were the IREDs from Streptomyces aurantiacus (SaIR), Streptomyces sp. GF3587 (RGF3587IR), Streptomyces kanamyceticus (SkIR), Streptomyces ipomoeae 91-03 (SiIR), Streptomyces sp. GF3546 (SGF3546IR), and Paenibacillus elgii B69 (PeIR). The IREDs were overexpressed in Escherichia coli (E. coli) cells and used directly after lyophilization. Several organic solvents and buffer amounts were screened for the reduction of the two substrates β-carboline harmane and 1-methyl-3,4-dihydroisoquinoline to the corresponding amines. Cyclopentyl methyl ether (CPME) proved to be the best solvent choice for the envisaged reduction. In addition, CPME is currently referred to as an environmentally benign solvent. Optimized reaction conditions were applied to 20 mM of the hardly water soluble substrates, leading to good conversions (up to 96%) and excellent enantiomeric excesses (>99%) in the best cases. The use of micro-aqueous reaction systems opens the way to further applications of IREDs with hardly water soluble substrates. (Figure presented.).

Full text of DOI:10.1002/adsc.201501154

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DOI: 10.1002/adsc.201501154
Application of Imine Reductases (IREDs) in Micro-Aqueous
Reaction Systems
Zaira Maugeria,* and Dçrte Rothera
a
Jꢀlich GmbH, Institute of Bio- and Geosciences 1, 52428 Jꢀlich, Germany
Fax : (+49)-2461-61-3870; phone: (+49)-2461-61-5199; e-mail: z.maugeri@fz-juelich.de
Received: January 6, 2016; Revised: June 9, 2016; Published online: && &&, 0000
Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/adsc.201501154.
Abstract: Here we present the applicability of dif-
ferent imine reductases (IREDs) in micro-aqueous
reaction systems. Subjects of the study were the
IREDs from Streptomyces aurantiacus (SaIR),
Streptomyces sp. GF3587 (RGF3587IR), Streptomy-
ces kanamyceticus (SkIR), Streptomyces ipomoeae
or resolving agents. A broad set of asymmetric cata-
lytic methods has been developed including organoca-
talysis and transition metal catalysis, and intense ef-
forts have been taken to increase both yields and ste-
reoselectivities.[4] Recently published syntheses show
the potential of biocatalytic methods as a valid alter-
native for the asymmetric synthesis of chiral amines.
For many years the only relevant option has been the
kinetic resolution of racemic amines using lipases.[5]
Nowadays, enzymes from several classes such as
transaminases, monoamine oxidases (MAO-N), amine
dehydrogenases, and phenylalanine ammonia lyases
are known to catalyze the production of chiral
amines.[6–9] With the exception of MAO-N, all the
above mentioned classes of enzymes generate primary
amines.
Imine reductases (IREDs) represent a novel class
of biocatalysts permitting asymmetric synthesis of sec-
ondary and tertiary amines by using nicotinamide ad-
enine dinucleotide phosphate (NADPH) as cofactor.
Their discovery dates back to the last decade but
a real breakthrough was made by Mitsukura and co-
workers in 2011 when they purified and characterized
the (R)-IRED from Streptomyces sp. GF3587.[10] They
investigated the reduction of the 2-methyl-1-pyrroline
(2-MPN), being able to gain 9.8 mM out of 10 mM of
(R)-2-methylpyrrolidine [(R)-2-MP] with excellent an
enantioselectivity of 99%. Two years later the same
research group purified and characterized the (S)-
IRED from Streptomyces sp. GF3546.[11] Therewith
they broadened the substrate scope by testing 1-
methyl-3,4-dihydroisoquinoline and 6,7-dimethoxy-1-
91-03
(SiIR),
Streptomyces
sp.
GF3546
(SGF3546IR), and Paenibacillus elgii B69 (PeIR).
The IREDs were overexpressed in Escherichia coli
(E. coli) cells and used directly after lyophilization.
Several organic solvents and buffer amounts were
screened for the reduction of the two substrates b-
carboline harmane and 1-methyl-3,4-dihydroisoqui-
noline to the corresponding amines. Cyclopentyl
methyl ether (CPME) proved to be the best solvent
choice for the envisaged reduction. In addition,
CPME is currently referred to as an environmental-
ly benign solvent. Optimized reaction conditions
were applied to 20 mM of the hardly water soluble
substrates, leading to good conversions (up to 96%)
and excellent enantiomeric excesses (>99%) in the
best cases. The use of micro-aqueous reaction sys-
tems opens the way to further applications of
IREDs with hardly water soluble substrates.
Keywords: imine reductases; imines; micro-aqueous
reaction system; organic solvent; reduction; whole
cells
Chiral amines represent very important building methyl-3,4-dihydroisoquinoline and obtaining stereo-
blocks for the synthesis of biologically active pharma- selectivities higher than 90% in all cases. Just recently,
ceutical drugs, agro and fine chemicals, when high Schrittwieser and co-workers published a thorough
chemical and stereoisomeric purities are required. It review on biocatalytic imine reductions collecting all
is estimated that 40% of all pharmaceuticals contain results obtained in the last years utilizing IREDs.[3]
chiral amine moieties.[1–3] Besides their application in Based on the protein sequences of the first character-
pharmaceuticals and agrochemicals, optically pure ized IREDs, more enzymes have been discovered via
amines, amino acids, and amino alcohols are frequent- sequence homology[12] and an electronic library of
ly employed in chemical syntheses as chiral auxiliaries about a thousand putative imine reductases, the Imine
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Reductase Engineering Database (available on the
webpage https://ired.biocatnet.de/), was generated.[13]
Crystal structures of several IREDs were ob-
tained[13–17] and possible reaction mechanisms pro-
posed.[14,17] Several new substrates such as 5-, 6-, and
7-membered cyclic imines, 3,4-dihydroisoquinolines,
3,4-dihydro-b-carbolines, and 3H-indoles were tested,
and good conversions and excellent stereoselectivities
observed.[18–21] Besides the broad acceptance of cyclic
imines (predominantly investigated because of their
good stability in aqueous solutions), also acyclic
imines were identified as substrates for IREDs-medi-
Scheme 1. Asymmetric reduction of the b-carboline (1) and
isoquinoline (3) substrates to the corresponding amines (2
ated catalysis by testing the NADP+-dependent oxida- and 4) by using whole cells expressing IREDs in a micro-
tion of several acyclic amines with different IREDs.[16]
aqueous reaction system as model reactions.
Another feature of IREDs, although at the moment
only partially investigated, is their ability to reduce
open-chain imines through a so-called reductive ami-
In Table 1 the six selected IREDs are listed. The
nation. This reaction was investigated for several ke- IREDs were overexpressed in E. coli cells and used
tones. Despite the fact that reaction rates were rather in a lyophilized form without any further purification.
slow (potentially due to the hydrolytic instabilities of d-Glucose was added to the reaction as co-substrate
the intermediate imines), it was possible to access for cofactor regeneration using the glucose dehydro-
amines with good enantiomeric excesses representing genase (GDH) present in the cells.
a proof of principle of the great potential of IRED
applications.[15,22]
Table 1. IREDs screened for the reduction of the imine sub-
strates 1 and 3. GI=GenInfo Identifier, NCBI database.
It is frequently published that enzymes, apart from
few exceptions (e.g., lipases), show highest activities
in buffer, mimicking their natural environment. How-
ever, when hydrophobic or water unstable substrates
(e.g., some acyclic imines) are involved, pure aqueous
solutions are difficult to apply (especially when
a second phase formation should be avoided) render-
ing the use of solubility enhancers essential. The ap-
plication of biocatalysts in organic solvents,[23–25]
micro-aqueous systems,[26–28] or in neat substrates,[29]
might overcome these problems and in addition facili-
tate downstream processes. At the same time, overall
enzyme activity might be reduced. Successful applica-
IRED
Organism
GI
SaIR
RGF3587IR
SkIR
SiIR
SGF3546IR
PeIR
Streptomyces aurantiacus[1]
Streptomyces sp. GF3587[2]
Streptomyces kanamyceticus[3]
Streptomyces ipomoeae 91-03[2]
Streptomyces sp. GF3546[2]
Paenibacillus elgii B69[2]
514923777
460838084
123248375
496688866
460838082
498183793
In preliminary experiments, carried out to find suit-
tions of whole cell biotransformations in micro-aque- able reaction conditions, the imine reductase from
ous reactions systems have been lately published,[30–32] Streptomyces aurantiacus (SaIR) was chosen as model
allowing substrate concentrations up to 0.5M and sim- enzyme. Aiming for the identification of the most
plified downstream processing. Whole cell catalyst suitable buffer for its later application in the micro-
formulation can alleviate the problem of reduced aqueous reaction system, four different buffer species
activity in the above-mentioned non-conventional with varying pHs were screened for the reduction of
media. The remaining cell envelope seems to protect the imine substrate (1). The SaIR catalyzed the reac-
the enzyme inside the cell to a certain extent guaran- tion in all four buffers giving best performance in
teeing enhanced stability. Moreover, by using whole HEPES and TEA buffer at pH 7.5 and 10, respective-
cells, no time- and money-consuming purification is ly (Figure 1). As HEPES buffer pH 7.5 led to 99%
needed and, most importantly, expensive cofactor ad- conversion, this buffer was chosen for following ex-
dition becomes obsolete as delivered by the cell, periments.
hence cutting overall production costs.[33]
Further investigations were performed to identify
We herein describe the potential of E. coli whole the best organic solvent among seven selected ones
cells expressing IREDs operated in micro-aqueous re- with varying properties (e.g., different polarities)
action systems. The reductions of the hydrophobic b- (Table 2). To restore the catalytic activity of the
carboline harmane (1) and 1-methyl-3,4-dihydroiso- lyophilized cells, 10% (v/v) of 1M HEPES buffer
quinoline (3) to the corresponding amines (2 and 4) pH 7.5 was added to the reaction mixture. The high
were used as test reactions (Scheme 1).
buffer concentration was chosen based on the results
lately reported by our group[30] in which whole cells
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vents on the imine reduction activity has been further
investigated using the imine 2-methyl-1-pyrroline. The
data are reported in the Supporting Information. Be-
sides this drawback, in sum enhanced substrate solu-
bility and facilitated downstream processing can
render such a system advantageous.
Once MIBK and CPME were selected, a more de-
tailed study was conducted to determine whether
both media are suitable for the micro-aqueous reac-
tion system or if some limitations might favor one
over the other one. In 1987 Yamane et al. introduced
the concept of ꢂmicro-aqueousꢃ for the first time.[28,34]
They proposed a model explaining how much water is
bound to the protein depending on the use of water-
miscible or water-immiscible solvents. In water-misci-
ble solvents the excess of water will be dissolved,
whereas in water-immiscible solvents it will remain as
free water. Since the water bound to the protein is in
equilibrium with free water molecules, for each
system a specific amount of water molecules might
cause cell clumping. In this work several amounts of
1M HEPES buffer pH 7.5 [range of 5–15% (v/v)]
were screened in micro-aqueous reaction systems with
both MIBK and CPME. In all cases a monophasic
system was formed, since the cells absorbed complete-
ly the buffer added to the reaction.
Clearly visible differences could be observed when
the two organic solvents had been applied with vary-
ing buffer amounts in the micro-aqueous reaction
system (Figure 2). At the beginning of the reaction,
no cell clumping occurred and in both organic sol-
vents the biocatalysts showed higher activity in the
presence of increased buffer amounts. After 24 h, in
vials containing MTBE higher percentages of buffer
led to cell clumping, impeding good catalyst disper-
sion in the reaction media, making quantification of
both substrate and product difficult. Although no sub-
strate could be detected after 24 h anymore, it could
not be excluded nor confirmed that cells are still
active after 24 h due to the clumping problem. The
product amount decreased from 8 to 24 h, correlating
Figure 1. Preliminary screening of SaIR with four different
buffers for the reduction of the b-carboline harmane 1. Re-
action conditions: 50 mg of lyophilized E. coli cells overex-
pressing SaIR, 5 mM of 1, 50 mM of co-substrate (d-glu-
cose), buffer concentration 100 mM, 2% (v/v) of dimethyl-
formamide (DMF), total volume 500 mL, 258C, 16 h.
Table 2. Organic solvent screening for the reduction of 1 cat-
alyzed by E. coli cells overexpressing the SaIR.[a]
Organic solvent
Observed activity (conversion)
DMC
no
2-MTHF
EtOAc
MIBK
CPME
no
no
yes (34%)
yes (25%)
[a]
Reaction conditions: 50 mg of lyophilized E. coli cells
overexpressing the SaIR, 5 mM of 1, 50 mM of co-sub-
strate (d-glucose), 10% (v/v) of 1M HEPES buffer
pH 7.5, total volume 500 mL, 258C, 8 h. MTBE=methyl
tert-butyl ether; DMC=dimethyl carbonate; 2-MTHF=
2-methyltetrahydrofuran;
EtOAc=ethyl
acetate;
MIBK=methyl isobutyl ketone; CPME=cyclopentyl
methyl ether.
expressing the benzaldehyde lyase (BAL) from Pseu- to the cell clumping occurring during this time. In
domonas fluorescens and the alcohol dehydrogenase fact, the mass balance was closed only at the begin-
from Ralstonia sp. (RADH), showed highest activities ning of the reaction, when cells were still freely dis-
in micro-aqueous system in the presence of 1M persed. It can be assumed that both substrate and
buffer concentration. Aiming to dissolve at least product either adhered directly to the cells or re-
20 mM of the substrate 1 in a micro-aqueous reaction mained inside them when clumping occurs. In CPME,
system, two of the chosen solvents, specifically methyl the cells, even with buffer concentrations up to 15%
tert-butyl ether (MTBE) and toluene, had to be ex- (v/v), remained well distributed and after 24 h the
cluded from the screening, due to the low solubility of mass balance could still be closed. Two factors might
the substrate (up to 10 mM) in these media. Among influence the different behaviors of the systems. First,
the other five organic solvents, the cells showed activ- the solubility of water has been reported to be 2%
ity only in two of them, namely the methyl isobutyl (m/m) in MIBK and 0.3% (m/m) in CPME.[35,36]
ketone (MIBK) and the cyclopentyl methyl ether Therefore, MIBK adsorbs more water molecules from
(CPME, Table 2). As mentioned above, the catalytic the air humidity in comparison to CPME. The effec-
activity is often reduced when the enzyme is applied tive higher concentration of water in the system
in unconventional media. The effect of organic sol- might cause cell clumping. Second, depending on the
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Figure 3. Screening of different buffer amounts in micro-
aqueous reaction systems using CPME as organic solvent.
Reaction conditions: 50 mg of lyophilized E. coli cells over-
expressing SaIR, 10 mM of 1, 100 mM of co-substrate (d-
glucose), 5–15% (v/v) of 1M HEPES buffer pH 7.5, total
volume 500 mL, 258C.
amount of water in the system and good catalytic ac-
tivity.
The selected IREDs (Table 1) were tested under
optimized reaction conditions [10% (v/v) of 1M
HEPES buffer pH 7.5 and CPME as organic solvent]
in the presence of 20 mM of substrate concentration.
In Table 3 the results for the reduction of both imine
substrates 1 and 3 are summarized.
Moderate to excellent enantioselectivities were ach-
ieved confirming the observations by previous au-
thors.[15–16,18] The IREDs from Streptomyces aurantia-
cus (SaIR) and Paenibacillus elgii B69 (PeIR) showed
the best stereoselectivities for both substrates yielding
in both cases enantiomeric excesses ꢀ99% of the (S)-
product. With 96% conversion 1-methyl-2,3,4,9-tetra-
Figure 2. Screening of different buffer amounts in micro-
aqueous reaction system using both MIBK (A) and CPME
(B) as organic solvents. Reaction conditions: 50 mg of
lyophilized E. coli cells overexpressing the SaIR, 10 mM of
1, 100 mM of co-substrate (d-glucose), 5–15% (v/v) of 1M
HEPES buffer pH 7.5, total volume 500 mL, 258C, 24 h.
organic solvent chosen, different interactions with the hydro-1H-pyrido[3,4-b]indole (2) could be gained
cell membrane can be expected, leading to morpho- using SaIR (Table 3). Best conversion for 1-methyl-
logical changes and aggregation.
1,2,3,4-tetrahydroisoquinoline (4) could be detected
Recently, CPME has been promoted as a new and
alternative process medium to the classical ethereal
solvents (e.g., diethyl ether, tetrahydrofuran, dioxane,
1,2-dimethoxyethane) due to its peculiar properties
such as high boiling point (1068C), low formation of
peroxides, and relative stability under acidic and basic
conditions.[35]
Results obtained applying the optimal solvent
(CPME) and buffer (1M HEPES buffer pH 7.5) to
the reduction of 1 with SaIR are shown in Figure 3.
MIBK has been omitted due the impossibility of fol-
lowing the reaction satisfactorily. The reaction pro-
ceeded poorly with 5% (v/v) of buffer and slowly
with 7.5%, but addition of 10–15% (v/v) of buffer led
to very good results. The buffer amount of 10% was
finally chosen for the optimized reaction set-up, rep-
resenting a good compromise between minimum
Table 3. Reduction of the imine substrates 1 and 3 catalyzed
by E. coli cells overexpressing the IREDs.[a]
1
3
IRED
Conv. %.
ee %
Conv. %
ee %
RGF3587IR
SkIR
0
0
–
–
48
0
(R)-63
–
SiIR
SaIR
SGF3546IR
PeIR
0
–
54
48
67
51
(R)-46
(S)-99
(S)-94
(S)-99
96
36
71
(S)-99
(S)-99
(S)-99
[a]
Reaction conditions: 50 mg of lyophilized E. coli cells
overexpressing the IRED, 20 mM of substrate, 200 mM
of co-substrate (d-glucose), 10% (v/v) of 1M HEPES
buffer pH 7.5 in CPME, total volume 500 mL, 258C, 24 h.
Conv.=conversion.
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490 mL of buffer in a 1.5-mL siliconized vial containing
50 mg of lyophilized E. coli overexpressing the various
IREDs and 50 mM of the co-substrate (d-glucose). The re-
action was performed at 258C. After 16 h the reaction was
stopped, basified with 10M sodium hydroxide (NaOH) and
centrifuged to remove the cells. The supernatant was ex-
tracted 3 times with 0.5 mL of ethyl acetate. The organic
phases were collected and the solvent evaporated under
vacuum. The sample was dissolved in 100 mL of ethyl acetate
and conversions and stereoselectivities determined by
normal phase chiral HPLC.
with SGF3546IR. Although the IRED RGF3587
from Streptomyces sp. yielded lower conversion of 3
and an enantiomeric excess of 63%, the catalyst is an
interesting candidate for future optimization as the
(R) stereoisomer is gained, complementing the access
to both possible products.
In summary, we have demonstrated the applicabili-
ty of whole cells expressing IREDs in a micro-aque-
ous reaction system. Best reaction conditions in terms
of optimal buffer species and amount, and suitable or-
ganic solvent, were applied to the reduction of the b-
carboline harmane and 1-methyl-3,4-dihydroisoquino- Experimental Details for Bioreduction in the Micro-
line. Most of the screened IREDs were able to reduce
both substrates to the corresponding amines in
CPME by the addition of 10% (v/v) 1M HEPES
buffer pH 7.5. Excellent ee (99%) values were ach-
ieved by the IREDs from Streptomyces aurantiacus
and Paenibacillus elgii B69 for both products. The re-
Aqueous Reaction System
In a typical bioreduction in micro-aqueous reaction system,
450 mL of organic solvent containing 10 mmol of the imine
substrate were transferred to a siliconized vial containing
50 mg of lyophilized E. coli overexpressing the IRED and
100 mmol of the co-substrate (d-glucose). 50 mL of 1M
sults now open the possibility to expand the substrate HEPES buffer pH 7.5 were finally added and the reaction
mixture performed at 258C. Samples were taken at specific
time intervals, basified with 10M NaOH, diluted 1/5 in the
same organic solvent and centrifuged to remove the cells.
Conversions and stereoselectivities were determined by
normal phase chiral HPLC.
scope of IREDs to more hydrophobic compounds
such as complex heterocyclic aromatic molecules.
Experimental Section
Cloning and Expression of the IREDs
Acknowledgements
The gene sequences encoding for SaIR and SkIR, optimized
as reported in the literature,[14,15] were ordered as synthetic
genes from Thermo Fisher Scientific and cloned in pET28a
(SkIR) or pET22b (SaIR). The genes of the other IREDs
(RGF3587IR, SiIR, SGF3546IR and PeIR) were purchased
from Enzymicals AG (Greifswald, D) and obtained in
vector pET28b. E. coli DH5a and BL21 (DE3) cells were
used for cloning and expression of the recombinant protein.
Recombinant cells were precultivated in lysogeny broth
(LB) for 16 h at 378C with shaking at 150 rpm, transferred
into auto-induction medium (AI-medium) and cultivated for
another 48 h at 208C with shaking at 90 rpm. Finally, cells
were harvested by centrifugation (48C at 7000 rpm for
30 min) and lyophilized.
This work was financially supported by the Helmholtz Young
Investigators Group program of the Helmholtz Association.
We thank Enzymicals AG for providing us four of the tested
IREDs. Moreover we thank Prof. Dr. Michael Mꢀller for his
precious advice and Jorge Olivares Rivas for his practical
work.
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Adv. Synth. Catal. 0000, 000, 0 – 0
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ÞÞ
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COMMUNICATIONS
Application of Imine Reductases (IREDs) in Micro-
Aqueous Reaction Systems
7
Adv. Synth. Catal. 2016, 358, 1 – 7
Zaira Maugeri,* Dçrte Rother
Adv. Synth. Catal. 0000, 000, 0 – 0
7
ꢁ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ÞÞ
These are not the final page numbers!