Continuous flow synthesis of chiral amines in organic solvents: Immobilization of E. coli cells containing both ω-transaminase and PLP

Article abstract of DOI:10.1021/ol502712v

E. coli cells containing overexpressed (R)-selective ω-transaminase and the cofactor PLP were immobilized on methacrylate beads suitable for continuous flow applications. The use of an organic solvent suppresses leaching of PLP from the cells; no addition

Full text of DOI:10.1021/ol502712v

Letter
pubs.acs.org/OrgLett
Continuous Flow Synthesis of Chiral Amines in Organic Solvents:
Immobilization of E. coli Cells Containing Both ω‑Transaminase and
PLP
Leandro H. Andrade,†,§ Wolfgang Kroutil,‡ and Timothy F. Jamison*,†
†Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, United States
‡Department of Chemistry, Organic and Bioorganic Chemistry, University of Graz, Heinrichstraße 28, 8010 Graz, Austria
S
* Supporting Information
ABSTRACT: E. coli cells containing overexpressed (R)-
selective ω-transaminase and the cofactor PLP were
immobilized on methacrylate beads suitable for continuous
flow applications. The use of an organic solvent suppresses
leaching of PLP from the cells; no additional cofactor was
required after setting up the packed-bed reactor containing the
biocatalyst (ω-TA-PLP). Non-natural ketone substrates were
transformed in flow with excellent enantioselectivity (>99%
ee). Features of this novel system include high-throughput
(30−60 min residence time), clean production (no quench, workup, or purification required), high enzyme stability (the packed-
bed reactor can be continuously operated for 1−10 days), and excellent mass recovery.
ptically pure amines enable the synthesis of several
classes of biologically active compounds, such as
in MTBE, and thus, we can expect that neither enzyme nor
PLP dissociation would be observed.
O
agrochemicals and pharmaceuticals.1 Numerous methods to
prepare chiral amine derivatives have been described,2 but the
direct preparation of free amines themselves is still not a simple
task, since most of the described methods do not provide such
requirement. An efficient synthetic approach for this purpose is
the use of enzymatic reactions. In particular, asymmetric
amination of ketones catalyzed by ω-transaminases can produce
chiral primary amines.3−5 ω-Transaminases are pyridoxal 5′-
phosphate (PLP)-dependent enzymes that can catalyze the
transfer of amino groups from amine donors to ketone groups.
The need for enzyme recyclability and stability is an
important goal in biocatalysis, and the use of a heterogeneous
system is one possible means to this end. For example, ω-
transaminases have been immobilized within sol−gel materi-
als.6,7 We and others have also described the entrapment of ω-
transaminase and cells on sol−gel materials, prepared with
poly(vinyl alcohol)s.7
Having this concept in mind, we have developed an efficient
system to produce chiral amines continuously by asymmetric
amination of ketones catalyzed by ω-transaminase-PLP
complex immobilized on methacrylated beads. E. coli cells
containing the overexpressed (R)-selective ω-transaminase
from Arthobacter,9 was selected for this study.
Methacrylate polymeric resin (beads) was used as support for
the E. coli cells containing (R)-selective ω-TA and PLP
(Scheme 1). Despite the presence of naturally occurring PLP
Scheme 1. Immobilization of E. coli Cells and PLP on
Methacrylate Polymeric Resin
Flow chemistry is an important tool useful for increasing the
product throughput of enzymatic processes, especially with
lipases, which are not cofactor-dependent enzymes.8 The
immobilized system that we describe herein now enables the
use of cofactor-dependent enzymes under flow conditions with
organic solvents; an additional cofactor (PLP) beyond that
which is already found in the naturally expressed enzyme is not
required.
We envisioned the immobilization of a ω-transaminase
together with its cofactor PLP on beads and its application
under flow conditions using a packed-bed reactor in organic
solvent (methyl tert-butyl ether, MTBE). The use of MTBE is
desirable because transaminase, PLP, and cells are not soluble
inside the cell, we added an additional amount (0.008 M) to
the immobilization solution, only to ensure the presence of
sufficient PLP in the system. We further reasoned that the
peptidoglycan layer in the E. coli cell wall (containing functional
groups such as amide, alcohol, and ether) could easily bind to
the methacrylate polymer primarily via hydrogen bonding.
After E. coli and PLP immobilization, the enzyme activity
(transaminase) for ω-TA-PLP-beads was evaluated by using the
asymmetric amination of methoxyacetone (0.05 M) with
Received: October 5, 2014
© XXXX American Chemical Society
A
dx.doi.org/10.1021/ol502712v | Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
Letter
isopropylamine (0.1 M) in MTBE at 30 °C for 24 h. (For
details, see Table S1, Supporting Information.)9
hour under continuous flow operation, it was possible to obtain,
in all cases, the same conversion and enantioselectivity.
The ketone concentration was decreased to 0.01 M, and the
conversion was excellent (95%); however, by increasing 5-fold
the ketone concentration (0.05 M), an 85% conversion was
observed (Table 1, entry 2).
The ratio between ketone concentration and isopropylamine
was also evaluated under flow conditions (1:2, 1:4 and 1:10;
Table 1), and the optimum ratio was found to be 1:4 (ketone/
isopropylamine). To lengthen enzyme the lifetime, the
continuous flow system was operated at 20 and 30 °C (Table
1, entries 4−10). At 20 °C and reduced ketone concentration
(0.01 and 0.025 M), 42−65% conversion and >99% ee were
observed (Table 1, entries 7−10). At 30 °C and 60 min, tR
comparable results (85% conversion, >99% ee; Table1, entry 5)
to those obtained at 50 °C with a tR of 30 min (85% conv,
>99% ee; Table 1, entry 1). All reactions were carried out under
continuous flow operation for at least 2 h. In all cases, the
transaminase activity was evaluated every hour and maintained
efficacy and selectivity, indicating a stable system.
The enantiopure (R)-amine 2a was prepared in excellent
conversion (96%) and enantiomeric excess (>99%). To explore
enzyme recyclability, the beads were used four more times and
the results were the same, indicating excellent enzyme stability
under these nonaqueous conditions. As expected, the presence
of an additional PLP (0.0005 M) did not increase the enzyme
performance. As further addition of PLP to the reaction in
organic solvent does not yield an active enzymatic system (ω-
TA-PLP-beads), it would appear after attachment of ω-
transaminase to the support that, under aqueous conditions,
it is and remains saturated with PLP once organic solvents are
used.
To reduce the reaction time (24 to 2 h), the temperature was
increased to 50 °C. By applying these conditions, the chiral
amine was obtained with 72% conversion and >99% ee. These
results demonstrated that this system is efficient and stable at
elevated temperature.
The design of the flow system is depicted in Table 1. By
using two syringes, substrates (ketone and amino donor) in
To evaluate the reaction scope for the immobilized (R)-
selective ω-transaminase (ω-TA-PLP-beads) under flow
conditions in an organic solvent, different α-alkoxy and α-
aryloxy acetones (1b−d) were investigated, with isopropyl-
amine as the amine donor (Table 2). Reaction conditions were
optimized to maximize amine production. The asymmetric
Table 1. Asymmetric Amination under Flow Conditions in
a
Packed-Bed Reactor
Table 2. Asymmetric Amination of α-Alkoxy- and α-Aryloxy
a
Acetones under Flow Conditions
b
entry 1a (M) isoPA (M)
cfo (h)
temp (°C) (R)-2a (%)
1
2
3
4
5
6
7
8
9
0.05
0.01
0.05
0.05
0.05
0.05
0.025
0.025
0.01
0.01
0.2
0.04
0.1
0.2
0.2
0.1
0.1
0.1
0.04
0.1
1, 2, 3, 4
1, 2
50
50
50
30
30
20
20
20
20
20
85
94
77
67
84
42
56
58
65
45
1, 2
1, 2, 3, 4
1, 2, 3, 4
1, 2
c
1, 2, 3, 4
1, 2, 3
1, 2, 3, 4
1, 2, 3, 4
10
a
Continuous flow operation (cfo): methoxyacetone (0.01, 0.025, or
0.05 M) and isopropylamine (isoPA: 0.040, 0.1, or 0.2 M) in MTBE
(saturated with water); temp 20, 30, or 50 °C. Residence time (tR): 30
min. ω-TA-PLP-beads (280 mg) were packed into a stainless steel
tubing (1/4 in. × 4.6 mm × 10 cm; reactor volume with beads: 0.5
b
mL). Back pressure regulator (BPR): 75 psi. Conversion and ee were
c
determined by GC analysis after amine derivatization to acetamide. tR:
60 min.
organic solvent (MTBE) can be injected into a packed-bed
reactor (PBR) containing the immobilized enzymatic system
(ω-TA-PLP) attached to methacrylate beads.
Two syringes were charged with solutions of methoxy ketone
(0.1 M) and isopropylamine (0.4 M) and connected to the
packed-bed reactor through a Y-adaptor. After mixing these two
solutions, the final ketone concentration is 0.05 and 0.2 M for
isopropylamine. The residence time (tR) for the flow system
was established at 30 min, and continuous flow asymmetric
amination of methoxyacetone at 50 °C was efficiently achieved.
The chiral amine was obtained with 85% conversion and >99%
ee (Table 1, entry 1). By analyzing the reaction effluent every
a
Continuous flow operation (cfo): ketones 1a−d and isopropylamine
(isoPA) in MTBE (saturated with water); temp: 50 °C. tR: 30 min. ω-
TA-PLP-beads (280 mg) were packed into a stainless steel tubing (1/4
in. × 4.6 mm × 10 cm; reactor volume with beads: 0.5 mL). BPR: 75
b
psi. tR: 60 min.
B
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Organic Letters
Letter
amination of methoxyacetone 1a (0.05 M) was also performed
under continuous operation for 20 h, and transaminase activity
remained constant (85% conv, >99% ee; Table 2, entry 1).
Phenoxyacetone 1b at a 0.01 M concentration was
transformed into the corresponding chiral amine with good
conversion (71%) and high enantiomeric excess (>99%) (Table
2, entry 2), and increased ketone concentration (0.05 M) led to
a slight reduction in conversion, but with a higher amine
production per unit time (Table 2, entry 3). Increasing the
residence time to 60 min increased the conversion (81% conv,
>99% ee; Table 2, entry 4).
Another important feature of this enzymatic PBR system was
that enzyme activity was maintained, even after 20 h of
continuous operation (Table 2, entry 4). The same reaction
conditions (tR: 30 and 60 min) and ketone concentration (0.01
and 0.04 M) were applied for the asymmetric amination of
benzyloxyacetone 1c. Once again, the chiral amine was
obtained in excellent enantiomeric excess (>99%) and with
high conversion (76−90%) (Table 2, entries 5−7). (2,6-
Dimethylphenoxy)acetone 1d was also transformed to the
corresponding chiral amine (mexiletine 2d) with excellent
results (Table 2, entries 8 and 9). It is noteworthy that the
amine 2d is a therapeutically important chiral drug used as
antiarrhythmic, antimyotonic, and analgesic oral drug.5e
We also studied the stability of the immobilized cell during
PBR continuous operation for several days (Figures 1 and 2).
continuous flow conditions with only a small decrease in
enzyme activity (<10%), but with the same enantioselectivity
(>99% ee for amine 2d) (Figure 1). As this concept was
applied for a small-scale process (reactor volume: 0.5 mL), after
5 days operation, the reaction effluent was 120 mL.
Considering the small amount of E. coli cells used to prepare
the packed bed reactor (50 mg/reactor), the chiral amine
productivity was quite high.
Despite excellent performance of the system at 50 °C, we
decreased the temperature to 30 °C and increased the residence
time to 60 min to increase both enzyme lifetime under
extended continuous operation (10 days, Figure 2) and
productivity. As before, the chiral amine 2d was produced
with >99% ee, and only a small decrease in enzyme activity was
observed (<10%) after 10 days of operation. Based on these
results, we conclude that for higher amine production, a shorter
residence time at 50 °C should be used.
To isolate the free chiral amine from the reaction product
stream, we designed a catch-and-release system, which consists
of a silica gel cartridge attached to the packed-bed reactor. As
the reaction solvent is MTBE, the amines 2a−d have higher
retention factors in silica than do the starting materials (1a−d)
under these conditions. Thus, the desired amine is retained,
thus separating any remaining ketone continuously from the
effluent (Scheme 2).
Scheme 2. Continuous Flow Production and Catch-and-
Release System for Chiral Amines
Figure 1. Continuous flow production of mexiletine 2d. tR: 30 min;
temp: 50 °C.
We performed the continuous flow production of all chiral
amines 2a−d for 5 h, and then the silica gel cartridge was
removed and, for amine release, the cartridge was rinsed with 4
mL of methanol. After evaporation of the solvent and the more
volatile isopropylamine, it was possible to isolate the chiral
amines in good yields [75% (amine 2a), 78% (amine 2b, tR 60
min), 70% (amine 2c), 79% (amine 2d)].
In conclusion, a highly stable enzymatic system attached to
methacrylate beads was developed to produce chiral amines
under flow conditions using organic solvents. Immobilization of
E. coli cells containing overexpressed ω-transaminase and the
cofactor PLP on beads gave a biocatalyst (ω-TA-PLP-Beads)
that was able to perform the asymmetric amination of non-
natural ketones in flow with excellent enantioselectivity (>99%
ee). The use of an organic solvent suppresses leaching of PLP
from the cells; no additional cofactor was required after setting
up the packed-bed reactor containing the biocatalyst. Other
features of this novel system include high throughput (tR 30−
60 min), clean production (no quench, workup, or purification
required), high enzyme stability (the packed-bed reactor can be
used for several days), and excellent mass recovery.
Figure 2. Continuous flow production of mexiletine 2d (tR: 60 min;
temperature: 30 °C).
The first set of continuous operation was selected to have the
same flow conditions described in Table 2 (tR: 30 min; temp:
50 °C; ketone 0.05 M; isopropylamine 0.2 M). As shown in
Figure 1, the immobilized cells were used for 5 days under
C
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1129. (d) Mallin, H.; Menyes, U.; Vorhaben, T.; Hohne, M.;
Bornscheuer, U. T. ChemCatChem 2013, 5, 588.
̈
ASSOCIATED CONTENT
■
S
* Supporting Information
(7) (a) Koszelewski, D.; Muller, N.; Schrittwieser, J. H.; Faber, K.;
̈
Kroutil, W. J. Mol. Catal. B: Enzym. 2010, 63, 39. (b) Car
́
denas-
ez, C.; Alvaro, G.; Tufvesson, P.;
Woodley, J. M. Biotechnol. Prog. 2012, 28, 693. (c) Casablancas, A.;
Experimental procedures, characterization data, screening table,
and continuous flow system pictures. This material is available
free of charge via the Internet at http://pubs.acs.org.
́
́ ́
Fernandez, M.; Neto, W.; Lop
́
Car
Mas, C.; Gonzal
Biotechnol. 2013, DOI: 10.2225/vol16-issue3-fulltext-4. (d) Paivio, M.;
́
denas-Fernan
́
dez, M.; Alvaro, G.; Benaiges, M. D.; Caminal, G.; de
́
ez, G.; Lopez, C.; Lopez-Santín, J. Electron. J.
́
́
AUTHOR INFORMATION
■
̈
̈
Corresponding Author
*E-mail: tfj@mit.edu.
Present Address
§Institute of Chemistry, University of Sao Paulo, Av. Prof.
Lineu Prestes 748, SP 05508-900, Sao Paulo, Brazil.
Kanerva, L. T. Process Biochem. 2013, 48, 1488.
(8) (a) Anderson, N. G. Org. Process Res. Dev. 2012, 16, 852.
(b) Itabaiana, I., Jr.; de Mariz e Miranda, L. S.; de Souza, R. O. M. A. J.
Mol. Catal. B: Enzym. 2013, 85−86, 1.
(9) (a) Mutti, F. G.; Kroutil, W. Adv. Synth. Catal. 2012, 354, 3409.
(b) Yamada, Y.; Iwasaki, A.; Kizaki, N. Kaneka Corporation. EP
0987332A1, 2000.
̃
̃
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
L.H.A thanks FAPESP (Sao Paulo Research Foundation; Grant
̃
No: 2013/04540-0) for financial support.
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