Synthesis of (1R,3R)-1-amino-3-methylcyclohexane by an enzyme cascade reaction

Article abstract of DOI:10.1016/j.tet.2015.11.005

Amine transaminases (ATAs) are powerful enzymes for the synthesis of chiral amines. Although the request for amines with more than one chiral center is increasing, their synthesis is still challenging. Here we show a casacde reaction combining an enoate r

Full text of DOI:10.1016/j.tet.2015.11.005

Tetrahedron xxx (2015) 1e5
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Synthesis of (1R,3R)-1-amino-3-methylcyclohexane by an enzyme
cascade reaction
*
Lilly Skalden, Christin Peters, Lukas Ratz, Uwe T. Bornscheuer
Institute of Biochemistry, Biotechnology and Enzyme Catalysis, Greifswald University, Felix-Hausdorff-Strasse 4, 17487 Greifswald, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Amine transaminases (ATAs) are powerful enzymes for the synthesis of chiral amines. Although the
request for amines with more than one chiral center is increasing, their synthesis is still challenging. Here
we show a casacde reaction combining an enoate reductase (ERED) and an amine transaminase (ATA-
VibFlu), which allows access to optically pure (1R,3R)-1-amino-3-methylcyclohexane. Because all known
wildtype EREDs show a (S)-selectivity for 3-methylcyclohex-2-enone and the ATA-VibFlu only showed
a modest enantioselectivity, different variants of EREDs and ATAs were investigated and suitable mutant
enzymes were identified. In whole cell biocatalyses using the ERED YqjM Cys26Asp/Ile69Thr and the
ATA-VibFlu Leu56Ile (1R,3R)-1-amino-3-methylcyclohexane was obtained at high optical purity (97% de).
Ó 2015 Elsevier Ltd. All rights reserved.
Received 9 October 2015
Received in revised form 3 November 2015
Accepted 5 November 2015
Available online xxx
Keywords:
Amine transaminase
Enoate reductase
Cascade reaction
Protein engineering
Small cyclic substrates
1. Introduction
asymmetric synthesis, which starting from a prochiral precursor
allows a theoretical yield of 100%.
The interest for chiral amines increased dramatically in the last
years. More than 80% of the top 200 current drugs contain amino
functions1 showing the importance of chiral amines as precursors
for the pharmaceutical and fine-chemical industry.2e4 Although
several chemical methods to produce chiral amines have been
developed,5 the production by biocatalytic routes became more and
more into the focus.6e9,2,10 Compared to chemical routes bio-
catalysts mostly work at mild conditions, i.e., in aqueous phase at
physiological pH, at ambient temperature and normal pressure.
Furthermore, the high selectivity of enzymes can replace expensive
chiral metal catalysts, which are required to achieve a chiral envi-
ronment for chemical routes;5 also the eco-efficiency can be in-
creased.11,12 One of the most famous example is the production of
Sitagliptin, an antidiabetic compound. In this case the rhodium-
catalyzed asymmetric enamine hydrogenation was replaced by
biotransformation using an amine transaminase (ATA), which was
optimized by protein engineering for this synthesis.13,14 Indeed,
transaminases became highly popular biocatalysts for the pro-
duction of various chiral amines in the last decade.2e4,15,16 Next to
their ability to produce optically pure amines from a racemic
mixture through kinetic resolution, they also can catalyze an
Transaminases are pyridoxal-50-phosphate (PLP) dependent
enzymes and catalyze the transfer of an amino group from an
amino donor to an amino acceptor.17e19 Depending on their sub-
strate scope transaminases can be divided into
transaminases.20
-Transaminase convert substrates with a car-
boxylate in -position to the carbonyl function, whereas for u-
a-, u- and amine
a
a
transaminases at least one C-atom is in-between. Amine-trans-
aminases substrates can lack completely the carboxylic group and
hence ATA are the preferred enzymes to synthesize chiral amines.
ATA usually show high enantioselectivity21e23 and this facili-
tates to synthesize compounds with multiple stereocenters by
combining several enzymes in cascade reactions. Compared to
chemical routes, the compatibility of different biocatalysts to each
other is easier and they can be adapted to a certain range of reaction
conditions.12 Furthermore a missing selectivity of one enzyme for
a certain chiral center can be overcome by the combination of
various selective enzymes.24
Recently, we reported the synthesis of two 1-amino-3S-meth-
ylcyclohexane diastereomers by the combination of an enoate re-
ductase and an amine transaminase.25 The flavin-mononucleotide
(FMN) containing and NAD(P)H-dependent enoate reductases
(EREDs) catalyze the selective reduction of a,b-unsaturated ketones
or aldehydes.26 Whereas the wildtype enzyme of the ATA from
Vibrio fluvialis (ATA-VibFlu) only showed a moderate selectivity for
the conversion of racemic 3-methylcyclohexanone 2, the enoate
reductase Old Yellow Enzyme exhibited excellent (S)-selectivity for
* Corresponding author. Tel.: þ49 3834 86 4367; fax: þ49 3834 86 794367;
e-mail address: uwe.bornscheuer@uni-greifswald.de (U.T. Bornscheuer).
http://dx.doi.org/10.1016/j.tet.2015.11.005
0040-4020/Ó 2015 Elsevier Ltd. All rights reserved.
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2
L. Skalden et al. / Tetrahedron xxx (2015) 1e5
3-methyl-cyclohex-2-enone. 3DM guided protein engineering of
the ATA-VibFlu led to two improved variants. 3DM is a structure-
based database, which connects structure-guided sequence align-
ments with various other bioinformatic tools.27,28 Sequences,
structure information, protein ligands and mutational information
from literature are integrated into this software. The ATA variants
Leu56Ile and Leu56Val from V. fluvialis were generated guided by
3DM. In the application of the cascade reaction the diastereomeric
purity of (1R,3S)-1-amino-3-methylcyclohexane was enhanced
from 14% de to 66% de by the Leu56Val variant. In contrary, the
cascade reaction of the OYE and the variant Leu56Ile resulted in
70% de of the (1S,3S)-diastereomer. The diastereoselectivity for the
(1R,3S)-compound could be further enhanced by the addition of
30% DMSO to give 89% de. A recently published paper by Monti
et al. described the synthesis of (1R,3S)- and (1S,3S)-1-amino-3-
methylcyclohexane in a cascade reaction, too.29 They used the
Codexis ATA Screening kit (Codexis Inc., USA) and the OYE3 to
achieve diastereomerically pure compounds. Nevertheless also
they could only generate two out of the four diastereomers.
For the synthesis of the missing two diastereomers (3a and 3b,
Scheme 1) an enoate reductase with opposite enantioselectivity is
required. The described wildtype enoate reductases all exhibit (S)-
selectivity for 2.26,30,31 Only a mutant of the enoate reductase YqjM
from Bacillus subtilis, described by Bougioukou et al., led to a switch
in the selectivity.32 The application of this mutant in a cascade re-
action was already shown by Agudo et al. for the synthesis of 3-oxo-
cyclohexane carboxylic acid methyl ester.33 In this contribution, we
aimed for using this enoate reductase to access the required two
further diasteromers 3a and 3b.
and hence all reactions were now conducted as whole cell bio-
catalyses, which has the advantage that cofactor recycling is more
easily facilitated. Whole cell biotransformation performed then
using E. coli containing the YqjM Cys26Asp/Ile69Thr variant gave
full conversion of 3-methylcyclohex-2-enone after 1.5
h
(4 mmol Lꢀ1) or after 4.5 h (10 mmol Lꢀ1).
2.3. Cascade reaction of the YqjM variant and the amine
transaminase VibFlu Leu56Ile
Thanks to our previous studies with different variants of the
ATA-VibFlu using racemic 3-methylcyclohexanone 2, the absolute
configurations
of
the
diastereomers
of
1-amino-3-
methylcyclohexane was already known25 and hence we could
easily determine the diastereomeric ratio between the (1S,3R)- and
(1R,3R)-diastereomers after the cascade reaction: for all three var-
iants of ATA-VibFlu (Leu56Ala, Leu56Ile and Leu56Val) the (1R,3R)-
diastereomer was preferred over the (1S,3R)-diastereomer. As the
Leu56Ile variant was the most stereoselective ATA in the synthesis
of the (1R,3R)-diastereomer this variant was used in subsequent
cascade reaction studies. The initial experiment was performed
using E. coli whole cells harboring YqjM Cys26Asp/Ile69Thr to
which the purified ATA-VibFlu Leu56Ile mutant was added. This
biocatalysis resulted as expected in the formation of the (1R,3R)-1-
amino-3-methylcyclohexane diastereomer and we were pleased to
find that this resulted in excellent optical purity (97% de). The use of
purified enzyme dramatically reduces the economic value of such
a reaction and furthermore requires addition of LDH/GDH to shift
the transaminase reaction towards product synthesis. Hence, we
next transformed both plasmids encoding YqjM Cys26Asp/Ile69Thr
and ATA-VibFlu Leu56Ile into E. coli BL21 (DE3) and after expression
at 30 ꢁC could obtain both enzymes in soluble form (Fig. 1).
Scheme 1. Synthesis of 1-amino-3-methylcyclohexane in a cascade reaction combin-
ing an enoate reductase (ERED) and an amine transaminase (ATA). The LDH/GDH
enzymes are required to shift the equilibrium of the ATA-catalyzed reaction.
2. Results and discussion
2.1. Choice of appropriate enoate reductases
From literature data,32 the two double mutants YqjM Cys26Asp/
Ile69Thr and YqjM Cys26Asp/Ala104Trp were chosen as prime
candidates. In addition, we also introduced these mutations into
the xenobiotic reductase A (XenA) from Pseudomonas putida ATCC
1745331 (corresponding positions: Cys25Asp, Ile66Thr and
Ala101Trp) as this enzyme belongs as YqjM to the group of ther-
mophilic like enoate reductases although they share only 38.4%
sequence identity. Unfortunately, no soluble protein could be ob-
tained. Nevertheless, the double mutants YqjM Cys26Asp/Ile69Thr
and YqjM Cys26Asp/Ala104Trp could be generated via QuikChange
mutagenesis and expressed in active form as reported.32 Bio-
catalyses confirmed the (R)-selectivity described for both mutants,
but as variant YqjM Cys26Asp/Ile69Thr was better expressed, all
further experiments were performed with this variant.
Fig. 1. SDS-PAGE of cultivation samples of the co-expression of the ERED YqjM
Cys26Asp/Ile69Thr and the ATA VibFlu Leu56Ile. Lanes 1 and 4: crude extract, lanes 2
and 5: insoluble protein, lanes 3 and 6: soluble protein. Lines 1e3 show the protein
content at the time of induction and lanes 4e6 show the protein content 5 h after
induction. The upper frame shows the ATA, the lower frame the ERED.
Cascade reactions with resting cells containing the ERED as well
as the ATA were performed in the presence or absence of LDH/GDH
enzymes and the cofactor NADH. The comparison gave that the
synthesis of 1-amino-3-methylcyclohexane was eight-fold higher
after 60 h in the presence of the LDH/GDH system (83% conversion)
compared to biocatalysis without it (11% conversion). This indicates
that without this system pyruvate accumulates and the amine
transaminase reaction is slowed down because of an unfavored
2.2. Biocatalyses with the enoate reductase variant
In our earlier work purified EREDs were used,25 but for YqjM it
was reported that different tags impair the activity of the enzyme34
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L. Skalden et al. / Tetrahedron xxx (2015) 1e5
3
equilibrium. Pyruvate is excreted by E. coli by an overflow pro-
duction35,36 and perhaps the added LDH/GDH system is able to
reduce the excreted amount of pyruvate to balance its extracellular
and intracellular concentration and thus enables a shift of the
equilibrium to the desired asymmetric synthesis of the chiral
methylcyclohexanone to the (1S,3R)-diastereomer, although the
synthesis of (1R,3S)-1-amino-3-methylcyclohexane did not suffer
from this. Further investigations on the interplay between both
binding pockets as well as mutations within each binding pocket of
the ATA are hence required to explain and alter the selectivity of
this amine transaminase from V. fluvialis.
€
amines. A similar system was described by Borner et al. for whole
cell biocatalyses with ATAs to shift the reaction to the product
site.37 It was also observed that after 60 h biocatalysis in the
presence of LDH/GDH, substrate 1 was nearly completely con-
sumed, whereas the intermediate 2 was still present. The supple-
mentation with LDH/GDH at this time point led to >99% conversion
after 89 h. Similar to the first test with purified ATA, stereo-
selectivity of both enzymes was not altered and the product
(1R,3R)-1-amino-3-methylcyclohexane was again obtained with
97% de.
3. Conclusion
In this work we have achieved the highly diastereoselective
two-step synthesis of (1R,3R)-1-amino-3-methylcyclohexane by
the combination of the enoate reductase variant YqjM Cys26Asp/
Ile69Thr and the amine transaminase variant Leu56Ile from V. flu-
vialis. Mutagenesis studies for ATA-VibFlu to produce also the
missing (1S,3R)-diastereomer unfortunately failed pointing out the
complex design of enzyme stereopreference.
2.4. Investigation of further amine transaminases to produce
(1S,3R)-1-amino-3-methylcyclohexane
4. Experimental section
4.1. Materials
To identify amine transaminases, which prefer to produce the
(1S,3R)-diastereomer instead of the (1R,3R)-diastereomer, both
enantiomers of 3-methylcyclohexanone were docked with
YASARA38 into the crystal structure of the amine transaminase from
V. fluvialis (pdb-code: 4E3Q). Additionally to Leu56, three further
residues (small binding pocket: Phe19, Val153, large binding
pocket: Ala228) were identified, which could influence the binding
of (R)-3-methylcyclohexanone. These three residues were also
targeted in other protein engineering approaches of this amine
transaminase to alter its substrate scope.39,22 With the help of 3DM
suitable mutations on this positions were chosen, which led to 14
further variants in addition to the previous reported ones:
Phe19Tyr/Cys/Val, Leu56/Met/Ser, Ala228Ie/Gly/Val/Cys/Ser/Thr
and Val153Ala/Ile/Ser. All mutants could be expressed as soluble
proteins. The screening against racemic 3-methylcyclohexanone
showed unfortunately that none of these mutants produce
(1S,3R)-1-amino-3-methylcyclohexane in excess compared to the
(1R,3R)-diastereomer (Table 1).
All chemicals were purchased from Fluka (Buchs, Switzerland),
Sigma (Steinheim, Germany), Merck (Darmstadt, Germany), VWR
(Hannover, Germany), or Carl Roth (Karlsruhe, Germany) and were
used without further purification unless otherwise specified.
Polymerases were obtained from New England Biolabs GmbH (NEB,
Beverly, MA, USA) and primers were ordered from Invitrogen (Life
Technologies GmbH, Darmstadt, Germany).
4.2. Bacterial strains and plasmids
E. coli TOP10 [F’lacIq, Tn10(TetR) mcrA D(mrr-hsdRMS-mcrBC)
F80 LacZDM15 DlacX74 recA1 araD139 D(ara leu)7697 galU galK
rpsL (StrR) endA1 nupG] was obtained from Invitrogen (Carlsbad,
CA, USA). E. coli BL21 (DE3) [fhuA2 [lon] ompT gal (l DE3) [dcm]
DhsdS] was purchased from New England Biolabs (Beverly, MA,
USA). The plasmid pET24b bearing the gene encoding the ATA from
V. fluvialis (accession no. F2XBU9) was kindly provided by Prof.
Byung Gee Kim (Seoul National University, South-Korea).
Table 1
Composition of the diastereomers of 1-amino-3-methylcyclohexane produced by
different variants of the ATA from V. fluvialis using rac-3-methylcyclohexanone as
substrate
4.3. Cloning
The enoate reductase variants were generated via QuikChange
PCR with specific primers for each variant:
XenA:
Variant
WTa
Conversion [%]
(1R,3R)
(1S,3S)
(1S,3R)
(1R,3S)
99
16
2
12
15
3
8
5
4
99
99
40
34
36
55
45
41
43
38
42
45.5
39
26
50
38
29
44
42
44
30
39
47
16
4
4
2
2
3
7
5
6
30
12
24
14
8
10
8
26
9
Phe19Cysb
Phe19Valb
Val153Alab
Val153Ileb
Val153Serb
Leu56Metb
Leu56Serb
Ala228Thrb
Leu56Ilea
Leu56Vala
Cys25Asp fw:50eCAT TCC GCC CGA TTG CCA GTA CAT Ge30
Cys25Asp rv:50eCAT GTA CTG GCA ATC GGG CGG AAT Ge30
Ile66Thr fw:50eGAA GGG CGC ACC ACC CCT GGe30
Ile66Thr rv:50eCCA GGG GTG GTG CGC CCT TCe30
Ala101Trp fw:50eGCA TCC AGA TTT GGC ACG CCGe30
Ala101Trp rv:50eCGG CGT GCC AAA TCT GGA TGCe30
10
0.5
5
7
40
a
Results from previous work with purified amine transaminase.25
YqjM:
b
The screening against rac-3-methylcyclohexanone (10 mmol Lꢀ1) was per-
formed in deep-well plates (0.6 mL per well) with crude extract of the ATA variants
Cys26Asp fw:50eCATGTCGCCAATGGATATGTATTCTTCTCe30
Cys26Asp rv:50eGAGAAGAATACATATCCATTGGCGACATGe30
Ile69Thr fw:50eCCC TCA AGG ACG AAC CAC TGA CCA AGA Ce30
Ile69Thr rv:50eGTC TTG GTC AGT GGT TCG TCC TTG AGG Ge30
Ala104Trp fw:50eCGG CAT TCA GCT TTG GCA TGC CGG ACGe30
Ala104Trp rv:50eCGT CCG GCA TGC CAA AGC TGA ATG CCGe30
for 78 h at 30 ꢁC and 750 rpm.
Hence, the creation of an ATA variant showing the desired
(1S,3R)-enantiopreference turned out to be not possible so far. The
reason for this could be the orientation of the amino- and methyl-
group at the cyclohexane ring. In the cis-diastereomer both sub-
stituents are positioned on one site of the cyclohexane ring,
whereas in the trans-configuration, both are placed on opposite
sites. These orientations seems to prevent the conversion of (R)-3-
The plasmid (2
(1.5
L), Pfuþ polymerase (0.5
tilled water (35.5 L). The PCR program included the following
temperature steps: hold at 95 ꢁC for 5 min, afterwards 25 cycles of
m
L) was mixed with Pfuþ buffer (5
L), primers (2 L of each) and dis-
mL), dNTPs
m
m
m
m
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4
L. Skalden et al. / Tetrahedron xxx (2015) 1e5
the following: hold at 95 ꢁC for 45 s, 53 ꢁC for 45 s, 72 ꢁC hold for
7.5 min. Finally hold at 72 ꢁC for 10 min.
All ATA-VibFlu variants were generated via MegaWhop PCR
with specific forward or reverse primer for each variant. The other
corresponding primer was either the T7 forward or T7 reverse
primer.
(0.9 U) and LDH (0.15 U) was added. Biocatalyses with ATA and rac-
2 had a total volume of 0.6 mL and were performed in deep well
plates. The biocatalyses contained: the crude extract of the amine
transaminases, GDH (0.9 U), LDH (0.15 U),
L-alanine
(200 mmol Lꢀ1), glucose (1 mmol Lꢀ1), substrate (10 mmol Lꢀ1) and
NADH (1.1 mmol Lꢀ1) dissolved in sodium phosphate buffer
(50 mmol Lꢀ1, pH 7.5) including PLP (0.1 mmol Lꢀ1). The substrate
solutions were prepared as stock solution (1 mol Lꢀ1) in DMSO. The
reactions were incubated for 78 h at 30 ꢁC at 750 rpm. Biocatalyses
Phe19Cys:50eG CTC TAT GGT TGC ACC GAC ATG Ce30
Phe19Val:50eG CTC TAT GGT GTG ACC GAC ATG Ce30
Phe19Tyr:50eG CTC TAT GGT TAT ACC GAC ATG Ce30
Leu56Ser:50eGCC AAC TCG GGC AGC TGG AAC ATG Ge30
Leu56Met:50eCGC AAC TCG GGC ATG TGG AAC ATG Ge30
Val153Ile:50eGCC TAT CAC GGC ATC ACC GCC GTT TCe30
Val153Ser:50eGCC TAT CAC GGC AGC ACC GCC GTT TCe30
Val153Ala:50eGCC TAT CAC GGC CGT ACC GCC GTT TCe30
Ala228Cys:50eCGG TGA TGG GCT GCG GCG GCG TGe30
Ala228Ile:50eGTG ATG GGC ATC GGC GGC GTGe30
Ala228Gys:50eCG GTG ATG GGC GGC GGC GGC GTGe30
Ala228Ser:50eCG GTG ATG GGC AGC GGC GGC GTGe30
Ala228Thr:50eCG GTG ATG GGC ACC GGC GGC GTGe30
Ala228Val:50eCG GTG ATG GGC GTG GGC GGC GTGe30
samples (300
extracted at first with ethylacetate (300
(150 L). The organic phases were combined and dried with an-
mL) were mixed with NaOH (150
m
L of 1 mol Lꢀ1) and
mL) and then with hexane
m
hydrous sodium sulfate before GC analysis was performed.
4.7. Docking studies
The docking studies were performed with YASARA38 using the
structure of the ATA from V. fluvialis (pdb-code 4E3Q). (R)- and (S)-
3-methylcyclohexanone were alternatively docked into the active
3
ꢀ
site. The chosen simulation cell was defined to be 18ꢂ17ꢂ18 A . All
residues of the active site and the active site loop were included.
The plasmid (4
(1.5
L), Pfuþ polymerase (0.5
(2 L) and distilled water (35.5
m
l) was mixed with Pfuþ buffer (5
L), specific primer (1 L), T7 primer
L). The PCR program included the
mL), dNTPs
m
m
m
4.8. Analytics
m
m
following temperature steps: hold at 95 ꢁC for 5 min, afterwards 30
cycles of the following: hold at 95 ꢁC for 45 s, 54.8 ꢁC for 45 s, 72 ꢁC
hold for 6 min. Finally hold at 72 ꢁC for 10 min. The PCR products
The determination of the conversion, the ratio of the
diastereomers or enantiomers were identified via GC as described
previously.25 The derivatisation of the samples was performed with
(8
(1
m
m
L) were mixed with 10x Pfuþ buffer (5
L), Pfuþ polymerase (1
mL), plasmid (2
mL), dNTPs
5 mL trifluoroacetic acid anhydride.
m
L) and distilled water (33 L). The fol-
m
lowing program was used: hold 68 ꢁC for 5 min, hold 95 ꢁC for
1 min, 10 cycles of the following three steps: 95 ꢁC for 30 s, 53 ꢁC for
30 s and 68 ꢁC for 7 min. Afterwards 10 cycles of the following three
steps were made: 95 ꢁC hold for 30 s, 55 ꢁC hold for 30 s and 68 ꢁC
hold for 11 min.
Acknowledgements
We thank the European Union (KBBE-2011-5, grant no. 289350)
and the DFG (grant no. Bo1862/6-1) for financial support. We are
grateful to Prof. Byung-Gee Kim (Seoul National University, Korea)
for the gene encoding the ATA from V. fluvialis.
4.4. Cultivation, expression and purification
The cultivation and expression of the EREDs and the ATAs were
performed as described elsewhere.32,31,25 The purification of the
ATA VibFlu Leu56Ile was performed as described previously.25 After
the co-transformation of the plasmids bearing the genes encoding
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