ω-Transaminases for the amination of functionalised cyclic ketones

Article abstract of DOI:10.1039/c5ob01204j

The potential of a number of enantiocomplementary ω-transaminases (ω-TAms) in the amination of cyclic ketones has been investigated. After a preliminary screening of several compounds with increasing complexity, different approaches to shift the equilibrium of the reaction to the amine products were studied, and reaction conditions (temperature and pH) optimised. Interestingly, 2-propylamine as an amine donor was tolerated by all five selected ω-TAms, and therefore used in further experiments. Due to the higher conversions observed and interest in chiral amines studies then focused on the amination of α-tetralone and 2-methylcyclohexanone. Both ketones were aminated to give the corresponding amine with at least one of the employed enzymes. Moreover, the amination of 2-methylcyclohexanone was investigated in more detail due to the different stereoselectivities observed with TAms used. The highest yields and stereoselectivities were obtained using the ω-TAm from Chromobacterium violaceum (CV-TAm), producing 2-methylcyclohexylamine with complete stereoselectivity at the (1S)-amine position and up to 24 : 1 selectivity for the cis : trans [(1S,2R) : (1S,2S)] isomer.

Full text of DOI:10.1039/c5ob01204j

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Cite this: DOI:
ω-Transaminases for the amination of functionalised
cyclic ketones
10.1039/x0xx00000x
N. Richter,a,b R. C. Simon,c H. Lechner,c W. Kroutil,b,c J. M. Wardd and H. C. Hailesa,*
Received 00th January 2012,
Accepted 00th January 2012
The potential of a number of enantiocomplementary
ωꢀtransaminases (ωꢀTAms) in the
amination of cyclic ketones has been investigated. After a preliminary screening of several
compounds with increasing complexity, different approaches to shift the equilibrium of the
reaction to the amine products were studied, and reaction conditions (temperature and pH)
DOI: 10.1039/x0xx00000x
www.rsc.org/
optimised. Interestingly, 2ꢀpropylamine as an amine donor was tolerated by all five selected
TAms, and therefore used in further experiments. Due to the higher conversions observed and
interest in chiral amines studies then focused on the amination of ꢀtetralone and 2ꢀ
ωꢀ
α
methylcyclohexanone. Both ketones were aminated to give the corresponding amine with at
least one of the employed enzymes. Moreover, the amination of 2ꢀmethylcyclohexanone was
investigated in more detail due to the different stereoselectivities observed with TAms used.
The highest yields and stereoselectivities were obtained using the
Chromobacterium violaceum (CVꢀTAm) producing 2ꢀmethylcyclohexylamine with complete
stereoselectivity at the (1 )ꢀamine position and up to 24:1 selectivity for the cis trans
[(1 ,2 ):(1 :2 )] isomer.
ωꢀTAm from
,
S
:
S
R
S S
Introduction
An alternative method to generate enantiopure amines that is
currently attracting significant interest is the use of
transaminases Despite the improved
ꢀTAms).16ꢀ20
ω
ꢀ
Single isomer chiral amines are one of the most common
functional groups found in natural products and pharmaceutical
compounds including, sitagliptin the antihyperglycemic drug
for the treatment of diabetes,1 the antihypertensive dilevalol,2
(
ω
sustainability with this biocatalytic approach, one problem has
been the issue of shifting the reaction equilibrium towards the
amine product. However, in recent years efforts have been
focussed on the development of methods to overcome this
unfavourable equilibrium, via the chemical or enzymatic
removal of the coꢀproduct or use of an excess of amine
donor.1,21–30 The incorporation of enzymatic cascades has been
particularly successful, including reuse of the coꢀproduct in a
multiꢀenzymatic cascade with a carboligation step.31 Several of
and (S)ꢀrivastigmine used for the treatment of Alzheimer’s
disease.3,4 Enantiopure amines are therefore highly desirable
building blocks for the development of new pharmaceuticals.
The frequent occurrence of such chiral amines has also
highlighted interest in their preparation as synthons for use in
the pharmaceutical sector. For instance, the aminotetraline
motif is present in a variety of pharmaceuticals, such as the
these studies used
ωꢀTAms for the preparation of
pharmaceutical intermediates or bioactive compounds,24,25,27–36
and have also lead to an industrial process.1
antidepressants sertraline and norsertraline with an
aminoteraline core,5–7 and rotigotine a treatment for Parkinson’s
disease containing a
ꢀaminotetraline core unit.8,9
αꢀ
β
Here we describe the use of several
ωꢀTAms in the
Traditionally single isomer chiral amines are generated
from racemic mixtures using crystallisation methods, or they
can be synthesised using chiral auxiliaries.7,10–12 In addition
more recently a variety of organocatalytic, metalꢀdependent as
well as chemoꢀenzymatic dynamic kinetic resolution methods
have been developed to produce enantiopure amines.13–15 The
requirement for metals in some of these systems such as lipaseꢀ
catalysed dynamic kinetic resolutions is however a major
backdraw when considering the sustainability of the process.14
asymmetric amination of several cyclic substrates. Moreover,
different methods to shift the equilibrium towards the desired
amine product were compared and reaction parameters
optimised with the model compound cyclohexanone
1. The
amination of two selected substrates was then investigated in
further detail, to establish the different stereoselectivities of the
ω
ꢀTAms used.
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Figure 1. Initial screening results giving conversions for a range of cyclic ketone substrates employing the selected ω-TAms. (R)- or (S)-MBA 11 were used as amine
donors and the product acetophenone was detected by HPLC analysis (see Scheme). All reactions were performed in triplicate and standard deviations were less than
10%.
Results and discussion
method highlighted substrates for further investigation.
Control reactions were performed in the absence of amine
acceptor and low levels of acetophenone were detected
which were subtracted from assay results with amine
acceptor present. The results indicated that several ketones
Our aim was to test a panel of promising and recently
reported
ketones, including sterically challenging substrates, to
determine substrate type acceptance by the selected
ωꢀTAms against a range of functionalised cyclic
ω
ꢀ
showed good levels of conversion with the
selected, particularly CVꢀTAm, PpꢀTAm and ArRMut11.
Cyclohexanone was the best cyclic substrate for most ωꢀ
TAms with conversions of up to 40%. Interestingly, while
the substitution at the ꢀposition on the sixꢀmembered ring
in 2ꢀmethylcyclohexanone was particularly well tolerated
ωꢀTAms
TAms. These were (S)ꢀωꢀTAms from Chromobacterium
violaceum DSM30191 (CVꢀTAm),37 Vibrio fluvialis (Vfꢀ
TAm),38 Klebsiella pneumonia KPN_0799 (KpꢀTAm),39 and
1
Pseudomonas putida PP_3718 (PpꢀTAm)36. Also the (
R)ꢀωꢀ
α
TAms from Mycobacterium vanbaaleni (MvꢀTAm),40 and a
variant from Arthrobacter sp. (ArRMut11).1 CVꢀTAm and
VfꢀTAm were selected as they have been used with a range
of acyclic substrates and cyclic compounds such as
cyclohexanone,19,20,31,37,38,41,42 while PpꢀTAm has recently
been reported to accept dopamine36 and KpꢀTAm39 was
selected as a promising TAm from screening our UCL TAm
library. MvꢀTAm has been used almost exclusively with
ketones/amines as acyclic moietes,40,43,44 and ArRMut11
3
with only slightly lower conversions, the presence of a
conjugated C=C double bond led to significantly less
activity with
with the fiveꢀmembered rings: while cyclopentanone
readily accepted by several ꢀTAms, the corresponding
enone had negligible reactivity with all the ꢀTAms used.
This presumably reflects the modified steric demands and
reactivity in the ꢀenones and less electrophilic carbonyl
moiety. The diketones and , and bicyclic compound 10
had negligible levels of acceptance. The bicyclic systems αꢀ
tetralone (KpꢀTAm, ArRMut11) and 8aꢀmethylꢀ3,4,8,8aꢀ
tetrahydroꢀ1,6(1 ,7 )ꢀnaphthalenedione were accepted
4
. A similar reactivity pattern was observed
5
was
ω
7
ω
α,β
with 1,3ꢀketoamides to generate the (R)ꢀfunctionality in
sitagliptin as well as for example bicyclic tetralone and
chromone substrates and a carbazolamine.1,28,34,45
2
6
8
Ten ketone substrates
generate the corresponding amines 1a
cyclohexanone and cyclopentanone to determine the
influence of ring size, as well as diketones ( ),
unsaturated ketones ( ), ꢀtetralone and ketones with
ꢀmethyl groups ( , and camphor 10). Initial assays used the
ꢀTAms (crude cell lysates) and either ( )ꢀ or ( )ꢀαꢀ
1
–
10 were selected, which would
H
H
9
–
10a including
,
with conversions at levels of 5ꢀ10% (ArRMut11), with even
lower conversions for several of the other TAms. Tetralone
1
5
2
,
6
α,βꢀ
8
has previously been used as a substrate with ArRMut11
4
,
7
,
9
α
8
together with coꢀproduct removal to shift the equilibrium
toward the desired amine, so these results using two TAms
and no coꢀproduct removal were promising.28,45 The reaction
α
3
ω
R
S
methylbenzylamine (MBA) 11 as the amine donor,
of ArRMut11 with
9 has also recently been reported, and
depending on the selectivity of the transaminase, with
substrates 1–10: the product acetophenone was detected by
HPLC at 254 nm (Figure 1).37 This preliminary assay
although products were observed by LCꢀMS analysis no
products could be isolated.30 With the exception of the Vfꢀ
TAm which showed in general poor activity with cyclic
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substrates, the five other ωꢀTAms were studied in more
detail, initially with ketone
(a) Enzyme-coupled shifted equilibrium
1
.
O
O
NH2
O
To facilitate a better comparability of the different TAm
enzymes, the specific activity of crude cell extracts in the
TAm, PLP
OH
+
OH
+
phosphate buffer pH 8
NH2
O
amination of
1 was investigated. In addition, freeze dried
1a
1
A
LDH/GDH
NAD+
O
samples of clarified lysate were prepared as convenient
preparations for storage and usage. The highest activity was
obtained using crude cell extracts of CVꢀTAm and
ArRMut11, with 0.58 U/mg and 0.39 U/mg of total protein,
respectively (see experimental). After lyophilisation the
activity was decreased in most cases, though not
significantly, with residual activities in the range of 55ꢀ
100%. For this reason and ease of usage lyophilised cell
extracts were used in all of the following studies.
To identify optimal reaction conditions, as well as
investigating the reaction temperature and pH, a study
comparing the use of MBA 11 to both enzymeꢀcoupled and
excess amine donor methods to shift the equilibrium towards
B
AlaDH/FDH
NAD+
OH
OH
(b) Amine donor shifted equilibrium
O
NH2
O
NH2
TAm, PLP
+
+
phosphate buffer pH 8
1
12
1a
Scheme 1. Methods used to shift the TAm reaction equilibrium toward the
desired amine product, using as the ketone acceptor. (a) Enzyme-coupled
shifted equilibrium; (b) amine donor shifted equilibrium
1
the product amine was conducted using cyclohexanone
(Scheme 1). Two enzymeꢀcoupled systems were used
(Scheme 1a), where ꢀ or ꢀalanine was used as the amine
donor and the coꢀproduct pyruvate was removed by either a
ꢀlactate dehydrogenase (LDH) (Scheme 1a ) or recycled
by an alanine dehydrogenase (AlaDH) for ( )ꢀselective
TAms (Scheme 1a
).18,28,46 The nicotinamide cofactor
1
L
D
Table 1. A comparison of the different methods used to shift the
equilibrium toward the product amine 1a and conversions observed after
24 h using 1 as substrate at pH 8, 30 °C.
L
A
S
ωꢀ
TAm
MBA 11
amine donor
(Fig. 1)
A
B
IPA 12
amine
donor
(Sch. 1b)
conv. (%)
B
LDH/GDH
(Sch. 1a)
conv. (%)
AlaDH/FDH
(Sch. 1a)
conv. (%)
(NADH) required was recycled by employing standard
techniques using formate dehydrogenase (FDH) or glucose
dehydrogenase (GDH).18,28,46 An alternative enzymeꢀ
independent method was investigated using 2ꢀpropylamine
(isopropylamine, IPA) 12 as the amine donor which
generates acetone as a coꢀproduct (Scheme 1b).1,37,42
Most enzymatic reactions proceeded with good
conversions of up to 95% (Scheme 1, Table 1), especially
compared to the conversions observed in the initial
screening experiments using MBA 11 (7ꢀ39%), confirming
conv. (%)
CVꢀTAm
KpꢀTAm
PpꢀTAm
MvꢀTAm
ArRMut11
36
7
39
24
39
92
0
88
0
88
50a
0
94
22a
95
91
31a
0
39a
93
a48 h reaction time. Reactions were performed in triplicate with standard
deviations of less than 10%. Product 1a was detected by GC analysis for
methods A, B and IPA 12, and acetophenone was detected by HPLC as
previously using MBA 11 as the amine donor (MBA 11 was not used in a
large excess as high numbers of equivalents have been found to have a
detrimental effect on the transaminase reaction).42
the benefits of using shifting systems in
ωꢀTAm reactions.
Interestingly, four of the ꢀTAm reactions showed similar
ω
or higher conversions using the IPA (12)ꢀamine donor
reaction systems compared to the use of enzyme coupledꢀ
systems. Only the MvꢀTAm showed a slightly lower
conversion (39%) compared to the AlaDH/FDHꢀsystem
(50%). In general, this broad acceptance of IPA 12 as amine
donor was unexpected since IPA does not appear to be an
Another interesting observation was that for the Kpꢀ
TAm and ArRMut11 no conversions were observed
employing the enzymeꢀcoupled systems, indicating that
alanine was not accepted as an amine donor. While in
engineering the Arthrobacter sp.
ωꢀTAm to accept high IPA
amine donor for many
ω
ꢀTAms.47 To date, only a few have
(
12) and coꢀsolvent concentrations the ability to use alanine
demonstrated high tolerance towards IPA 12 such as the
engineered variant ArRMut11, and also CVꢀTAm which
was used in the synthesis of (2S,3S)ꢀ2ꢀaminopentaneꢀ1,3ꢀ
has been lost, for the native KpꢀTAm enzyme not to accept
alanine as an amine donor is unexpected. However, since Kp
is a member of the Class III transaminases and there is
diol, facilitating the use of this low cost amine donor to shift
the equilibrium towards the product.1,42 The observation that
variable use of
αꢀamino acids amongst the Class III
transaminases, this finding is not too unusual.16
all five of the ωꢀTAms investigated can be used with IPA 12
All further experiments were conducted using the IPA 12
shifting system because of the advantages outlined above.
is notable, especially with respect to the applicability of
these enzymes, due its low cost and more facile optimisation
of reaction condition since only one enzyme is required.
Moreover, the highly volatile coꢀproduct acetone can be
readily removed, as recently demonstrated in the synthesis
of sitagliptin.1
The amination of
1 was then performed at two different pHs
and temperatures typically used in transamination reactions
(Table 2). Optimal conditions which were used for further
experiments were pH 8 and 30 °C for CVꢀTAm and Ppꢀ
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TAm, pH 8 and 45 °C for KpꢀTAm and MvꢀTAm, while the
best conditions for ArRMut11 were at pH 10 and 30 °C.
A more detailed study of the amination of
3
therefore performed, in order to evaluate the full product
stereochemistry with the three most productive ωꢀTAms
(Table 4). Samples were taken after 2 h, 4 h, 24 h and 48 h
was
Table 2. The pH and temperature optimisation of the TAm reaction using
1, and IPA 12 as the amine donor equilibrium shifting method, to give 1a
after 24 h.
and conversions, cis:trans ratios and enantioselectivities
were determined by GC analysis (Table 4). The data
confirmed the stereoselectivities observed before (Table 3),
but additionally by monitoring the amination over a period
of time it became apparent that some selectivities decreased
with increasing conversions/time. While CVꢀTAm was very
selective for generating the cisꢀisomer the other enzymes
seem to show lower selectivities. For example the
ArRMut11 only showed a strong preference towards the
formation of the transꢀisomer at very low conversions (6%
conversion and a cis:trans ratio of 20:80) while at a
conversion of 20% the cis:trans ratio had increased to 40:60.
The absolute configurations of the 3a stereoisomers were
TAm
pH 8, 30 °C pH 8, 45 °C
pH 10,
30 °C
conv. (%) conv. (%)
pH 10,
45 °C
conv. (%)
conv. (%)
CVꢀTAm
KpꢀTAm
PpꢀTAm
MvꢀTAm
ArRMut11
94
5
91
18
0
8
0
7
94
n.d.
14
n.d.
27
94
17
93
10
41
n.d.
90
n.d. – not determined. Conversions were determined in triplicate with
errors below 10%. Product 1a detected by GC analysis.
The amination of 1, 3 and 8, to give 1a, 3a and 8a was
determined using known (
(ArRMut11 and CVꢀTAm respectively) with (2
racemic . The amine products 3a from the four reactions
R
)ꢀ and (
S
)ꢀselective TAms
then studied in more detail as initial experiments indicated
reasonable levels of conversion (Table 3): compound 8a is
an important chiral product and the potential to establish two
stereogenic centres in 3a in a single step is particularly
R
)ꢀ3 and
3
were then correlated to the isomeric amine products by
chiral GCꢀanalysis to establish the stereochemical outcome
of the reactions.
In all cases the amine was formed in exceptionally high
stereoselectivities (>99% ee) while the variable cis:trans
interesting. Ketones
enzymes, however ketone
ArRMut11 to give exclusively the αꢀaminotetraline (
as determined by chiral GC analysis. This was consistent
with previous reports using this
ꢀTAm with substrate 8 28,45
1
and
3
were well accepted by the TAm
was only accepted by
)ꢀ8a
8
R
,
ratios resulted from the ability of the ωꢀTAms to distinguish
between the stereocentre at the αꢀmethyl position. Racemic
ω
.
Only traces of 8a were observed with KpꢀTAm and Mvꢀ
TAm (Table 3).
samples of
3 were used in all experiments, other than when
establishing absolute configurations. For reactions where
high conversions and/or high diastereomeric ratios were
observed, some racemisation at the αꢀcarbon of 3 will have
occurred at pH 8 used with CVꢀTAm and PpꢀTAm and pH
10 with ArRMut11. Such dynamic asymmetric
Amination of the αꢀsubstituted ketone
dynamic kinetic resolution due to the chiral αꢀmethyl group.
Ketone was accepted by all the selected ωꢀTAms, but
conversion yields varied from 8% to 91%. Analysis of the
products by GC indicated that CVꢀTAm preferentially gave
3 involves a
3
transaminations involving
received little attention in the literature to date. Recently
they have been described with an ꢀsubstituted ketone
possessing a large
ꢀphenethylether group.33 However here
we have the much less sterically differentiating methyl
group at the ꢀposition where notably for CVꢀTAm
αꢀsubstituted ketones have
cisꢀ3a, while the transꢀ3a isomer was formed preferentially
by PpꢀTAm and ArRMut11ꢀTAm.
α
Table 3. ωꢀTAm catalysed amination of cyclic ketones 1, 3, and 8 using
IPA 12 as the amine donor to give 1a, 3a, and 8a.
α
NH2
NH2
NH2
α
excellent ees and high diastereoselectivities were observed.
The change in cis/trans product ratio over time probably
reflects the consumption of the prefered isomer of
shorter reaction times. Notably, for CVꢀTAm a high
preference was observed for the (2 )ꢀmethyl group.
3 at
1a
conv.
(%)
3a
amine
config.
8a
TAm
conv.
(%)
cis:trans conv.
amine
R
ratio
(%)
config.
In light of the high stereoselectivities for CVꢀTAm,
docking calculations were performed in order to gain
insights into the stereopreference observed. Calculated
binding affinities of all four PLPꢀimine quinonoid
intermediates of the reaction were determined and results are
summarised in Table 5.
CVꢀTAm
KpꢀTAm
PpꢀTAm
MvꢀTAm
ArRMut11
94
18
94
41
94
58
8
90
10
91
(1S)
(1S)
(1S)
(1R)
(1R)
88:12
63:37
43:57
53:47
36:64
0
1
0
2
19
ꢀ
n.d.
ꢀ
n.d.
(R)
n.d. – not determined. The reactions were performed under the optimised
conditions in triplicate with a standard deviation of under 10%. Products
detected by GC and chiral GC analysis. Conversions were determined
after 24 h (1a), 48 h (3a) and 144 h (8a).
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Table 4. Transamination of 3 to 3aꢀisomers using IPA 12 as the amine donor, and conversions, cis:trans ratios and ees over a reaction time of 48 h.
NH2
NH2
NH2
NH2
NH2
NH2
TAm
time (h)
conv. (%)
(S)
(R)
(R)
(R)
(S)
(R)
(S)
(S)
:
cis:trans ratio
96:4
cis ee (%)
trans ee (%)
CVꢀTAm
PpꢀTAm
2
4
24
48
2
23
33
47
58
31
49
90
90
6
93:7
all
all
88:12
88:12
61:36
54:46
42:58
43:57
20:80
40:60
38:62
36:64
>99 (1S,2R)
>99 (1S,2S)
4
all
all
24
48
2
4
24
48
>99 (1S,2R)
>99 (1S,2S)
ArRMut11
20
76
91
all
all
>99 (1R,2S)
>99 (1R,2R)
Conversions and cis:trans ratios were detected by achiral GC, while eeꢀvalues were determined using chiral GC; results were determined in triplicate with a
standard deviation of less than 10%.
Table 5. Calculated relative binding affinities of the quinonoid
Interestingly, the best calculated relative affinity (~ ꢀ8
intermediates (with the methyl group both axial and equatorial).
kcal/mol) was observed with the (R)ꢀconfigured ligand,
which is preferentially transformed by the CVꢀTAm (Table
5, entry 1 and 2). In contrast, the minor product of the
entry
configuration
(methyl)a
final conformation of relative binding
methyl group
affinity
(kcal/mol)
transamination reaction (1S,2S)ꢀ3a had a lower calculated
affinity (ꢀ6.4 kcal/mol, entry 3 and 4). Thus the trend
generally matched the observed experimental data. For more
1
2
3
4
R
R
S
equatorial
axial
equatorial
axial
ꢀ8.1
ꢀ7.9
ꢀ6.4
ꢀ6.4
insights, both equatorial quinonoid intermediates (
R)ꢀ (entry
S
1) and ( )ꢀ (entry 3) were evaluated in more detail after
S
a The PLP structure in the docking experiments was overlayed with the
PLP as reported in the crystal structure.
modelling into the active site using the holo structure of CVꢀ
TAm (4AH3).48 As shown in Figure 2, the position of the
sixꢀmembered ring varied depending on the methyl group
stereochemistry. Moreover, the docked structures show that
the (2
the binding pocket (indicated in grey). However, apart from
slightly better positioning of the (2 )ꢀ vs. the (2 )ꢀquinonoid
R)ꢀquinonoid (green), better fits the space available in
R
S
no additional steric factors could be determined to explain
the stereochemistries observed. In general, the residue
Lys288 is known to play a crucial role in the catalytic
mechanism of TAms.48,49 When no substrate is in the active
site it forms a Schiffs base with the PLP cofactor, and during
the reaction the amine donor replaces the Lys288, which is
released as a consequence and changes its position. It is
therefore possible that the dynamic repositioning of Lys288
further influences the stereopreference observed.
Figure 2. Docking of (2S) (pink) and (2R) (green) quinonoid intermediate into
the active site of CV-TAm (4AH3). The binding pocket is indicated as grey
shadow, and amino acids involved in the transamination reaction are
labelled.
Conclusions
Several (S)ꢀ and (R)ꢀωꢀtransaminases have been investigated
for the transamination of a range of cyclic ketones. In a
preliminary screen suitable TAms were identified to
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J. Name., 2012, 00, 1-3 | 5

Organic & Biomolecular Chemistry
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VJieowuArrtnicalelONnlainme e
DOI: 10.1039/C5OB01204J
transform the substrates of interest and the amine donor 2ꢀ
propylamine 12 was accepted by all five selected ꢀTAms
for further study. The amination of cyclohexanone , 2ꢀ
methylcyclohexanone , and ꢀtetralone were studied in
more detail: yields of up to 94% were achieved with selected
transaminases. When using 2ꢀmethylcyclohexanone
site
ω
Kp_forward CATATGACACTGGACGATCTCGC
Kp_reverse CTCGAGTTCGCTAAAAAATGTTTC
Nde
I
I
1
Xho
-
α
8
Mv_forward
ATATACATATGGGCATTG
ACACCGGAACCTCGAATCTC
ATAGCGGCCGCTCAG
NdeI
3
Mv_reverse
NotI
enantioꢀ and diastereoselectivities were investigated. The
highest selectivities were obtained when using CVꢀTAm:
TACTGGATCGCTTCGATCAG
Restriction sites used for cloning are underlined
(1
selectivity at the amine position and up to 24:1
diastereoselectivity for the cis (1 ,2 ) isomer. This high
S)ꢀ2ꢀmethylcyclohexylamine was formed with complete
Heterologous enzyme expression and preparation
S
R
The
ωꢀTAms from Chromobacterium violaceum and Vibrio
reaction selectivity is extremely interesting as it enables the
cyclic amine 3a containing twoꢀdefined chiral centres to be
formed in one step from racemic starting material. Such
fluvialis were expressed as described previously.37,38 All
other
ωꢀTAms were expressed in E. coli BL21(DE3)pLysS
in lysogeny broth medium containing ampicillin (100 mg Lꢀ
1). Cultures were grown at 37 °C until an OD600 of 0.5ꢀ0.7
was reached. Enzyme expression was induced by addition of
dynamic asymmetric transaminations with
received little attention to date, and the high selectivities
achieved by CVꢀTAm with ꢀsubstituted substrates will be
αꢀketones have
α
isopropylꢀβꢀDꢀthiogalactopyranoside (0.5 mM), and the
explored in future work with CVꢀmutants.
temperature was reduced to 25 °C. After 16ꢀ20 h cells were
harvested by centrifugation and stored at ꢀ20 °C. To prepare
cell free crude extract cells (20% v/w) were suspended in
HEPES buffer (100 mM, pH 7.5) containing pyridoxalꢀ5ꢀ
phosphate (PLP) (0.5 mM), and disrupted by ultra sonication
(2 x 1 min, 40% output). The crude extract was cleared by
centrifugation (20 min, 16.000 x g) and either directly used
or freeze dried and stored at ꢀ20 °C.
Experimental
General
All starting materials were obtained from commercial
suppliers and used as received unless otherwise stated.
DNAꢀmodifying enzymes were obtained from Thermo
scientific (Germany) or New England Biolabs (USA). The
enzymes used for shifting the equilibrium were
commercially available from: Lꢀlactate dehydrogenase from
General procedure for TAm screening
For the HPLCꢀbased TAm screening
(R)ꢀ or (S)ꢀαꢀ
rabbit
muscle
(SigmaꢀAldrich,
Austria),
glucose
methylbenzylamine (MBA) 11 (25 mM) and pyridoxalꢀ5ꢀ
dehydrogenase (Xꢀzyme, Germany), and formate
dehydrogenase from Candida boidinii (Codexis, USA). The
Lꢀalanine dehydrogenase was prepared as reported
phosphate (PLP) (0.5 mM) were dissolved in HEPES buffer
(100 mM, pH 8). E. coli crude extract (12.5
the overexpressed ωꢀTAm were added to 212.5
solution. The reaction was started by the addition of 25
ꢀ
L) containing
ꢀ
L of this
previously.46
)ꢀ
was prepared as previously described.50
(R)ꢀ and (S)ꢀ8a are commercially available.
ꢀ
L
(
R 3
of substrate in DMSO (final concentration 10 mM, 10
vol%). After an incubation for 21 h at 30 °C and 180 rpm
Cloning of TAm genes in suitable expression vectors
A synthetic gene for the previously reported ꢀTAm from
the reaction was stopped by the addition of 250
ꢀL of
ω
acetonitrile containing 0.2% TFA. Denaturated protein was
removed by centrifugation, and the supernatant was
analysed by HPLC (Agilent) using a Discovery®Bio Wide
Pore C18 column (Supelco, 25 x 4.6 mm, 10 µm beads) with
UV detection at 254 nm. Concentrations of acetophenone
were determined using a linear gradient: 30% ꢀ 60% B over
10 min (A = water, B acetonitrile, both containing 0.1%
TFA). The acetophenone produced eluted at a retention time
of 8.6 min.
Arthrobacter sp. variant (ArRMut11)1 was designed, as
codonꢀoptimised genes (DNA 2.0, U.S.A.), and
subsequently cloned into the expression vector pET29a
(Invitrogen, Germany) using standard techniques.
Additionally, plasmids containing codonꢀoptimised genes of
the
fluvialis 38
pneumoniae39 were used. In addition, the
ω
ꢀTAms from Chromobacterium violaceum,37 Vibrio
Pseudomonas putida (PpꢀTAm),36 and Klebsiella
ꢀTAm from
,
ω
Mycobacterium vanbaalenii Mvan4516 (MvꢀTAm) (gene
bank accession no. 119958286; ABM15291)40 was
amplified from genomic DNA using the primers listed
below and standard PCR procedures. Restriction sites were
introduced to enable the cloning in the desired expression
vector pET29a.
Determination of initial rates of the five selected TAms
Initial rates were determined using the HPLCꢀbased method
described above using cyclohexanone
Cyclohexanone (10 mM), ( )ꢀ or ( )ꢀMBA 11 (10 mM) and
PLP (1 mM) were solved in sodium phosphate buffer (100
mM, pH 8). The reaction was started by addition of 100
1 as the substrate.
R
S
ꢀ
L
of enzyme solution (crude cell extract or freeze dried cells).
Sequence of the primers used for the amplification of Kp and Mv.
Samples were taken after 5 min, 10 min, 15 min, and 30
Name
Sequence
restriction
6 | J. Name., 2012, 00, 1-3
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ARTICLE
DOI: 10.1039/C5OB01204J
min, and the linear slope was used for the calculation of
initial rates and specific activities.
Determination of enantiomeric purity
The enantiomeric exessess of amines 3a and 8a were
determined by GC using a modified ꢀcyclodextrin column
(CPꢀChirasilꢀDEX CB, 25 m, 0.32 mm, 0.25 m) after
derivatisation to the corresponding trifuoroacetamides. For
the derivatisation trifluoroacetic anhydride (5 L) was added
to the extracted sample, and after an incubation (800 rpm) at
30 °C the reaction was quenched with NaHCO3 and the ee
was analysed. Temperature programme for 3a: 100 °C hold
2 min, 2 °C min–1 to 135 °C, 25 °C min–1 to 180 °C.
β
Specific activities of selected TAms using cyclohexanone 1 as substrate
and (R)ꢀ or (S)ꢀMBA 11 as amine donor
ꢀ
TAm
Crude cell extract
[U mg–1 total protein]
Freeze dried cell
ꢀ
extract
[U mg–1 total protein]
CVꢀTAm
KpꢀTAm
PpꢀTAm
MvꢀTAm
ArRMut11
0.58
n.d.
0.32
0.008
0.11
0.13
0.006
0.39
Retention times: (1
S
,2
R
)ꢀ3a 5.5 min, (1R,
2S)ꢀ3a 5.7 min,
0.006
0.24
(1S, )ꢀ3a 6.7 min and (1R,2R
2
S
)ꢀ3a 7 min. Temperature
programme for 8a: 120 °C, 5 °C min–1 to 160 °C, 20 °C
min–1 to 180 °C. Retention times: (
8.5 min.
S
)ꢀ8a 8.2 min and (R)ꢀ8a
n.d. – not determined.
General procedure for the TAm reactions using different
equilibrium shifting systems
Docking studies
For docking experiments the holo crystal structure of the ωꢀ
Transamination reactions using the a) AlaDH/FDHꢀ, b)
LDH/GDH or c) 2ꢀpropylamine IPA 12 system were
performed as follows: a) substrate (20 mM), Dꢀ or Lꢀalanine
(200 mM), ammonium formate (60 mM), PLP (1 mM),
NAD+ (0.5 mM), formate dehydrogenase (11 U) and Lꢀ
alanine dehydrogenase (12 U) were dissolved in sodium
phosphate buffer (1 mL, 100 mM, pH 8); b) substrate (20
mM), Dꢀ or Lꢀalanine (200 mM), glucose (60 mM), PLP (1
mM), NAD+ (0.5 mM), glucose dehydrogenase (30 U) and
Lꢀlactate dehydrogenase (90 U) were dissolved in sodium
phosphate buffer (1 mL, 100 mM, pH 8); c) substrate (20
mM), IPA 12 (200 mM), and PLP (1 mM) were dissolved in
sodium phosphate buffer (1 mL, 100 mM, pH 8 or 10). All
biotransformations were started by the addition of
lyophilised E. coli crude cell extract containing the
TAm from C. violaceum was used (pdb: 4AH3). The apoꢀ
structure of 4AH3 as well as the structure of the quinonoid
intermediate51 of all conformers were generated with
Maestro and the energy optimisation of the ligands was
performed using the MacroModel “Minimization” followed
by a “Conformational Search” (all Schrödinger LLC). The
two conformations with the lowest energy were used for the
docking experiments. Optimisation of the protein structure
after removal of the bound PLP was performed with
autodock tools
v
1.5.6. Docking calculations were
performed using AutoDock Vina52 with a x=24/y=24/z=24
grid box centred on x=4.8, y=ꢀ0.5, z=7.4. Lowest energy
clusters were selected and visualised using pymol v0.99rc6.
Acknowledgements
overexpressed ωꢀTAm corresponding to an activity of 0.5 U
in the amination of cyclohexanone (CVꢀTAm, PpꢀTAm and
ArRMut11 see Table 1), while the maximum amount of 10
mg was added in the case of the KpꢀTAm and MvꢀTAm.
The mixture was incubated at 30 °C or 45 °C and 800 rpm in
a thermoshaker (Eppendorf, Germany).
This work has been supported by a postdoctoral fellowship
of the German academic exchange service (DAAD) to N. R.,
the Austrian BMWFJ, BMVIT, SFG, Standortagentur Tirol
and ZIT through the Austrian FFGꢀCOMETꢀ Funding
Program. H. L. was funded by the Austrian Science Fund
(FWF, project W9ꢀ01 DK Molecular Enzymology).
Samples were taken at different time points, and the
reaction was stopped by addition of 10 vol% of NaHCO3,
and extracted with ethyl acetate (2
to the amine were measured by GC (Agilent 7890 A system
equipped with a FID detector). The biotransformations of
× 500 ꢀL). Conversions
Notes and references
Department of Chemistry, University College London, 20 Gordon
a
Street, London WC1H 0AJ London, UK. Tel: +44 (0)20 7679 7463.
Eꢀmail: h.c.hailes@ucl.ac.uk.
cyclohexanone
monitored using an Agilent DBꢀ1701 column (30 m, 0.25
mm, 0.25 m) using the following temperature programmes:
60 °C, hold for 5 min, 15 °C min–1 to 150 °C; retention
times cyclohexylamine 1a 6 min and cyclohexanone 7.9
min.
60 °C, hold for 5 min, 5 °C min–1 to 80 °C, 60 °C
min–1 to 250 °C; retention times trans
min and 8.4 min. Conversions for αꢀtetralone
1 and 2ꢀmethylcyclohexanone 3 were
b
ACIB GmbH, c/o Department of Chemistry, University of Graz,
ꢀ
Heinrichstrasse 28, 8010 Graz, Austria.
A
c
Department of Chemistry, University of Graz, NAWI Graz,
1
Heinrichstrasse 28, 8010 Graz, Austria.
B
d
The Advanced Centre for Biochemical Engineering, Department of
ꢀ
3a 5.9 min, cis
ꢀ
3a 6.4
Biochemical Engineering, University College London, Bernard Katz
Building, Gordon Street, London, WC1H 0AH, UK.
3
8
were
determined using an Agilent HP5 (30 m, 0.32 mm, 0.25 ꢀm)
and the following programme: 120 °C, 5 °C min–1 to 160 °C,
1
C. K. Savile, J. M. Janey, E. C. Mundorff, J. C. Moore, S. Tam,
W. R. Jarvis, J. C. Colbeck, A. Krebber, F. J. Fleitz, J. Brands, P.
50 °C min–1 to 300 °C, retention times 1ꢀaminotetraline 8a
4.4 min and αꢀtetralone 8 4.7 min.
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Organic & Biomolecular Chemistry
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VJieowuArrtnicalelONnlainme e
ARTICLE
DOI: 10.1039/C5OB01204J
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