Application of Transaminases in a Disperse System for the Bioamination of Hydrophobic Substrates

Article abstract of DOI:10.1002/adsc.201901434

The challenging bioamination of hydrophobic substrates has been attained through the employment of a disperse system consisting of a combination of a low polarity solvent (e. g. isooctane or methyl-tert-butylether), a non-ionic surfactant and a minimal amount of water. In these conditions, amine transaminases (ATA) were shown to efficiently carry out the reductive amination of variously substituted cyclohexanones, providing good conversions often coupled with a superior stereoselectivity if compared with the corresponding chemical reductive amination. An array of synthetically useful 4-substituted aminocyclohexanes was consequentially synthesized through biocatalysis, analyzed and stereochemically characterized. (Figure presented.).

Full text of DOI:10.1002/adsc.201901434

Title: Application of Transaminases in a Disperse System for the
Bioamination of Hydrophobic Substrates
Authors: Andrea Fiorati, Per Berglund, Maria Svedendahl Humble, and
Davide Tessaro
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To be cited as: Adv. Synth. Catal. 10.1002/adsc.201901434
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10.1002/adsc.201901434
Advanced Synthesis & Catalysis
FULL PAPER
DOI: 10.1002/adsc.201((will be filled in by the editorial staff))
Application of Transaminases in a Disperse System for the
Bioamination of Hydrophobic Substrates
Andrea Fiorati,a Per Berglund,b Maria S. Humble,b,c Davide Tessaroa,*
a
Politecnico di Milano, Department of Chemistry, Materials and Chemical Engineering “G. Natta”, p.za L. da Vinci 32,
I-20133 Milano, Italy
E-mail: davide.tessaro@polimi.it
KTH Royal Institute of Technology, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH),
b
Department of Industrial Biotechnology, AlbaNova University Center, SE-10691 Stockholm, Sweden
Pharem Biotech AB, Biovation Park, Forskargatan 20 J, SE-15136 Södertälje, Sweden
c
Received: ((will be filled in by the editorial staff))
Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/adsc.201######.((Please
delete if not appropriate))
Abstract. The challenging bioamination of hydrophobic An array of synthetically useful 4-substituted
substrates has been attained through the employment of a aminocyclohexanes was consequentially synthesized
disperse system consisting of a combination of a low polarity through biocatalysis, analyzed and stereochemically
solvent (e.g. isooctane or methyl-tert-butylether), a non-ionic characterized.
surfactant and a minimal amount of water. In these
Keywords: biotransformations; amination; multiphase
conditions, amine transaminases (ATA) were shown to
catalysis; enzyme catalysis; diastereoselectivity;
efficiently carry out the reductive amination of variously
chemoenzymatic synthesis
substituted cyclohexanones, providing good conversions
often coupled with a superior stereoselectivity if compared
with the corresponding chemical reductive amination.
with a minor amount of hydrophilic solvents (e.g.
DMSO) when an increase in solubility of a
Introduction
hydrophobic substrate is sought. In contrast, there are
only a few reports covering the application of ATAs
in pure organic solvents.[2] The first ATA
4-Substituted cyclohexylamines are important
building blocks in medicinal chemistry.[1] A highly
selective and general synthesis methodology for this
class of compounds is therefore desired. Here, a
biocatalytic approach for the stereoselective
conversion of 4,4-disubstituted cyclohexanones into
the corresponding amines using amine transaminases
(ATAs, EC 2.6.1.x) as biocatalysts (Scheme 1) is
presented. The reactions are performed in a non-
conventional disperse system in order to overcome
the solubility issues associated with such
hydrophobic substrates.
successfully employed in a substantial amount of
organic solvent (i.e. 50 % DMSO) was reported in
2010 for the synthesis of sitagliptin,[3] where the
immobilization of the enzyme permitted even the
employment of neat organic solvents.[4] Since then, a
number of reports on immobilized transaminases
have been published.[5] However, the employment of
immobilized ATAs for synthetic purposes is far from
being of wide use. The main drawbacks may be
related to mass transfer problems or to the scarce
solubility of the cofactor (pyridoxal 5'-phosphate,
PLP) in organic solvents.[6]
Recently, the application of some lyophilized
ATAs in ethyl acetate and methyl-tert-butyl ether
(MTBE) was shown.[2a] Increased stability of the
enzyme and the absence of substrate inhibition were
underlined. The key factor of this work is the activity
of water in the organic medium; this is in line with
the
generally
accepted
mechanism
of
transamination,[7] where a water molecule is involved
in the catalytic cycle. The same authors also report on
the use of this approach in the stereoselective
synthesis of valinol,[8] and for 3-substituted
cyclohexylamine derivatives.[9] The use of surfactants
(i.e. Brij® C10 and octyl β-D-glucopyranoside) as an
Scheme 1. Biocatalytic reductive amination of achiral
cyclohexanone derivatives.
The reactions catalyzed by ATAs are commonly
carried out in aqueous media, often in combination
1
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10.1002/adsc.201901434
Advanced Synthesis & Catalysis
additive prior to the lyophilization of the enzyme for
employment in organic solvent was recently reported
for the biocatalytic reductive amination of
methoxyacetone. In that study, the ATA from
Chromobacterium violaceum (Cv-ATA) was applied
as a suspension of surfactant-stabilized lyophilized
powder for the stereoselective conversion of
methoxyacetone to the corresponding amine in dry
isooctane employing (S)-phenylethylamine ((S)-PEA)
as amino donor (Scheme 2).[10] Unfortunately, this
approach led to unsatisfactory results when applied to
4-substituted-cyclohexanones, in particular in terms
of reaction rate. In order to address this issue, a novel
approach for the application of ATAs in organic
solvents was developed. In this procedure, an
aqueous solution of the enzyme, a non-ionic
surfactant, the substrate, and an appropriate organic
solvent are mixed to form a macroscopically
homogeneous system. Such procedure was
successfully applied to the reductive amination of a
time brought some beneficial effects in terms of
catalyst activity (see SI), even if the reaction rate still
was too low to be of practical use. Since the water
activity in the organic solvent generally plays a major
role in the enzyme performance,[2a] the effect of small
additions of water into the reaction mixture was
explored and established to have, indeed, some
beneficial effects on both the reaction rate and the
final conversion, also by preventing the imine
formation, as demonstrated by GC-MS (see SI).
However, the high hydrophobicity of isooctane
allows the addition of only a tiny amount of water
before a new liquid phase appears (water activity
reaches 1), preventing an efficient mixing of the
components and complicating the downstream
processing.
As an alternative approach stemming from the
same components,
a ternary reaction mixture
library
of
hydrophobic
4,4-disubstituted
containing a hydrophobic organic solvent, a small
amount of water and an appropriate surfactant, with
the aim to generate amphiphilic supramolecular
assemblies was designed. Such systems have been
extensively described in the 1980s by Luisi and
coworkers,[13] and are based on the formation of
reverse micelles encapsulating the enzyme. Thus, a
water-in-oil disperse environment suitable for ATAs
was designed and employed. According to literature,
in this work, we refer to micelles (oil in water) and
reverse micelles (water in oil) when the assemblies
dimensions are around 5-10 nm, microemulsions
when droplets dimensions are 10-100 nm and
emulsions when the droplets size ranges 0.5-200
cyclohexanones using different ATAs, and led to
interesting results also in terms of stereoselectivity.
Results and Discussion
At first, following the procedure described by
Chen et al.,[10] Cv-ATA was employed as a
surfactant-stabilized powder in dry isooctane at 37 °C
using 4-tert-butyl-cyclohexanone (1a) as a model
substrate (Scheme 2). This resulted in a very low
reaction rate, and only 40 % conversion was observed
after 8 days. On the other hand, the diastereomeric
ratio (24/76 cis/trans) of the newly formed amine was
the opposite with respect to the product obtained
through metal-mediated reductive amination.[11]
mm.[14] Briefly,
a
non-ionic surfactant (e.g.
Polyethylene-glycolhexadecyl-ether, Brij® C10) is at
first dissolved in a hydrophobic organic solvent
(isooctane or MTBE) together with the substrates at
37 °C. After complete dissolution, a small amount of
aqueous enzymatic preparation (1 % v/v) was added
to the reaction mixture and then vigorously shaken. In
order to find suitable reaction conditions for
Scheme 2. Model reaction: bioamination of 4-tert-
butylcyclohexanone (1a).
screening our library of ketones,
a
model
transamination system was explored with the use of
1a (25 mM), (S)-PEA, Brij® C10 (as surfactant) and
Cv-ATA (0.95 U) as biocatalyst. Through a
preliminary characterization of the system, the
following parameters were evaluated: donor/acceptor
ratio, nature of organic solvent and surfactant,
reaction temperature, nature of amino donor.
When the reaction solvent is isooctane and the
surfactant employed is Brij® C10 (50 mM) at 37 °C
the reaction mixture is perfectly clear and transparent.
The addition of 1.0 % v/v of the aqueous enzymatic
preparation, upon vigorous shaking, turns the mixture
into a macroscopically homogeneous milky emulsion.
This mixture is stable as long as the necessary
agitation is provided. Interruption of the shaking
leads to phase separation and to a drastic slowdown
A MS analysis of the crude reaction mixture showed
that (S)-PEA spontaneously reacted with the substrate
giving the corresponding Schiff base as a by-product.
This reaction is reasonably favored by the low water
activity in the reaction media. Despite the
reversibility of the Schiff base formation, the
depletion of the ketone from the mixture is probably
responsible for the reduction of the reaction rate and,
consequently, of the overall conversion. In order to
increase the reaction efficiency, the reaction
parameters were tuned, in particular focusing on the
lyophilization time and the water activity in the
solvent.[12] Indeed, an increase of the freeze-drying
2
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10.1002/adsc.201901434
Advanced Synthesis & Catalysis
Table 1. Effect of water concentration on the system self-
assembly. Micelles shape is only based on the measured
hydrodynamic radius. Conditions: 25 mM ketone 1a; 50
mM (S)-PEA; 50 mM decane; 50 mM Brij® C10. A proper
amount of Hepes buffer pH 8.2, 50 mM, with or without
13 mg/mL Cv-ATA.
of the conversion rate. On the other hand, when
employing no more than 0.2 % v/v of water, a stable,
transparent and clear solution is obtained. In this case,
we assume that such a configuration may be
considered as a micellar system, where the enzyme
solution is entrapped in thermodynamically stable
reverse micelles. Such a hypothesis is confirmed by
dynamic light scattering (DLS) experiments (Figure 1,
green peaks), which show that the reaction system is
composed of a homogeneous population of particles
with a mean hydrodynamic radius of 16 nm.
Water
Solvent
concentration
Enzyme
isooctane
MTBE
v/v [%]
Spheric
micelles
Spheric
micelles
Spheric
micelles
Stable
Oblong
micelles
Stable
emulsion
Stable
emulsion
Unstable
emulsion
0
No
No
0.2
0.2
1.0
Yes
Yes
emulsion
In order to evaluate the performance of this system,
the conversion of 1a to the corresponding amine was
studied. Two different experiments were carried out
employing the same amount of enzyme (0.13 mg,
0.82 U) diluted in order to obtain a final
concentration of the aqueous solution of 1.0 and
0.2 % v/v, respectively. A third experiment was
performed using 1.0 % v/v of the enzymatic
preparation at a higher concentration (0.45 mg, 2.8 U).
Figure 1. Dynamic light scattering showing the mean
hydrodynamic radius distributions of the particles obtained
using 50 mM of Brij® C10 in isooctane at 37 °C. Dashed
line, green peaks: 0.2 % v/v of aqueous enzymatic
preparation. Continuous lines, read peaks: 1.0 % of
aqueous enzymatic preparation.
This is consistent with the hypothesis of a solution
containing spherical inverse micelles with a confined
aqueous phase encapsulating the enzyme, as
illustrated in the literature.[13] An increase of the
buffer concentration leads to a destabilization of the
micelles, which coalesce in droplets and form an
emulsion stabilized by the surfactant (Figure 1, red
peaks). When isooctane is substituted with MTBE,
DLS analysis indicates the presence of two
Figure 2. Effect of water content. A. 0.2 % v/v of buffer
containing 0.82 U Cv-ATA. B. 1.0 % v/v of buffer
containing 0.95 U Cv-ATA. C. 1.0 % v/v of buffer
containing 2.8 U Cv-ATA. Reaction conditions: 25 mM
ketone 1a; 50 mM (S)-PEA; 50 mM decane; 50 mM Brij®
C10; enzyme preparation in HEPES buffer (pH 8.2, 50
mM); isooctane to final volume 1 mL.
supramolecular populations, the former on
a
nanometric scale and the latter in the range of
hundreds of nm, even employing a small amount
(0.2 % v/v) of aqueous solution (data shown in SI).
According to the aforesaid definitions, the system
should be in this case described as an emulsion, even
though the mixture looks macroscopically like a
transparent and clear solution. The different
assemblies are summarized in Table 1
The results are reported in Figure 2. According to
these data, the emulsion systems possess better
performances when compared to the corresponding
micellar systems. Moreover, as expected, employing
about 3.5 times more enzyme leads to an
approximately three-fold faster reaction.
3
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10.1002/adsc.201901434
Advanced Synthesis & Catalysis
Another interesting aspect is that the reaction workup
is quick and easy if compared to a standard extractive
protocol. By just centrifuging the cooled (4 °C)
reaction mixture it is possible to separate the organic
phase, containing substrates and products, from the
solids, composed of salts, enzyme, cofactor and
surfactant.
donors by Cv-ATA,[16] no conversion was observed in
all cases.
The stability of Cv-ATA in the studied reaction
medium is remarkably high: the enzyme retains its
activity all along the explored reaction time (187 h, >
1 week). The addition of a different substrate, i.e.
methoxyacetone, to the solution after reaching the
conversion plateau immediately reactivates the
reaction: a further aliquot of (S)-PEA is consumed
and the corresponding amount of acetophenone is
released (Figure 3). This also demonstrates that the
conversion observed at the end of the reaction
depends on the assessment of the thermodynamic
equilibrium and is not due to enzyme inactivation.
Figure 4. Influence on conversion by the ratio between
ketone 1a and amino donor. Reaction conditions: 50 mM
(S)-PEA; 50 mM decane; 50 mM Brij® C10; 10 µL of Cv-
ATA preparation (0.95 U) in HEPES buffer (pH 8.2, 50
mM); 37 °C; isooctane to a final volume of 1 mL.
Next, different solvents; MTBE, isooctane, ethyl
acetate (AcOEt), cyclohexane, chloroform, toluene
and dimethoxyethane (DME) were explored. Among
them, only DME is miscible with water. In our
experiments, isooctane allowed the highest
conversions. Good results were also obtained using
MTBE and cyclohexane, while ethyl acetate was less
efficient (Figure 5).
Figure 3. Stability of the enzymatic preparation: Addition
of methoxyacetone (25 mM) after 144 h of reaction.
Reaction conditions: 25 mM ketone 1a; 50 mM (S)-PEA;
50 mM decane; 50 mM Brij® C10; 10 µL enzyme
preparation (0.95 U) in HEPES buffer (pH 8.2, 50 mM);
isooctane to a final volume of 1 mL.
Figure 4 reports the results of a set of experiments
aimed at investigating the influence and proper ratio
between amino donor and amino acceptor. These
experiments were carried out with
a
fixed
concentration of 1a, while the concentration of (S)-
PEA was varied. Apparently, while a slight excess of
the amino donor has a positive effect, higher
concentrations of (S)-PEA lead to significant
inhibition. However, for the purpose of screening the
ketones library (see later), 50 mM of (S)-PEA was
applied to push the thermodynamic equilibrium
towards the product.[15] At the same time,
experiments were performed using different amino
donors, in particular benzylamine and isopropylamine
at different concentrations. Although both amines are
known in the literature for being accepted as amino
Figure 5. Influence of organic solvent. Reaction
conditions: 25 mM ketone 1a; 50 mM (S)-PEA; 50 mM
decane; 50 mM Brij c10; 10 µL enzyme preparation (0.95
U) in HEPES buffer (pH 8.2, 50 mM); organic solvent to a
final volume of 1 mL; 37°C.
No conversion was detected using either
chloroform, toluene or DME. We point out that this
solvent screening was focused on improving the
4
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10.1002/adsc.201901434
Advanced Synthesis & Catalysis
activity of Cv-ATA, and the solvent tolerability may
differ for other ATAs. Moreover, the identity of each
organic solvent might also affect the final position of
the reaction equilibrium.
achieved conversions were in all cases disappointing
(3-27 %). Moreover, any increase in the surfactant
concentration led to a decrease in the conversion.
The use of AOT resulted in no conversion in all the
After selecting the best performing organic solvent, performed experiments. Such a result is somehow
the model reaction was evaluated in isooctane at
different temperatures (see SI). The highest
conversions were obtained at 37 °C. Temperatures
lower than 30 °C lead to the incomplete dissolution
of Brij® C10 in the medium, while higher
temperatures resulted in lower conversions. Albeit the
enzyme in other conditions showed good
thermostability,[17] it is nevertheless known that the
surprising since AOT is the most common surfactant
reported in the literature for enzymatic micellar
systems.[13]
In order to explore the versatility of the disperse
system, a library of twelve 4-substituted and 4,4-
disubstituted cyclohexanones (Figure 6) was selected
and employed.
presence of (S)-PEA significantly lowers its melting
18]
point.[10,
This fact, in combination with the
particular environment we describe, might explain the
thermal inactivation we observed.
Table 2. Employed surfactants. AOT stands for Aerosol
OT, or dioctyl sodium sulfosuccinate.
Surfactant
Type
HLB
Brij® C10
TritonTM X-100
Brij® 58
Non-ionic
Non ionic
Non-ionic
Non-ionic
Ionic
12.0
13.5
15.7
16.7
-
Tween® 20
AOT
Once set up the reaction conditions, five different
surfactants were investigated (Table 2, structures in
SI). The surfactants were evaluated for the
conversion of 25 mM of 1a to the corresponding
amine (Table 3).
Table 3. Effect of surfactant on final conversion [%] (n.c.
= no conversion).
Figure 6. Ketones library.
Ketones 1d-k were synthesized starting from the
Concentration [mM]
Surfactant
commercial
precursor
1,4-cyclohexanedione
1.0
2.5
5.0
10
25
50
75
100
1.5
monoethylene acetal (Scheme 3A). The latter is
reacted with the appropriate Grignard reagent as the
first step.
Brij® C10
Brij® 58
TritonTM X- 27
< 3 < 3 < 3 < 3 6.5
65
25
25
27
21
20
24
22
8.1
10
6.0
8.2
3.9
100
Tween 20
AOT
22
25
15
6.1
11
n.c. n.c. n.c. n.c. n.c. n.c. n.c. n.c.
Reaction conditions: 25 mM ketone 1a; 50 mM (S)-
PEA; 50 mM decane; 10 µL of enzyme preparation (0.95
U) in HEPES buffer (pH 8.2, 50 mM); 37 °C; isooctane to
a final volume of 1 mL.
The use of Brij® C10, at a concentration of 25 mM
or lower, did not result in any detectable conversion.
However, a higher concentration (50 mM) of the
same surfactant gave acceptable results (65 %
conversion). A further increase in concentration is
restricted by its solubility in isooctane at 37 °C.
Conversely, all the other non-ionic surfactants (Brij®
58, TritonTM-X100 and Tween 20) activated the
reaction at very low concentrations (1-5 mM), but the
5
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10.1002/adsc.201901434
Advanced Synthesis & Catalysis
Scheme 3. A. Synthesis of ketones 1d-k. B. Metal-
mediated synthesis of the reference amines 3a-k.
trans isomers in a ratio varying from 60:40 to 95:5
(Table 4). The two isomers can be distinguished and
quantified by 1H-NMR analysis as described in the SI.
Then, the protective group is easily removed by
mild acid hydrolysis, and quantitative yields of the
desired ketone (1d-k) are obtained after purification
by flash chromatography or by distillation. Ketone 1l
is a key intermediate for the synthesis of the antiviral
drug radalbuvir (coded as GS-9669),[19] and was
kindly provided by Flamma SpA (Chignolo D’Isola,
Italy).
All the corresponding amines (3a-l) were chemically
synthesized through metal-mediated reductive
amination in order to be employed as references for
the enzymatic reaction (Scheme 3B), with the
exception of amine 3c, which is commercially
available as a mixture of cis/trans. The ketones (1a-l)
were reacted with hydroxylamine to form the oximes
(2a-l), which were in turn reduced using
NaBH4/MoO3 in methanol at 0 °C.[11] After the
appropriate workup, the resulting amines (3a-l) were
pure enough for 1H-NMR characterization. Such
reductive aminations result in mixtures of cis and
Table 4. Observed stereoselectivity for the reduction of
the oximes using NaBH4 and MoO3.
Product
cis [%]
trans [%]
62
59
88
87
88
89
95
77
90
93
85
38
41
12
13
12
11
5
23
10
3
3a
3b
3d
3e
3f
3g
3h
3i
3j
3k
3l
15
In total, four different enzymes were screened: Wild
type Cv-ATA,[20] the variant Cv-ATA W60C,[21] wild
type ATA from Vibrio fluvialis (Vf-ATA) and ATA
113 (a commercial ATA from Codexis®).
Table
5.
Bioamination
of
4,4-disubstituted-cyclohexanones:
Cv-ATA Cv-ATA-W60
conversions
and
cis/trans
ratios.
Vf-ATA
Substrate Solvent
conv. [%] cis [%] trans [%] conv. [%] cis [%] trans [%] conv. [%] cis [%] trans [%]
MTBE
1a
50
60
55
83
45
70
55
28
50
-
98.6
98.3
95.1
97.6
68.5
21.0
24.3
12.1
1.7
-
1.4
1.7
4.9
2.6
31.5
79.0
75.7
87.9
98.3
-
15
-
98.7
1.3
3
98.6
96.8
98.8
95.7
20.7
37.0
18.0
7.1
1.4
3.2
isooctane
-
-
56
3
MTBE
1b
79
-
99.6
0.4
1.2
isooctane
-
0.5
-
-
80
8
4.3
MTBE
1c
10
-
99.5
79.3
63.0
82.0
92.9
92.3
88.0
98.2
99.7
> 99.9
99.8
99.6
99.8
22.9
22.5
95.2
98.3
99.1
99.4
99.5
99.8
isooctane
-
85
10
30
13
40
14
47
9
MTBE
1d
22
-
4.0
-
96.0
isooctane
-
MTBE
1e
21
-
0.9
-
99.1
7.7
isooctane
-
12.0
1.8
MTBE
1f
60
-
1.5
-
98.5
-
26
-
0.6
-
99.4
isooctane
-
0.3
MTBE
1g
46
-
0.4
-
99.6
-
15
-
0.2
-
99.8
< 0.1
0.2
isooctane
-
50
3
MTBE
1h
58
-
< 0.1
-
> 99.9
-
33
-
0.6
-
99.4
0.4
isooctane
-
50
10
47
3
0.2
MTBE
1i
41
-
77.7
-
22.3
-
19
-
77.5
-
22.5
77.1
77.5
4.8
isooctane
-
MTBE
1j
15
-
0.8
-
99.2
-
10
-
13.8
-
86.2
isooctane
-
30
3
1.7
MTBE
1k
n.c.
n.c.
35
15
-
-
n.c.
n.c.
22
-
-
-
0.9
isooctane
-
-
-
-
99.4
-
18
14
29
0.6
MTBE
1l
7.1
2.7
92.9
97.3
0.6
-
0.5
isooctane
0.2
Reaction conditions: 25 mM ketone; 50 mM (S)-PEA; 50 mM decane; 50 mM Brij® C10; 10 µL of enzyme preparation
(0.95 U) in HEPES buffer (pH 8.2, 50 mM); isooctane or MTBE: final volume 1 mL. (n.c. = no conversion).
6
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10.1002/adsc.201901434
Advanced Synthesis & Catalysis
The W60C variant of Cv-ATA shows an increased
enantiospecificity for (S)-PEA compared to the wild
type.
The ketone library (Figure 6) was explored using 25
mM of ketone, 50 mM of (S)-PEA, 50 mM of decane,
50 mM of Brij® C10, 1.0 % v/v HEPES buffer
solution (50 mM, pH 8.2) containing 0.95 U of ATA.
The final volume of the reaction solutions was
adjusted to 1 mL using MTBE or isooctane as
solvents. The mixtures were vigorously shaken at
37 °C and left to react for 48 hours (results in Table
5). Despite the preliminary solvent exploration had
shown that isooctane was the most suitable solvent
for these reactions, some of the synthesized ketones
are poorly soluble in this solvent, and only MTBE
was employed. The reported conversions were
measured after 48 hours. At the end of the reaction,
the solution was cooled to 4 °C to obtain phase
separation. The organic supernatant essentially
contains both the remaining substrates and the
products, whereas salts, enzyme, cofactor and most of
the surfactant are collected in the aqueous phase.
When ATA 113 was employed, no conversions were
detected in any case. However, interesting results
were obtained with the other ATAs. In general, the
explored enzymes showed high cis selectivity
towards 4-monosubstituted-cyclohexanones, whereas
4-substituted-4-hydroxyl-cyclohexanones
are
selectively transformed into trans amines. This
apparent switch of selectivity is simply due to
nomenclature: as a matter of fact, both the groups
have the 4-alkyl chain positioned on the same side of
the cyclohexene ring with respect to the amino
moiety (Figure 7).
Figure 7. Comparison of the structure of the two groups of
amines obtained after biotransformation.
The selectivity of the ATAs towards ketone 1i is
reversed, despite the similarity between phenyl and
thienyl rings. In addition, the hydroxyl moiety seems
not to affect the enzyme selectivity. In general, the
biocatalytic approach leads to a good stereoselectivity.
In addition, the ATA-catalyzed transaminations result
in the production of the more unusual trans isomer as
the major product, while the chemical approach using
NaBH4 and MoO3 favors the cis isomer.
7
This article is protected by copyright. All rights reserved.

10.1002/adsc.201901434
Advanced Synthesis & Catalysis
GC-FID analyses were performed on a Hewlett Packard
5890 Series II gas chromatography apparatus equipped
with a CP-ChiraSil-DEX CB 25 m x 0.32 mm x 0.25 mm
or Chrompack CP-SIL 5CB column (25 m x 0.32 mm).
The inlet and the detector were set to 250 °C. The
following method was used: initial temperature 100 °C,
hold for 0.5 min, 10 °C min-1 to 200 °C and hold 2-25 min.
Conclusion
A novel approach for the use of ATAs in the
transformation of hydrophobic substrates was
developed and employed, carrying out the reductive
amination of a library of 12 cyclic ketones. This
approach involves the use of surfactants for obtaining
UV spectra and absorbance measurements were performed
on a Jasco V-560 UV/VIS spectrophotometer.
Dynamic light scattering (DLS) measurements were
conducted on a Zetasizer Nano ZS instrument (Malvern,
UK), at 37 °C, with an equilibration time of 120 s.
a
macroscopically homogeneous water-in-oil
emulsion, where the enzymatic preparation is finely
dispersed in the organic phase. Such a system was
characterized with dynamic light scattering analysis
and different parameters were explored in order to
optimize the process (e.g. temperature, solvents and
type of surfactant). The highest conversions were
obtained using isooctane or MTBE as solvent, Brij®
C10 as surfactant at 37 °C and 1.0 % v/v of aqueous
enzymatic preparation. Among the evaluated amino
donors, only (S)-PEA was effective in this approach.
Moreover, the solvent was proven to be an important
parameter. MTBE dissolved the starting materials
more efficiently than isooctane, even if the latter is
better tolerated by the enzyme. In the end, this
approach possesses many useful advantages. First,
the use of organic solvents and surfactants permits to
address the frequent solubility issues of hydrophobic
substrates. At the same time, enzyme stability in the
reaction mixture is remarkably high (stable for more
than a week), the reaction workup is easy and
practical since it mainly consists in cooling of the
reaction mixture and followed by centrifugation to
recover the final product. Lastly, this protocol can be
employed with different substrates and ATAs.
HPLC Conditions
The HPLC analyses of the cyclohexylamine derivatives
were
performed
after
derivatization
with
o-
phthaldialdehyde (OPA) and N-protected-L-cysteine (N-
acetyl-L-cysteine or N-tert-butyloxycarbonyl-L-cysteine,
respectively NAC and TBC).[22] The OPA-NAC reagent
was prepared by dissolving 8 mg of OPA and 10 mg of
NAC in 1 mL of HPLC grade methanol, while OPA-TBC
was prepared by dissolving 10 mg of OPA and 10 mg of
TBC in 1 mL of HPLC grade methanol. Derivatizations
were performed by adding 175 µL of borate buffer (0.4 M,
pH 10.4) to 25 µL of samples and then adding 50 µL of
OPA-NAC or OPA-TBC reagent. The samples were left to
react for 1 minute in the dark before the injection. Due to
the different nature of each amine, different HPLC
conditions were employed; these are summarized in Table
5.
Table 5. HPLC running conditions for the analysis of the
produced amines, after derivatization. A: acetate buffer 10
mM pH 5.2 (45%), MeOH (55%); B: MeOH (100%); C:
acetate buffer pH 5.2 (95%), MeOH (5%)
Derivatizing Flow
A
B
C
cis
trans
agent
[mL
min-1]
0.85
0.5
[%] [%] (%)
r.t.
r.t.
[min] [min]
3a
OPA-NAC
3b OPA-NAC
87
13
-
-
22.9
34.1
26.7
23.9
36.1
24.2
92.5 7.5
Experimental Section
3c
OPA-TBC
0.85
87
13
Reagents and instruments
3d
3e
3f
OPA-TBC
OPA-TBC
OPA-NAC
OPA-NAC
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.85
0.5
100
100
100
100
92.5 7.5
92.5
92.5 7.5
-
-
-
-
-
-
-
-
-
7.5
-
-
9.4
13.1
5.4
9.2
4.6
7.5
6.1
13.9
10.6
18.5
28.3
13.0
27.2
9.2
16.1
14.2
42.3
16.2
All the necessary chemicals were purchased from Sigma-
Aldrich. All solvents employed in reactions were of
analytical grade and, where indicated, were dried on
activated 4 Å molecular sieves prior to use. The solvents
employed in HPLC analysis were of HPLC grade and the
prepared eluents were filtered through a 0.45 µm
membrane prior to use. Silica gel 60 F254 plates (Merck)
were used for analytical TLC. Detection was achieved with
UV light followed by ninhydrin, potassium permanganate,
or cerium molybdate stains.
3g
3h OPA-NAC
3i
3j
3k OPA-NAC
3l OPA-TBC
OPA-NAC
OPA-NAC
-
70
100
30
-
-
4-methyl-4-hydroxycyclohexanone (1d).
HPLC analyses were performed on a Jasco apparatus
equipped with a diode array detector and a fluorimeter,
fitted with a Kinetex 2.6 µm C18 100 Å column,
length/internal diameter = 100/4.6 mm (Phenomenex). The
excitation and emission wavelengths for fluorescence
detection were 350 nm and 450 nm, respectively.
An ethereal solution (30 mL) of CH3I (598 μL, 9.6 mmol)
was added dropwise to a suspension of Mg turnings (233
mg, 9.6 mmol) in dry diethyl ether (2.5 mL) under
vigorous stirring. The mixture was left to react at r.t. (a
reflux condenser was added in order to control the reaction
and prevent solvent evaporation) until complete dissolution
of Mg; then, a solution of 1,4-cyclohexanedionone
monoethylene ketal (1.5 g, 9.6 mmol) in dry diethyl ether
(30 mL) was gradually added to the reaction mixture. The
consumption of the ketone was followed by TLC. When
the ketone was completely consumed, 25 mL of water
were added to the reaction, the organic phase was
separated and the aqueous phase was extracted with diethyl
ether (2x30 mL). The organic phase was dried over anh.
Na2SO4 and evaporated under reduced pressure obtaining
1.36 g of a pale yellow oil. 25 mL of aqueous 0.05 M HCl
were added to the product and the resulting mixture was
1H and 13C-NMR spectra were recorded on Bruker ARX
1
400 instrument operating at the H resonance frequency of
400 MHz. Chemical shifts (d, ppm) are reported relative to
tetramethylsilane (TMS) as the internal standard. All
spectra were recorded in CDCl3 or DMSO-d6 at 305 K.
Coupling constants (J) are reported in Hz. Mass spectra
were acquired on BRUKER Esquire 3000 PLUS equipped
with ESI Ion Trap LC/MS System.
8
This article is protected by copyright. All rights reserved.

10.1002/adsc.201901434
Advanced Synthesis & Catalysis
stirred overnight at r.t. When the hydrolysis was complete
the reaction medium was saturated with NH4Cl and then
extracted with AcOEt (4x25 mL). The organic phase was
dried over Na2SO4 and evaporated under reduced pressure.
The obtained pale yellow oil was purified by flash
chromatography (AcOEt/Hexane/MeOH 2/8/0.1) obtaining
790 mg of a white solid corresponding to compound 1d in
dissolved in MeOH (25 mL), MoO3 was added and the
resulting mixture was stirred at 0 °C. Then, NaBH4 (209
mg, 5.52 mmol) was portion-wise added in 2 hours. The
depletion of the oxime was followed by TLC. When the
substrate was completely consumed, the solvent was
evaporated under reduced pressure and the residue was
suspended in ethyl acetate (10 mL); a solution of
ammonium hydroxide (1 M, 5 mL) was added and the
resulting mixture was filtered through a Celite pad. After
phase separation, the aqueous phase was saturated with
NaCl and extracted with AcOEt (4 x 10 mL). The
combined organic fractions were dried over Na2SO4 and
the solvent distilled off to obtain 50 mg of a yellowish oil
(yield 71 %), which is a mixture of cis/trans isomer of the
compound 3d.
1
64% yield. H NMR (CDCl3): 1.33 (s, 3H), 1.61 (s, 1H),
1.75-1.87 (dt, 2H, J=5, 13.3), 1.89-1.98 (m, 2H), 2.15-2.24
(m, 2H), 2.70-2.74 (m, 2H).
According to the procedure described above, starting from
1,4-cyclohexanedionone monoethylene ketal and the
opportune alkyl chloride or bromide, the following ketones
were synthesized. Yields are calculated based on the
starting ketale.
Yield 96%
1H NMR (CDCl3): 1.13-1.20 (m, 3H), 1.30-1.79 (m, 9H),
2.50-2.59 (m, 1H, cis isomer), 2.70-2.80
4-ethyl-4-hydroxycyclohexanone (1e).
(m, 1H, trans isomer). ESI-MS m/z 130 [M + H+].
White solid. 655 mg, yield 48 %.
1H-NMR (CDCl3): 0.90-0.97 (t, 3H, J=7.5), 1.39 (s, 1H),
1.52-1.62 (q, 2H, J=7.5), 1.70-1.80 (dt, 2H, J=5.1, 8.4),
1.87-1.95 (m, 2H), 2.17-2.25 (m, 2H), 2.63-2.74 (m, 2H).
According to the procedure described above and starting
from the corresponding ketones, the following amines
were synthetized. Yields are calculated based on the
starting ketone.
4-hydroxy-4-propylcyclohexanone (1f).
White solid. 252 mg, yield 17 %.
1H-NMR (CDCl3): 0.91-0.97 (t, 3H, J=7.2), 1.34-1.56 (m,
5H), 1.71-1.83 (m, 2H), 1.88-1.97 (m, 2H), 2.18-2.26 (m,
2H), 2.64-2.65 (m, 2H).
4-tert-butyl-cyclohexyl-1-amine (3a).
Yellowish oil. 57 mg, yield 81%
1H-NMR (CDCl3): 0.83-0.86 (s, 9H), 0.91-1.13 (m, 5H),
1.58.1.97 (m, 6H), 2.50-2.62 (m, 1H, trans isomer), 2.79-
2.92 (m, 1H, cis isomer). ESI-MS m/z 156 [M + H+].
4-butyl-4-hydroxycyclohexanone (1g).
White solid. 392 mg, yield 24 %.
1H-NMR (CDCl3): 0.88-0.94 (t, 3H, J=7.4), 1.26-1.42 (m,
5H), 1.51-1.57 (m, 2H), 1.76-1.82 (dt, 2H, J=5.2,13.1),
1.88-1.97 (m, 2H), 2.17-2.24 (m, 2H), 2.64-2.73 (m, 2H).
13C NMR (CDCl3): 13.88, 23.10, 25.53, 36.76, 36.94,
42.22, 70.03, 212.40.
4-phenyl-cyclohexyl-1-amine (3b).
Yellowish oil. 67 mg, yield 95%
1H-NMR (CDCl3):1.19-1.29 (m, 2H), 1.46-1.63 (m, 4H),
1.86-2.02 (m, 4H), 2.42-2.52 (m, 1H), 2.59-2.69 (m, 1H,
trans isomer), 2.96-3.04 (m, 1H, cis isomer), 7.12-7.35 (m,
5H). ESI-MS m/z 176 [M + H+].
4-phenyl-4-hydroxycyclohexanone (1h).
White solid. 603 mg, yield 33%
4-amino-1-ethylcyclohexan-1-ol (3e).
1H-NMR (CDCl3): 1.722 (s, 1H), 2.17-2.26 (m, 2H), 2.29-
2.52 (m, 4H), 2.87-3.00 (dt, 2H, J=5.2, 13.7), 7.29-7.34 (d,
1H, J=7.29), 7.38-7.43 (t, 2H, J=7.32), 7.53-7.56 (d, 2H,
J=7.29)
Yellowish oil. 65 mg, yield 92%
1H-NMR (CDCl3): 0.81-0.87 (t, 3H, J=7.5), 1.24-1.68 (m,
14H), 2.51-2.60 (m, 1H, cis isomer), 2.76-2.84 (m, 1H,
trans isomer). ESI-MS m/z 144 [M + H+].
4-hydroxy-4-(thiophen-2-yl)cyclohexanone (1i).
Yellowish solid. 999 mg, yield 53 %
4-amino-1-propylcyclohexan-1-ol (3f).
Yellowish oil. 63 mg, yield 90%
1H-NMR (CDCl3): 2.18-2.35 (m, 4H), 2.70-2.85 (m, 4H),
6.89-6.93 (m, 1H), 6.95-6.98 (dd, 1H, J= 1.2, 3.6), 7.16-
7.20 (dd, 1H, J= 1.2, 5.0). 13C NMR (CDCl3): 37.22, 39.38,
70.58, 122.54, 124.38, 126.88, 152.15, 211.10.
1H-NMR (CDCl3): 0.80-0.91 (m, 3H), 1.15-1.80 (m, 16H),
2.53-2.63 (m, 1H, cis isomer), 2.75-2.85 (m, 1H, trans
isomer). ESI-MS m/z 158 [M + H+].
4-amino-1-butylcyclohexan-1-ol (3g).
Yellowish oil. 64 mg, yield 91%
4-benzyl-4'-hydroxyl-cyclohexanone (1j).
White solid. 588 mg, yield 30 %
1H-NMR (CDCl3): 0.82-0.94 (m, 3H), 1.12-1.86 (m, 18H),
2.52-2.64 (m, 1H, cis isomer), 2.85-2.97 (m, 1H, trans
isomer). ESI-MS m/z 172 [M + H+].
1H-NMR (CDCl3): 1.74-1.93 (m, 5H), 2.13-2.22 (m, 2H),
2.57-2.68 (m, 2H), 2.80 (s, 2H), 7.14-7.30 (m, 5H). 13C
NMR (CDCl3): 36.79, 36.85, 48.63, 69.86, 126.90, 128.44,
130.39, 136.40, 211.78.
4-amino-1-phenylcyclohexan-1-ol (3h).
Yellowish oil. 67 mg, yield 95%
1H-NMR (DMSO-d6): 1.54-1.87 (m, 9H), 2.64-2.70 (m,
1H, cis isomer), 7.05-7.11 (t, 1H, J=7.3), 7.17-7.23 (t, 2H,
J=7.3), 7.35-7.40 (d, 2H, J=8.1). ESI-MS m/z 192 [M +
H+].
4-decyl-4'-hydroxyl-cyclohexanone (1k).
White solid. 977 mg. yield 40 %
1H-NMR (CDCl3): 0.84-0.88 (t, 3H, J=6.7), 1.14-1.42 (m,
17H), 1.49-1.56 (m, 2H), 1.71-1.82 (dt, 2H, J=5.2, 13.5),
1.88-1.97 (m, 2H), 2.17-2.26 (m, 2H), 2.62-2.75 (m, 2H).
13C NMR (CDCl3): 14.23, 22.58, 23.69, 29.23, 29.51,
30.08, 31.82, 36.84, 36.96, 42.60, 70.21, 212.01.
4-amino-1-(thiophen-2-yl)cyclohexan-1-ol (3i).
Yellowish oil. 63 mg, yield 89%
1H-NMR (DMSO-d6): 1.51-1.70 (m, 4H), 1.71-1.82 (td,
2H, J=4.2, 13.4)1.88-1.95 (m, 2H), 2.59-2.69 (m, 1H, cis
isomer), 2.74-2.82 (m, 1H, trans isomer), 2.61 (s, 2H),
2.58-2.65 (m, 1H, trans isomer), 6.80-6.84 (dd, 1H, J=3.6,
4.9), 6.84-6.87 (dd, 1H, J=1.2, 3.6), 7.09-7.11 (dd, 1H,
J=1.2, 4.9). ESI-MS m/z 198 [M + H+].
4-amino-1-methylcyclohexan-1-ol (3d).
A mixture of 1d (70 mg, 0.55 mmol), hydroxylamine
hydrochloride (57 mg, 0.82 mmol), sodium acetate (90 mg,
1.09 mmol), water (1 mL) and MeOH (4.5 mL) was stirred
at r.t. for 1 h. Most of the solvent was evaporated under
reduced pressure; the residue was resuspended in 5 mL
water and extracted with AcOEt (4 x 10 mL). The
combined organic fractions were dried over NaSO4 and the
solvent distilled off to give 79 mg of the corresponding
oxime as a white solid, which was used in the next
reactions without purification. To the obtained oxime,
4-amino-1-benzylcyclohexan-1-ol (3j).
Yellowish oil. 66 mg, yield 96%
9
This article is protected by copyright. All rights reserved.

10.1002/adsc.201901434
Advanced Synthesis & Catalysis
1H-NMR (DMSO-d6): 1.16-1.65 (m, 11H), 2.38-2.47 (m,
1H, cis isomer), 7.01-7.17 (m, 5H). ESI-MS m/z 206 [M +
H+].
evaluated by GC-FID following the concentration of
acetophenone, while the cis/trans ratio of the formed
amine was evaluated by HPLC. For the GC-FID, 20 μL
samples were diluted in 0.5 mL of cyclohexane and then
injected without further derivatization. HPLC analysis was
performed at the end of each experiment; the reaction
medium was evaporated under reduced pressure, then
about 2.5 mg of the residue was dissolved in 1 mL HCl
0.05 M. The resulting solution was analyzed as previously
described.
4-amino-1-decylcyclohexan-1-ol (3k).
Yellowish oil. 57 mg, yield 65%
1H-NMR (CDCl3): 0.87-0.92 (t, 3H, J=6.7), 1.22-1.95 (m,
29H), 2.87-2.97 (m, 1H, cis isomer). ESI-MS m/z 256 [M
+ H+].
4-amino-1-(((tetrahydrofuran-2-
Bioaminations in disperse systems.
yl)oxy)methyl)cyclohexan-1-ol (31).
Yellowish oil. 43 mg, yield 61%
1H-NMR (CDCl3): 1.28-1.38 (td, 2H, J= 3.6, 13.0), 1.46-
1.58 (qd, 2H, J= 3.3, 12.5, 23.7), 1.66-1.75 (m, 4H), 1.87-
1.94 (bs, 3H), 1.95-2.01 (m, 2H), 2.58-2.68 (m, 1H), 3.19-
3.27 (q, 2H, J=8.8), 3.75-3.91 (m, 4H), 4.09-4.14 (m, 1H).
ESI-MS m/z 216 [M + H+].
Firstly, the dry solvent (isooctane or MTBE) was heated to
37 °C. Secondly, the surfactant (50 mM) was added and
dissolved. Then, ketone (25 mM), (S)-PEA (50 mM),
decane (50 mM) and the enzymatic solution (1 % v/v)
were sequentially added. 10 μL of the enzyme preparation
contained 0.95 U of enzyme dissolved in HEPES buffer
(50 mM, pH 8.2 or 7.0 depending on the type of enzyme).
The enzyme solution was incubated at 37 °C for at least 30
minutes before use. The reaction mixture was vigorously
shaken. Conversions were evaluated by GC-FID following
the concentration of acetophenone while the cis/trans ratio
of the formed amine was evaluated by HPLC. For the GC-
FID analyses, 20 μL samples were diluted in 0.5 mL
cyclohexane and then injected without any further
derivatization. HPLC analyses were performed at the end
of each experiment. Prior to the HPLC analysis, the
reaction medium was evaporated under reduced pressure,
then about 2.5 mg of the residue was dissolved in 1 mL
HCl 0.05 M. The resulting solution was analysed as
previously described.
Enzyme preparation.
The genes encoding for the Cv-ATA-wt, Cv-ATA-W60C,
Cv-ATA-F88A/A231F, Cv-ATA-W60C/F88A/A231F and
Vf-ATA were inserted in a pET28a(+) vector including an
N-terminal His6-tag. The plasmids were transferred to the
host BL21(DE3) Escherichia coli strain. Cells were grown
at 37 °C in TB medium (Terrific Broth) supplemented with
50 mg/L kanamycin. Protein expression was induced at an
OD600 ∼ 0.7-0.9 by addition of IPTG (1 mM). After 24 h
of incubation at 20 °C and 220 rpm, the cells were
harvested by centrifugation (30 min, 8000 rpm, 4 °C) and
resuspended in IMAC binding buffer (50 mM NaH2PO4,
300 mM NaCl, 10 mM imidazole, pH 8.0). The cells were
disrupted by sonication and the cell debris was removed by
centrifugation (30 min, 20,000 rpm, 4 °C) followed by
filtration (0.45 μm). The purification process was
performed on a Ni-NTA Sepharose column. For Cv-ATA-
wt, Cv-ATA-F88A/A231F and Vfl-ATA the fraction
containing ATA activity was dialyzed against HEPES
buffer (50 mM, pH 8.2) on a PD10 column (GE
Healthcare), according to the manufacturer's protocol.
After addition of PLP (3 mM), the enzyme was incubated
for 24 h at 4 °C. Thereafter, it was dialyzed against HEPES
buffer (50 mM, pH 8.2) to remove the excess of PLP, after
When product isolation was needed, at the end of the
reaction, the solution was brought to 4 °C, incubated for 30
min and centrifuged to facilitate the phase separation. The
supernatant was evaporated and the residue was purified
by column chromatography.
incubation at 37 °C for
1
h. For Cv-ATA-
Acknowledgements
W60C/F88A/A231F and Cv-ATA-W60C the same
procedure was applied using HEPES buffer (50 mM, pH
7.0). The protein concentration of the enzyme preparation
was estimated by absorbance at 280 nm. The enzyme was
stored at 4 °C and was incubated for 30 min at 37 °C prior
to using.
The authors wish to deeply thank Prof. Francesca Baldelli-
Bombelli (Politecnico di Milano) for DLS experiments and
Flamma SpA (Chignolo d’Isola, ITALY) for the kind gift of
substrate 11a.
This project was carried out in the frame of the COST Action
CM1303 “SysBiocat” and dr. Fiorati could benefit from the
Surfactant-protected lyophilized enzyme powders were
prepared as follows: the surfactants, Brij® C10 (15 mg) and
octyl-β-D-glucopyranoside (15 mg), were dissolved in 3
mL HEPES buffer (50 mM, pH 8.2) at 37 °C. Then, the
enzyme was added (3 mL, 6 mg/mL). After mixing, the
solution was rapidly frozen in liquid N2 and lyophilized for
the established time. For side-to-side comparison, half of
the enzyme batch was lyophilized without the addition of
surfactants.
funding for
a “Short Term Scientific Mission” in Prof.
Berglund’s laboratories.
References
Enzyme activity.
The enzymatic activity was assayed spectrophotometrically
at 25 °C by monitoring the formation of acetophenone
from PEA; 1 mL of reaction mixture containing 2 μg (or
150 μg) enzyme, 2.5 mM PEA (S or R depending on the
enantiopreference of the enzyme), 2.5 mM pyruvate and
HEPES buffer (50 mM, pH 8.2) was used. The reaction
was monitored for 2 min following the absorbance increase
at 245 nm (ε = 12 mM-1 cm-1). One unit (1 U) was defined
as the amount of enzyme converting 1 μmol of PEA in 1
min.
[1]
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This article is protected by copyright. All rights reserved.

10.1002/adsc.201901434
Advanced Synthesis & Catalysis
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10.1002/adsc.201901434
Advanced Synthesis & Catalysis
FULL PAPER
Application of Transaminases in a Disperse System
for the Bioamination of Hydrophobic Substrates
Adv. Synth. Catal. Year, Volume, Page – Page
Andrea Fiorati, Per Berglund, Maria S. Humble,
Davide Tessaro*
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