Amine dehydrogenases: Efficient biocatalysts for the reductive amination of carbonyl compounds

Article abstract of DOI:10.1039/c6gc01987k

Amines constitute the major targets for the production of a plethora of chemical compounds that have applications in the pharmaceutical, agrochemical and bulk chemical industries. However, the asymmetric synthesis of α-chiral amines with elevated catalytic efficiency and atom economy is still a very challenging synthetic problem. Here, we investigated the biocatalytic reductive amination of carbonyl compounds employing a rising class of enzymes for amine synthesis: amine dehydrogenases (AmDHs). The three AmDHs from this study-operating in tandem with a formate dehydrogenase from Candida boidinii (Cb-FDH) for the recycling of the nicotinamide coenzyme-performed the efficient amination of a range of diverse aromatic and aliphatic ketones and aldehydes with up to quantitative conversion and elevated turnover numbers (TONs). Moreover, the reductive amination of prochiral ketones proceeded with perfect stereoselectivity, always affording the (R)-configured amines with more than 99% enantiomeric excess. The most suitable amine dehydrogenase, the optimised catalyst loading and the required reaction time were determined for each substrate. The biocatalytic reductive amination with this dual-enzyme system (AmDH-Cb-FDH) possesses elevated atom efficiency as it utilizes the ammonium formate buffer as the source of both nitrogen and reducing equivalents. Inorganic carbonate is the sole by-product.

Full text of DOI:10.1039/c6gc01987k

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Amine dehydrogenases: Efficient biocatalysts for the reductive
amination of carbonyl compounds
Tanja Knaus,† Wesley Böhmer† and Francesco G. Mutti*
Received 00th January 20xx,
Accepted 00th January 20xx
Amines constitute the major targets for the production of a plethora of chemical compounds that have applications in the
pharmaceutical, agrochemical and bulk chemical industries. However, the asymmetric synthesis of α‐chiral amines with
elevated catalytic efficiency and atom economy is still a very challenging synthetic problem. Here, we investigated the
biocatalytic reductive amination of carbonyl compounds employing a raising class of enzymes for amine synthesis: the
amine dehydrogenases (AmDHs). The three AmDHs from this study ‐ operating in tandem with a formate dehydrogenase
from Candida boidinii (Cb‐FDH) for recycling of the nicotinamide coenzyme ‐ performed the efficient amination of a range
of diverse aromatic and aliphatic ketones and aldehydes with up to quantitative conversion and elevated turnover
numbers (TONs). Moreover, the reductive amination of prochiral ketones proceeded with perfect stereoselectivity, always
affording the (R)‐configured amines with more than 99% enantiomeric excess. The most suitable amine dehydrogenase,
the optimised catalyst loading and the required reaction time were determined for each substrate. The biocatalytic
reductive amination with this dual‐enzyme system (AmDH – Cb‐FDH) possesses elevated atom efficiency as it utilizes the
ammonium formate buffer as the source of both nitrogen and reducing equivalents. Inorganic carbonate is the sole by‐
product.
DOI: 10.1039/x0xx00000x
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catalysed by a lipase; however, this process is limited by a
maximum of 50% theoretical yield.5 Quantitative yield into
enantiopure amines can be obtained through dynamic kinetic
Introduction
Amines are the most widely used chemical intermediates for
the production of active pharmaceutical ingredients, fine
chemicals, agrochemicals and a significant number of bulk
chemicals.1,2 Nowadays, the production of amines in the
laboratory, as well as in industrial scale, relies principally on
the reductive amination of carbonyl containing compounds.3
Nonetheless, the chemocatalytic reductive amination requires
precious, and sometimes toxic, metal catalysts coordinated to
sophisticated organic ligands that operate at high pressure of
hydrogen gas. Furthermore, the overall process is quite
lengthy as various protection and deprotection steps are
involved. Finally, a follow‐up recrystallization of the amine
product is often needed in order to improve the enantiomeric
excess, and the traces of heavy metals from the catalyst have
to be removed to comply with legislative requirements. On the
other hand, tremendous advancements in biocatalytic
methods for chiral amines have been reached during the past
two decades.4 An industrially applied method is the kinetic
resolution of racemic amines via the selective acylation
resolution (e.g. combining a hydrolase with a metal‐catalyst)6
or deracemisation and desymmetrisation (e.g. combining an
amine oxidase with a chemical reducing reagent or artificial
metal‐enzymes or Pd nanoparticles)7‐16 Besides these earlier
established methods, the arsenal of enzymes for asymmetric
amine synthesis has been enriched, for instance encompassing
wild‐type as well as engineered ω‐transaminases17‐22 However,
the formal reductive amination of carbonyl compounds by ω‐
transaminases requires supra‐stoichiometric amounts of an
amine donor (e.g. 5 equivalents of alanine or ca. 10
equivalents of 2‐propylamine) and strategies for shifting the
unfavorable thermodynamic equilibrium. For example, the use
of an additional enzyme for removing the co‐product
pyruvate19 or special equipment for the selective evaporation
of the co‐product acetone.21 Whilst the use of alternative
amine donors has demonstrated to provide an improved
thermodynamic driving force, these molecules are expensive
and require multi‐step chemical synthesis or generate co‐
products that polymerise and therefore complicate the work‐
up of the reaction.23‐25 More recently imine reductases have
gained interest, but the substrate scope of these enzymes is
practically limited to cyclic secondary imines.26‐30 Finally, other
enzymes have been applied for chiral amine synthesis such as
ammonia lyases,31‐36 Pictet‐Spenglerases,37‐40 berberine bridge
enzymes41‐43 and engineered P450 monooxygenases.44‐46
Van ‘t Hoff Institute for Molecular Sciences (HIMS), University of Amsterdam,
Science Park 904, 1098 XH, The Netherlands. E‐mail: f.mutti@uva.nl
† These authors contributed equally to the work.
Electronic Supplementary Information (ESI) available: full list of the substrates with
related structures, detailed descriptions of the expression and purification of
the enzymes, full data sets and explanation for the biocatalytic reactions,
NMR spectra, GC analytical methods and data as well as GC traces. See
DOI: 10.1039/x0xx00000x
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However, all these last mentioned classes of enzymes are time in buffers at different pH (see SupportingVieInwfAortricmleaOtniloinne
DOI: 10.1039/C6GC01987K
S5.1). No significant decrease of the absorbance for NADH was
active on rather specific, yet valuable, families of substrates.
In this context, amine dehydrogenases (AmDHs) are a new detected in buffer at pH 6.5 for 24 h, indicating that the
class of enzymes that possess tremendous potential for the coenzyme is stable under this condition. At pH 8.8, the
development of the next generation of processes for the absorbance started to diminish smoothly after 5 h, but it was
synthesis of α‐chiral amines.47 The applicability of this class of still two thirds of the initial value after 24 h. In contrast, NADH
enzymes in organic synthesis has been demonstrated in our was significantly more unstable at pH 10. In fact, the
notable biocatalytic dual‐enzyme hydrogen‐borrowing absorbance decreased linearly and it was halved just after 2 h
amination of alcohols.48,49 In general, AmDHs catalyse the and depleted after 24 h. Finally, the absorbance of NADH in
reductive amination of ketone and aldehyde substrates using NaOH (0.1 N) was reduced to 20% of the initial value in 3
NAD(P)H as the hydride source. The study and exploitation of minutes and fully depleted within 1 h. These data showed that
AmDHs for
biocatalytic
processes
is, however, the ideal pH of 8.2 – 8.8 for the biocatalytic reductive
underdeveloped. In particular, only one report on a natural amination might also be a consequence of a diminished
occurring amine dehydrogenase has been reported about two stability of the nicotinamide coenzyme at higher values of pH.
decades ago but experiments have not been reproduced Hence, ammonium chloride and ammonium formate buffers at
again.50 As a consequence, a narrow panel of AmDHs have pH 8.5 – 8.7 were used for the continuation of our studies.
been created recently through protein engineering starting
from wild‐type amino acid dehydrogenases as scaffold51‐53 or
DNA shuffling of first generation variants.54 Although initial
reaction rates have been determined for the amination of a
limited number of ketones, a systematic investigation on the
substrate acceptance, optimal reaction conditions as well as
chemo‐ and stereoselectivity of the known AmDHs has not
been undertaken until to date. This works aims at providing
Scheme 1. Amine dehydrogenases catalyze the reductive amination of
this knowledge and at showing the potential of AmDHs for the
ketones and aldehydes (50 mM) to chiral amines. Catalytic amount of
efficient asymmetric synthesis of α‐chiral amines.
nicotinamide coenzyme (1 mM) is applied. The reducing equivalents as well
as the nitrogen source are originated from the buffer of the reaction:
ammonia/ammonium formate (pH 8.5, 1 M). The only by‐products are water
Results and discussion
Optimisation of the reaction conditions
and carbonate.
In our previous study, we also determined that ca. 700 mM of
ammonium cation/ammonia was required to achieve > 99%
In our previous study the most elevated reaction rates for the
reductive amination of (para‐fluorophenyl)acetone
(1a),
conversion, at 30 °C, for substrate 1a (20 mM). In that case,
NAD+ was applied in catalytic amount (1 mM) and recycled
using glucose (60 mM) and a commercial engineered GDH.48
Nevertheless, the reaction with glucose as cosubstrate
generates a stoichiometric amount of gluconic acid, hence
reducing the atom economy of the reaction.59 Furthermore,
we had to employ a large amount of GDH (300 U mL‐1) for
sustaining the amination in ammonium buffer (pH 8.7, > 700
mM), due to its mediocre stability under the reaction
conditions. Consequently, in the present study, we envisaged
the recycling system based on formate and FDH (recombinant
enzyme from Candida boidinii)60 to be the preferable
alternative because formate was already present in the
reaction buffer as counteranion of the ammonium species.
Moreover, these new experiments showed that an extremely
low amount of FDH (2.0 – 3.0 U mL‐1) was sufficient to obtain a
quantitative amination. Therefore, we compared the
performance of the reductive amination in the following cases:
i) glucose/GDH (150 U) as system for the recycling of NADH in
ammonium chloride buffer (pH 8.7, 1 M); ii) glucose/GDH (150
U) in ammonium formate (pH 8.5, 1 M) and iii) formate/FDH
(purified, 14 μM equal to 1.5 U) in ammonium formate (pH
8.5, 1 M), (for details, Supporting Information S5.2 and S5.3).
This investigation was extended to the three amine
catalysed by the AmDH variant originated from the wild‐type
L‐phenylalanine dehydrogenase from Bacillus badius (for the
sake of clarity, referred in this work as Bb‐PhAmDH), were
observed in ammonium chloride and ammonium formate at
pH between 8.2 and 8.8.48 Besides the elevated activity and
stability of the enzymes under the operational reaction
conditions, another important and often neglected parameter
to be considered for the reductive amination using AmDHs is
the stability of the coenzyme NADH/NAD+ in solution at more
basic pH. In fact, in practical biocatalytic reactions, the
coenzyme has to be applied in catalytic amounts and recycled
at the expense of a sacrificial substrate such as, among the
others, formate in combination with formate dehydrogenase
(FDH, Scheme 1) or glucose in combination with glucose
dehydrogenases (GDH, not shown).
Surprisingly, studies on the stability of nicotinamide
coenzymes dissolved in aqueous buffers at different pH and
temperature are only a few in the literature. Although NADH is
reported to be fairly stable at basic pH, other publications
suggest that the decomposition of NADH in solution occurs at
55
ambient temperature in alkali or even at nearly neutral pH
with certain type of buffers.56‐58 Hence, in this study, the
structural stability of the reduced form of the coenzyme,
NADH, was monitored spectrophotometrically (λ 325 nm) over
2 | J. Name., 2012, 00, 1‐3
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dehydrogenases that are available in our collection: i) the Bb‐ notable that the stereoselective outcome of the VrieewacAtrtiioclne Ownlianse
DOI: 10.1039/C6GC01987K
PhAmDH, ii) a variant originated from the L‐phenylalanine perfect in all the cases (Table 1, > 99% (R)).
dehydrogenase from Rhodococcus sp. M4 (in this work Aim at understanding the overall catalytic efficiency of the
indicated as Rs‐PhAmDH)53 and iii) a previously described reductive amination under the optimized reaction conditions,
chimeric AmDH (in this work indicated as Ch1‐AmDH)48,54 we increased gradually the concentration of the substrate up
obtained by domain shuffling of the Bb‐PhAmDH with a variant to 50 mM and maintaining the same concentration of AmDH
from
the
leucine
dehydrogenase
from
Bacillus (80 ‐ 130 μM), NAD+ (1 mM), FDH (14 μM), ammonium buffer
stearothermophilus. The reductive aminations were carried (1 M). The Rs‐PhAmDH and Ch1‐AmDH converted the related
out with the representative best substrate for each AmDHs, substrates 24a and 10a (50 mM), respectively, with > 99%
according to our own data and other data from literature (1a
)
conversion and perfect stereoselectivity (> 99% (R)) within 21
for Bb‐PhAmDH, 4‐phenylbutan‐2‐one (24a) for Rs‐PhAmDH h (Supporting Information S5.4). In contrast, Bb‐PhAmDH
and 2‐heptanone (10a) for Ch1‐AmDH). The initially tested turned out to be a less efficient catalyst in this regard as the
reaction conditions were: substrate concentration (20 mM), conversion of 1a at 50 mM concentration slightly dropped to
NAD+ concentration (1 mM), AmDH concentration (80 ‐ 130 88% within 21 h reaction time (Table 1 entry 4 and full data set
μM) and ammonium buffer (1 M), at 30 °C, for 24 h. Under in Supporting Information S5.4). Finally, the catalyst loading
these reaction conditions it was not possible to reach was reduced for the reductive amination employing Rs‐
quantitative conversion (within 21 h) using the glucose/GDH PhAmDH and Ch1‐AmDH. The amination of 24a and 10a (50
recycling system in ammonium chloride buffer with any of the mM) proceeded quantitatively within 21 h using 50 μM of Rs‐
three AmDHs, despite the use of 3 equivalents of glucose PhAmDH and 32 μM of Ch1‐AmDH, respectively. The
(Table 1, entries 1, 5 and 9). Switching from ammonium calculated turnover number (TON)61 was equal or more than
chloride to formate and maintaining the same composition of 1000 and therefore comparable, or even superior, to the
the reaction mixture resulted in quantitative conversion for values previously obtained for the amination of ketones in
the amination of substrates 24a and 10a with Rs‐PhAmDH and aqueous buffers with other enzymes such as ω‐
Ch1‐AmDH (Table 1, entries 6 and 10). However, no transaminases.21,62,63 Moreover, compared to the bio‐
improvement was observed in the case of Bb‐PhAmDH (Table amination with ω‐transaminases, AmDHs do not require an
1, entry 2). When the third, preferred, option with formate as enantiopure amine donor (e.g. L‐ or D‐alanine)19 and inhibition
cosubstrate was tested, all the amination reactions afforded phenomena are not observed (i.e. inhibition due to
the related product with >99% conversion (Table 1, entries 3, 7 cosubstrate alanine and/or coproduct pyruvate).64‐66 As an
and 11). Hence, we deduced that the employed FDH60 can additional parameter, the concentration of FDH could be
recycle NADH more efficiently than the GDH, likely due to its lowered to only 9.5 μM, still providing the same conversion.
higher stability in ammonium/ammonia buffer at pH 8.5. It is
Table 1. Optimization of the reductive amination using AmDHs. The influence of the composition of the buffer solution, the enzyme loading and the
substrate concentration was investigated.
Substrate
concentration
[mM]
Enzyme
concentration
[µM]
Entry
Enzyme
Substrate
coenzyme/buffer system
Conversion [%]
ee% (R)
1
2
Bb‐PhAmDH
Bb‐PhAmDH
Bb‐PhAmDH
Bb‐PhAmDH
Rs‐PhAmDH
Rs‐PhAmDH
Rs‐PhAmDH
Rs‐PhAmDH
Ch1‐AmDH
Ch1‐AmDH
Ch1‐AmDH
Ch1‐AmDH
1a
1a
20
20
20
50
20
20
20
50
20
20
20
50
115
115
115
115
130
130
130
50
GDH/NH4Cl
GDH/HCOONH4
Cb‐FDH/HCOONH4
Cb‐FDH/HCOONH4
GDH/NH4Cl
79
76
>99
>99
>99
>99
>99
>99
>99
>99
>99
>99
>99
>99
3
1a
>99
88
4
1a
5
24a
24a
24a
24a
10a
10a
10a
24a
72
6
GDH/HCOONH4
Cb‐FDH/HCOONH4
Cb‐FDH/HCOONH4
GDH/NH4Cl
>99
>99
>99
61
7
8
9
80
10
11
12
80
GDH/HCOONH4
Cb‐FDH/HCOONH4
Cb‐FDH/HCOONH4
99
80
>99
98
32
Reaction conditions: NAD+ (1 mM); recycling enzyme (GDH or Cb‐FDH) in ammonium chloride buffer (1 M, pH 8.7) or ammonium formate buffer (1.005 M, pH 8.5);
reaction volume 0.5 mL, temperature 30 °C, reaction time 24 h; agitation on an orbital shaker 190 rpm.
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Influence of the temperature and time studies
PhAmDH was affected at 60 °C as the conversion raised
smoothly, reaching a maximum of 93% only after 30 h.
Conversely, the chimeric enzyme Ch1‐AmDH performed the
amination of 10a almost equally well in the range of
temperatures investigated that spans from 30 °C to 60 °C. In
fact, after 5 h the conversion was above 90% for the
aminations at 30 °C, 40 °C and 50 °C and reached 82% at 60 °C.
The rate of the reductive amination, instead, was lower at 20
°C.
Under the selected reaction conditions (ammonium formate
buffer pH 8.5, 1M; substrate concentration 50 mM; NAD+ 1
mM; FDH 14 μM; varied concentration of AmDHs), we studied
the influence of the temperature on the progress of the
reductive amination. In fact, we postulated that an increase in
the temperature might accelerate the kinetics of the reaction,
whereas an excessive temperature may be detrimental for the
stability of the enzymes.
The progress for the reductive amination of 1a (50 mM) using
Bb‐PhAmDH (46 μM) showed a consistent increase in the
reaction rate when the temperature was raised from 20 °C, to
30 °C and finally 40 °C (Figure 1A). It is interesting to note that,
for this enzyme, the conversion increased almost linearly over
time for every temperature tested. Furthermore, the final
conversion (taken after 24 h) at 40 °C doubled the value
observed at 20 °C (83% vs. 37%). Nonetheless, the progress of
the reaction at 50 °C was worse than at 20 °C, leading to a
mediocre conversion of 21 % after 24 h. A further increase of
the temperature up to 60 °C provoked a complete loss of the
enzymatic activity (Supporting Information S5.6). The lack of
conversion at 60 °C cannot be attributed to the deactivation of
the FDH or the decomposition of the coenzyme NAD since the
reductive amination well performed up to 60 °C with Ch1‐
AmDH as the biocatalyst (Figure 1C). Furthermore, our data
are in agreement with the profile of activity vs. stability of Bb‐
PhAmDH that shows a rapid denaturation of the enzyme
above 50 °C.52
The reaction profiles for the reductive amination of substrates
24a and 10a (50 mM) with Rs‐PhAmDH (48 μM) and Ch1‐
AmDH (33 μM) respectively, were significantly different from
the previous one. For both Rs‐PhAmDH and Ch1‐AmDH, the
conversions increased hyperbolically over time (Figure 1B and
1C). In particular, Rs‐PhAmDH is an extremely active enzyme
on its preferred substrate 10a. Considering the first hour of the
reaction, wherein the conversion correlated linearly with time,
the maximum turnover frequency (TOF) was reached already
at 20 °C. In particular the increase of the temperature in the
range 20 °C, 30 °C, 40 °C and 50 °C led always to the same
conversion after 1 h (varying from 80% ‐ 83%). Quantitative
conversion (>98%) of 24a was obtained at 20 °C and 30 °C
within 3 h (Figure 1B and Supporting Information S5.7). The
efficiency at 40 °C was slightly lower as the reaction required 5
h to overcome 99% conversion. In contrast, the kinetics of the
reaction was negatively influenced at 50 °C with a drop of the
catalytic activity after 1 h. In fact, additional 29 h were
required to increase the conversion from 83% (after 1h) to
98% (after 30 h) at this temperature. The activity of Rs‐
Figure 1. Progress of the reaction versus the time for the reductive
amination of: A) 1a using Bb‐PhAmDH (46 μM), B) 24a using by Rs‐PhAmDH
(48 μM), and C) 10a using Ch1‐AmDH (33 μM). The study was carried out at
different temperatures: 20 °C (black), 30 °C (yellow), and 40 °C (blue), 50 °C
(green), and 60 °C (red). Reaction conditions: 0.5 mL, 50 mM substrate,
ammonium formate buffer (1.00 M, pH 8.5), FDH (14 μM).
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Nevertheless, only with Ch1‐AmDH, quantitative conversion (> deamination of a few amines catalysed by AmDHs have been
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98%) was obtained at every temperature from 20 °C to 60 °C at previously measured.51,52 However, a study describing the
DOI: 10.1039/C6GC01987K
the end of the reaction (30 h, Supporting Information S5.8). substrate scope of these enzymes for the organic synthesis of
The Ch1‐AmDH/Cb‐FDH dual enzyme system for the reductive amines from prochiral ketones and aldehydes has not been
amination was instead inapplicable at 70 °C, albeit a mediocre published so far. Moreover, the information regarding the
conversion (8%) was observed at this temperature after 18 h. stereoselectivity for the amination with AmDHs is limited to a
Our observation is in agreement with the previously very few compounds. Therefore, in this research, we tested an
determined half‐life of 40 min for Ch1‐AmDH at 70 °C.54
extensive library of structurally diverse prochiral ketones such
Regardless to the degree of conversion, the AmDH enzyme and as phenylacetone derivatives and phenylacetaldehyde (1‐7a),
the substrate employed, the enantiomeric excess was not aliphatic methylketones and aldehydes (8‐13a), acetophenone
affected by the reaction time or the temperature. The derivatives (14‐19a) and, finally, a selection of more sterically
stereoselectivity remained always perfect (> 99% (R)).
demanding ketones (20‐25a) (Figure 2). The substrate
concentration was kept at 50 mM, whereas the amount of
enzyme and the reaction time was varied in order to achieve
the maximum efficiency (i.e. highest ratio [S]/[E] with highest
conversion).
Current substrate scope of the reductive amination using
AmDHs
The initial reaction rates for the reductive amination of a
limited number of carbonyl compounds and the oxidative
Figure 2. Substrate scope tested by amine dehydrogenases.
First, we examined the family of phenylacetone derivatives other substituted phenylacetones. Indeed, Bb‐PhAmDH
(Figure 2, Table 2). It was previously shown that Bb‐PhAmDH converted ortho‐, meta‐ and para‐ methoxy substituted
accepts (para‐fluorophenyl)acetone
(
1a
)
as the best phenylacetone derivatives (2‐4a), but the conversion was
substrate.52 In our independent experiment (Table 2, entry 2), mediocre (from 3% to 21%, Supporting Information Table S7)
Bb‐PhAmDH (50 μM) converted 1a (50 mM) to 93% of the despite the reaction time was prolonged up to 48 h. Bb‐
amine product 1b within 48 h and perfect stereoselectivity PhAmDH also accepted (para‐methyl)phenylacetone (5a, 7%
(>99% (R)). Hence, it may be logical to assume that Bb‐ conversion), whilst (meta‐trifluoromethyl)phenylacetone (6a
)
PhAmDH can also be a useful biocatalyst for the amination of was not converted at all (Supporting Information Table S7).
Table 2. Reductive amination of phenyl 2‐propanone derivatives and phenyl‐acetaldehyde employing AmDHs.
Enzyme
concentration [µM]
Time
[h]
Conversion
substrate [%]
ee%
(R)
Entry
Nr.
Enzyme
1
2
3
4
5
6
7
8
1a
1a
2a
3a
4a
5a
6a
7a
Ch1‐AmDH
Bb‐PhAmDH
Ch1‐AmDH
Rs‐PhAmDH
Rs‐PhAmDH
Rs‐PhAmDH
Rs‐PhAmDH
Bb‐PhAmDh
30
50
24
48
48
24
24
48
48
48
93
93
>99
>99
>99
>99
>99
>99
>99
n.a.
130
50
>99
98
50
>99
98
130
130
50
98
34
Reaction conditions: substrate 50 mM, AmDH 30‐130 µM, Cb‐FDH 14 µM, ammonium formate buffer (1.005 M, pH 8.5), T 30°C, agitation orbital shaker 190 rpm.
n.a not applicable
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Surprisingly the chimeric enzyme Ch1‐AmDH, known to be equal to 98%) and excellent stereoselectivity (> 99% (R)),
active on aliphatic ketones48 and acetophenone derivatives,54 (Table 2, entries 4‐7). On the other hand, Bb‐PhAmDH proved
was a superior catalyst for the amination of 1a. Compared to to be still a useful catalyst for the amination of phenyl‐
the amination with Bb‐PhAmDH, Ch1‐AmDH afforded the acetaldehyde (7a), (Table 2, entry 8). In contrast, both Rs‐
product 1b with the same conversion but in half of the PhAmDH and Ch1‐AmDH were inactive on this type of
reaction time (24 h) and at a significantly lower enzyme aldehyde.
loading (30 μM, Table 2, entry 1). Ch1‐AmDH was also the best A similar scenario was revealed also in the case of the
catalyst for the amination of 2a that reached quantitative reductive amination of aliphatic ketones and aldehydes (Figure
conversion in 48 h (Table 2, entry 3).
2, Table 3). Initially, we investigated the less sterically
The third AmDH from this study, Rs‐PhAmDH, has been demanding alkyl methylketones. Ch1‐AmDH and Rs‐PhAmDH
developed and tested only for the reductive amination of 4‐ were the most active biocatalysts on this family of substrates.
phenylbutan‐2‐one as substrate (24a, Table 5, entry 6).53 In Substrates (50 mM) bearing a medium length linear chain such
this study we disclosed that Rs‐PhAmDH has a much wider as 2‐hexanone (9a) and 2‐heptanone (10a) were efficiently
substrate scope than expected and reported before. converted by Ch1‐AmDH (30 μM) within 24 h (Table 3, entries
Interestingly Bb‐PhAmDH and Rs‐PhAmDH were engineered 2 and 3). Ketones bearing a shorter linear chain (2‐pentanone,
from their respective parent wild‐type phenylalanine 8a) or a branched chain (4‐methylpentan‐2‐one, 12a) were
dehydrogenases by mutating similar positions in the active site converted with lower turnover numbers by Ch1‐AmDH and,
(K78, N277 for Bb‐PhAmDH and K66, S149 and N262 for Rs‐ therefore, an elevated concentration of enzyme was necessary
PhAmDH). In fact, in the wild‐type enzymes, the side chains of (130 μM, Table 3, entries 1 and 7). A ketone bearing a longer
the K and N residues located in the active site generate chain such as 2‐octanone (11a) was a challenging substrate for
hydrogen bond interactions with the oxygens of carboxylic Ch1‐AmDH (Table 3, entry 5). Fortunately, Rs‐PhAmDH seems
moiety of the natural substrate. Furthermore, the two parent to be a complementary enzyme in this respect: Rs‐PhAmDH
phenylalanine dehydrogenases have 34% identity. Despite (50 μM) aminated 2‐octanone (50 mM, 11a) with elevated
these similarities, we have shown before (Figure 1) that the conversion (93%, table 3, entry 6). Notably, the enantiomeric
two variants have a different thermal stability and reactivity excess was perfect for all the reductive aminations (>99% (R)).
measured on their respective preferred substrate: Rs‐PhAmDH Interestingly, Ch1‐AmDH and Rs‐PhAmDH rapidly converted 3‐
can operate efficiently at 60 °C, whereas Bb‐PhAmDH is methylbutanal (13a, Table 3, entry 9 and 10) even though both
completely deactivated above 50 °C. Table 2 reveals that Rs‐ enzymes were inactive on the arylaliphatic aldehyde 7a. These
PhAmDH is a superior biocatalyst than Bb‐PhAmDH also in data demonstrate that all the known AmDHs are capable of
terms of substrate acceptance. In fact, Rs‐PhAmDH was the converting ketones and aldehydes although with different
optimal AmDH for the conversion of 3a, 4a, 5a and 6a substrate scope.
affording the related amines in elevated conversion (more or
Table 3. Reductive amination of aliphatic methyl ketones and aldehydes employing AmDHs.
Enzyme
concentration [µM]
Time
[h]
Conversion
substrate [%]
ee%
(R)
Entry Nr.
Enzyme
1
2
3
4
5
6
7
8
9
8a
9a
Ch1‐AmDH
Ch1‐AmDH
Ch1‐AmDH
Rs‐PhAmDH
Ch1‐AmDH
Rs‐PhAmDH
Ch1‐AmDH
Rs‐PhAmDH
Ch1‐AmDH
130
30
48
24
24
24
48
48
48
48
24
75
92
98
99
50
93
96
91
>99
>99
>99
>99
>99
>99
>99
>99
>99
n.a
10a
30
50
11a
12a
13a
30
50
130
130
30
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10
Rs‐PhAmDH
130
48
99
n.a
View Article Online
DOI: 10.1039/C6GC01987K
Reaction conditions: 0.5 mL, 50 mM substrate, 30‐130 µM enzyme, ammonium formate buffer (1.005 M, pH 8.5), 14 µM Cb‐FDH.
n.a not applicable
Acetophenone derivatives (Figure 2) are discussed separately 4, the para‐methyl substituent in 14a is the one possessing the
as the type and position of the substituents on the phenyl ring most intense electron donating character, while the meta‐
affect considerably the reactivity of these substrates, due to fluoro in 19a is the strongest electron withdrawing
the existence of resonance and field effects.67 This substitutions. Furthermore para‐hydroxy acetophenone,
phenomenon is not always considered for enzymatic reactions, whose hydroxyl substituent has an even higher electron
for which a low catalytic rate is often solely ‐ and sometimes donating impact than a para‐methyl,67,68 was not converted at
misleadingly ‐ interpreted as the result of a poor affinity of the all (Supporting Information Table S6).
substrate to the active site of the enzyme or the intrinsic low Although not based on a more rigorous determination of the
enzymatic turnovers (kcat). In contrast,
a number of initial reaction rates, this initial observation may suggest that
publications on the reactivity of acetophenone derivatives the enzymatic reductive amination with AmDHs is favored by
with other oxidoreductases such as alcohol dehydrogenases the delocalization of a higher partial positive charge on the
showed that resonance and field effects can play a major, and reactive carbonyl carbon during the reaction. This assumption
sometimes unexpected, role.68‐70
is corroborated by the fact that the same influence of the
In this regard, the chimeric Ch1‐AmDH and Rs‐PhAmDH turned substituents was reveled for the reduction of acetophenone to
out to be the most efficient enzymes for the amination of alcohols with alcohol dehydrogenases (ADH). In fact, both
acetophenone derivatives (Table 4 and Supporting Information ADHs and AmDHs belong to the class of the oxidoreductases
Table S8). The reductive amination of these substrates (50 (EC1) and share the same cofactor (NAD) and a similar reaction
mM) required generally a higher amount of enzyme (up to 130 mechanism.68‐71 Additionally, none of the AmDHs from this
μM)
Methylacetophenone (14a) was the less converted substrate indicating that steric effect might also play a significant role in
(9%, table 4, entry 1) whereas meta‐fluoroacetophenone (19a the enzymatic reductive amination (Supporting Information
for
obtaining
moderate
conversions.
para‐ study accepted ortho‐methyl acetophenone as a substrate,
)
afforded the most elevated conversion (43%, table 4, entry 6). Table S6).
Along the series of acetophenone derivatives reported in table
Table 4. Reductive amination of acetophenone derivatives employing AmDHs.
Enzyme
concentration [µM]
Time
[h]
Conversion
substrate [%]
ee%
(R)
Entry Nr.
Enzyme
1
2
3
4
5
6
14a
15a
16a
17a
18a
19a
Ch1‐AmDH
Ch1‐AmDH
Ch1‐AmDH
Ch1‐AmDH
Rs‐PhAmDH
Ch1‐AmDH
130
50
48
48
48
48
48
48
9
>99
>99
>99
>99
>99
>99
39
34
22
33
43
130
130
100
130
Reaction conditions: 0.5 mL, 50 mM substrate, 30‐130 µM enzyme, ammonium formate buffer (1.005 M, pH 8.5), 14 µM Cb‐FDH.
Finally, the reactivity of the AmDHs was investigated on more phenyl ring such as 1‐phenylpropan‐1‐one, 1‐phenylbutan‐1‐
sterically demanding substrates. Bulky‐bulky ketones (20‐23a
)
one and 1‐phenylpentan‐1‐one were either not accepted or
(Figure 2) are challenging substrates for the amination afforded mediocre conversions (Supporting Information Table
catalysed by other enzymes such as ω‐transaminases. Natural S6). Conversely, when the carbonyl moiety was positioned
occurring ω‐transaminases, that are suitable for applications in further from the aromatic ring, the ketones (50 mM) were
biotechnology, seem to accept mainly ketones possessing a converted efficiently by Rs‐PhAmDH (90‐130 μM). For
bulky group on one side and a small methyl group on the other instance, 1‐phenylbutan‐2‐one (21a), 1‐phenylpentan‐2‐one
side.19,20,72‐77 Alternatively, wild‐type ω‐transaminases are
(22a) and 1‐phenylpentan‐3‐one (23a) afforded the amine
active on bicyclic ketones.78 To the best of our knowledge, only product up to >99% conversion and perfect stereoselectivity
one scaffold from a wild‐type ω‐transaminase was engineered (>99% (R)), (Table 5, entries 3, 4 and 5). In contrast, Bb‐
for accepting bulky‐bulky substrates.21,76
PhAmDH and Ch1‐AmDH were poorly active on these
Thus, we were intrigued to understand whether AmDHs also substrates or not active at all (Supporting Information Table S7
share the same limitation in relation to the range of ketones and S8).
that can be converted. Interestingly, all the tested bulky‐bulky As an example of an aliphatic and more sterically demanding
ketones bearing the carbonyl moiety conjugated with the ketone, 3‐heptanone (20a) was tested. In this case Ch1‐AmDH
This journal is © The Royal Society of Chemistry 20xx
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Journal Name
was the most active enzyme (Table 5, entry 1) in agreement (96%, Table 5, entry 7). The Ch1‐AmDH also quantitatively
View Article Online
with the general trend for this enzyme (i.e. elevated converted 25a. However, surprisingly,DbOeI:s1i0d.1e0s39t/hCe6GCd0e1s9ir8e7dK
conversions for aliphatic ketones, table 3).
product
3‐phenylpropan‐1‐amine
25b
(70%),
the
For the sake of completeness, 4‐phenyl‐butan‐2‐one (24a) and hydrocinnamic alcohol was obtained as side‐product (30%). To
an aldehyde such as hydrocinnamaldehyde (25a) were assayed the best of our knowledge, this is the only documented case
for the reductive amination. Rs‐PhAmDH (50 μM) was capable wherein an amine dehydrogenase reduces
a carbonyl
of quantitatively aminating 24a at 50 mM scale within 24 h compound leading to the formation of significant amount of
(Table 5, entry 6) with more than 99% ee. Rs‐PhAmDH was also alcohol as by‐product.
the optimal biocatalyst for the reduction of the aldehyde 25a
Table 5. Reductive amination of bulky‐bulky ketones employing AmDHs.
Enzyme
concentration [µM]
Time
[h]
Conversion
substrate [%]
ee%
(R)
Entry Nr.
Enzyme
1
2
3
4
5
6
7
20a
Ch1‐AmDH
Rs‐PhAmDH
Rs‐PhAmDH
Rs‐PhAmDH
Rs‐PhAmDH
Rs‐PhAmDH
Rs‐PhAmDH
90
48
48
48
48
48
24
48
57
32
>99
>99
>99
>99
>99
>99
n.a.
100
130
100
100
50
21a
22a
23a
24a
25a
>99
71
87
>99
96
100
Reaction conditions: 0.5 mL, 50 mM substrate, 30‐130 µM enzyme, ammonium formate buffer (1.005 M, pH 8.5), 14 µM Cb‐FDH.
n.a not applicable
Representative biocatalytic reductive amination in
preparative scale
Additionally, amine (R)‐4b is the optically active intermediate
for the synthesis of the blockbuster pharmaceutical formoterol
80, 81
sold under various trade names including Foradile and
In order to ascertain that our optimized reaction conditions at
analytical scale are applicable in preparative scale biocatalytic
reactions, we attempted the asymmetric amination of (para‐
methoxy)phenylacetone (4a, 208 mg) using Rs‐AmDH (Scheme
2).
Oxeze.
Conclusions
In this work, we have shown that amine dehydrogenases hold
the premise for the development of the next generation of
chemical processes for the synthesis of α‐chiral amines.47 The
applicability of amine dehydrogenases was demonstrated for
the asymmetric amination of a range of structurally diverse
prochiral ketones and aldehydes. The most suitable enzyme,
the optimal catalyst loading and reaction times were
determined for each substrate from this study. The majority of
the substrates tested were aminated with elevated conversion
and elevated TONs; moreover, all the α‐chiral amine products
were obtained with perfect optical purity (>99% R). This fact is
of particular interest as ω‐transaminases capable of giving
access to (R)‐configured amines are rare in nature. Only a very
The reaction was successfully performed with ca. 50 mM
substrate (208 mg), 43 µM of Rs‐AmDH, 15 µM FDH, 1 mM of
NAD+ in ammonium formate buffer (1 M, pH 8.5) at the
temperature of 30 °C. The substrate was converted into the
optically pure amine (R)‐4b with 91% conversion and >99% ee
within 24 h reaction time. After work‐up, (R)‐4b was isolated
with 82% yield. The purity and authenticity of the product was
confirmed by NMR and GC (Supporting Information S6). Amine
(R)‐4b was recently reported as an important building block for
the synthesis of tacrine–selegiline hybrids that possess
cholinesterase and monoamine oxidase inhibition activities for
the treatment of Alzheimer's disease.79
82,83
few (R)‐selective ω‐transaminases have been discovered
and only one enzyme was engineered to convert a specific
bulky‐bulky ketone with elevated stereoselectivity.21,62From
this study, it became also evident that a single amine
dehydrogenase capable of accepting a large variety of
substrates is not available. For instance, the chimeric Ch1‐
AmDH is very active on aliphatic ketones and acetophenone
derivatives whereas Rs‐PhAmDH is an excellent biocatalyst for
the amination of phenylacetone derivatives and more
sterically demanding ketones. Additionally, the influence of
the temperature on the biocatalytic reductive amination with
Scheme 2. Preparative reductive amination of (para‐methoxy
phenyl)acetone (4a, 208 mg) using Rs‐PhAmDH. Reaction conditions:
ammonium formate buffer (1 M, pH 8.5), T 30 °C, agitation on an orbital
shaker (190 rpm), reaction time 24 h.
8 | J. Name., 2012, 00, 1‐3
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the three AmDHs variants was determined. In particular, a pH After centrifugation, the organic layer was driedVwiewithArtiMclegOSnOlin4e.
DOI: 10.1039/C6GC01987K
in the range of 8.2 – 8.8 is preferable as a consequence of the Enantiomeric excess was determined by GC with a Variant
improved stability of the enzymes (AmDHs and FDH) and, Chiracel DEX‐CB column. Details on the GC analysis and
especially, of the coenzyme (NAD). Finally, the optimised methods are reported in the Supporting Information
reaction parameters were applied to the synthesis of an paragraph S7.
important drug precursor on a scale of multi‐hundreds of
milligrams.
Preparative biocatalytic reductive amination for the synthesis
)‐4b
The reductive amination catalyzed by amine dehydrogenases of (
R
operating in tandem with formate dehydrogenase possesses
NAD+ (final concentration 1 mM) was dissolved in ammonium
formate buffer (30 mL, 1.005 M, pH 8.5) in a 50 mL round
bottom flask. Ketone 4a (195 µL, 1.27 mmol), FDH (233 µL
from stock solution 80.7 mg mL‐1, final concentration 15 µM,),
and Rs‐PhAmDH (1.02 mL from stock solution 48.8 mg mL‐1,
final concentration 43 µM) were added and the reaction
mixture was shaken in an incubator at 30 °C for 24 hours. The
progress of the reaction was monitored by TCL and GC. When
quantitative conversion was achieved, the reaction mixture
was acidified to pH 2‐4 via addition of HCl (1M). The water
layer was washed with methyl tert‐butyl ether (15 mL) to
remove any possible remaining ketone starting material. The
pH of the water phase was increased to basic pH via KOH (10
M) while cooling in an ice bath. The water layer was extracted
with methyl tert‐butyl ether (2 x 15 mL). The organic fractions
containing the amine product were combined and dried over
MgSO4. After filtration and evaporation of the solvent, the
product was obtained in pure form. Column chromatography
was not required. The authenticity of the product was
an elevated atom efficiency as the ammonium formate buffer
is simultaneously the source nitrogen and reducing
equivalents. Stoichiometric inorganic carbonate is the sole by‐
product. Additionally, the reductive amination catalyzed by
AmDH / FDH is performed under atmospheric pressure. In
contrast, the amination catalysed by ω‐transaminases in
industrial scale, in aqueous system and using 2‐propylamine
(ca. 10 equivalents) as amine donor requires a removal of the
co‐product acetone by employing reduced pressure and
nitrogen sweep. Hence, besides the requirement of supra‐
stoichiometric amount of 2‐propylamine and the generation of
one equivalent of acetone, a significant amount of energy is
consumed to operate at low pressure. We expect therefore
that the herein described reductive amination will be applied
increasingly in the future when new amine dehydrogenase
variants possessing expanded substrate scope and
complementary stereoselectivity will become available. Co‐
expression of AmDH/FDH in a single host organism and / or co‐
immobilisation of both enzymes will enhance the practical
applicability of the biocatalytic process.84‐86
1
1
confirmed by H‐NMR (Supporting Information Figure S4). H
NMR (400 MHz, CDCl3): δ 7.12 (d, J = 8.6 Hz, 2H), 6.86 (d, J =
8.7 Hz, 2H), 3.81 (s, 3H), 3.13 (m, 1H), 2.67 (dd, J = 13.4, 5.3 Hz,
1H), 2.47 (dd, J = 13.4, 8.1 Hz, 1H), 1.12 (d, J = 6.3 Hz, 3H).
Experimental
General
Keywords
The AmDH variants and the Cb‐FDH were expressed as
recombinant enzymes in E. coli BL21 (DE3). Details are amine dehydrogenases, stereoselective reductive amination,
reported in the Supporting Information paragraph S4.
biocatalysis,α‐chiral amine synthesis
General optimized procedure for the biocatalytic reductive
amination on analytical scale
Competing financial interest
The authors declare to have no competing interests, or other
interests that might be perceived to influence the results
and/or discussion reported in this article.
The reactions were conducted in ammonium formate buffer
(1.005 M, pH 8.5, final volume 0.5 mL) containing NAD+ (final
concentration 1 mM). Enzymes AmDH (30 ‐ 130 µM) and Cb‐
FDH (14.1 µM) and the substrate (50 mM) were also added.
The reactions were run at 30 °C in an incubator for 21 hours
(190 rpm) or longer if required in selected cases. Work‐up was
performed by the addition of KOH (100 µL, 10 M) followed by
the extraction with dichloromethane (600 µL). The water layer
was removed after centrifugation and the organic layer was
dried with MgSO4. Conversion was determined by GC with an
Agilent DB‐1701 column. The enantiomeric excess of the
amine product was determined after derivatization to
acetamido. Derivatization of the samples was performed by
adding 4‐dimethylaminopyridine in acetic anhydride (40 µL of
stock solution 50 mg mL‐1). The samples were shaken in an
incubator at RT for 30 minutes. Afterwards water (300 µL) was
added and the samples were shaken for additional 30 minutes.
Acknowledgements
This project has received funding from the European Research
Council (ERC) under the European Union’s Horizon 2020
research and innovation programme (grant agreement No
638271, BioSusAmin).
Dutch funding from the NWO Sector Plan for Physics and
Chemistry is also acknowledged.
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Page 11 of 11
Green Chemistry
View Article Online
DOI: 10.1039/C6GC01987K
The optimised dual-enzyme (AmDH-FDH) reductive amination of a broad range of carbonyl
compounds affords enantiopure amines with conversion up to 99% using ammonia as amine
donor and formate as reducing reagent.