Formal asymmetric biocatalytic reductive amination

Article abstract of DOI:10.1002/anie.200803763

All for one: A combination of three biocatalysts (ω-transaminase, alanine dehydrogenase, and an enzyme such as formate dehydrogenase for cofactor recycling) catalyze a cascade to achieve the asymmetric transformation of a ketone into a primary α-chiral unprotected amine through a formal stereoselective reductive amination (see scheme). Only ammonia and the reducing agent (formate) are consumed during this reaction. (Chemical Equation Presented).

Full text of DOI:10.1002/anie.200803763

Angewandte
Chemie
DOI: 10.1002/anie.200803763
Chiral Amines
Formal Asymmetric Biocatalytic Reductive Amination**
Dominik Koszelewski, Ivꢀn Lavandera, Dorina Clay, Georg M. Guebitz, David Rozzell, and
Wolfgang Kroutil*
Asymmetric methods to prepare optically active a-chiral
primary amines are highly demanded in asymmetric synthesis
owing to the biological/pharmacological activity of many
amines.[1] Various techniques have been reported, such as
asymmetric 1,2-addition to imines and asymmetric amination
of a,a-disubstituted aldehydes,[2] transformation of allylic
alcohols into amines,[3] (dynamic) kinetic resolution,[4] and
cyclic deracemization[5] employing racemic amines as sub-
strates. Asymmetric reductive amination of ketones has been
investigated with transition-metal catalysts[6] and organo-
catalysts,[7] as well as via sulfinyl imine intermediates.[8]
Although tremendous progress in organo/metal catalysis has
been achieved for the asymmetric reductive amination of
ketones to access a-chiral amines, improved protocols are still
required that are simple, green, and economically viable and
that lead to high enantiomeric excesses.
Biocatalytic reductive amination or transamination is well
established for accessing a-amino acids from the correspond-
ing a-keto carboxylic acids.[9] However, the situation is
different for primary amines that are not adjacent to a
carbonic acid moiety. w-Transaminases[4a–c,10] have recently
received attention for the preparation of such a-chiral
unprotected amines. w-Transaminases are employed mainly
in one way, namely for the kinetic resolution of racemic chiral
amines;[11] only a few reports deal with asymmetric syn-
thesis[12] by starting from a prochiral ketone, probably due to
problems in shifting the equilibrium to the product side, as
well as due to the moderate stereoselectivity of the employed
w-transaminases. These asymmetric synthetic processes usu-
ally require at least stoichiometric amounts of an amine donor
(for example, alanine). The latter leads to a side product
(pyruvate), which has to be removed during the transforma-
tion by using, for instance, pyruvate decarboxylase[12a] or
lactate dehydrogenase.[12c] Additionally, limitations due to
inhibition by the product amine and by pyruvate have been
reported. An ideal process would use ammonium as the
amine donor, together with a cheap reducing agent (for
example, formate, hydrogen, or glucose; see Scheme 1). Even
Scheme 1. Ideal reductive amination of ketones to form a-chiral
amines at the expense of glucose or formate as a reducing agent.
fewer reports employing biocatalysts can be found in the
literature for such a reductive amination of ketones (excluding
a-keto carbonic acids and aldehydes) by employing an
enzyme, nicotinamide adenine dinucleotide (phosphate) in
the reduced form (NAD(P)H), and ammonia.[13] Unfortu-
nately, to the best of our knowledge, no enzyme catalyzing
this reaction has been identified by DNA sequencing.
Therefore, we envisaged a cascade that emulates the
reaction scheme outlined in Scheme 1. For this purpose, we
combined three enzymes: 1) an w-transaminase transfers the
amino group from alanine to the substrate to be converted
(Scheme 2), to give the desired amine and pyruvate; 2) an
amino acid dehydrogenase (for example, alanine dehydro-
genase) recycles alanine from pyruvate by consuming ammo-
nium and NAD(P)H; 3) finally, the cofactor is recycled[14] by
using standard methods (for example, formate dehydrogenase
and formate, glucose dehydrogenase and glucose).[15] In this
concept, alanine is not consumed but recycled.
[*] Dr. D. Koszelewski, Dr. I. Lavandera, D. Clay, Prof. W. Kroutil
Research Centre Applied Biocatalysis, c/o Department of Chemistry,
Organic and Bioorganic Chemistry, University of Graz, Heinrich-
strasse 28, 8010 Graz (Austria)
Fax: (+43)316-380-9840
E-mail: wolfgang.kroutil@uni-graz.at
Prof. G. M. Guebitz
Institute of Environmental Biotechnology, Research Centre Applied
Biocatalysis, University of Technology, Petersgasse 12, 8010 Graz
(Austria)
Dr. D. Rozzell
Codexis, Inc., Redwood City, CA (USA)
Scheme 2. Outline of the formal reductive amination concept for the
preparation of optically pure amines. DMSO: dimethylsulfoxide;
AADH: a-amino acid dehydrogenase; NAD(P)+: nicotinamide adenine
dinucleotide (phosphate) in the oxidized form; FDH: formate dehydro-
genase; GDH: glucose dehydrogenase.
[**] Financial support by the FFG and the Province of Styria is gratefully
acknowledged. Codexis is thanked for providing various enzymes.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.200803763.
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In a first experiment, we tried this approach by using
acetophenone as the substrate and commercial w-transami-
nase from Vibrio fluvialis; however, we could not detect any
conversion (< 1%) into the desired amine.
Therefore, we moved back one step and searched for
better suitable w-transaminases. We tested 19 commercially
available w-transaminases for the transamination of 2-penta-
none (1a) by employing alanine (5 equiv) as the amine donor
and lactate dehydrogenase for the removal of pyruvate. Only
two w-transaminases, ATA-113 and ATA-117, showed high
activity and stereoselectivity for the transamination of this
ketone. ATA-113 led to the S enantiomer (> 99% ee) and
ATA-117 led to the R enantiomer (> 99% ee). In the absence
of the lactate dehydrogenase, no measurable conversion was
detected; this can be attributed to the nonfavored equilibrium
between pyruvate/amine and alanine/ketone, which is
strongly on the side of alanine/ketone.[12a]
Having identified a suitable w-transaminase, we tried the
reductive amination concept (Scheme 2) on 2-pentanone (1a)
at a preparative concentration (50 mm) by employing ATA-
113 with l-alanine dehydrogenase and cofactor recycling. To
increase the solubility of the lipophilic substrates in aqueous
solution, DMSO (15% v/v) was added. The reaction was
started with just half an equivalent of l-alanine. To our
delight, a conversion value of 56% clearly indicated that the
l-alanine was recycled;[16] however, the reaction time was
rather long (68 h), whereby we identified the w-transaminase
as catalyzing the rate-limiting reaction step. To decrease the
reaction time, either a larger amount of w-transaminase or a
larger amount of alanine could be used. Since the latter
option seemed to be cheaper, we measured the conversion at
various l-alanine concentrations (Figure 1).
Scheme 3. Amines 2a–i were prepared by formal asymmetric reductive
amination from the corresponding ketones.
Table 1: Formal biocatalytic reductive amination according to Scheme 2
by employing w-transaminase ATA-113.
Substrate
Product
c [%][a]
ee [%][b]
1a
(S)-2a
(S)-2b
(S)-2c
(S)-2d
(S)-2e
(S)-2 f
(S)-2g
(S)-2h
2i
92
>99
89
93
>99
>99
>99
34
1b
1c[c]
1d
6
1e[c]
1 f[c]
1g[c]
1h
>99
>99
50
>99
>99
>99
86
94
1i[c]
n.a.[d]
[a] The reaction was performed by employing ATA-113, l-alanine
dehydrogenase, formate dehydrogenase, NAD+, and l-alanine (see the
Supporting Information); conversions were determined after 24 h by GC
analysis. [b] The enantiomeric excess was measured, after derivatization,
by GC analysis with a chiral stationary phase. [c] 2.76 U of w-trans-
aminase were applied. [d] n.a.: not applicable.
concept (ee 93%), whereas optically pure amine (ee > 99%)
was obtained in the experiment employing lactate dehydro-
genase for pyruvate removal. On the other hand, ketones 2-
butanone (1b) and 2-octanone (1c) were converted into
optically pure (S)-2b and (S)-2c (ee > 99%), respectively.
For aromatic and aryl-alkyl ketones, the conversion and
optical purity varied. For instance, acetophenone (1d) was
reductively aminated with complete stereoselectivity (ee >
99%). 4-Phenyl-2-butanone (1e) was transformed very
efficiently (> 99% conversion), although with reduced ste-
reoselectivity (34%). In the case in which only one methylene
unit was between the carbonyl moiety and the aromatic
system (1 f), complete stereoselectivity was again observed
(ee > 99%).
Chiral b-amino acid derivatives are valuable building
blocks for the synthesis of numerous biologically active
compounds, such as b-peptides, b-lactam antibiotics, and
other drugs.[17] Thus, formal reductive amination of acetoace-
tate 1h yielded the enantioenriched b-amino ester (S)-2h with
complete conversion (> 99%, 94% ee). Additionally, the
cyclic ketone 1i was fully converted, a result indicating the
high flexibility of the utilized enzyme to accept different types
of substrates.
Figure 1. Conversion (c) of 2-pentanone (1a; 50 mm) into the corre-
sponding amine after 24 h at various l-alanine concentrations by a
formal reductive amination according to Scheme 2.
As expected, higher concentrations of l-alanine led to a
faster total reaction. Since alanine is cheap, is internally
recycled in the system, and does not interfere with the other
enzymes in the system, we used it in our further experiments
at a concentration of 250 mm.
To show the applicability of this concept, various ketones,
1a–1i, were successfully transformed into the corresponding
(S)-amines (Scheme 3) by employing w-transaminase ATA-
113 (Table 1). Interestingly, the optical purity slightly
decreased for (S)-2a by using the reductive amination
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solvent of the combined organic phases was evaporated under
To access the opposite R enantiomer, one can imagine the
same concept but employing the R-stereoselective w-trans-
aminase ATA-117 in combination with d-alanine and a d-
alanine dehydrogenase. As a further option, we found that
ATA-117 not only accepted d-alanine but also l-alanine. To
our delight, the optical purity was not affected by switching
the enantiomer of the amine donor. For instance, ATA-117
reduced 2-butanone (1a) into (R)-2-butylamine ((R)-2a) with
> 99% ee by using d- or l-alanine; however, the reaction rate
was 20 times slower with l-alanine than with d-alanine.
Nevertheless, as a demonstration, the formal reductive
amination of ketone 1g by employing R-selective ATA-117,
l-alanine, and l-alanine dehydrogenase gave enantiomeri-
cally pure (R)-1-phenoxy-2-propylamine (2g; > 99% ee, 46%
conversion), which shows that ATA-117 possesses excellent
stereoselectivity with this substrate. Amine (R)-2g is a
valuable building block for the synthesis of several antiepi-
leptic agents.[18]
reduced pressure and the amine was purified by flash column
chromatography (hexane/ethyl acetate and methanol) to yield (S)-2 f
(98.6 mg, 98% yield; 98% ee): [a]2D0 = + 27.8 (c = 0.7, CHCl3; liter-
ature value:[22] + 35.2, c = 0.95, CHCl3, for the S enantiomer, 98% ee);
the 1H NMR spectrum was in agreement with that in the literature.[18c]
Received: July 31, 2008
Published online: October 29, 2008
Keywords: amines · asymmetric catalysis · biocatalysis ·
.
cascade reactions · reductive amination
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Experimental Section
Representative example of the preparation of (S)-amines: (4-
methoxyphenyl)acetone (1 f; 100 mg, 0.61 mmol) was transformed
in phosphate buffer (17 mL, 100 mm, pH 7.0, 1 mm NAD+, 1 mm
PLP) containing a crude preparation of w-transaminase ATA-113
(18.4 U, 40 mg, Codexis), l-alanine (272 mg, 3.05 mmol), l-alanine
dehydrogenase (41 U, 48.09 UmgÀ1
; Fluka, no. A7653-100UN),
ammonium formate (115 mg, 1.83 mmol), formate dehydrogenase
(200 mL, 40 U, 200 UmLÀ1; Jꢀlich, now Codexis, no. 24.11), and
DMSO (15% v/v) at 308C. After 24 h, the conversion was > 99%, the
pH value of the mixture was adjusted to pH 1 with HCl (5m), and any
possible remaining ketone was extracted 5 times with dichloro-
methane (5 ꢁ 10 mL). The pH value was adjusted to pH 12 and the
amine was extracted 4 times with dichloromethane (4 ꢁ 10 mL). The
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