Monoamine oxidase-ω-transaminase cascade for the deracemisation and dealkylation of amines

Article abstract of DOI:10.1002/cctc.201300990

Herein we report a one-pot protocol for the deracemisation of chiral benzylic amines employing a novel monoamine oxidase-ω-transaminase cascade, allowing access to enantiopure compounds in >99 % ee. We also demonstrate that the same enzymatic cascade can be employed for the dealkylation of secondary amines with >99 % conversion. Cascade ball: A monoamine oxidase- ω-transaminase cascade has been developed for the deracemisation of chiral benzylic amines, allowing access to the enantiopure compounds in >99 % ee. The same system was also employed for the efficient dealkylation of secondary amines.

Full text of DOI:10.1002/cctc.201300990

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DOI: 10.1002/cctc.201300990
Monoamine Oxidase–w-Transaminase Cascade for the
Deracemisation and Dealkylation of Amines
Elaine O’Reilly,[a] Cesar Iglesias,[b] and Nicholas J. Turner*[a]
Herein we report a one-pot protocol for the deracemisation of
chiral benzylic amines employing a novel monoamine oxidase–
w-transaminase cascade, allowing access to enantiopure com-
pounds in >99% ee. We also demonstrate that the same
enzymatic cascade can be employed for the dealkylation of
secondary amines with >99% conversion.
catalyse the reversible aminations of carbonyl compounds
such as aldehydes and ketones, providing an attractive route
for the preparation of chiral primary amines.[1,5] A number of
reports describing the deracemisation of amines employing w-
TAs have also emerged.[3,5] Kroutil and co-workers reported
a two-step procedure for the deracemisation of a range of pri-
mary amines by exploiting enantiocomplementary TAs to ach-
ieve an initial resolution followed by an asymmetric synthesis.[6]
Yun and co-workers recently developed a one-pot, one-step
deracemisation method by using enantiocomplementary w-TAs
in which the (R)-TA had negligible activity towards the amine
acceptor.[7]
Chiral amines are prevalent in bioactive natural products and
active pharmaceutical ingredients (APIs), leading to a demand
for simple and cost-effective methods for their production in
optically pure form. Although the chemical industry has tradi-
tionally relied on established chemical methods for the synthe-
sis of optically pure amines, there is growing interest in the de-
velopment of biocatalytic routes for their preparation.[1] The
exceptional selectivity of enzymes and their compatibility
under similar reaction conditions has allowed multi-enzyme
cascade processes to be designed, avoiding the use
of protecting groups and costly intermediate purifica-
tion steps. Although the simplicity of enzyme-mediat-
ed kinetic resolution means that it is one of the most
common methods for accessing optically enriched
amines or their derivatives from racemates, the major
limitation of this approach is the maximum 50%
yield achievable. Dynamic kinetic resolution (DKR) ap-
proaches remain of limited use for the production of
optically pure amines and few examples have been
reported.[2] Additionally, a number of elegant enzy-
matic and chemo-enzymatic routes for the deracemi-
sation of amines have been developed.[3]
MAO-N from Aspergillus niger, in combination with the non-
selective chemical reducing agent ammonia borane (NH3·BH3),
has been exploited for the chemoenzymatic deracemisation of
a range of structurally diverse amines.[8] The approach relies on
selective oxidation of the (S)-amine by MAO-N followed by
Recent advancements in protein engineering have
led to a significant increase in the number of en-
zymes with suitable properties for industrial biocata-
lytic applications.[4] This was highlighted by the
recent development and application of engineered
transaminase (TA) and monoamine oxidase (MAO-N)
variants for the asymmetric syntheses of APIs sitaglip- Scheme 1. MAO-N/w-TA cascade for the deracemisation of primary amines 1a–h.
tin and boceprevir.[4c,d] w-TAs have been shown to
[a] Dr. E. O’Reilly,+ Prof. N. J. Turner
non-selective reduction with NH3·BH3, resulting in accumula-
tion of the (R)-enantiomer over time. Although this approach
School of Chemistry, University of Manchester
Manchester Institute of Biotechnology
131 Princess Street, Manchester, M1 7DN (UK)
has been successfully applied for the deracemisation of cyclic
amines, its utility with acyclic structures is limited by compet-
ing hydrolysis of the imine under aqueous conditions.[8e]
Fax: (+44)0161-275-1311
E-mail: nicholas.turner@manchester.ac.uk
Homepage: http://www.coebio3.manchester.ac.uk/
In this Communication, we overcome this limitation by ex-
ploiting the rapid hydrolysis of imines to give the correspond-
ing ketone and, subsequently, employing an w-TA to regener-
ate the chiral amine. Our approach (Scheme 1) relies on selec-
tive MAO-N mediated oxidation of the (S)-enantiomer of the
[b] C. Iglesias+
Facultad de Quꢀmica
Universidad de la Repfflblica
(Uruguay)
[+] Both authors contributed equally to this work.
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amine to the imine which undergoes spontaneous hydrolysis
to the corresponding ketone (2a–h). Subsequent reductive
amination mediated by an w-TA provides access to the optical-
ly pure (R)-amines. The application of this one-pot MAO-N/w-
TA cascade for the selective N-dealkylation of secondary
amines is also described.
cytochromes P450 are known to catalyse this reaction with ex-
cellent selectivity[14] but often display limited substrate scope.
We have previously demonstrated that MAO-N variants selec-
tively oxidise amines substituted with bulky alkyl groups in
preference to those with smaller groups.[8b,e–g] Hence, we ex-
posed secondary amines 3a–c to the MAO-N/w-TA combina-
tion with the aim of accessing the dealkylated product, benzyl-
amine (Scheme 2). As expected, MAO-N D9 mediated the oxi-
a-Methylbenzyl amine (a-MBA) 1a was used as a model
compound to assess the efficiency of the deracemisation
method (Scheme 1). We have previously reported the kinetic
resolution of a similar panel of MBA derivatives by using com-
mercially available w-TA, ATA117.[9]
Optically pure MBA analogues have been employed as chiral
auxiliaries,[10] for chiral derivatisation[11] and for the synthesis of
peptides.[12] Amine rac-1a was subjected to the purified MAO-
N D9 variant in phosphate buffer, providing a 50:50 mixture of
2a and (R)-1a after 16 h. Despite the high conversion of aceto-
phenone to (R)-a-MBA catalysed by ATA117, subsequent reduc-
tive amination by the TA in the same pot initially resulted in
poor conversion of 2a to (R)-1a (ꢀ70%), with 30% of the
ketone remaining. We hypothesised that the H2O2 by-product
from the MAO-N reaction was negatively affecting the TA reac-
tion, thus, the cascade was repeated in the presence of a bio-
catalytic peroxide scavenger. Co-addition of catalase with the
TA and lactate dehydrogenase/glucose dehydrogenase (LDH/
GDH) system following MAO-N oxidation and hydrolysis pro-
vided (R)-1a in >99% conversion and >99% ee. Exposure of
racemic a-MBA derivatives 1b–h to the same deracemisation
protocol provided the (R)-enantiomers in good conversion and
excellent ee (Table 1). We have also used MBA derivative 1e to
Scheme 2. MAO-N/w-TA cascade for the overall dealkylation of amines 3a–c.
dative N-debenzylation of 3a–c selectively, leading to the syn-
thesis of benzaldehyde 4, which was converted to benzylamine
5 by using ATA117 and an excess of d-alanine, following perox-
ide scavenging. The overall transformation proceeded with
>99% conversion, formally representing a method for selec-
tive amine dealkylation in aqueous media under benign condi-
tions.
The selectivity displayed by engineered MAO-N variants in
the oxidation of either bulky, lipophilic (MAO-N D11) or sterical-
ly less demanding, alkyl substituted amines (MAO-N D5 or
D9)[8e,f] suggests that this methodology is not limited to aryl–
alkyl systems but may be effective for mediating regioselective
N-dealkylations of unsymmetrical dialkyl secondary amines.
Further optimisation is required to reduce the quantity of ex-
pensive alanine donor employed, which includes the possibility
of recycling the methyl-, ethyl- and isopropylamine by-prod-
ucts formed during the dealkylation of 3a–c as alternative
amine donor sources, representing an atom efficient and mild
approach to the dealkylation of secondary amines.
Table 1. Deracemisation of racemic amines 1a–h by using a MAO-N/
w-TA cascade process.
Rac-1
Conversion[a] [%]
(R)-enantiomer ee [%]
1a
1b
1c
1d
1e
1 f
99
87
91
90
99
90
90
81
>99
>99
>99
>99
>99
>99
>99
>99
1g
1h
[a] Based on conversion of the ketone.
In summary, we have developed a one-pot enzymatic cas-
cade process for the deracemisation of chiral amines, providing
the (R)-amines in >99% ee. This one-pot protocol exploits five
commercially available biocatalysts for selective amine oxida-
tion (MAO-N), peroxide scavenging (catalase), asymmetric re-
ductive amination (w-TA), pyruvate removal (LDH) and cofactor
recycling (GDH). Significantly, all five biocatalysts can be added
together without affecting conversion or selectivity. This ap-
proach has potential to enhance the ee of poorly selective TA
biotransformations by recycling the undesired (S)-enantiomer.
We have also demonstrated that the MAO-N/w-TA combination
can be applied to the selective oxidation and hydrolysis of sec-
ondary amines followed by reductive amination, formally rep-
resenting a biocatalytic route for N-dealkylation. The broad
substrate spectrum of MAO-N and increasing substrate scope
demonstrate that the deracemisation can be performed in one
pot with all five biocatalysts and reagents present from the be-
ginning of the biotransformation without compromising con-
version or ee.
Many N-alkylamines and their corresponding dealkylated de-
rivatives, including members of the opioid family, have potent
biological activity and as such, N-dealkylation is an important
transformation in the pharmaceutical industry. A number of
chemical approaches for the dealkylation of amines have been
reported, often requiring harsh reaction conditions.[13] N- and
O-demethylation is a pivotal transformation in drug metabo-
lism and natural biosynthetic pathways and enzymes such as
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adjusting it to pH 12 with 5m NaOH, extracted with EtOAc (1ꢁ
300 mL) and analysed by chiral HPLC without derivatisation.
of w-TAs means that this approach has the potential to
become an efficient route to a diverse range of optically active
amines.
Acknowledgements
Experimental Section
The research leading to these results has received funding from
the European Union Seventh Framework Programme (FP7/2007–
2013) under grant agreement n8 266025 BIONEXGEN and grant
agreement n8 245144 AMBIOCAS.The authors also acknowledge
funding from ANII MOV_CA_2012_1_7759 and CSIC-UdelaR-Uru-
guay.
General
GC flame ionization detector (FID) analysis was performed on Agi-
lent 6850 equipped with a Gerstel MultiPurpose Sampler MPS2L
and a Varian CP CHIRASIL-DEX CB column (25 mꢁ0.25 mm, df =
0.25 mm). Amines were acetylated by using acetic anhydride and
triethylamine at RT prior to GC-FID analysis. HPLC analysis was per-
formed on an Agilent system equipped with a G1379A degasser,
G1312A binary pump, G1329 auto sampler unit, G1315B diode
array detector and G1316A temperature-controlled column com-
partment with a CHIRALPAK IC analytical column (250 mm length,
4.6 mm diameter, 5 mm particle size).
Keywords: biotransformations · chiral amines · dealkylation ·
deracemisation · enzyme catalysis
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Materials
Commercially available reagents were used throughout without
further purification. All reagents and solvents were purchased from
Sigma Aldrich or Alfa Aesar. Escherichia coli BL21(DE3) were pur-
chased from Invitrogen (Carlsbad, CA). Cells were grown routinely
in baffled flasks and harvested at 8000 rpm at 48C for 20 min.
Commercially available ATA117, LDH-103 and GDH (CDX-9012)
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Biotransformations
One-pot sequential procedure
Purified MAO-N D9 (25 mL, 0.52 mgmLÀ1) was added to 1 mL of 4-
(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer
(100 mm, pH 7.5) containing the racemic amine substrate (5 mm,
from a 200 mm stock in DMSO) and incubated at 378C for 20 h.
Following complete conversion of the (S)-enantiomer to the corre-
sponding ketone (monitored by chiral GC-FID), catalase
(5 mgmLÀ1) was added and the reaction incubated for 5 min at RT.
Commercially available ATA117 (2.5 mgmLÀ1), pyridoxal phosphate
(2.02 mm), NAD+ (1.5 mm), glucose (10 mgmLÀ1, 55.5 mm), GDH
(50 U), LDH (113 U) and d-alanine (45 mgmLÀ1, 500 mm) were then
added to the biotransformation and the pH of the mixture was ad-
justed to 7.5. Following incubation at 378C for a further 16 h, the
mixture was basified by adjusting it to pH 12 with 5m NaOH and
extracted with EtOAc (1ꢁ300 mL) prior to derivatisation.
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One-pot procedure
Commercially available ATA117 (2.5 mgmLÀ1) was rehydrated in
HEPES buffer (1 mL, 100 mm, pH 7.5) containing PLP (2.02 mm),
NAD+ (1.5 mm), glucose (10 mgmLÀ1, 55.5 mm), GDH (50 U), LDH
(113 U) and d-alanine (45 mgmLÀ1, 500 mm). Purified MAO-N D9
(25 mL, 0.52 mgmLÀ1) and catalase (5 mgmLÀ1) were added with
racemic 1e (5 mm from a 200 mm stock in DMSO) and the mixture
incubated at 378C, 250 rpm for 24 h. The mixture was basified by
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Published online on February 12, 2014
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