Asymmetric Synthesis of Primary and Secondary β-Fluoro-arylamines using Reductive Aminases from Fungi

Article abstract of DOI:10.1002/cctc.201901999

The synthesis of chiral amines is of central importance to pharmaceutical chemistry, and the inclusion of fluorine atoms in drug molecules can both increase potency and slow metabolism. Optically enriched β-fluoroamines can be obtained by the kinetic resolution of racemic amines using amine transaminases (ATAs), but yields are limited to 50 %, and also secondary amines are not accessible. In order to overcome these limitations, we have applied NADPH-dependent reductive aminase enzymes (RedAms) from fungal species to the reductive amination of α-fluoroacetophenones with ammonia, methylamine and allylamine as donors, to yield β-fluoro primary or secondary amines with >90 % conversion and between 85 and 99 % ee. In addition, the effect of the progressive introduction of fluorine atoms to the α-position of the acetophenone substrate reveals the effect of mono-, di- and tri-fluorination on the proportion of amine and alcohol in product mixtures, shedding light on the promiscuous ability of imine reductase (IRED)-type dehydrogenases to reduce fluorinated acetophenones to alcohols.

Full text of DOI:10.1002/cctc.201901999

Accepted Article
Title: Asymmetric Synthesis of Primary and Secondary ß-Fluoro-
arylamines using Reductive Aminases from Fungi
Authors: Gideon Grogan, Daniel Gonzalez-Martinez, Anibal Cuetos,
Mahima Sharma, Marina Garcia-Ramos, Ivan Lavandera, and
Vicente Gotor-Fernandez
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To be cited as: ChemCatChem 10.1002/cctc.201901999
Link to VoR: http://dx.doi.org/10.1002/cctc.201901999
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10.1002/cctc.201901999
ChemCatChem
COMMUNICATION
Asymmetric Synthesis of Primary and Secondary -Fluoro-
arylamines using Reductive Aminases from Fungi
Daniel González-Martínez,[a],[b] Aníbal Cuetos,[a] Mahima Sharma,[a] Marina García-Ramos,[b] Iván
Lavandera,*[b] Vicente Gotor-Fernández*[b] and Gideon Grogan*[a]
Abstract: The synthesis of chiral amines is of central importance to
pharmaceutical chemistry, and the inclusion of fluorine atoms in drug
molecules can both increase potency and slow metabolism. Optically
enriched -fluoroamines can be obtained by the kinetic resolution of
racemic amines using amine transaminases (ATAs), but yields are
limited to 50%, and also secondary amines are not accessible. In
order to overcome these limitations, we have applied NADPH-
dependent reductive aminase enzymes (RedAms) from fungal
species to the reductive amination of -fluoroacetophenones with
ammonia, methylamine and allylamine as donors, to yield -fluoro
primary or secondary amines with >90% conversion and between 85
and 99% ee. In addition, the effect of the progressive introduction of
fluorine atoms to the -position of the acetophenone substrate reveals
the effect of mono-, di- and tri-fluorination on the proportion of amine
and alcohol in product mixtures, shedding light on the promiscuous
ability of imine reductase (IRED)-type dehydrogenases to reduce
fluorinated acetophenones to alcohols.
followed by nucleophilic addition using a Grignard reagent,
providing the final compounds in high dr (>20:1).
More recently, we showed that amine transaminases
(ATAs) can be applied to the 100 mg scale asymmetric synthesis
of -fluoroamines, with up to 99% ee., through the kinetic
resolution of racemic substrates.13 A series of racemic -fluoro
arylethylamines including 1b and 2b (Scheme 1) was converted
by both (S)- and (R)- selective ATAs to give the corresponding
enantiopure -fluoroamines, in addition to acetophenones 3a and
4a, co-products that arise through the enantioselective tandem
hydrodefluorination-deamination process that is catalyzed by the
enzymes.
Chiral amines are significant functional groups that feature
prominently in many pharmaceuticals. There are many
asymmetric methods for their synthesis that use transition metal
catalysis coupled to chiral ligands,1,2 but considerations of
selectivity, sustainability and green chemistry have dictated that
biocatalytic methods have achieved prominence in recent years.3-
5 The synthesis of fluoroamines represents a special case of chiral
amine synthesis, as the addition of the fluorine atom can improve
the efficiency of bioactive molecules through increased potency
or slower metabolism.6,7 -Fluoroamines are very interesting
derivatives as they can be excellent pyridoxal 5’-phosphate
(PLP)-dependent enzyme inhibitors.8 They can be synthesized
simply through the ring opening of N-tosyl aziridines with TBAF,9
but their asymmetric synthesis is rare, and complicated if chirality
at both the amine and fluorine-bearing carbons is under
consideration. In a recent example, Vara and Johnston used
Brønsted base catalysts, such as (MeO)2PBAM·HNTf, for the
enantioselective synthesis of Boc-protected -amino--fluoro-
nitroalkanes from -fluoro arylnitromethane and aldimine
precursors.10 Other examples using organocatalytic catalysts
have been provided by Lindsley and co-workers,11,12 who
performed the enantioselective fluorination of N-sulfinyl aldimines,
Scheme 1. Kinetic resolution of β-fluoroamines by
a
promiscuous
hydrodefluorination-deamination process catalyzed by ATAs.25
However, the applicability of the method is compromised, owing
to the maximum 50% yields that may be achieved, and the
concomitant inhibition of the ATAs used, due to the formation of a
covalent adduct with PLP in the active site of the enzyme.
A solution to the limited yield of the ATA-catalyzed process
is presented by imine reductases (IREDs).14,15 When presented
with ketones, such as acetophenone, and a large excess of
ammonia or other amine, these NADPH-dependent enzymes
catalyze the asymmetric reduction of imines formed in solution, to
give chiral amine products such as -methylbenzylamine (3b) in
a theoretically quantitative yield and with high enantiomeric
excess (Scheme 2). A subgroup of IRED enzymes, reductive
aminases (RedAms)16-18 has also been shown to catalyze the
formation of the iminium ion with certain ketone and amine
partners, with the consequence that the coupling reaction is
achieved with the supply of only an equimolar ratio of the
substrates.16,19 Interestingly, when presented with ,,-
trifluoroacetophenone 5a (Scheme 2), a promiscuous reduction
of the ketone to the alcohol 5e is performed by an IRED from
Streptosporangium roseum (SrIRED),20 a reaction which has thus
far not been observed in any other IRED/RedAm-catalyzed
transformation of any carbonyl substrate.
These investigations prompted us to study the activity of
RedAms as catalysts for the production of -fluoro-arylamines
such as 1b from -fluoroketones and amine precursors. We
demonstrate that these enzymes are indeed effective catalysts for
the production of optically enriched (N-substituted) -fluoroamine
products. We have also conducted a detailed analysis of the
catalytic promiscuity of IRED-type dehydrogenases for the
reduction of ketones fluorinated at the -position, to the alcohol,
in addition to amine, products.
[a]
[b]
Dr Aníbal Cuetos, Dr Mahima Sharma, Prof. Gideon Grogan
Department of Chemistry
University of York
Heslington, York, YO10 5DD UK
e-mail: gideon.grogan@york.ac.uk
Mr Daniel González-Martínez, Ms Marina García-Ramos, Prof. Iván
Lavandera, Prof. Vicente Gotor-Fernández
Department of Organic and Inorganic Chemistry
University of Oviedo
Avenida Julián Clavería 8, 33006 Oviedo (Asturias), Spain
e-mail: lavanderaivan@uniovi.es; vicgotfer@uniovi.es
Supporting information for this article is given via a link at the end of
the document.
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10.1002/cctc.201901999
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COMMUNICATION
Scheme 2. RedAm-catalyzed transformations described in this study.
were stopped at 20 h. Hence acetophenone (3a) was transformed
only to amine products when incubated with AdRedAm and
ammonia, methylamine or allylamine; -fluoroacetophenone (1a)
was converted into a mixture of products only when ammonia was
the donor; while ,-difluoroacetophenone (6a) was transformed
to predominantly the alcohol when ammonia was the donor, but
to more equal proportions of alcohol and amine with methylamine
or allylamine; finally, ,,-trifluoroacetophenone (5a) was
transformed exclusively into the alcohol 5e in each case.
Biotransformations of fluorinated acetophenones by fungal
RedAms
In initial experiments, -fluoroacetophenone 1a at 5 mM initial
concentration, and on an analytical scale, was transformed using
AdRedAm from Ajellomyces dermatitidis16 as the catalyst, in the
presence of a large excess (250 mM) of methylamine (MeNH2)
and with glucose-6-phosphate (G6P) and glucose-6-phosphate
dehydrogenase (G6PDH) to regenerate the NADPH cofactor.
Under these conditions, we assume that the RedAms are acting
in an IRED mode, binding and reducing the imine formed in
solution, as acetophenones are not good substrates for equimolar
reductive amination reactions.16,19 Details on gene cloning and
protein purification can be found in the Supporting Information (SI,
Sections III-IV). The synthesis of substrates and product
standards is detailed in SI Section XI. In initial experiments,
conversions of up to 65% to the amine product 1c were achieved,
with only 3% evolution of alcohol product 1e at 30 ºC (Scheme 2,
Table 1, entry 1).
After optimization (SI, Table S1), 37 ºC was selected as the
best temperature (Table 1, entry 2) and also an increase of the
amine donor up to 500 mM (Table 1, entry 3) afforded a
conversion of 92% into 1c. Experiments (Table 1, entries 4-9)
also showed that -fluoroacetophenone (1a) was a superior
substrate to acetophenone (3a) when aminated with either
ammonia or methylamine, giving 18% and 46% conversion to
products 1b and 1c respectively after 20 h, compared to 6% and
28% for the transformation of 3a into 3b or 3c. However, in the
case of the reaction of 1a with ammonia, the fluorinated alcohol
1e was detected in a significant amount of 14% (Table 2, entry
4). The proportion of amine to alcohol product was not only
influenced by the amine donor, but also by the extent of
fluorination on the acetophenone substrate (Table 1, Figure 1).
In order to better compare the results, enzymatic transformations
Table 1. Transformations of -fluoroacetophenone (1a, X=CH2F) with
AdRedAm and several amine donors. Reactions between acetophenone
(3a, X=CH3) and amine donors have been added for comparison.
Entry
Ketone
Amine
Donor
t (h)
Amine
b-d (%)[a]
Alcohol
e (%)[a]
1
2
3
4
5
6
7
8
9
1a[b]
1a
MeNH2
MeNH2
64
65
77
92
18
46
27
6
3
4
[c]
1a
MeNH2
5
1a
NH3
20
20
14
<1
<1
<1
<1
<1
MeNH2
AllylNH2
NH3
3a
MeNH2
AllylNH2
28
35
[a] Conversions were determined by GC-FID analysis of the reaction crude.
Reaction conditions: ketone (5 mM), amine (50 eq.), AdRedAm (1 mg/mL),
NADP+ (1 mM), G6P (30 mM), G6PDH (2 U), MeCN (2% v/v), buffer
Tris·HCl (100 mM, pH 9.0, 500 µL) at 37 ºC. [b] Reaction was carried out at
30 ºC. [c] 100 eq. of amine donor were used.
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COMMUNICATION
Figure 1. Percentages of amine (black) and alcohol (grey) products obtained by biotransformation (t = 20 h) of fluorinated acetophenone derivatives by AdRedAm
(left) and NfRedAm (right). These results are tabulated in Table S2 in the Supporting Information.
A
similar trend was observed for equivalent
by AdRedAm with only poor conversion (<5%) using either
ammonia or methylamine.
biotransformations catalyzed by NfRedAm from Neosartorya
fumigatus (Figure 1). Additional results obtained for AtRedAm
from Aspergillus terreus,19 NfisRedAm from Neosartorya fischeri,
and AspRedAm from Aspergillus oryzae16 can be found in the
Supporting Information (SI, Section VII, Figure S5, Table S2). It
is important to note that in control reactions, which were
performed in the absence of RedAm, but with both the cofactor
and the cofactor recycling system, neither amination reactions,
nor reduction of the ketones to alcohols, were observed. The
stereoselectivity of the RedAm-catalyzed biotransformations is
shown in Table 2 and in the SI (Table S3), and the analytical
methods and results detailed in the Supporting Information (SI,
Section X), with details on the assignment of absolute
configuration (SI, Section XII). In most cases the
enantioselectivity of the reactions was very high (up to 99%), and
although these enzymes have been described as (R)-
selective,16,19 products were of the (S)-enantiomeric series,
except amine 3b, due to the change in Cahn-Ingold-Prelog (CIP)
priority. The ee values for amines 1b, 6c and 6d were especially
high, with products obtained in enantiopure form for most
examples (SI, Table S3).
AdRedAm and NfRedAm appeared to be the best
biocatalysts overall for the transformations (Table 2), giving
amine products with high conversions and in excellent ee using
an excess of the amine donor (80-100 eq.) and in the presence of
a small percentage of an organic co-solvent. Alcohols that were
formed in the reactions were also of the (S)-enantiomeric series
and displayed ee values >97% in many cases (Table S3).
RedAms also catalyze the amination of para-halogenated -
fluoroacetophenones with F (7a), Cl (8a), Br (2a) and I (9a) as
substituents with ammonia (SI, Section IX). Although activities
decreased with increasing size of the halogen atom,
enantioselectivity was largely conserved, obtaining (S)-amine
products with high to excellent ee (Table 2). With these substrates,
in no case were the corresponding alcohol derivatives observed
The p-iodo derivative 9a was only poorly transformed using
ammonia, as was the p-methyl derivative 10a (<2% conversion).
2-chloro -fluoroacetophenone (not shown) was also transformed
The biotransformation of 1a to 1c by AdRedAm was
performed on a 70 mg scale, giving a GC conversion of 68% and
a crude yield of 55% following extractive work up as described in
SI Section XIII). Column chromatography yield the pure product
in 44% yield with 96 % ee..
Table 2. Conversions and ee in the reductive amination of fluorinated
acetophenones using RedAms and different amine donors (b: NH3, c:
MeNH2, d: AllylNH2).
Product
1b[b]
1c
Enzyme
AdRedAm
AdRedAm
NfRedAm
NfRedAm
NfRedAm
NfisRedAm
NfRedAm
NfRedAm
eq.
amine
Co-
solvent
t
(h)
c
ee
(%)[a]
(%)[a]
80
MeOH
(1%)
44
20
20
70
20
40
40
40
81
84
69
71
65
76
76
34
99
96
93
99
99
99
99
85
100
100
100
100
100
100
100
MeOH
(1%)
1d
MeOH
(1%)
6c[c]
6d
MeCN
(2%)
MeCN
(2%)
7b
MeOH
(1%)
8b
MeOH
(1%)
2b
MeOH
(1%)
[a] Conversions and enantiomeric excess values were determined by GC-
FID analysis of the reaction crude after in situ derivatisation with acetic
anhydride. [b] 10 mM 1a. [c] 0.05 mM NADP+ used to minimise the alcohol
6e formation (16%).
Studies on the promiscuous reduction of ketones fluorinated at
the α-position by IREDs/RedAms
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In addition to expanding the applicability of RedAms to the
production of primary and secondary β-fluoro-arylamines, this
study also sheds more light on the observation that ,,-
trifluoroacetophenone 5a is reduced exclusively to the alcohol by
a new reaction to the available biocatalytic toolbox, and provides
a platform for improvement of these processes through both
enzyme and reaction engineering.
a
promiscuous reaction catalyzed by the SrIRED from
Experimental Section
Streptosporangium roseum.20 We observed this to be a general
trend for other IRED/RedAm enzymes, particularly for 5a, but also
to a lesser extent when the mono- and di-fluorinated substrate
analogues 1a and 6a are used. A number of factors may
contribute to the promiscuous activity observed. The results
suggest that the hydride transfer process is easier for
Details of gene cloning and expression, enzyme purification and assay,
synthesis of substrates and product standards, GC and HPLC analyses,
biotransformation protocols and modelling/thermodynamics studies can be
found in the Supporting Information.
fluoroacetophenones than for non-fluorinated analogs, as the Acknowledgements
electrophilicity of the carbonyl carbon is steadily increased with
A.C. and M.S. were funded by grants BB/P005578/1 and
successive fluorine substitutions. When comparing the Gibbs free
energies of the carbonyl reduction using DFT calculations, we
observed that reduction of1a, 6a and 5a were 20.2, 37.0 and
40.1 kJ mol-1 more favorable, respectively, than for the reduction
of 3a (SI Section XIV, Table S11). On the other hand, the imine
formation from 1a, 6a and 5a was also more favorable with
respect to acetophenone (SI, Tables S11-S12), but in this case
the influence of the number of fluorine atoms was not significant
(15.8-17.9 kJ mol-1). Overall, these energetic values are in
agreement with the formation of the alcohols, especially for di-
and tri-fluorinated ketones, when compared to non- and also
mono-fluorinated ketones. However, the different amine:alcohol
product ratios obtained when using different amine donors for the
same ketone substrate, such as 1a and 6a suggests that
molecular recognition of both substrates may also play a part in
switching the chemoselectivity of the reaction. For the reactions
presented, where a large excess of amine is used, a preference
for alcohol over amine production (or vice versa) must reflect the
preferred binding of the ketone substrate over the imine recruited
from solution within the active site. In a recent study by one of our
groups together with Nestl and co-workers, we determined the
structure of the imine reductase SrIRED in complex with the
hydrate of ,,-trifluoroacetophenone.21 This revealed that, in
the presence of this fluorinated substrate, two of the -fluorine
atoms make a direct interaction with one of the ribose hydroxyl
groups of NADP+, and may, in the case of the fluoroketone
substrate 5a, draw the electrophilic carbon of the substrate within
a suitable distance for reduction by the cofactor. These
interactions may be less pronounced in either ,-
difluoroacetophenone 6a or -fluoroacetophenone 1a and of
course absent for acetophenone 3a, in which series a decreasing
proportion of alcohol products are observed. The increasing
proportion of amine products obtained when 1a or 6a are
aminated with ammonia, methylamine and allylamine is therefore
a reflection of the increasingly successful competition of the N-
alkylated imine for the active site over the fluoroketone. These
phenomena therefore constitute an interesting example of
substrate-directed enzyme promiscuity dependent upon both
electronic and thermodynamic effects.
BB/M006832/1 respectively, from the UK Biotechnology and
Biological Sciences Research Council (BBSRC). Financial
support from the Spanish Ministry of Economy and
Competitiveness (MEC, Project CTQ2016-75752-R) is gratefully
acknowledged. D.G.-M. thanks the Asturian regional government
for a predoctoral fellowship inside the Severo Ochoa program.
Keywords: oxidoreductase
• imine reductase • reductive
amination • fluoroamine • biocatalysis
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The synthesis of chiral amines from readily available
prochiral substrates remains a challenge for asymmetric catalysis,
and IRED/RedAm-type enzymes can offer sustainable and atom-
efficient solutions to some of these problems. The recognition that
-fluorinated ketones can serve as substrates for alkyl-amination
by RedAms, as well as for reduction to chiral alcohols, introduces
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Entry for the Table of Contents (Please choose one layout)
COMMUNICATION
Daniel González-Martínez, Aníbal
Cuetos, Mahima Sharma, Marina
García-Ramos, Iván Lavandera,*
Vicente Gotor-Fernández* and Gideon
Grogan*
Page No. – Page No.
A series of enantioenriched primary and secondary β-fluoroamines can be obtained
through a stereoselective reductive amination process catalyzed by different
reductive aminases with a selection of amine donors.
Asymmetric Synthesis of Primary and
Secondary β-Fluoro arylamines using
Reductive Aminases from Fungi
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