Combination of the Suzuki–Miyaura Cross-Coupling Reaction with Engineered Transaminases

Article abstract of DOI:10.1002/chem.201804366

The combination of enzymatic and chemical reaction steps is one important area of research in organic synthesis, preferentially as cascade reactions in one-pot to improve total conversion and achieve high operational stability. Here, the combination of the Suzuki–Miyaura reaction is described to synthesize biaryl compounds followed by a transamination reaction. Careful optimization of the reaction conditions required for the chemo- and biocatalysis reaction enabled an efficient two-step-one-pot reaction yielding the final chiral amines with excellent optical purity (>99 % ee) in up to 84 % total conversion. Key to the success was the protein engineering of the amine transaminases from Asperguillus fumigatus (4CHI-TA) where single alanine mutations increased the conversion up to 2.3-fold. Finally, the transfer to a continuous flow system after immobilization of the best 4CHI-TA variant is demonstrated.

Full text of DOI:10.1002/chem.201804366

A Journal of
Accepted Article
Title: Combination of the Suzuki-Miyaura-cross coupling reaction with
engineered transaminases
Authors: Ayad W.H. Dawood, Jonathan Bassut, Rodrigo O.M.A. de
Souza, and Uwe Bornscheuer
This manuscript has been accepted after peer review and appears as an
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To be cited as: Chem. Eur. J. 10.1002/chem.201804366
Link to VoR: http://dx.doi.org/10.1002/chem.201804366
Supported by

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COMMUNICATION
Combination of the Suzuki-Miyaura-cross coupling reaction with
engineered transaminases
Ayad W.H. Dawood,[a] Jonathan Bassut[b], Rodrigo O.M.A. de Souza,[b] Uwe T. Bornscheuer*[a]
Abstract The combination of enzymatic and chemical reaction steps
is one important area of research in organic synthesis, preferentially
as cascade reactions in one-pot to improve total conversion and
achieve high operational stability. Here, we describe the combination
of the Suzuki-Miyaura reaction to synthesize biaryl compounds
followed by a transamination reaction. Careful optimization of the
reaction conditions required for the chemo and biocatalysis reaction
enabled an efficient two-step-one-pot reaction yielding the final chiral
amines with excellent optical purity (>99% ee) in up to 84% total
conversion. Key to success was the protein engineering of the amine
transaminases from Asperguillus fumigatus (4CHI-TA) where single
alanine mutations increased conversion up to 2.3 fold. Finally, we
enzymes which is at the same time the basis for the
classification of their stereoselectivity. The substrate recognition
is ensured by two binding pockets: a small one, naturally only
able to accommodate a methyl or ethyl group and a large one,
with space for e.g. a phenyl or a carboxyl moiety. Additionally
they are divided into six subclasses, depending on the natural
substrate and the position of the transferred amine group and/or
carboxyl moiety.[6] Amine transaminases (ATA), a subgroup of
ω-TAs (class III transaminase family), are of special interest for
the chemo-enzymatic application. ATAs tolerate substrates
without a carboxyl moiety and therefore a wide range of ketones
and aldehydes. Hence, in the last decade ATA became very
attractive targets for enzyme engineering mainly with the aim to
enlarge the small binding pocket in order to allow the
acceptance of sterically more demanding substrates.[5,7]
demonstrated the transfer to
a continuous flow system after
immobilization of the best 4CHI-TA variant.
The Suzuki-Miyaura-cross coupling reaction has received
increasing attention over the last two decades mainly because of
the access to biaryl compounds and the introduction of
functional groups.[8–10] This type of transition-metal-dependent
reaction catalyzes the C-C-bond formation between halides and
boronic acids under transmetalation as key reaction step.
Originally the Suzuki-Miyaura reaction was performed in organic
solvents and employed phosphine ligands to reduce the metal
catalyst – palladium – to the active Pd(0) species which often
meant that the Pd-catalyst was water and air sensitive.[8,11] Very
intensive research in this field generated a great number of
reports where the Suzuki-Miyaura reaction took place in
phosphine-free and water-solvent systems in order to address
the demands of green chemistry.[8,10,12–16] Especially the
compatibility of the Suzuki-Miyaura reaction with water was
meaningful because water is one of the most attractive solvents
in terms of costs, availability, toxicity, safety and environmental
protection.[8,10,13] One beneficial aspect for the field of
biocatalysis is certainly the “enzyme-compatibility” of water as
“natural solvent”. However, first successful one-pot-chemo-
enzymatic approaches combining Suzuki-Miyaura reaction and
enzymes included the activity of an alcohol dehydrogenase,[17–19]
whereby Burda et al. and Borchert et al. gained the chiral biaryl-
alcohol through asymmetric synthesis with very good conversion
and enantiomeric excess values (91–97%, >99%ee).[18,19]
Furthermore, Ahmad et al. produced bi-phenylalanine via the
involvement of a phenylalanine ammonia lyase prior to the
cross-coupling reaction.[20] Interestingly, France et al. included
both, enzymatic transamination via ATA013 and Suzuki-Miyaura
reaction, to facilitate their route to dibenzazepines.[21] However,
the latter work contained intermediate purification steps and
solvent changes and therefore did not met our targeted reaction
compatibility and the envisaged benefits of a one-pot- reaction
setup.
In the last few decades enzymes played an increasing role
as catalysts in organic chemistry. The access to a large number
of compounds or intermediates in pharmaceutical and chemical
industries was realized by the utilization of e.g. reductases,
hydrolases, oxygenases, dehalogenases or transaminases.[1]
Enzymatic routes are an environmentally benign alternative to
classical chemical synthesis strategies because of their mild and
less waste intensive operation conditions.[2] Nevertheless, the
application of enzymes in synthetic chemistry suffers very often
from limitations e.g. in operational stability, substrate scope and
stereo- or regioselectivity which of course hampers the industrial
relevance of a biocatalyst.[3] To address these limitations
enzyme engineering became a highly relevant field of organic
chemistry and more recently to combine also chemo- and
biocatalysis in the form of cascades, preferentially in one-pot-
reactions.[4]
Transaminases (TA) are certainly one of the most versatile
and widely used biocatalysts for the production of chiral amines
which are important compounds or building blocks for the
pharmaceutical and agrochemical industry.[5] TA catalyze the
transfer of an amine group from an amine donor to a ketone or
aldehyde acceptor, following
a
Ping-Pong Bi-Bi-reaction
mechanism utilizing pyridoxal-5’-phosphate (PLP) as co-factor.
TAs are grouped into the fold types I and IV of PLP-dependent
[a]
[b]
M.Sc. Biochem. A.W.H. Dawood, Prof. Dr. U.T. Bornscheuer
Dept. of Biotechnology & Enzyme Catalysis
Institute of Biochemistry, Greifswald University,
Felix-Hausdorff-Str. 4, 17487 Greifswald (Germany)
E-mail: uwe.bornscheuer@uni-greifswald.de
Web: http://biotech.uni-greifswald.de
Dr. J. Bassut, Prof. Dr. R.O.M.A. de Souza
Biocatalysis and Organic Synthesis Group, Institute of Chemistry
Federal University of Rio de Janeiro (Brazil)
Inspired from those works we considered the approach of a
compatible combination of a cross-coupling reaction with a
transamination reaction as evident.
Supporting information for this article is given via a link at the end of
the document
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Table 1. Comparison of 4CHI-TA variants in the synthesis of biphenyl amines 1c – 4c with isopropylamine (IPA) as amine donor. The ATA reaction
was initiated after the Suzuki-Miyaura cross-coupling reaction by adding of ATA crude lysate and IPA/PLP-containing reaction solution (Scheme 1).
ATA/ variant
Substrate conversion [%][a]
1b
2b
3b
4b
4CHI-wt
38.5 ± 3.8
45.6 ± 3.2
72.5 ± 1.0
37.6 ± 2.7
87.5 ± 1.4
83.8 ± 4.2
39.7 ± 4.2
48.6 ± 3.1
75.0 ± 2.6
18.2 ± 2.8
79.1 ± 3.7
79.8 ± 3.5
38.9 ± 1.6
21.6 ± 2.7
19.2 ± 2.9
46.8 ± 3.1
23.6 ± 3.4
17.4 ± 3.2
27.2 ± 2.4
27.1 ± 2.5
18.4 ± 2.5
35.7 ± 2.8
14.8 ± 2.0
17.4 ± 4.2
4CHI-H53A
4CHI-F113A
4CHI-R126A
4CHI-I146A
4CHI-F113A-I146A
[a] Assay conditions for the asymmetric synthesis (final concentrations): 1 mM ketone (1b – 4b), 0.75 M IPA, 30% (v/v) DMF, HEPES/Na2CO3 buffer (27 mM) pH
10, 0.1 mM PLP, 30 °C, 0.37 – 6.79 U mL-1 of ATA (crude cell lysate with overexpressed ATA, given activities refer to (R)-PEA as amine donor and pentanal as
amine acceptor). Because of fluctuations in activity in the acetophenone assay obtained after mutagenesis, the expression level was also checked via SDS-PAGE
(Fig. S1, Supporting Information). Samples were taken in triplicate from three parallel reactions. Conversion was determined after 20 h via gas chromatography
(GC) with 2-iodoacetophenone as internal standard for the quantification of ATA-substrate consumption (1b – 4b). Corresponding amines were identified via
GCMS. Retention times for all compounds are given in Table S3 and enantiomeric excess values for 3c in Table S5.
Thus, we present a successful two-step-one-pot-reaction
including ligand-free, palladium-catalyzed Suzuki-Miyaura
cross-coupling combined with an engineered ATA activity to
obtain chiral biaryl amines.
of buffer traces and PLP, we considered the part of the cross-
coupling reaction as a determining factor in our setup (Table
S2). Simultaneously, we examined the ATA reaction part in
terms of reaction conditions (presence or absence of reactants
or reagents of the Suzuki-Miyaura reaction) and acceptance of
sterically demanding biphenyl ketones. We tested those ATA
mentioned above with 4-acetyl-biphenyl 3b and 1-(1,1’-biphen-4-
yl)-ethanamine 3c and indeed 4CHI-TA turned out as interesting
candidate with good starting activity (Table 1). Moreover, the
activity of 4CHI-TA revealed as quite robust in the presence of
DMF, sodium carbonate, PdCl2 and even in the absence of
buffer reagents (Fig. S5 and S6, Supporting Information). So, we
decided to design our reaction setup accordingly, namely to start
with the Suzuki-Miyaura cross-coupling reaction to produce the
biphenyl ketones 1b–4b and perform the transamination
reaction subsequently by addition of a solution containing the
amine donor, PLP and 4CHI-TA (Scheme 1, Table 1). We used
isopropylamine (IPA) as amine donor, because it is certainly one
of the most favored amine donors for asymmetric syntheses
since it is cheap, achiral (the enantioselectivity of the used ATA
has not to be considered) and a co-product removal is not
critical in contrast to the usage of alanine. 4CHI-TA wild type
showed indeed quite well starting activity for all investigated
biphenyl ketones 1b–4b but conversion did not exceed 40% in
any case.
a
Reports of phosphine-free palladium-catalyzed Suzuki-
Miyaura reactions usually contained the replacement of the
original ligand with other stabilizing additives or activating
supports, like palladium on carbon (Pd/C)[22] or mesoporous
silica[23], ethylene glycol or EDTA.[14,15,24] Also water-soluble
ligands on phosphine basis were presented (see[18,25]).
Interestingly, we found several works which showed a simplified
ligand-free application of the Suzuki-Miyaura reaction in N,N-
dimethylformamide (DMF)-water-mixtures and/or in the
presence of an inorganic base.[12,16,22,26] Liu et al. underlined the
robustness of their method for the fast reaction of aryl bromides
with arylboronic acids at ambient temperature and in the
presence of water and air.[16] DMF-water-mixtures were a good
starting point for our claim of ATA compatibility. Recently, we
reported the successful production of chiral halogenated amines
in DMF-water-mixtures using the ATA from Aspergillus
fumigatus (4CHI-TA) and Silicibacter pomeroyi.[27] We choose
four biphenyl model compounds which cover several aspects
regarding position and substitution of the biphenyl moiety as well
as the presence of heterocycles (Scheme 1). Starting with the
aryl bromides 5-bromo-3-acetyl-pyridine 1a as well as 4’-
bromoacetophenone 3a and (fluor-substituted) arylboronic acids
we were able to synthesize the desired biphenyl ketones 1b–4b
with very good conversion according to the mentioned method
from Liu et al. using sodium carbonate[14,19,28] as activating base
and using free PdCl2 as catalyst (Table S1, Fig. S2, Supporting
Information). However, after attempts to convert 3a to 3b failed
in the presence
We performed in silico studies to identify possible amino acid
residues around the active site of 4CHI-TA which might hamper
the acceptance of bulky biphenyls. Molecular docking studies
indicated in total four amino acid residues in the large binding
pocket of 4CHI-TA which possibly cause interferences with the
substrates: H53, F113, R126 and I146 (according to the PDB
numbering, see Fig. S7). Alanine variants were produced via the
QuikChange protocol to generate more space at the respective
position in the active site. The application of the 4CHI variants in
the asymmetric synthesis of amines 1c–4c showed in fact a
remarkable effect of mutations F113A and I146A for the
conversion of compounds 1b and 2b. For both substrates high
conversion values of 72–75% with 4CHI-F113A as well as 79–
87.5% with 4CHI-I146A could be reached keeping in mind that a
single alanine mutation was sufficient in both cases to double
total conversion. The ‘flipping arginine’ in 4CHI, R126[29]
obviously plays a slight detrimental role for the acceptance of 3b
and 4b but the exchange to alanine did not achieve the desired
impact.
Scheme 1. Starting from aryl halides chiral biaryl amines could be obtained by
the sequential order of Suzuki-Miyaura and ATA reaction. The crude Suzuki-
Miyaura reaction product was diluted with an ATA/IPA/PLP-containing solution
achieving the final conditions for the ATA reaction. Molar equivalents were
given in relation to 1a and 3a.
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COMMUNICATION
O
OH
B
O
Br
NH2
PdCl2
added
OH
+ PLP
+
3 eq. Na2CO3, 50% DMF
30oC
N
1.5 eq.
1b
1a
750 eq.
N
crude reaction mixture
0.1mL/min
NH2
4CHI-I146A@EziG
1c
N
HEPES/Na2CO3 pH 8
30% DMF, 30oC
tR = 3.5 h
43% conversion
Scheme 2. Setup of the continuous flow synthesis of 1-(5-phenylpyridin-3-yl)ethanamine 1c. Molar equivalents are given in relation to 1a. The crude Suzuki-
Miyaura reaction product was added in flow to an IPA and PLP containing solution and subsequently pumped through a column containing the immobilized variant
4CHI-I146A at a flow rate of 0.1 mL h-1. After pumping 0.45 mL, samples for GC analysis were withdrawn. The conversion was calculated on the basis of ATA
substrate conversion (1-(5-phenylpyridin-3-yl)ethanone 1b) and set in relation to a blank experiment with empty carrier instead of immobilisate.
In fact, this highly conserved residue is supposed to be very
flexible because it is known to facilitate the dual substrate
recognition of transaminases by salt bridge formation to the
carboxylate function of respective substrates. On the other hand
it has to ‘flip out’ when hydrophobic substrates need to be
coordinated.[6,30] So, by a simple replacement of this residue
sustainable effects on the activity towards bulky substrates are
probably not expectable. In contrast to the in silico indication the
mutation of H53 had a rather detrimental effect on the synthesis
of 3c and 4c and only a slight improving effect on substrates 1b
and 2b. In response to the results shown in Table 1 a double
mutant of 4CHI-TA containing the mutations F113A and I146A
was produced in the hope to further increase the conversion, but
no significant difference in conversion could be detected in
comparison to each single mutant (Table 1). The excellent
stereoselectivity of 4CHI-TA wild type and the variants was
confirmed for amine 3c, whereby in any case an enantiomeric
excess of >99%ee was reached (Table S5, Supporting
Information). This was indeed not unexpected because the small
binding pocket was not changed and additionally the demand on
the large binding pocket was even increased by substrates 1b–
4b. Therefore, changes in enantioselectivity were indeed
considered unlikely.
recombinant proteins without pre-treatment or activating of
supports with e.g. cross-linking agents. At the same time it
allows a more specific loading with the recombinant target
protein. We decided to use EziGTM carriers[38] and ran initial
experiments regarding efficiency, selectivity and stability of the
4CHI-TA-immobilisates: 10 U g-1 support were reached and no
enzyme leaching was found in the presence of 30% DMF (Fig.
S8 and S9, Supporting Information). The reaction setup for the
continuous flow experiment is shown in Scheme 2. The two
reaction solutions – first, the crude Suzuki-Miyaura reaction
mixture producing 1b and second an IPA/PLP-containing
solution – were pumped synchronically through a column with
the immobilized variant 4CHI-I146 resulting in a total conversion
of 43% from 1a to 1c representing a proof-of principle study.
Herein, we presented the access to chiral biaryl amines via
the combination of the Suzuki-Miyaura cross-coupling with a
transaminase-catalyzed reaction under ligand-free and mild
conditions. Major achievements were the excellent compatibility
of chemo- and biocatalysis to achieve the cross-coupling
reaction in the presence of oxygen and DMF-water mixtures
while maintaining high robustness of the 4CHI-TA in the
presence of reagents required for the Suzuki-Miyaura reaction.
Furthermore, the starting activity of the (R)-selective 4CHI-TA
towards biaryl ketones could be increased up to 2.3-fold
(substrate 1b and 4CHI-I146A) by simple alanine mutations of
four amino acid residues around the active site as derived from
in silico studies. This complements a recent example for the
engineering of the (S)-selective ATA from Vibrio fluvialis to
accept biphenyl ketone substrates.[39] Our concept to use the
biaryl cross-coupling products directly for the transamination
reaction represents certainly a great advantage as amine group
protection strategies could be avoided (free amines are known
Having improved biocatalysts and a compatible reaction
system for the combination of the Suzuki-Miyaura coupling and
the ATA reaction established we next transferred this to a
continuous flow system as exemplified for the production of 1c.
A large variety of flow systems were already reported in the field
of biocatalysis in general, but also for the production of biaryl
compounds via the Suzuki-Miyaura reaction since continuous
reaction systems have several of advantages in terms of
economic aspects e.g. the increase of the space-time-yield,
lowering of reaction times and decreased waste production.[31]
Another aspect is the possible compartmentalization of several
sub-reactions. For such processes, efficient immobilization of
the enzyme becomes significant. Several reports dealt with the
successful immobilization of ATAs using different supports (e.g.
as Pd-chelating functional groups). Additionally,
a Suzuki-
Miyaura reaction with N-protected educts is known to be less
effective.[13] In addition, we provided a proof-of-concept for a
transfer of the system to continuous flow conditions.
For experimental details see Supporting Information.
Chitosan[32]
,
functionalized Cellulose[33]
,
polystyrene[34] or
methacrylate beads[35] as well as concepts for the co-
immobilization of the cofactor PLP,[35,36] For our example here,
the immobilization via metal affinity resins[37] was choosen
because this is a convenient method for the immobilization of
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COMMUNICATION
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thanks the Alexander-von-Humboldt foundation for a CAPES-
Humboldt research fellowship.
Keywords: Amine transaminases, Suzuki-Miyura-cross
coupling, enzyme engineering, chiral amines, isopropylamine,
continuous flow, enzyme immobilization
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The combination of amine
Ayad W.H. Dawood, Jonathan
transaminase (ATA) activity and the
Suzuki-Miyaura cross-coupling
reaction in a two-step-one-pot
fashion is presented. The biaryl
amines could be obtained with high
conversion and excellent
enantiomeric excess (99%ee) using
isopropylamine as the amine donor
after engineering of the
Bassut, Rodrigo O.M.A. de Souza,
Uwe T. Bornscheuer*
Page No. – Page No.
Combination of the Suzuki-
Miyaura-cross coupling reaction
with engineered transaminases
transaminase from Asperguillus
fumigatus.
This article is protected by copyright. All rights reserved.