Chemo-Enzymatic Synthesis of Pyrazines and Pyrroles

Article abstract of DOI:10.1002/anie.201810555

Herein we report the biocatalytic synthesis of substituted pyrazines and pyrroles using a transaminase (ATA) to mediate the key amination step of the ketone precursors. Treatment of α-diketones with ATA-113 in the presence of a suitable amine donor yielded the corresponding α-amino ketones which underwent oxidative dimerization to the pyrazines. Selective amination of α-diketones in the presence of β-keto esters afforded substituted pyrroles in a biocatalytic equivalent of the classical Knorr pyrrole synthesis. Finally we have shown that pyrroles can be prepared by internal amine transfer catalyzed by a transaminase in which no external amine donor is required.

Full text of DOI:10.1002/anie.201810555

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Accepted Article
Title: Chemo-enzymatic synthesis of pyrazines and pyrroles
Authors: Nicholas John Turner, Anthony Green, and Jin Xu
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201810555
Angew. Chem. 10.1002/ange.201810555
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Chemo-enzymatic synthesis of pyrazines and pyrroles
Jin. Xu, Anthony P. Green,* and Nicholas J. Turner*
Abstract: Herein we report the biocatalytic synthesis of substituted
pyrazines and pyrroles using a transaminase (ATA) to mediate the
key amination step of the ketone precursors. Treatment of α-
diketones with ATA-113 in the presence of a suitable amine donor
yielded the corresponding α-amino ketones which underwent
oxidative dimerization to the pyrazines. Selective amination of α-
diketones in the presence of β-keto esters afforded substituted
pyrroles in a biocatalytic equivalent of the classical Knorr pyrrole
synthesis. Finally we have shown that pyrroles can be prepared by
internal amine transfer catalyzed by a transaminase in which no
external amine donor is required.
Amine transaminases (ATAs) have emerged in recent
years as highly valuable biocatalysts for the conversion of a
broad range of ketones and aldehydes to the corresponding
[
1]
primary amines. ATAs have found application especially in the
synthesis of chiral amine building blocks for natural products,
pharmaceuticals and agrochemicals and have been used for
large scale manufacture of APIs (e.g. sitagliptin). ATA catalyzed
reactions are reversible and hence strategies have been
developed to ensure high conversion of carbonyl substrate to
amine product including (i) use of excess amine donor; (ii)
removal of products in situ and (iii) use of ‘smart’ amine donors
[
5]
[4]
Figure 1. (a) Two important drugs (Atorvastatin and Sunitinib
)
containing
pyrrole functional group and a pyrazine (Polycartine B ) used as flavour
enhancer. (b) biocatalytic retrosynthetic approach to the synthesis of
[
8]
A
[9]
pyrazine and pyrrole mediated by ATAs.
[
2]
to displace the equilibrium of the reaction. ATAs have also
been extensively engineered to broaden substrate scope,
enhance selectivity and improve stability under process
Initially two symmetrical α-diketones 1-2 and one non-
symmetrical α-diketone 3 were examined as substrates for ATA-
13 using either (S)-aminotetralin (1.1 equiv) or isopropylamine
10 equiv) as amine donor (Scheme 1). All reactions proceeded
smoothly with α-diketones fully consumed (GC-MS) after 72 h to
[
3]
conditions. However to date their application beyond the
production of chiral amines has been limited. Herein we show
that ATAs can be employed for the synthesis of N-
heteroaromatic compounds, specifically pyrazines and pyrroles,
and demonstrate advantages of using biocatalysis over
conventional methods for the preparation of these compounds.
N-Heterocyclic motifs, both saturated and unsaturated,
feature widely in pharmaceutical compounds and constitute
1
(
yield pyrazine products (see SI).
[
4]
privileged scaffolds. Amongst the plethora of different systems,
pyrroles feature widely (e.g. Atorvastatin and Sunitinib (Figure
[
5]
[6]
[7]
1
a) as well as substituted piperidines , pyrrolidines
and
indoles. Pyrazines are important components of aroma and
[
8]
flavor compounds including Polycartine B. Based upon a
[
9]
retrosynthetic analysis, we envisaged using ATAs to catalyze
amination of α-diketones into the corresponding α-amino
ketones which could then serve as key building blocks for both
pyrazine (path a) and pyrrole (path b) synthesis (Figure 1b). A
key challenge in this approach is to be able to control pyrazine
versus pyrrole synthesis by appropriate selection of reaction
conditions.
Scheme 1. A chemo-enzymatic approach for the synthesis of symmetric
substituted pyrazines by employing (S)-selective ATA-113.
[*]
Dr. A. P. Green, Prof. N. J. Turner
School of Chemistry, University of Manchester
Manchester Institute of Biotechnology
1
31 Princess Street, Manchester M1 7DN (UK)
With isopropylamine as amine donor, pyrazine products
were directly extracted from aqueous phase in pure form, with
no significant by-product or impurity observed (see SI).
E-mail: anthony.green@manchester.ac.uk
nicholas.turner@manchester.ac.uk
Supporting information for this article is given via a link at the end of
the document.
[
8b]
Pyrazines 4 and 5 are used as flavor enhancers and for the
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treatment of coronary atherosclerotic disease and ischemic
α-diketone 7 was chosen as a candidate substrate to couple with
β-keto ester a (10 equiv). ATA-117 catalyzed regioselective
amination of α-diketone 7 at C-3 with high efficiency, the product
of which underwent spontaneous coupling with β-keto ester a to
yield pyrrole 7a (Entry 1, Table1) with no observable pyrazine
formation. Reaction progress was determined by both GC-MS
[
10]
cerebrovascular disease respectively. Pyrazine 6 was isolated
as single regioisomer, reflecting the selectivity of the
a
biocatalytic amination step. Having established the viability of
using ATAs for pyrazine synthesis, our attention turned to the
Knorr pyrrole synthesis in which an α-amino ketone is
condensed with a β-keto ester. A biocatalytic approach to
pyrrole synthesis presents two key challenges: i) the prevention
of undesired oxidative dimerization of α-amino carbonyl
intermediates (pyrazine synthesis) and ii) the chemo-selective
amination of α-diketones in the presence of β-keto ester co-
substrates.
1
analysis (see SI) and H NMR analysis with the addition of 1,3-
dinitrobenzene (1 equiv) as internal standard (Table 1a) for
quantitative analysis of reaction components. The conversion of
α-diketone 7 in the presence of β-keto ester a proceeded
smoothly (see SI) and after 72 h a 55% conversion was
1
observed via H NMR analysis. A preparative scale reaction
yielded pyrrole 7a in 38% isolated yield (Entry 1, Table 1). The
generality of the process was investigated by examining a series
of aryl substituted α-diketones 7-12 in combination with two β-
keto esters a and b using ATA-117 with (R)-aminotetralin as
amine donor (Table 1b). All preparative scale reactions were
Table 1. Conversion of aryl α-diketones 7-12 to pyrroles 7-12a and 7b in the
presence of β-keto esters a and b.
1
(
a)
determined to be successful by both GC analysis and H NMR
Internal
Tetralone
Pyrrole
analysis (see SI), with isolation of pyrrole products subsequently
standard
by-product
product
(
Entries 2-7, Table 1).
Having established an effective means of synthesizing
pyrroles from aryl α-diketones, we next attempted the more
challenging synthesis of pyrroles from dialkyl α-diketones in
which the corresponding α-amino ketones are more susceptible
to oxidative dimerization to form pyrazines).
(b)
Table 2. Conversion of dialkyl α-diketone 2 to pyrazine 5 and pyrrole 2b in the
presence of β-keto ester b.
[
a]
1
2
[a]
Entry
Product
7a
R
H
R
NMR Conv.[%]
Yield [%]
38
1
2
3
4
5
6
7
Et
Et
Et
Et
Et
Et
Me
55
36
26
42
60
78
[
b]
Entry
pH
Equiv of b
Pyrrole/pyrazine ratio [%]
8a
3’-CF
4’-CF
4’-Cl
3
34
1
2
3
4
5
6
7
5
10
10
10
10
3
99:1
9a
3
21
6
96:4
10a
11a
12a
7b
36
7.4
9
81:19
69:31
97:3
4’-MeO
3’-Cl, 5’-Cl
H
31
48
[
b]
5
n.d.
42
5
2
89:11
87:13
1
[
a] Determined by H NMR analysis with the addition of 1,3-dinitrobenzene (1
equiv) as internal standard after 72 h. [b] n.d. = not determined, pyrrole 7b
directly isolated by flash chromatography after extraction.
5
1
[
a] Reaction condition: 20 mM α-diketone 2 and 20-200 mM β-keto ester b. [b]
Determined by GC-MS analysis after 24 h.
During our initial studies above, the products of amination of
aryl-substituted α-diketones were observed not to spontaneously
dimerize to pyrazines by GC-MS analysis (see SI). Hence, aryl
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Initially the simplest dialkyl α-diketone 2 was chosen as the
starting material to couple with β-keto ester b and the reaction
monitored by GC-MS analysis (see SI). Using the original
reaction conditions, the formation of both pyrazine 5 and pyrrole
In summary, we have developed biocatalytic methodology
for the synthesis of pyrazines and pyrroles using transaminases.
Pyrazine synthesis relied on the oxidative dimerization of an α-
amino ketone which was generated in situ from the
corresponding α-diketone via (regioselective) amination
catalyzed by ω-transaminases. The conversion of α-diketones to
pyrroles in the presence of β-keto ester was achieved via Knorr
pyrrole synthesis and the prevention of undesired dimerization of
the α-amino carbonyl intermediate was controlled by pH
modification. In addition, a self-sufficient approach for pyrrole
synthesis was realized using a β-amino ester as a substrate,
which simultaneously served as amine donor. This latter
approach exploited the reversible nature of biocatalytic
amination to shuttle amine functionality across reactions
partners with the reaction driven by irreversible pyrrole
formation.
2
b was detected with an 81:19 pyrrole/pyrazine ratio (Entry 3,
Table 2) and a 30% yield of pyrrole product was obtained.
Decreasing the pH of the reaction led to higher
pyrrole/pyrazine ratio (Entries 1-4, Table 2) with the
a
pyrrole/pyrazine ratio (99:1) optimal at pH 5 (Entry 1, Table 2).
Further optimization with reduced equivalents of β-keto ester b
at pH 5 (Entries 5-7, Table 2) yielded a pyrrole/pyrazine ratio
(
1
97:3) with 3 equiv of β-keto ester b (Entry 5, Table 2). Even with
equiv of β-keto ester b, the pyrrole/pyrazine ratio was still
maintained at a high level (87:13) (Entry 7, Table 2).
The ATA mediated synthesis of pyrroles relies on
amination of the α-diketone substrate in the presence of β-keto
ester, raising the question as to whether the ATAs is strictly
selective for α-diketones, or whether undesired amination of the
β-keto esters proceeds in a reversible fashion with the reaction
driven by the thermodynamically favourable pyrrole formation.
To probe the kinetic substrate selectivity, the reaction was
initiated using the corresponding β-amino ester 13 as substrate
in the presence of α-diketone but no additional amine donor. GC
Acknowledgements
NJT is grateful to the ERC for the award of an Advanced Grant
(
Grant number 742987). APG is grateful to the Biotechnology
and Biological Sciences Research Council for the award of a
David Phillips Fellowship (Grant number BB/M027023/1) and to
the ERC for the award of a Starter Grant (Grant number 757991)
1
and H NMR analysis (see SI) both confirmed pyrrole synthesis
with a conversion of 58% after 72 h. Under these conditions, the
amino group of the β-amino ester is transferred to α-diketone 7
to give the corresponding α-amino ketone precursor which then
reacts with the β-keto ester a to form pyrrole 7a via Knorr pyrrole
synthesis (Scheme 2). Control reactions in the absence of ATA
failed to produce any pyrrole product, confirming that the
transformation is enzyme catalyzed. This internal amine transfer
system provides an alternative disconnection for biocatalytic
pyrrole synthesis exploiting the reversible nature of biocatalytic
amination to shuttle amine functionality across reactions
partners. Ideally, a stoichiometric ratio of β-amino ester and α-
diketone would be consumed to access stoichiometric pyrrole
product in the absence of external amine donor, while the only
by-product is water. Previously, a similar mechanism has been
proposed to shuttle the amine functionality across the whole
Experimental Section
Detailed experimental procedures are presented in the supplementary
information.
Keywords: -transaminase • pyrrole • pyrazine • biocatalytic
retrosynthesis • N-heterocycle.
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Entry for the Table of Contents
Layout 2:
COMMUNICATION
Jin. Xu, Anthony P. Green,* and
Nicholas J. Turner*
Page No. – Page No.
Chemo-enzymatic synthesis of
pyrazines and pyrroles
Transaminases (ATAs) have been
employed in the chemo-enzymatic
synthesis of both pyrazines and pyrroles
by exploiting regioselective amination
and condensation reactions.
Text for Table of Contents
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