CaC-Ene-Reductases Reduce the CaN Bond of Oximes

Article abstract of DOI:10.1021/acscatal.0c03755

Although enzymes have been found for many reactions, there are still transformations for which no enzyme is known. For instance, not a single defined enzyme has been described for the reduction of the CaN bond of an oxime, only whole organisms. Such an enzymatic reduction of an oxime may give access to (chiral) amines. By serendipity, we found that the oxime moiety adjacent to a ketone as well as an ester group can be reduced by ene-reductases (ERs) to an intermediate amino group. ERs are well-known enzymes for the reduction of activated alkenes, as of α,β-unsaturated ketones. For the specific substrate used here, the amine intermediate spontaneously reacts further to tetrasubstituted pyrazines. This reduction reaction represents an unexpected promiscuous activity of ERs expanding the toolkit of transformations using enzymes.

Full text of DOI:10.1021/acscatal.0c03755

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Research Article
CC-Ene-Reductases Reduce the CN Bond of Oximes
Stefan Velikogne, Willem B. Breukelaar, Florian Hamm, Ronald A. Glabonjat, and Wolfgang Kroutil*
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ABSTRACT: Although enzymes have been found for many
reactions, there are still transformations for which no enzyme is
known. For instance, not a single defined enzyme has been
described for the reduction of the CN bond of an oxime, only
whole organisms. Such an enzymatic reduction of an oxime may
give access to (chiral) amines. By serendipity, we found that the
oxime moiety adjacent to a ketone as well as an ester group can be
reduced by ene-reductases (ERs) to an intermediate amino group. ERs are well-known enzymes for the reduction of activated
alkenes, as of α,β-unsaturated ketones. For the specific substrate used here, the amine intermediate spontaneously reacts further to
tetrasubstituted pyrazines. This reduction reaction represents an unexpected promiscuous activity of ERs expanding the toolkit of
transformations using enzymes.
KEYWORDS: biocatalysis, ene-reductases, oxime, amine, pyrazine
investigated activated oxime 1a, the CN double bond
INTRODUCTION
■
conjugated to a carbonyl reminded us of α,β-unsaturated
The repertoire of chemical reactions gets constantly
expanded,1 including the spectrum of biocatalytic trans-
carbonyl compounds, which are typical substrates for flavin-
dependent ERs.14 In general, ERs have been described to
reduce CC double bonds, which are activated via a broad set
of electron-withdrawing functionalities such as carbonyls
(aldehydes, ketones, carboxylic acids, and esters), nitriles,
cyclic imides, and nitro groups. Although 1a does not possess a
CC but a CN double bond, several purified ERs15−19
were screened for their activity toward the model compound
1a (Scheme 1 and Table 1). Surprisingly, all ERs investigated
transformed substrate 1a in the presence of a NADPH
recycling system [glucose dehydrogenase (GDH)/glucose].
Isolation of the obtained product revealed the formation of the
tetrasubstituted pyrazine 2a as the sole detectable product.
Pyrazines have previously been obtained, for example, by
chemoenzymatic reaction sequences involving transaminases.20
In the absence of GDH/glucose, product formation of 2a
corresponded to the amount of NADPH present (Table S2).
In the absence of NADPH, no product formation was
detectable at all. Furthermore, no product was detected (i)
when omitting the ER, (ii) when the ER was substituted for a
catalytic amount of FMN, and (iii) when the ER was
substituted for a stoichiometric amount of NADPH. This
indicated that this transformation is catalyzed by the ERs.
formations by, for example, (i) exploiting light2 or (ii) evolving
enzymes for new reactions.3 However, the biocatalytic
transformation of an oxime to an amine has not been observed
using a defined enzyme.
The oxime functionality can be chemically transformed via
various established reactions4 such as rearrangements5 or
chemical reductions, the latter leading to amines,6 imines,7
aziridines,8 or hydroxylamines.9 Biocatalytic transformations of
oximes have been reported to give carbonyl compounds or
alcohols only using baker’s yeast, alcohol dehydrogenases, or
ene-reductases (ERs).10 In these cases, the oxime was most
likely transformed enzymatically to the corresponding imine,
which was subsequently hydrolyzed spontaneously to the
carbonyl compound. Other reports indicated that whole-cell
organisms such as Baker’s yeast or anaerobic organisms may
enable the reduction of oximes to afford the corresponding
hydroxylamines and/or amines or even pyrazines,11 but the
involved enzymes have never been identified. Here, we report
the first enzymatic reduction of an oxime to an amine using
ERs.
RESULTS AND DISCUSSION
■
Searching for a defined enzyme to reduce the CN double
bond of an oxime, we suspected first that imine reductases12
may be well-suited enzymes. Six randomly selected imine
reductases (see the Supporting Information for details) were
tested for the reduction of the model compound 1a. However,
no transformation was observed in any case, which supports a
previous report on unsuccessful reduction of oximes with
imine reductases.13 Looking closer to the structure of the
Received: August 28, 2020
Revised: October 21, 2020
https://dx.doi.org/10.1021/acscatal.0c03755
© XXXX American Chemical Society
ACS Catal. 2020, 10, 13377−13382
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Research Article
general, the data in Table 1 show that all substrates were
converted approximately equally well by OYE1, 2, and 3 and
FOYE. Although XenA led to only moderate results for 1a−g,
it showed best performance for the ethyl ketone-derived
substrate 1h in comparison to all other enzymes tested.
Preparative biotransformations were performed at the 200
mL scale using the enzyme that gave the highest product
formation on analytical scale. Products 2a and 2d gave the best
isolated yields with 61 and 62%, respectively, whereas the
isolated yield of pyrazines of 2g and 2h was 40%. Control
experiments indicated that the formed products are stable in an
aqueous solution under the conditions used and can be
recovered quantitatively.
Scheme 1. Reductive Transformation of Activated Oximes
by ERs
Next, we turned our attention to the possible reaction
pathway. As described for pyrazine formation employing
transaminases,20 an α-amino keto motif can be expected to
be an intermediate; thus, for the reaction of 1a, α-amino-β-
keto ester 3 might be the intermediate (Scheme 2).
Table 1. Transformation of the Oximes with Purified ERs
a
enzyme
Scheme 2. Proposed Reaction Pathway from 1a to Pyrazine
2a through ER-Catalyzed Oxime Reduction and
Spontaneous Oxidation
bcd
, ,
product formation 2 [%]
#
sub OYE1
OYE2
OYE3
66
OPR3
XenA
FOYE
61
64 (42)
53 (53)
51
77
53
62 (35)
14
1
2
3
4
5
6
7
8
1a
1b
1c
1d
1e
1f
64
64
48
50
61
56
59
18
68 (61)
58
49
51
63
59
56
17
40
43
38
41
49
45
49
16
15
<1
9
17
<1
<1
29
38 (39)
62
39
53 (62)
77 (57)
59 (50)
51
1g
1h
28
a
The enzymes originate from Saccharomyces pastorianus (OYE1),15
Saccharomyces cerevisiae (OYE2 and OYE3),16 Arabidopsis thaliana
(OPR3),17 Pseudomonas putida (XenA),18 and Ferrovum sp. JA12
(FOYE).19 Reaction conditions: ER (200 μg/mL, 4 μM), 10 mM
b
substrate, 0.5 mM NADPH, 50 mM glucose, 4 mg/mL GDH, 5%
DMSO (v/v), 50 mM phosphate buffer, pH 7.5, 30 °C, 24 h, and 120
c
rpm. Product formation is defined by the amount of substrate
The formation of compound 3 from the oxime (in
equilibrium with the corresponding nitroso compound)
formally requires two hydrides originating from NAD(P)H.
Either an imine or a hydroxylamine might be formed after the
first hydride transfer. Dimerization of 3 to X, followed by
spontaneous oxidation, leads to the final product. As a first
attempt to detect the intermediates, the biotransformation of
1a was followed via NMR over a period of 30 min. When
analyzing the obtained spectra, the peaks could be assigned to
the oxime substrate, buffer components, glucose, and the
pyrazine product (see the Supporting Information, Figure
S48); however, no intermediate was detected with this
method. In addition, following the reaction by HPLC−
HRMS did not lead to any hint either.
In an attempt to get hold of the proposed intermediate 3, a
cascade reaction was envisioned to reduce the carbonyl group
of the possible formed intermediate 3 by an alcohol
dehydrogenase to obtain the threonine ester 4 (Scheme 3).
Consequently, the cyclization of 3 would be circumvented and
the formation of 4 would give an indirect proof for the
formation of compound 3. For this purpose, an ADH is
required, which does not transform oxime 1a but may
transform any derivative. Fortunately, it turned out that
ADH-A from Rhodococcus ruber21 does not transform oxime
1a. Therefore, any new product observed in the cascade must
be due to ADH-catalyzed transformation of a compound
formed by reduction with the ER.
transformed to 2 as deduced from GC analysis using calibration
d
curves. Numbers in brackets represent isolated yield.
Subsequently, a library of β-keto α-oximo ester substrates
and one β-keto α-oximo amide were investigated by varying
the size of the alcohol part of the ester (R′) and the substituent
R at the ketone side (substrates 1b−1h, Table 1). Increasing
the size of the ester (R′) moiety from ethyl (1a) to bigger
groups such as benzyl (1f) led to a comparable amount of
product formation for most enzymes, except XenA, which
behaved differently and led to very low product formation for
some esters. Substrate 1g, a N-phenylamide, was converted in a
comparable fashion like the related ester 1f. Again XenA was
different because for this enzyme, 1g was the substrate, leading
to the second best value for product formation. In general, it
can be stated that the accepted scope of ester moieties is rather
broad, as far as tested.
In contrast, extending the methyl group at the ketone moiety
to an ethyl group (substrate 1h) led to low product formation
with almost all enzymes investigated (entry 8). The only
exception was the ER XenA, which gave decent product
formation of 2h for this substrate (38%). However, when the
size of the substituent at the ketone moiety (R) was extended
further to an n-butyl or phenyl group (substrates 1i and 1j, see
the Supporting Information for details), no pyrazine formation
or any other product was detected with any enzyme. In
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Research Article
Scheme 3. Cascade Reaction to Give D-Threonine Ethyl Ester 4 for the Indirect Proof of Intermediate 3
Performing the cascade and analyzing the formed product by
HR-MS (Figures S59 and S60) as well as after derivatization
[as a N-benzoyl derivative (5)] by HPLC on a chiral stationary
phase allowed to identify the newly formed product as a
threonine derivative and to determine the absolute config-
uration of the stereocenters of the product 4 by comparison
with independently derivatized stereoisomers of threonine
(Figures S3 and S4). As expected, the ADH-A led to the
formation of an (S)-configured alcohol moiety.21 Interestingly,
D-configuration was observed for the α-carbon for all ERs
investigated with excellent optical purity (>98% ee and de,
Table 2). Thus, the CN bond was reduced in a
candidate for clinical studies. Using the approach described
here, liguzinediol (6) was synthesized in a two-step chemo-
enzymatic reaction sequence (Scheme 4).
In the first step, the ER produced the pyrazine ester 2d,
which was isolated and subjected to chemical reduction using
sodium borohydride giving 80 mg of liguzinediol (6), which
corresponds to an overall yield of 45% over two steps. This
example demonstrates the applicability of the promiscuous
activity26 of ERs for the transformation of oximes.
CONCLUSIONS
■
Herein, we report the reduction of an oxime to an amine by a
specific enzyme, namely, ERs. Selected biocatalytic trans-
formations of oximes resulted in general in the formation of
alcohols or aldehydes when using defined enzymes; few cases
report the formation of amines using wild-type cells, however,
without knowing the involved enzyme(s). This study presents
the first biocatalytic reduction of oximes to an amine using a
defined enzyme. The reaction was found to work using a
variety of structurally different ene-reductases and eight β-keto
α-oximo ester or amide substrates. The oxime moiety of the
substrates tested was reduced to amine and then reacted
further spontaneously to pyrazines because of the nature of the
substrate. The intermediate amine was trapped in situ by
reducing the neighboring ketone group to an alcohol after
amine formation, thereby avoiding the spontaneous cyclization.
Therefore, the reaction sequence is suggested to involve a 2-
fold reduction of the oxime moiety, yielding an α-amino-β-keto
ester intermediate. Both enzymatic steps (carried out by ADH
and ER) showed very high stereoselectivity, yielding the
trapped intermediate in >98% ee and de and showing the ERs
to be (R)-selective toward these substrates. The reaction was
also shown to be feasible on a preparative scale (2 mmol),
providing the pyrazine products in decent yields. The
application of this reaction was demonstrated in a chemo-
enzymatic synthesis of liguzinediol. The reduction of oximes to
amines exploiting the promiscuous activity of ER demonstrates
the potential of biocatalysis for non-natural reactions extending
the biocatalytic toolbox for synthetic applications.27
Table 2. Product Formation (4) and Stereochemical
Outcome of the Cascade Reactions
ab
,
c
c
enzyme
4 (%)
ee [%]
de [%]
abs. config. 4
OYE1
OYE2
OYE3
OPR3
XenA
FOYE
54
46
40
38
20
29
>98
>98
>98
>98
>98
>98
>98
>98
>98
>98
>98
>98
(2R,3S)
(2R,3S)
(2R,3S)
(2R,3S)
(2R,3S)
(2R,3S)
a
Reaction conditions: 10 mM substrate, ER (200 μg/mL, 4 μM),
ADH-A (2 mg/mL heat purified enzyme), 0.5 mM NADPH, 0.5 mM
NADH, 50 mM glucose, 4 mg/mL GDH, DMSO (5% v/v), 50 mM
b
phosphate buffer, pH 7.5, 30 °C, 24 h, and 120 rpm. Product
formation of 4 is defined as the amount of substrate transformed to 4
c
analyzed as a N-benzoyl derivative by HPLC. Ee and de were
determined by HPLC analysis on a chiral phase.
stereoselective fashion, as only one stereoisomer of 4 was
detected. The observed high stereospecificity again clearly
supports that the reduction of the oxime is enzyme-catalyzed.
The pyrazine core is present in a multitude of naturally
occurring flavor and fragrance compounds, which usually
contribute to a roasted, nutty, or chocolate-like aroma.22
Pyrazines have been synthesized via various chemical
methods23 including recent biocatalytic protocols using
transaminases.20a,b In addition to the olfactory properties,
various pyrazines have potential applications in the medical
sector.24 For instance, liguzinediol (5) [(3,6-dimethylpyrazine-
2,5-diyl)dimethanol] has recently been discovered as a
potential agent for the treatment of heart failure with fewer
side effects than traditional agents24a,25 and is hence a suitable
ASSOCIATED CONTENT
* Supporting Information
■
sı
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acscatal.0c03755.
Scheme 4. Chemoenzymatic Synthesis of Liguzinediol (6)
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AUTHOR INFORMATION
Corresponding Author
Wolfgang Kroutil − Institute of Chemistry, University of Graz,
NAWI Graz, 8010 Graz, Austria; Field of Excellence
BioHealth, University of Graz, 8010 Graz, Austria;
BioTechMed Graz, 8010 Graz, Austria; orcid.org/0000-
0002-2151-6394; Email: wolfgang.kroutil@uni-graz.at
■
Authors
Stefan Velikogne − Institute of Chemistry, University of Graz,
NAWI Graz, 8010 Graz, Austria
Willem B. Breukelaar − Institute of Chemistry, University of
Graz, NAWI Graz, 8010 Graz, Austria
Florian Hamm − Institute of Chemistry, University of Graz,
NAWI Graz, 8010 Graz, Austria
Ronald A. Glabonjat − Institute of Chemistry, University of
Graz, NAWI Graz, 8010 Graz, Austria; orcid.org/0000-
0003-3104-1940
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Complete contact information is available at:
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Author Contributions
S.V. and W.B.B. contributed equally.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The COMET center: acib: Next Generation Bioproduction is
funded by BMVIT, BMDW, SFG, Standortagentur Tirol,
Government of Lower Austria und Vienna Business Agency in
the framework of COMETCompetence Centers for
Excellent Technologies. The COMET-Funding Program is
managed by the Austrian Research Promotion Agency FFG.
The University of Graz, the Doctoral Academy and the Field of
Excellence BioHealth is acknowledged for the financial
support. We would also like to thank NAWI-Graz for
supporting the Graz Central LabMetabolomics. The
Austrian Science Fund (FWF) is thanked for funding within
doc.funds (DOC 46-821, Catalox).
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ABBREVIATIONS
ADH, alcohol dehydrogenase; ER, ene-reductase; GDH,
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■
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