Aerobic Baeyer–Villiger Oxidation Catalyzed by a Flavin-Containing Enzyme Mimic in Water

Article abstract of DOI:10.1002/anie.201810124

Direct incorporation of molecular oxygen into small organic molecules has attracted much attention for the development of new environmentally friendly oxidation processes. In line with this approach, bioinspired systems mimicking enzyme activities are of particular interest since they may perform catalysis in aqueous media. Demonstrated herein is the incorporation of a natural flavin cofactor (FMN) into the specific microenvironment of a water-soluble polymer which allows the efficient reduction of the FMN by NADH in aqueous solution. Once reduced, this artificial flavoenzyme can then activate molecular dioxygen under aerobic conditions and result in the Baeyer–Villiger reaction at room temperature in water.

Full text of DOI:10.1002/anie.201810124

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Accepted Article
Title: Aerobic Baeyer-Villiger Oxidation Catalyzed by a Flavin-
Containing Enzyme Mimic in Water
Authors: Yoan Chevalier, Yvette Lock Toy Ki, Didier le Nouen, Jean-
Pierre Mahy, Jean-Philippe Goddard, and Frederic Avenier
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10.1002/anie.201810124
Angewandte Chemie International Edition
COMMUNICATION
Aerobic Baeyer-Villiger Oxidation Catalyzed by a Flavin-
Containing Enzyme Mimic in Water
Yoan Chevalier,[a] Yvette Lock Toy Ki,[b] Didier le Nouen,[b] Jean-Pierre Mahy,[a] Jean-Philippe
Goddard,[b]* Frédéric Avenier[a]*
Abstract: Direct incorporation of molecular oxygen into small
organic molecules is attracting much attention for the development
of environmentally friendly new oxidation processes. In this line, bio-
inspired systems mimicking enzymes activities are of particular
interest since they may perform catalysis in aqueous media. Herein,
we demonstrate that the incorporation of a natural flavin cofactor
(FMN) into the specific microenvironment of a water-soluble polymer
allows the efficient reduction of the FMN by NADH in aqueous
solution. Once reduced, this artificial flavoenzyme can then activate
molecular dioxygen under aerobic conditions and perform the
Baeyer-Villiger reaction at room temperature in water.
and the second one a flavin unmodified at the N5 position but
substituted by a peptide at the N3 position.[14] In both cases, zinc
dust was added as a source of electrons and reactions were
performed in a mixture of organic solvents. The next challenge is
therefore to develop new systems using readily available
unmodified flavins performing catalysis using dioxygen in water.
Modified polyethyleneimines (PEI) are water soluble
polymers known as good mimics for the locally hydrophobic
microenvironment of enzyme’s active sites.[15–27] Using PEI
modified with guanidinium and octyl groups, we previously
showed that the incorporation of flavin mononucleotide (FMN)
into such a microenvironment generated artificial flavo-enzymes
capable of collecting electrons from nicotinamide adenine
dinucleotide (NADH) and then of reducing metallic cofactors
under anaerobic conditions.[26,27] Here, we demonstrate that
under aerobic conditions, similar artificial flavo-enzymes made of
FMN incorporated into PEI modified with guanidinium and octyl
groups (FMN-PEIguan-oct; Schemes 2) catalyze the oxidation of
NADH and perform aerobic BV reaction in water. To the best of
our knowledge, this is the first example of non-enzymatic BV
reaction performed in water using natural flavin cofactor and
dioxygen from the air.
Selective transformation of ketones into the corresponding
esters or lactones, also known as the Baeyer-Villiger (BV)
reaction, prevails as a major reaction in organic synthesis since
its discovery in 1899.[1] It was originally performed using
persulfate in concentrated sulfuric acid and evolved towards the
use of other strong oxidants such as mCPBA or H2O2 activated
by Lewis acids.[2–4] In the present context of sustainable growth,
the use of those hazardous materials, combined to the use of
organic solvents, has now become a major issue for these
transformations and historical chemical processes need to be
renewed. Therefore, performing selective oxidation reactions by
direct incorporation of an oxygen atom coming from molecular
dioxygen into organic molecules under mild condition has
become a priority, not only for the Baeyer-Villiger reaction, but
also for other oxidation processes.[5,6]
Scheme 1. Example of natural BVMO bearing a flavin cofactor in its active
site[28]
Baeyer-Villiger monooxygenases (BVMO) can activate
dioxygen thanks to the flavin cofactor located into their active
site (Scheme 1) and stand as an interesting solution to reduce
the impact on the environment.[3,7] However, biochemical issues
such as cloning, protein expression and substrate selectivity
reduce their scope of applications for organic synthesis and
chemical alternatives are still highly needed. In this sense, flavin
derivatives have been developed as bioinspired catalysts and
flavinium salts have demonstrated interesting oxidation activities
using H2O2 or even O2 in combination with a reductant, notably
for sulfoxidation reactions.[8,9] In the particular case of the BV
reaction, examples involving both a flavin derivative and H2O2
are very scarce.[10–12] So far, only two systems were described
using O2, the first one involving an N5-alkylated flavinium salt,[13]
Before preparing the artificial flavoenzyme, PEI was randomly
modified by the covalent incorporation of guanidinium and octyl
groups and purified by dialysis as previously described.[24] After
lyophilization from water, the so-formed polymer was
characterized by 1H NMR, which allowed the determination of
the guanidinium/octyl group ratio (Figure S1, S.I. section). The
FMN cofactor was then incorporated into the polymer thanks to
the specific electrostatic interaction between the negatively
charged phosphate groups and the guanidinium moieties. This
incorporation of the FMN into the confined hydrophobic
environment of the polymer was monitored by UV-Vis absorption
spectroscopy, showing a characteristic 10 nm hypsochromic
shift of the FMN band at 370 nm (Figure 1).[26,29,30] Importantly,
we also measured the absorption spectrum of FMN-PEIguan-oct
in the presence of an excess of bicylo[3.2.0]hept-2-en-6-one as
[a]
Yoan Chevalier, Prof. Jean-Pierre Mahy, Dr. Frédéric Avenier
Univ Paris Sud, Université Paris Saclay
LCBB, ICMMO, (UMR CNRS 8182), 91405 Orsay, France.
E-mail: frederic.avenier@u-psud.fr
[b]
Yvette Lock Toy Ki, Didier le Nouen, Prof. Jean-Philippe Goddard
Université de Haute-Alsace, Université de Strasbourg, CNRS
LIMA UMR 7042, F_68100 Mulhouse, France.
E-mail: jean-philippe.goddard@uha.fr
Supporting information for this article is given via a link at the end of
the document.((Please delete this text if not appropriate))
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10.1002/anie.201810124
Angewandte Chemie International Edition
COMMUNICATION
potential substrate, and the same 10 nm shift was observed
(Figure 1, red trace). This indicated that the excess of substrate
did not exclude the FMN from the hydrophobic environment of
the polymer and that the artificial flavoenzyme was not altered
by the presence of substrate. It is also worth to note that
previous fluorescence spectroscopy studies showed that about
90% of the FMN was incorporated inside the local
microenvironment of the polymer.[26]
agreement with previous observations made with FMN-PEIguan-
oct under anaerobic conditions using stoichiometric amounts of
NADH.[26] Here, the consumption of 200 equivalents of NADH
with respect to the FMN demonstrates the catalytic behavior of
the system, suggesting that during the process, the FMN is
successively reduced to FMNH2 and reoxidized by molecular
dioxygen. During such a process, the FMN cofactor should
therefore generate
a transient peroxo species, which is
reminiscent of the active intermediate formed within the catalytic
cycle of BVMO.[3,7]
Scheme 2. Artificial flavoenzyme (FMN-PEIguan-oct) obtained by
incorporation of FMN into
a
25 kDa multi-branched PEI modified with
2
1.5
1
guanidinium and octyl groups.
N
NH2
H2N
N
HN
N
N
HN
NH
N
N
HN
N
N
N
HN
N
NH
HN
H
0.5
0
NH2
HN
N
H2N
N
O
NH
O P OH
O
N
NH2
N
NH2
HN
HO
HO
OH
0
2
4
6
8
10
12
N
N
N
N
O
O
NH
Time (hours)
Figure 2. Evolution of the absorbance of NADH (400 µM) at 340 nm upon
addition either to an aqueous solution of FMN (2 µM) (black squares) or to an
aqueous solution of FMN-PEIguan-oct (red circles) (FMN (2 µM) and PEIguan-
oct (2 mM in monomer).
0,4
0,3
0,2
0,1
0
These observations prompted us to study the system for BV
reaction catalysis and NADH was added to an aqueous solution
of FMN-PEIguan-oct in the presence of bicylo[3.2.0]hept-2-en-6-
one as substrate (Table 1). After 12 hours stirring at room
temperature under ambient atmosphere in deionized water, the
solution was analyzed by GC-MS and 1H NMR. It is worth to
note that the FMN being light sensitive, all reactions were
performed in tinted glassware in order to avoid any side
photocatalytic reaction during the process. GC-MS analysis
revealed the formation of one major product with a retention time
corresponding to the 2-oxabicyclo[3.3.0]oct-6-en-3-one (lactone
1), as well as a minor product with close retention time, 3-
oxabicyclo[3.3.0]oct-6-en-2-one (lactone 2) (Figure S2, S.I.
section). In both cases, the mass detection allowed to clearly
300
350
400
450
500
550
nm
1
identify lactones 1 and 2 (Table 1), which was confirmed by H
Figure 1. UV-Visible absorption spectra of a 25 µM aqueous solution of FMN:
alone (black trace), in the presence of modified PEI (25 mM in monomer)
(green trace), and in the presence of both PEI (25 mM in monomer) and
bicylo[3.2.0]hept-2-en-6-one (50 mM) (red trace).
NMR analysis (Fig. S3 and S4, S.I. section). In addition, no
signal of epoxide was observed, neither by GC-MS nor by 1H
NMR analysis, clearly indicating the exclusive formation of the
Baeyer-Villiger products. Once the lactones identified, the
reaction was followed over time by GC (Figure 3) in 20 mM
HEPES buffer, pH 8.5. The concentration of lactones reached a
plateau after 10 h of reaction with a maximum yield of 70 % with
respect to NADH, together with an excellent lactone 1:lactone 2
ratio of 87:13. This global yield corresponds to 140 turnovers
and nicely correlates with the consumption of NADH observed in
Figure 2. In the absence of polymer, the FMN only yielded 3 %
of lactone, which is also in good agreement with the very low
consumption of NADH observed in Figure 2. Other controls in
absence of dioxygen, or simply using PEIguan-oct or NADH on
their own, did not yield any product. When the reaction was
performed under stoichiometric conditions (10 mM both in
substrate and NADH) a 42 % conversion of the substrate was
obtained, corresponding to 840 turnovers.
FMN-PEIguan-oct was then studied in the presence of NADH
and Figure 2 shows the consumption of NADH when it was
added either to a solution of FMN, or to a solution of FMN-
PEIguan-oct. In the absence of polymer, the concentration of
NADH remained stable over time, meaning that NADH was not
able to reduce efficiently the FMN in solution. Advantageously,
when the FMN was located inside the modified PEI, the kinetics
of NADH consumption was improving with the concentration of
PEIguan-oct, reaching
a maximum for a ratio of 1000
equivalents of PEIguan-oct (in monomer) with respect to the
FMN cofactor. For such a ratio, the 200 equivalents of NADH
were fully consumed over 10 hours. This clearly indicated that
the FMN could consume NADH thanks to its incorporation inside
the hydrophobic environment of the polymer. This result is in
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10.1002/anie.201810124
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COMMUNICATION
In order to gain more insight into this catalytic system, the pH
dependency was also investigated (Figure S5, S.I. section).
Below pH = 7, only the lactone 1 was obtained with a maximum
yield of 47 % at pH = 6.7. For higher pH(s), a mixture of lactones
1 and 2 was obtained reaching a maximum global yield of 72 %
at pH 9 (Table 1). This trend is in good agreement with the
mechanism of natural BVMO involving the nucleophilic attack of
a deprotonated 4a-peroxo-flavin intermediate on the carbonyl
group of the substrate. However, the selectivity for the formation
of lactone 1 at acidic pH is more difficult to rationalize. One may
suggest that the 4a-hydroperoxo-flavin intermediate is not
reactive enough to form the less favored lactone 2.
hydrogen peroxide. Interestingly, it also compares well with the
use of mCPBA, which was previously reported with only 29 %
selectivity for lactone 1 (Table 1).[32]
This artificial enzyme is the first catalytic system utilizing
unmodified natural flavin cofactor to activate dioxygen and
perform the B.V. reaction in water. Only one catalyst using
unmodified flavin at the N5 position and bearing an oligopeptide
at the N3 position was described so far.[14] In this case, Imada
and collaborators showed that efficient catalysis was dependent
on the presence of a carboxylic acid at the C-terminus of the
oligopeptide, and DFT calculations demonstrated that this
carboxylic acid could actually stabilize a putative flavin-4a-
hydroperoxo intermediate by hydrogen bonding. Based on these
calculations, one may suggest that similar flavin-4a-hydroperoxo
intermediate could be stabilized via hydrogen bonding with the
amine groups present in the microenvironment of the FMN-
PEIguan-oct system. This would explain the good catalytic
activity of such systems. However, neither Imada’s group, nor
ours, have yet been able to experimentally observe such flavin-
4a-hydroperoxo intermediate with N5 unmodified flavin. Finally,
apart from the stabilization of a flavin-4a-hydroperoxo species,
catalysis could also be explain by the in situ formation of
hydrogen peroxide, which was indeed observed during catalysis.
However, the control experiment using hydrogen peroxide in the
absence of FMN and dioxygen, only yielded 27% of lactone,
suggesting that both phenomenon were probably involved in the
production of lactone.
70
60
50
40
30
20
10
0
0
5
10
15
20
25
Overall, we have shown that the incorporation of the natural
FMN cofactors into the locally hydrophobic microenvironment of
a modified polyethyleneimine allows the flavin cofactors to
collect electrons from NADH, activate dioxygen and perform the
BV reaction in good yield and high selectivity. Such an artificial
flavo-enzyme stands therefore as potential alternative to the use
hazardous materials such as peroxides or peracids in organic
solvents and paves the way to the development of new
environmentally friendly catalysts capable of using dioxygen in
water.
Time (hours)
Figure 3. Kinetics for the total formation of lactones 1 and 2 catalyzed by
FMN-PEIguan-oct (FMN: 5 µM; PEIguan-oct: 5 mM) (black circles) or by FMN
only (FMN: 5 µM) (red squares) 20 mM HEPES, pH 8.5 with NADH: 1 mM;
substrate: 10 mM; at room temperature, under ambient atmosphere.
Table 1. Chemical and biomimetic oxidation of bicylo[3.2.0]hept-2-en-6-one.
O
O
O
O
O
O
O
[Ox]
O
Experimental Section
(1)
(2)
(3)
(4)
For experimental data, please see the supporting information section.
Oxidation
Products yields (%)
Selec.d
Ref.
Conditions
(1) (2) (3) (4)
(%)
Acknowledgements
FMN-PEIguan-oct
O2 /NADH (pH 6.7)a
O2 /NADH (pH 8.0)a
O2 /NADH (pH 9.0)a
H2O2 / acetic acidb
mCPBAc
47
56
-
-
-
-
-
-
-
-
100
87
83
85
29
This work
This work
This work
(31)
The authors thank the Agence Nationale de la Recherche (ANR-
16-CE29-0006) for financial support and JPG thanks the Institut
Universitaire de France for financial support.
8
60 12
77 14
-
Keywords: Dioxygen activation • Baeyer-Villiger reaction •
28
-
32 37
(32)
Catalysis • Artificial enzyme • Sustainable chemistry
aGC yields using [FMN] = 5 µM; [PEIguan-oct] = 5 mM in monomer ;
[substrate] = 10 mM; [NADH] = 1 mM; Atmospheric O2.; room temp. bIsolated
yield using conditions from ref. (31) with specific yields calculated by NMR
(see Supp. Info.). cData from ref. (32). dSelectivity for lactone 1.
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COMMUNICATION
Entry for the Table of Contents (Please choose one layout)
Layout 1:
COMMUNICATION
Incorporation of flavin cofactors (FMN)
within the locally hydrophobic
microenvironment of a modified
polyethyleneimine allows to activate
dioxygen in the presence of NADH
and to perform the Baeyer-Villiger
reaction in good yield, under ambient
atmosphere, at room temperature in
water.
Yoan Chevalier, Yvette Lock Toy Ki,
Didier le Nouen, Jean-Pierre Mahy,
Jean-Philippe Goddard,* Frédéric
Avenier*
Page No. – Page No.
Aerobic Baeyer-Villiger Oxidation
Catalyzed by a Flavin-Containing
Enzyme Mimic in Water
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