Visible light-driven and chloroperoxidase-catalyzed oxygenation reactions

Article abstract of DOI:10.1039/b915078a

Robust peroxidase-catalyzed enantiospecific oxyfunctionalizations can be achieved by simple light-driven in situ generation of hydrogen peroxide.

Full text of DOI:10.1039/b915078a

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Visible light-driven and chloroperoxidase-catalyzed oxygenation
reactionsw
Daniel I. Perez,z Maria Mifsud Grau,z Isabel W. C. E. Arends* and Frank Hollmann*
Received (in Cambridge, UK) 27th July 2009, Accepted 14th September 2009
First published as an Advance Article on the web 1st October 2009
DOI: 10.1039/b915078a
Robust peroxidase-catalyzed enantiospecific oxyfunctionaliza-
tions can be achieved by simple light-driven in situ generation
of hydrogen peroxide.
(e.g. flavine adenine dinucleotide FAD, -mononucleotide
FMN, or riboflavin Rf) by simple and cheap sacrificial
electron donors such as ethylenediaminetetraacetate (EDTA),
(2) high reactivity of reduced flavins (FADH2, FMNH2, RfH2)
with O2 yielding H2O2 (ESIw).17 We hypothesized that the
reaction sequence outlined in Scheme 1 might be useful to
promote CPO-catalysis.
Chloroperoxidase from Caldariomyces fumago (CPO, E.C.
1.11.1.10) is a versatile oxidation and oxyfunctionalization
catalyst.1 It catalyzes a broad range of synthetically useful
transformations such as oxidation of alcohols,2 hydroxylation
of allylic, propargylic and benzylic C–H bonds,3 CQC double
bond epoxidations,4 halohydroxylations,5 and heteroatom
oxygenation reactions.6 Many of these proceed with high to
exclusive regio-, chemo-, and enantioselectivity. Unlike
the related P450 monooxygenases,7 CPO does not rely on
prohibitively expensive nicotinamide cofactors and molecular
oxygen to regenerate its catalytically active oxyferryl heme
active site.1b Instead, CPO utilizes simple hydrogen peroxide,
which makes CPO an attractive catalyst for preparative
organic chemistry.
As a starting point for our light-driven in situ H2O2
generation system we (arbitrarily) chose FMN as photo-
catalyst and EDTA as sacrificial electron donor. An ordinary
slide projector equipped with a 250 W bulb (Philips 7748 XHP,
see ESIw for experimental setup) served as light-source.
Illumination of an anaerobic solution of FMN (0.1 mM in
potassium phosphate buffer (KPi) pH 5.1) in the presence of
EDTA resulted in fast (o1 minute) decolourization of the
solution indicating full reduction of FMN. The characteristic
yellow colour of oxidized FMN returned upon aeration
accompanied by the formation of H2O2. This sequence
could be repeated several times. From these experiments we
estimated a catalytic performance for the flavin photocatalyst
(TF) of approximately 1.6 turnovers per minute.
Its high synthetic potential however is impaired by its rather
poor operational stability, particularly towards H2O2.8
The exact inactivation mechanism is still under debate but it
is clear that oxidative degradation of the heme prosthetic
Next we combined the photocatalytic H2O2 generation with
CPO to perform a range of typical CPO-oxidation and
-oxyfunctionalization reactions (Table 1).
group plays a major role.9 Portionwise addition of H2O2
10,11
to maintain the H2O2 concentration at acceptable levels
significantly increased the total turnover number (TTN) of
CPO over stoichiometric use of H2O2 or less reactive organic
peroxides.3a,12 But the heterogeneous nature of the external
addition results in ‘hot spots’ comprising locally high H2O2
concentrations and fast CPO inactivation.6b This may be
circumvented by generating H2O2 in homogeneous phase
(in situ) by reduction of molecular oxygen. Chemical,13
electrochemical,6c,14 or enzymatic methods have been
reported.6b,15,16 But each approach comprises specific
disadvantages such as need for specialized equipment or a
second, costly enzyme. Furthermore, sufficiently high TTNs
have not been achieved yet for CPO. Consequently, the
quest for a simple, easily applicable, and robust in situ
H2O2-generation method continues.
In all cases, the photoenzymatic approach turned out to be
superior to the stoichiometric addition of H2O2 which we
attribute to an increased stability of CPO (vide infra).
Performing these experiments either in the absence of EDTA,
FMN, or in darkness yielded no conversion. Also in the
absence of CPO, no conversion was observed with the exception
of thioanisole where trace amounts of racemic sulfoxide were
found. The enantioselectivity of the CPO-catalyzed sulfoxidation
reaction was not impaired (Table 1).
The system is not confined to FMN as photocatalyst:
substituting FMN by FAD or Rf under otherwise identical
conditions influenced neither rate nor the stereochemical
outcome of the sulfoxidation reaction (data not shown).
Encouraged by these results, we further evaluated the
possibility to control the H2O2 formation rate via the photo-
catalyst concentration (Fig. 1). The overall rate correlated
with the flavin concentration applied. Thus, the sulfoxide
formation rate increased from 2.3 mM hꢀ1 to 11.8 mM hꢀ1
with increase of [FMN] from 10 mM to 250 mM. In terms of
TF(CPO) this corresponds to an increase from 9.8 minꢀ1 to
123 minꢀ1. Furthermore, significant accumulation of H2O2
was not observed. This indicates that the photocatalytic
generation of H2O2 was overall rate-limiting and H2O2 was
quickly consumed by CPO-catalyzed sulfoxidation. The very
low in situ H2O2 concentration also explains the significantly
Here, we report on a novel, light-driven approach for the
in situ generation of H2O2 to promote CPO-catalyzed
oxidation–oxyfunctionalization reactions. We make use of
(1) facile reduction of visible light-excited isoalloxazines
Department of Biotechnology, Biocatalysis and Organic Chemistry,
Delft University of Technology, Julianalaan 136, Delft, 2628 BL, The
Netherlands. E-mail: f.hollmann@tudelft.nl, i.w.c.e.arends@tudelft.nl;
Fax: +31 (0)152781415; Tel: +31 (0)1522781957
w Electronic supplementary information (ESI) available: Mechanistic
details, experimental setup. See DOI: 10.1039/b915078a
z These authors contributed equally.
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Scheme 1 Light-driven in situ H2O2 generation to promote CPO-catalyzed sulfoxidation reactions. Co-substrates used in this study comprise
EDTA and formate (see ESIw).
Table 1 Typical CPO-catalyzed oxidation–oxyfunctionalization
reactions driven by the proposed photochemical in situ H2O2 generation
approach compared to the stoichiometric use of H2O2
a
Conversion (%)/TN (CPO)
H2O2
FMN/hn/O2
Substrate
Product
30/6720
100/22 400
26/830
40/1270
96/3050
60/1910
a
Conditions: [thioanisole] = 8 mM, [CPO] = 0.357 mM, [H2O2] = 8 mM
or [EDTA] = 8 mM, [FMN] = 80 mM, hn, both methods yielded
(R)-sulfoxide in optical purity 499% ee; [indole] = [furfuryl alcohol] =
25 mM, [CPO] = 7.85 mM, [H2O2] = 25 mM or [EDTA] = 25 mM,
[FMN] = 250 mM, hn; no over oxidation products such as sulfone
Fig. 1 Influence of FMN concentration on the light-driven CPO-
catalyzed oxidation of thioanisole. Conditions: 25 mL tBuOH–50 mM
phosphate buffer pH = 5.1 (25/75), T = 25 1C, [thioanisole] = 50 mM,
[CPO] = 3.93 mM, oxidant: [H2O2] = 50 mM (J) or [EDTA] =
50 mM, [FMN] = 10 mM (B), 50 mM (&), 250 mM (n). In all samples,
the enantiomeric purity of the sulfoxide was greater than 98% ee.
or furfurylic acid were observed. TN
mol(product) ꢂ mol(CPO)ꢀ1
= turnover number =
.
increased operational stability of CPO: while with stoichio-
metric H2O2, CPO was fully inactivated within maximally
3 minutes, stable production of (enantiopure) sulfoxide
continued for at least 7 h in case of the photocatalytic in situ
formation of H2O2 corresponding to an at least 100-fold
improved operational stability of CPO. Thus, we were able
to demonstrate that the H2O2 supply rate can easily be
controlled via the photocatalyst concentration.
lower (78% ee, R) than using EDTA as the sacrificial electron
donor (499% ee, R). This reduction of enantioselectivity was
even more pronounced if H2O2 was added stoichiometrically
(ESIw). Similar effects have been reported recently for the
CPO-catalyzed epoxidation of limonene4d and can be rationalized
by assuming binding of formate to the heme-iron as suggested
by crystallographic data.19 Thus, presence of formate might
impair precise positioning of thioanisole within the CPO
active site.
Envisaging an environmentally benign photoenzymatic
reaction, EDTA may not be the sacrificial electron donor
of choice. Especially the generation of formaldehyde and
ethylene diamine as waste products is not desirable from
an environmental and toxicological point-of-view.18 Other
sacrificial electron donors such as methionine, adrenaline, or
nicotine17a are also not attractive as again significant amounts
of waste are generated. Therefore, we evaluated the suitability
of formate as simple and cheap sacrificial electron donor,
yielding CO2 as the sole by-product. In fact, EDTA could be
substituted by formate as sacrificial electron donor to promote
CPO-catalyzed sulfoxidation and full conversion was observed
(ESIw). Interestingly, here the enantioselectivity was somewhat
Overall, we have demonstrated that the proposed light-
driven approach for in situ generation of H2O2 is practicable
to promote CPO-catalyzed oxygenation reactions. The H2O2
generation rate can be easily controlled via the photocatalyst
concentration. Further characterization will result in an optimized
ratio of photo- and biocatalyst enabling high reaction rates
while minimizing H2O2-related inactivation of the enzyme.
Thus, we have turned a previously undesired side reaction17c,d
into a simple, robust, and easily applicable novel method for
peroxidase-catalyzed oxidation and oxyfunctionalization
reactions. Compared to established approaches (Table 2),
one major advantage of the photocatalytic generation of
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Table 2 CPO-catalyzed sulfoxidation of thioanisole using different H2O2 generation/dosage methods
TTN
CPO
Co-catalyst
H2O2 generation method
Yield (%)
ee (%)
Remark/requirements
a
Stoichiometric H2O2
Sensor-controlled2b
Glucose oxidase/glucose/O2
4900
—
—
30
n.d.
79
11–60
76
100
100
499
n.d.
99
9–36
93
—
Additional equipment
2nd enzyme
Autoclave/detonation gas
Electrochemical cell
Visible light
148 000
250 000
6500
58 900
22 400
22 000
1b,6b
n.d. (glucose oxidase)
12.8 (Pd/C)
—
1250 (FMN)
41000 (FMN)
13
Pd/H2/O2
Cathode/O2
6c
a
Flavin/EDTA/hn/O2
o99
a
Flavin/NaHCO2/hn/O2
78
Visible light
n.d. = not determined.a This study.
H2O2 lies in its simplicity. No specialized equipment or
catalysts are required, all reactions were performed at ambient
pressure and temperature using a readily available light-source
(also sunlight) and commercially available catalysts. Furthermore,
already under non-optimized conditions turnover numbers of
more than 1.000 and 22.000 have been achieved for the
photocatalyst and CPO, respectively. Current work ongoing
in our laboratory comprises full characterization and optimization
of the reaction setup and evaluation of further simple sacrificial
electron donors e.g. phosphite. Thus, we are convinced to
eventually obtain a simple, compatible, and environmentally
benign reaction setup. Furthermore, preliminary results suggest a
general applicability to heme enzyme-catalyzed oxidation
reactions such as horseradish peroxidase-catalyzed oxidative
C–C coupling reactions and cytochrome C-catalyzed oxygenations.
Also this approach might be used for simplified P450
oxygenation reactions via the hydrogen peroxide shunt
pathway20 and thereby become useful e.g. for screening and
drug metabolite synthesis.
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