Atom-Economic Electron Donors for Photobiocatalytic Halogenations

Article abstract of DOI:10.1002/cctc.201800886

In vitro cofactor supply and regeneration have been a major obstacle for biocatalytic processes, in particular on a large scale. Peroxidases often suffer from inactivation by their oxidative co-factor. Combining photocatalysis and biocatalysis offers an innovative solution to this problem, but lacks atom economy due to the sacrificial electron donors needed. Herein, we show that redox-active buffers or even water alone can serve as efficient, biocompatible electron sources, when combined with photocatalysis. Mechanistic investigations revealed first insights into the possibilities and limitations of this approach and allowed adjusting the reaction conditions to the specific needs of biocatalytic transformations. Proof-of-concept for the applicability of this photobiocatalytic reaction setup was given by enzymatic halogenations.

Full text of DOI:10.1002/cctc.201800886

Accepted Article
Title: Atom-Economic Electron Donors for Photobiocatalytic
Halogenations
Authors: Catharina J. Seel, Antonín Králík, Melanie Hacker, Annika
Frank, Burkhard Koenig, and Tanja Gulder
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10.1002/cctc.201800886
ChemCatChem
COMMUNICATION
Atom-Economic Electron Donors for Photobiocatalytic
Halogenations
Catharina Julia Seel,[a] Antonín Králík,[b] Melanie Hacker,[b] Annika Frank,[c] Burkhard König*[b] and Tanja
Gulder*[a]
Abstract: In vitro cofactor supply and regeneration has been a major
obstacle for biocatalytic processes, in particular on a large scale.
redox enzymes is photocatalysis.[8] The photocatalyst, like, e.g.,
flavin mononucleotide (FMN),[9] is excited to FMN* by blue light
Peroxidases often suffer from inactivation by their oxidative co-factor. followed by its reduction to FMNH2 at the expense of an electron
Combining photocatalysis and biocatalysis offers an innovative
solution to this problem, but lacks atom economy due to the
sacrificial electron donors needed. Herein, we show that redox-
active buffers or even water alone can serve as efficient,
biocompatible electron sources, when combined with photocatalysis.
Mechanistic investigations revealed first insights into the possibilities
and limitations of this approach and allowed for adjusting the
reaction conditions to the specific needs of biocatalytic
transformations. Proof-of-concept for the applicability of this
photobiocatalytic reaction setup was given by enzymatic
halogenations.
donor. FMNH2 is then re-oxidized by atmospheric O2 to form
H2O2 (Scheme 1). This continuous, light-driven process
guarantees a constant, but low hydrogen peroxide concentration
in solution and thus renders its combination with biocatalytic
processes ideal, especially if oxidation sensitive enzymes are
involved. The sacrificial electron donor, however, can constitute
an obstacle in this photobiocatalytic setup, as commonly
employed reductants, such as EDTA, formates, or 4-
methoxybenzyl alcohol, may suffer e.g. from incompatibility with
the enzyme and cumbersome separation from the products.[10]
Biocatalysts are playing
a
key role in establishing
environmentally friendly processes, as they offer high and often
unique reactivities and selectivities under mild reaction
conditions. Peroxidases (EC 1.11.1x) are a class of antioxidant
enzymes ubiquitous in Nature. They catalyze a plethora of
interesting transformations, like e.g., C,H-oxidations and
halogenations, by making use of cellular hydrogen peroxide
rather than utilizing complicated electron delivery chains via
nicotinamide cofactors.[1] Their sensitivity towards the inevitable
oxidant is a key limitation for their broad application. Heme-
dependent peroxidases, in particular, suffer from irreversible
oxidative inactivation of the prosthetic group by H2O2. Keeping
the H2O2 level low during the whole reaction is thus crucial for in
vitro biocatalytic processes.[2] Various methods providing
continuous oxidant supply have been developed to overcome
this drawback, such as slow dosage of hydrogen peroxide via a
syringe pump[3] or a H2O2-stat.[4] Additionally, in situ H2O2-
delivery systems were studied: electrochemical generation of
H2O2,[5] Pd/H2 in supercritical CO2,[6] or biocatalytic formation by
glucose oxidase (GOx).[7] The drawbacks of these procedures
reach from low atom economy to simply being not practical, in
particular on preparative scale.
Scheme 1. General mechanism of visible-light promoted electron transfer
employing flavin-derived redox mediators.
One innovative approach to acquire the electrons needed by the
However, intrigued by the advantages offered by this
photobiocatalytic concept,[11] we started to evaluate alternative
and more environmentally benign electron sources. We first
turned our attention on employing buffers as possible reductants.
As enzymatic transformations intrinsically require buffered
solutions, in order to adjust and maintain a distinct pH value at
which the enzyme is operating, these buffer components are
always present in any biocatalytic reaction mixtures. Employing
any other, external electron donors besides the buffer salt or the
[a]
C. J. Seel, Prof. Dr. T. Gulder
Department of Chemistry and Catalysis Research Center (CRC),
Technical University Munich, Lichtenbergstrasse 4, 85747 Garching,
(Germany)
E-mail: tanja.gulder@tum.de
[b]
[c]
A. Králík, Dr. M. Hacker, Prof. Dr. B. König
Institute of Organic Chemistry, University of Regensburg,
Universitätsstraße 31, 93053 Regensburg, (Germany)
E-mail: Burkhard.Koenig@chemie.uni-regensburg.de
Dr. A. Frank
solvent,
however,
constitutes
always
an
‘extra’,
Department of Chemistry, Center for Integrated Protein Science,
Technical University Munich, Lichtenbergstrasse 4, 85747 Garching,
(Germany)
(over)stoichiometric additive to the reaction mixture. While
screening different redox-active buffers, we observed that those
containing tertiary amines,[12] such as MOPS, MES, HEPES and
Supporting information for this article is given via a link at the end of
the document.
Tris, gave the best results
photocatalytic production of H2O2 in our system (see SI). Both
FMN and RFTA worked with equal success, but the hydrophilic
as electron donors for the
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COMMUNICATION
Reactions were carried out using 7.43 mM substrate, 0.5 mol% FMN, 3.77 µM
AmVHPO, 188 µM Na3VO4, 100 mM MES (pH 6.0), and 8.13 mM KBr at rt
under irradiation (455 nm). Yields are based on recovered starting material
and have been determined by aHPLC, bGC, or cNMR using dodecane, toluene
or phenol as internal standard. Conversions (shown in brackets) and ratios of
isomers were determined by aHPLC, bGC, or cNMR using dodecane, toluene
or phenol as internal standard. d50 mM MES buffer was used. e25 mM MES
buffer was used. f15 mol% FMN (15 min irradiation), additional 15 mol% FMN
nature of FMN makes its application more convenient. Kinetic
experiments (see Figure 2) revealed that the H2O2 production
catalyzed by FMN using MES buffer as an electron source is
very fast and reaches a plateau at 2 mM H2O2 after 10 min,
possibly due to an equilibrium between newly generated and
decaying hydrogen peroxide. For irradiation at the absorption
maximum of FMN (455 nm, blue LEDs), we did not observe any
impact of the FMN loading on the O2-consumption rate (see SI),
indicating that the energy output of the light source rather than
the catalyst loading determines the H2O2 generation rate, at
least at the FMN concentrations tested here (37 µM – 2 mM, see
SI). Consequently, the light intensity can be used to control the
H2O2 release and thus to fine tune the oxidative conditions
depending on the H2O2-sensitivity of the enzyme.
(15 min irradiation) followed by addition of
isomerization of the double bond was observed upon irradiation.
1
and enzyme. gPartial
with highly electron-rich substrates 1, however, was detectable
even at such low catalyst concentrations (see SI), resulting in
oxidative degradation of highly activated compounds such as 1c,
1i and 1j. To overcome this limitation, we slightly varied our
protocol and pre-generated H2O2 prior to the addition of the
substrate. This procedure resulted in good conversions of
oxidation-sensitive molecules and avoided substrate or product
decomposition.
The method was further advanced by evaluating its
application in aromatic chlorinations, which are generally more
difficult to achieve than the corresponding brominations.[17] Here,
the fungal VHPO from Curvularia inaequalis (CiVHPO)[18] was
employed as it offers a higher oxidation potential compared to
AmVHPO. By simply changing the added halide salt from KBr to
KCl the desired chlorination of activated arenes was likewise
possible, albeit, as expected, in lower chemical yields (Scheme
3). It is noteworthy that the regioselectivity for 3c with
predominant ortho-substitution differs from that observed during
the enzymatic bromination (see Scheme 2), hinting at a
We next turned our focus on combining this H2O2-
generating system with enzymatic halogenations. For these
photobiocatalytic studies we picked the vanadium-dependent
haloperoxidase (VHPO) AmVHPO from the cyanobacterium
Acaryochloris marina MBIC 11017.[13] AmVHPO shows
a
remarkable robustness towards organic solvents and heat
together with a broad substrate scope for aromatic bromination.
Nevertheless, sensitivity to high H2O2 concentrations, as already
reported for many VHPOs,[14] was also observed for AmVHPO
and thus makes it the perfect model enzyme to evaluate
photobiocatalytic halogenations.
Very low catalyst loadings of 0.5 mol% and buffer
concentrations as low as 25 mM turned out to be sufficient for
achieving halogenations of a manifold of different electron-rich
(hetero)aromatic compounds 1 in MES buffer (Scheme 2 and for
details SI) without any change in the pH during the course of the
reaction. No conversion of 1 was observed if the reaction was
conducted in the dark or in the absence of enzyme and of
FMN.[15] Lyophilized cell lysate of AmVHPO, constituting a more
time-efficient alternative to the purified enzyme, and the more
reactive isoenzyme AmVHPO II (cf. SI) were likewise applicable
under these conditions without any loss in reactivity. The
enzyme’s activity was not affected by 0.5 mol% FMN or
irradiation itself,[16] but inactivation became a critical factor at
higher FMN concentrations (cf. SI). Interaction of excited FMN*
mechanism
involving
an
N-chlorination
followed
by
intramolecular transfer of the chloro atom to the ortho position.
Scheme 3. Photobiocatalytic aromatic chlorinations using CiVHPO.
Reactions were carried out using 7.43 mM substrate, 0.5 mol% FMN, 1.60 µM
CiVHPO, 188 µM Na3VO4, 100 mM MES (pH 5.0) and 8.13 mM KCl at rt under
irradiation (455 nm). aHPLC, bGC, or cNMR using dodecane, toluene or phenol
as internal standard. Conversions (shown in brackets) and ratios of isomers
were determined by aHPLC, bGC, or cNMR using dodecane, toluene or phenol
as internal standard. d15 mol% FMN (15 min irradiation), additional 15 mol%
FMN (15 min irradiation) followed by addition of 1 and enzyme. eIsomerization
f
of the product was observed upon irradiation. Decomposition of the product
occurred during NMR measurement.
Although the use of buffer components as an electron source is
convenient, since the presence of buffer is necessary due to the
requirements of the enzymatic reaction, the ultimate goal of
sustainable chemistry is utilizing water to provide redox
equivalents.[19,20] During our studies,
heterogeneous photocatalytic H2O2 evolution from methanol[19]
a
method for
Scheme 2. Photobiocatalytic aromatic bromination using AmVHPO.
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10.1002/cctc.201800886
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COMMUNICATION
and water[22] oxidation in the presence of air using TiO2 doped
with gold nanoparticles[23] was reported. We thus set out to
evaluate the activity of this catalyst more closely (for details see
SI). For these studies, TiO2 was used in its rutile form, which
make the photocatalyst more enzyme-compatible due to its
hydrophobic properties that prevent hydrophilic enzymes from
adsorption to the TiO2 surface. This spatial separation might
protect the enzyme from highly reactive — but short-lived —
hydroxyl radicals formed by titanium dioxide in aerated aqueous
solution upon irradiation.[22] We first investigated the ability of
Au-TiO2 to generate H2O2 via water oxidation under irradiation
with different narrow-emission-band light sources, in particular,
UV-A (375 nm), blue (455 nm), green (535 nm), and white (as a
visible-light-only, broad-emission source) LEDs. Although Au-
TiO2 was previously described as a visible-light photocatalyst,[22]
hydrogen peroxide evolution exclusively occurred at 375 nm UV
irradiation in our hands (Figure 1).
Kinetic measurements of the catalyst’s activity showed a similar
profile of H2O2 generation as the FMN-containing system within
the first 240 s (Figure 2, black). Upon further irradiation the Au-
TiO2-based system reached a plateau[24] with an equilibrium
concentration of H2O2 3–4 times lower than that observed for
FMN (ca. 600 µM). This heterogeneous water-splitting system is
thus a milder source of in situ-generated hydrogen peroxide,
which can be favorable for more sensitive biocatalysts than
AmVHPO and CiVHPO.
In situ hydrogen-peroxide evolution using Au-TiO2 under
irradiation at 375 nm was then successfully applied to a
bromination of 2,6-dimethoxypyridine (1e) catalyzed by
AmVHPO reaching results superior to the above mentioned
flavin systems (Scheme 4). This shows a clear advantage of a
milder source of H2O2 in an enzymatic reaction. For this reaction,
non-oxidizable phosphate buffer was used to ensure that water
is the sole source of electrons. The results of the control
reactions (see SI) show a vital role of all reaction elements –
light, photocatalyst, oxygen, and enzyme. It is also worth
mentioning that the enzyme keeps its activity even in the
presence of phosphate (10 mM), which is an inhibitor[25] of
VHPOs due to its isosteric nature to the crucial vanadate
present in the active center of these enzymes. Moreover, the
tolerance of UV light underlines the high robustness of this class
of enzymes and makes it thus ideally suited for the use in
photobiocatalysis.
Figure 1. Schematic depiction of the photocatalytic generation of H2O2 using
Scheme 4. Application of the photocatalytic Au-TiO2-mediated generation of
H2O2 with an AmVHPO-catalyzed bromination. aYield is based on recovered
starting material and has been determined by HPLC chromatography using an
internal standard. bConversion and ratio of isomers were determined by HPLC
chromatography using an internal standard.
gold-nanoparticle-doped TiO2 as
hydrogen peroxide after irradiation (1h) of an Au-TiO2 suspension in pure
water using different light sources.
a photocatalyst and concentration of
When a degassed suspension of Au-TiO2 in water was irradiated
under N2 atmosphere, only traces of H2O2 and no oxygen were
detected in the solution and the gas phase, respectively.
AmVHPO-mediated bromination of 1e showed only low
conversion (4%), when performed under an inert atmosphere (cf.
SI). These observations correspond to a two-electron water
oxidation and a two-electron oxygen reduction taking place
under the given conditions.
In summary, we have shown that combining photocatalysis with
biocatalysis represents a mild and environmentally benign
approach to overcome the pending problem to supply oxidative
enzymes with their redox equivalents. The previously used,
wasteful and thus far from ideal sacrificial electron donors, have
been replaced by either redox-active buffers or even water itself,
both ubiquitously present in enzymatic reactions. These in-situ
H2O2-delivering setups stand out due to their practicability by
simply utilizing light with the help of low-concentrated organic or
heterogenous metal photocatalysts. Proof of concept towards
the applicability of the buffer/FMN/visible light as well as the
water/Au-TiO2/UVA approach was given by establishing efficient
photobiocatalytic halogenations using AmVHPO and CiVHPO as
convenient model enzymes. Both photochemical methods
proved to be beneficial for sustaining halogenation activity over
several hours. The described methods for oxidative H2O2
generation, however, are neither limited to (aromatic)
halogenations nor to the VHPO class of enzymes. Having now a
robust and biocompatible method in hands, expansion of this
versatile principle to different transformations by utilizing even
more delicate oxidation enzymes is currently conducted in our
laboratories.
2500
Au-TiO2
FMN
2000
1500
1000
500
0
0
200
400
600
800
1000
Time [s]
Figure 2. Kinetic of the H2O2 evolution by FMN in a MES-buffered solution and
Au-TiO2 in pure distilled water under standard conditions. H2O2 concentration
was determined by horseradish peroxidase assay.
Acknowledgements
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[20] M. Teranishi, R. Hoshino, S.-i. Naya, H. Tada, Angew. Chem. Int. Ed.
2016, 55, 12773-12777.
This work was funded by the Emmy-Noether (DFG, GU 1134-3)
and Heisenberg (DFG, GU 1134-4) program of the German
Research Foundation (DFG), the cluster of excellence CiPSM
and the BMBF (FKZ 031B0111A). C.J.S. thanks the Deutsche
Bundesstiftung Umwelt (grant 20015/400) and A.K. the Fonds
[21] a) Y. Ni, E. Fernandez-Fueyo, A. Gomez Baraibar, R. Ullrich, M.
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der Chemischen Industrie for
a
PhD fellowship. We
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acknowledge Prof. Dr. Kathrin Castiglione for providing genomic
DNA of A. marina and Prof. Dr. M. Groll for technical support
and advice.
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Keywords: Biocatalysis • Photocatalysis • Water splitting •
Sustainable Chemistry • Halogenation
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Bornscheuer, Nat. Catal. 2018, 1, 12-22.
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[15] For 1a halogenation was observed to some extend even in the absence
of FMN suggesting
a photocatalytic background reaction. Control
experiments revealed that H2O2 is generated by a direct auto-oxidation
of the electron-rich substrate 1a (see SI for details).
[16] Photochemically excited FMN can promote crosslinking of
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Photobiocatalysis constituted an
elegant way to overcome the pending
problem of cofactor inactivation in
peroxidase catalyzed transformations.
In this context, the utility of redox-
active buffers or even water alone as
efficient, biocompatible electron
sources has been demonstrated by
combining photocatalytic in situ H2O2
generation with enzymatic
Catharina Julia Seel,[a] Antonín Králík,[b]
Melanie Hacker,[b] Annika Frank,[c]
Burkhard König*[b] and Tanja Gulder*[a]
Page No. – Page No.
Atom-Economic Electron Donors for
Photobiocatalytic Halogenations
halogenations.
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