Deazaflavins as photocatalysts for the direct reductive regeneration of flavoenzymes

Article abstract of DOI:10.1016/j.mcat.2018.04.015

Deazaflavins are potentially useful redox mediators for the direct, nicotinamide-independent regeneration of oxidoreductases. Especially the O₂-stability of their reduced forms have attracted significant interest for the regeneration of monooxygenases. In this contribution we further investigate the photochemical properties of deazaflavins and investigate the scope and limitations of deazaflavin-based photoenzymatic reaction systems.

Full text of DOI:10.1016/j.mcat.2018.04.015

Molecular Catalysis 452 (2018) 277–283
Contents lists available at ScienceDirect
Molecular Catalysis
journal homepage: www.elsevier.com/locate/mcat
Deazaflavins as photocatalysts for the direct reductive regeneration of
flavoenzymes
a
a,b
a
a
c
a
M.M.C.H. van Schie , S.H.H. Younes , M.C.R. Rauch , M. Pesic , C.E. Paul , I.W.C.E. Arends ,
F. Hollmann
a
a,⁎
Department of Biotechnology, Delft University of Technology, van der Maasweg 9, 2629 HZ, Delft, The Netherlands
Sohag University, Faculty of Sciences, Department of Chemistry, Sohag 82524, Egypt
Wageningen University & Research, Laboratory of Organic Chemistry, Stippeneng 4, 6708 WE, Wageningen, The Netherlands
b
c
A R T I C L E I N F O
A B S T R A C T
Keywords:
Deazaflavins are potentially useful redox mediators for the direct, nicotinamide-independent regeneration of
Flavin
Photochemistry
Biocatalysis
Old yellow enzyme
Oxygen dilemma
oxidoreductases. Especially the O -stability of their reduced forms have attracted significant interest for the
regeneration of monooxygenases.
In this contribution we further investigate the photochemical properties of deazaflavins and investigate the
scope and limitations of deazaflavin-based photoenzymatic reaction systems.
2
Introduction
have developed a range of chemical [36–42], electrochemical [43–45],
and photochemical [46–51] approaches to target the flavin prosthetic
Oxidoreductases are amongst the most promising catalysts for pre-
parative organic synthesis for selective reduction, oxidation and oxy-
functionalisation reactions. Amongst them, flavin-dependent oxidor-
eductases are of particular interest due to the versatility of the flavin-
prosthetic group for selective reduction [1–5], and oxyfunctionalisation
reactions (Scheme 1) [6–9]. For most of these reactions, the catalytic
mechanism entails reductive activation of the enzyme-bound flavin
cofactor. The reduced flavin cofactor then either reduces a substrate
molecule (as in case of the so-called old yellow enzymes or ene re-
ductases) or it reacts with molecular oxygen forming a peroxoflavin
capable of selective oxyfunctionalisation reactions such as epoxidation
group directly while circumventing the natural nicotinamide cofactor
(together with the enzymatic regeneration system).
The mediators of choice for such NAD(P)H-independent regenera-
tion systems are flavins themselves. However, while flavin-based re-
generation systems perform well with O
the OYEs, their performance with O -dependent monooxygenases is
rather poor; the major limitation being the poor O -stability of reduced
2
-independent enzymes such as
2
2
flavins. One possibility to circumvent this Oxygen Dilemma [52] was
suggested by Reetz and coworkers [49], i.e. to utilise deazaflavins in-
stead of the ‘normal’ ones (Scheme 2) to promote a P450BM3-catalysed
aerobic hydroxylation of lauric acid. Compared to using ‘normal’ fla-
vins, significantly higher productivities were observed, which was at-
tributed to the higher oxidative stability of reduced deazaflavins. These
findings are in line with much earlier findings by Massey and coworkers
who demonstrated that fully reduced deazaflavins, in contrast to their
[
10–16], Baeyer-Villiger oxidations [17–20], or aromatic hydroxylation
reactions [13,21–32].
As mentioned above, all these structurally and mechanistically di-
verse enzymes have a reductive activation of the enzyme-bound flavin
in common. Generally, the reducing equivalents required for this re-
action are obtained from the natural nicotinamide cofactors (NAD(P)
H). For practical and economic reasons stoichiometric use of NAD(P)H
is not feasible, which is why in the past decades a myriad of different in
situ regeneration approaches have been developed allowing for the use
of NAD(P)H in catalytic amounts only [33–35].
2
‘normal’ analogues, exhibit a high stability against O [53].
Already in the 1970s, deazaflavins have been subject of extensive
research efforts. Especially Massey, Hemmerich and coworkers have
worked out the reactivity of deazaflavins revealing that the deaza-
semiquinone radical is significantly less stable compared to the ‘normal’
semiquinone [53–60]. This favours disproportionation and dimeriza-
Despite the success of these methodologies, a more direct approach
to regenerate the enzyme-bound flavin group could offer some ad-
vantages such as simplified reaction schemes. For this, we and others
tion reactions leading to non-radical products exhibiting low(er) O
2
reactivity. Furthermore, reduction of deazaflavins by exclusive 1 e-
donors like dithionite proceeds relatively slow compared to natural
⁎
Corresponding author.
E-mail address: f.hollmann@tudelft.nl (F. Hollmann).
https://doi.org/10.1016/j.mcat.2018.04.015
Received 21 February 2018; Received in revised form 15 April 2018; Accepted 16 April 2018
2468-8231/ © 2018 Elsevier B.V. All rights reserved.

M.M.C.H. van Schie et al.
Molecular Catalysis 452 (2018) 277–283
Scheme 1. Simplified scope of flavin-
dependent oxidoreductases for selec-
tive reduction of C]C-double bonds,
epoxidations, Baeyer-Villiger oxida-
tions, aromatic hydroxylations and ha-
logenations. All reactions depend on
the reduced nicotinamide cofactor,
which for economic reasons has to be
regenerated in situ using an (enzy-
matic) regeneration system.
in 1.5 ml glass vials. The reaction mixtures were illuminated from all
sides from a distance of 10 cm by a LED light source (SI for more detail).
The intensity spectrum of the LED light source was determined by a
calibrated spectrophotometer. Samples were gently stirred using Teflon
coated magnetic bars. Anaerobic experiments were performed in an
anaerobic chamber (on average 98% N
2 2
, 2% H ) with oxygen levels
below ppm levels. UV/Vis spectra were recorded using an Avantes DH-
Scheme 2. Simplified representation of flavins (left) and their deazaflavin
analogues (right). For reasons of clarity the 5 position wherein both differ are
marked.
2000 UV–vis-NIR light source and an Avispec 3648 spectrophotometer.
In a typical experiment 100 μM dRf were reduced by 10 mM sacri-
ficial single electron donor or 1 mM hydride donor in a 100 mM KPi
−1
−1
buffer at pH 6.0. The absorbance at 400 nm (ε = 12500 M cm ) was
followed to determine the oxidation state of dRf over time (Fig. S4.12).
Due to interference of absorption when using either hydride donors, the
oxidation state was determined by following the absorbance at 430 nm
flavins and via a covalent adduct intermediate.
These findings motivated us to further evaluate the applicability of
deazaflavins as photocatalysts/mediators to promote flavoenzyme cat-
alysed reactions.
−
1
−1
(ε = 5700 M cm ).
Due to the FMN, the YqjM has a typical absorbance spectrum with a
peak extinction coefficient at 455 nm which decreases as the FMN in the
active site is reduced. At 400 nm the extinction coefficient does not
change significantly with the redox-state of the enzyme, which made it
possible to determine the redox-state of the dRf and the YqjM si-
multaneously. For the reduction of YqjM, a dRf solution was first photo-
reduced by five equivalents of EDTA. Thereafter, 20 μM of YqjM was
mixed with 100 μM of the reduced dRf.
Photoenzymatic syntheses were performed using 5 μM YqjM in the
presence of 1 mM of 2-methyl cyclohexenone and 10 mM of EDTA in a
100 mM KPi buffer pH 6.0. The 2.5 ml reaction mixtures were extracted
with 0,5 ml ethyl acetate and analysed on a CP-wax 52 CB GC column
(50 m x 0,53 m x 2 μm)(GC method: 70 °C for 2 min. 2 °C/min–80 °C.
80 °C for 2 min. 2 °C/min–90 °C. 90 °C for 3 min. 25 °C/min–150 °C.
150 °C for 1 min. 25 °C/min–225 °C. 225 °C for 1 min). 5 mM Dodecane
was used as internal standard.
Materials and methods
Chemicals
Chemicals were purchased from Sigma-Aldrich, Fluka, Acros or
Alfa-Aesar with the highest purity available and used as received.
5
-deazariboflavin (dRf) was synthesized following a literature
method (SI for more detailed information) [61]. Stock solutions
0.2 mM in 100 mM phosphate buffer, pH 6) of the deazariboflavin
(
were prepared freshly every day.
The old yellow enzyme homologue from Bacillus subtilis (YqjM) was
produced and purified using a protocol recently established in our
group (SI for more detailed information) [36].
The NADH mimic N-benzyl nicotinamide (BNAH) was synthesized
following published procedures [39,62].
Experimental setup
Results and discussion
Photoreduction reactions were performed in 1 ml reaction mixtures
5-deazariboflavin (dRf) was synthesized from 3,4-dimethylaniline in
Scheme 3. Synthetic route for 5-deaza riboflavin (dRf).
278

M.M.C.H. van Schie et al.
Molecular Catalysis 452 (2018) 277–283
Fig. 1. Photochemical reduction of
deazariboflavin (dRf) using NADH or
EDTA as sacrificial electron donor. A:
UV/Vis spectra of the various dRf spe-
cies observed during the photoreduc-
ox
tion of dRf (red) with EDTA (blue)
and NADH (green). General conditions:
75 μM of dRf, 10 mM EDTA or 75 μM
NADH, 100 mM KPi buffer pH 6.0, Blue
LED light setup, max light intensity,
anaerobic conditions, RT. B: HPLC
chromatograms recorded of photo-
chemical reductions of dRf (red) using
NADH (green) or EDTA (blue) as stoi-
chiometric reductant. (For interpreta-
tion of the references to colour in this
figure legend, the reader is referred to
the web version of this article.)
Fig. 2. Dependency of the dRf reduction rate
(expressed as turnover frequency of dRf, TF) on
A: intensity of the blue LED light source, B:
ox
dRf concentration, C: Concentration of the
sacrificial electron donor and D: pH. General
ox
conditions: 60 μM of dRf
, 10 mM EDTA,
1
00 mM KPi buffer pH 6.0, blue LED light
setup, max light intensity, anaerobic condi-
tions, RT. TF = (initial dRf reduction rate [μM
−
1
−1
min ]) × (c(dRf) [μM])
.
a two-step synthesis following a recently published procedure (Scheme
being BNAH, with which also in the dark, significant formation of
2
H dRf was observed. One possible explanation for this may be the more
negative redox potential of BNAH compared to NADH(−0.36 V and
−0.315 V SHE, respectively) [63].
3
) [61]. Overall, 457 mg of pure dRf (28% overall yield) were pro-
duced.
Having dRf at hand, we next investigated its photoreduction using
either sodium ethylenediaminetetraacetate (EDTA, a single electron
donor) or NADH (a hydride donor) (Fig. 1). In addition to EDTA also 3-
The spectra of single electron donor- and hydride-donor reduced
dRfs differed significantly in shape suggesting different reduction pro-
ducts. This assumption was corroborated by chromatographic separa-
tion of the reaction products (Fig. 1B). While NADH-reduced dRf gave
(
(
N-morpholino)propanesulfonic acid (MOPS) and other amino alcohols
such as TRIS) also served as single electron donors (Fig. S4.4). Also
NADH can be replaced by the nicotinamide mimic benzyl dihy-
dronicotinamide (BNAH). It is worth mentioning here that in all cases
illumination was necessary to induce dRF reduction. The only exception
only one product (H
trum, Fig. S4.1) [56] EDTA-reduction of dRf yielded H
product (most likely assigned to the dimeric, half-reduced semiquinone
2
dRf, as confirmed by its characteristic UV spec-
2
dRf and another
279

M.M.C.H. van Schie et al.
Molecular Catalysis 452 (2018) 277–283
red
Fig. 3. Time course of the (dRf)
2
mediated
reduction of YqjM. A: spectra recorded over
time (1 min intervals) and B: time courses of
the characteristic absorption maxima for YqjM-
ox
ox
FMN (290 nm, ♦) and dRf (355 nm, ■).
ox
Conditions:
A
solution of 60 μM dRf
in
1
00 mM KPi buffer containing 0.5 mM EDTA
was illuminated (blue LED) until full reduction
was achieved, afterwards, this reaction mixture
was supplemented with the same volume of
4
0 μM YqjM (in the same buffer) and subse-
quently illuminated using a blue LED [71].
For interpretation of the references to colour
(
in this figure legend, the reader is referred to
the web version of this article.)
Fig. 4. Photoenzymatic reduction of 2-methyl cyclohexenone. General conditions: 5 μM YqjM, 1 mM 2-methyl cyclohexenone, 10 mM EDTA, 100 mM KPi pH 6.0,
blue LED light setup, blue LED at maximal light intensity, anaerobic conditions, RT. It should be mentioned, that the solubility limit of dRf was around 250 μM,
therefore, the sample at 300 μM dRf was turbid, which possibly influenced the regeneration reaction.
product) appearing simultaneously over time (Fig. S4.2). These findings
are in line with previous reports using EDTA or NaBH as reductants
58,64].
Next, we characterised the light-driven reduction of dRfox using
EDTA as a sacrificial electron donor in some more detail. It is worth
mentioning here that the photochemical reduction of dRf proceeded
approximately 14 times slower than the reduction of ‘normal’ ribo-
flavin. This is most likely attributed to the better overlap of the ribo-
flavin absorption spectrum with the emission spectrum of the LED used
in this study (Fig. S4.5) [65]. However, also differences in the redox
potentials of Rf (−0.146 V vs. SHE) and dRf (−0.237 V vs. SHE) may
contribute [66]. Using the Nernst equation, the redox potential of dRf
was estimated to be −0.332 V vs. SHE at pH 6.0 (see SI for the calcu-
lations).
The reduction rate of dRf was linearly dependent on the light in-
tensity applied (Figs. 2 A, S4.6). Likewise the overall rate of dRf re-
duction linearly depended on the dRf concentration itself (Fig. 2B).
Hence, we conclude that the in situ concentration of photoexcited dRf is
4
[
280

M.M.C.H. van Schie et al.
Molecular Catalysis 452 (2018) 277–283
Fig. 5. Photochemical reduction of
deazariboflavin using NADH (A) or
EDTA (B) as sacrificial electron donor
under aerobic (♦) and anaerobic (
)
conditions. Conditions: 10 mM EDTA
or 1 mM NADH, 100 mM KPi buffer pH
6.0, blue LED light setup, max light
intensity, RT. (For interpretation of the
references to colour in this figure le-
gend, the reader is referred to the web
version of this article.)
Table 1
higher electron densities around the N-atom of the donors facilitate the
electron transfer to dRf. These observations are in line with the oxi-
dation mechanism suggested by Kramer and coworkers [67]. These
authors suggested a methylene radical intermediate being formed after
single electron transfer of the amino acid donor and subsequent dec-
arboxylation. The extend of hyperconjugative stabilisation of this in-
termediate radical should increase with the N-substitution pattern as
well as with occurrence of a non-protonated N-substituent.
Aerobic reoxidation rates observed for deazariboflavin reduced by EDTA or
NADH.
Reoxidation rate [μM h−1]
Dark
Light
(
dRf)
2
1.7
0.78
96
30
H
2
dRf
red
To further examine the applicability of photoreduced dRf to re-
Conditions: 10 mM EDTA or 1 mM NADH, 100 mM KPi buffer pH 6.0, blue LED
light setup, max light intensity, RT.
generate oxidoreductases, we used it to reduce the FMN-dependent old
yellow enzyme homologue from Bacillus subtilis (YqjM) [36,68,69]. The
ox
different spectral properties of dRf (λmax = 390 nm) and YqjM-bound
ox
FMN (λmax = 455 nm) allow for the simultaneous determination and
red
quantification of the electron transfer between photoregenerated dRf
ox
and YqjM-bound FMN (Fig. 3). In accordance to previous findings by
Massey and Hemmerich [70] we found that photoregeneration of YqjM
was possible. Using e.g. 1.5 eq of (prior reduced) dRf full reduction of
YqjM was observed within 4 minutes. This reaction was observed under
blue light illumination only. Incubation of YqjM with prereduced dRf in
the dark or upon illumination with other wavelengths yielded no sig-
nificant reduction of the YqjM-bound FMN (Fig. S4.8). Currently, we
are lacking a satisfactory explanation for this observation. Possibly the
red
interaction of (dRf)
2
with the enzyme-bound FMN is sterically hin-
dered and photoexcitation of the latter may accelerated the long-dis-
tance electron transfer.
To test if catalytic turnover of both, dRf and YqjM is feasible, we
used the enantioselective reduction of 2-methyl cyclohexanone to (R)-
2-methyl cyclohexanone as model reaction (Fig. 4). In the absence of
dRf no conversion of the starting material was observed indicating that
direct photochemical reduction of YqjM-bound FMN by EDTA was not
efficient. However, already in the presence of 5 μM dRf (equimolar to
−
1
1
YqjM) a product formation rate of approximately 50 μM h
served. Hence, a turnover frequency of approximately 10 h
was ob-
was cal-
Fig. 6. EPR spectra recorded using the spin trap technique during the aerobic
reduction of dRf with BNAH under illumination (black) and in dark (red).
Conditions: 60 μM dRf, 1 mM BNAH, 1% v/v DMSO, 100 mM KPi pH 6.0, Kaiser
Fibre Optic Lighting system 15 V, 150 W, on half intensity, aerobic conditions.
−
culated for YqjM and dRf. This corresponds well to the YqjM-reduction
rate observed before (Fig. 3) indicating that the reduction of the bio-
catalyst was overall rate-limiting. The overall rate of the photoenzy-
matic reduction reaction increased steadily with increasing photo-
catalyst concentration (up to 200 μM, representing the solubility limit
(For interpretation of the references to colour in this figure legend, the reader is
referred to the web version of this article.)
−
1
for dRf). With it, the catalytic efficiency of YqjM increased to 40 h . It
is worth mentioning that in all experiments (R)-2-methyl cyclohex-
anone was formed almost exclusively (Fig. S4.9).
overall rate limiting. Varying the concentration of the sacrificial elec-
tron donor (EDTA) revealed a saturation-type dependency of the dRf
reduction rate on the EDTA concentration applied (Fig. 2C). Above
approximately 1.5–2 mM EDTA (pH 6), no further increase of the dRf
reduction rate was observed. Finally, there was a sigmoidal pH-de-
While these numbers are comparable to recently reported photo-
enzymatic systems,[46] the catalytic performance of YqjM falls back by
−1
orders of magnitude behind its potential (1.8 s
using NADPH as re-
ox
pendency of the dRf reduction rate with a turning point at approxi-
ductant) [36]. A plausible explanation for this is to assume an un-
favourable interaction of the reduced dRf mediators with the enzyme-
bound FMN resulting in poor electron transfer rates. Both, cofactor- and
enzyme engineering may generate artificial binding sites and thereby
accelerate the regeneration reaction [72,73].
mately pH 9 (Fig. 2D). Interestingly, using MOPS instead of EDTA
shifted this turning point to pH 7 (Fig. S4.7). Furthermore, the sub-
stitution pattern of the N-atom in the sacrificial electron donor had an
influence on the reduction rate (Fig. S4.4). Overall, it appears that
281

M.M.C.H. van Schie et al.
Molecular Catalysis 452 (2018) 277–283
Finally, we re-visited the O
above, significant reduction of YqjM-bound FMN and product forma-
tion in the photoenzymatic system was observed only under strictly
2
-stability of reduced dRfs. As mentioned
setups (anaerobic reduction of the flavin followed by aerobic biocata-
lytic reaction) as suggested by Schmid and coworkers [43] may be
doable.
2
anaerobic conditions. We therefore investigated the influence of O on
the photochemical reduction of dRf (Fig. 5). Independent from the sa-
crificial electron donor used (NADH or EDTA) reduction of dRf was
observed under anaerobic conditions only while it was negligible in the
Conflict of interest
None.
2 2 2
presence of O . In the latter cases, H O was detectable as well as NADH
red
oxidation (in case of Fig. 5A) suggesting dRf reoxidation occurring
simultaneously.
Acknowledgements
Therefore, we determined the reoxidation rates of photochemically
reduced dRf in the dark and under illumination (Table 1). Both, the
We thank the Netherlands Organisation for Scientific Research for
financial support through a VICI grant (No. 724.014.003). Excellent
technical support by M. Gorseling and support by Prof. W.R. Hagen
with the EPR measurements is gratefully acknowledged.
dimeric (dRf)
2 2
as well as the fully reduced H dRf were rather stable
against O in the dark whereas they swiftly reoxidized in the presence
2
of (blue) light. Similar observations had previously been made by
Hemmerich and coworkers [58].
Appendix A. Supplementary data
The influence of light can be rationalised assuming that (trace
amounts) of photoexcited, oxidised dRf synproportionate with H
2
dRf or
via
Supplementary data associated with this article can be found, in the
online version, at http://dx.doi.org/10.1016/j.mcat.2018.04.015.
(
dRf) forming a reactive semiquinone radical reacting with O
2
2
single electron transfer. This reaction should be autocatalytic, for which
we have found indications in the reoxidation time-course (Figure S
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Scheme 4. Aerobic reoxidation of reduced deazaflavin species. The reactive
.
semiquinone radical (dRf ) reacts fast with O
2
eventually leading to H
2
O
2
and
or
dRf with (photoexcited) dRf. For reasons of
simplicity, protonation equilibria have not been included in this scheme.
O
2
. dRf itself is formed either via the dimerization equilibrium of (dRf)
through synproportionation of H
2
9
525–9535.
2
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283