Biocatalytic C=C Bond Reduction through Carbon Nanodot-Sensitized Regeneration of NADH Analogues

Article abstract of DOI:10.1002/anie.201804409

Light-driven activation of redox enzymes is an emerging route for sustainable chemical synthesis. Among redox enzymes, the family of Old Yellow Enzyme (OYE) dependent on the nicotinamide adenine dinucleotide cofactor (NADH) catalyzes the stereoselective reduction of α,β-unsaturated hydrocarbons. Here, we report OYE-catalyzed asymmetric hydrogenation through light-driven regeneration of NADH and its analogues (mNADHs) by N-doped carbon nanodots (N-CDs), a zero-dimensional photocatalyst. Our spectroscopic and photoelectrochemical analyses verified the transfer of photo-induced electrons from N-CDs to an organometallic electron mediator (M) for highly regioselective regeneration of cofactors. Light triggered the reduction of NAD⁺ and mNAD⁺s with the cooperation of N-CDs and M, and the reduction behaviors of cofactors were dependent on their own reduction peak potentials. The regenerated cofactors subsequently delivered hydrides to OYE for stereoselective conversions of a broad range of substrates with excellent biocatalytic efficiencies.

Full text of DOI:10.1002/anie.201804409

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Accepted Article
Title: Biocatalytic C=C Bond Reduction through Carbon Nanodot-
Sensitized Regeneration of NADH Analogues
Authors: Jinhyun Kim, Sahng Ha Lee, Florian Tieves, Da Som Choi,
Frank Hollmann, Caroline Paul, and Chan Beum Park
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201804409
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Angewandte Chemie International Edition
10.1002/anie.201804409
(
(will be filled in by the editorial staff))
Photobiocatalysis
Biocatalytic C=C Bond Reduction through Carbon Nanodot-Sensitized
Regeneration of NADH Analogues**
Jinhyun Kim, Sahng Ha Lee, Florian Tieves, Da Som Choi, Frank Hollmann, Caroline E. Paul* and Chan Beum
Park*
Abstract: Light-driven activation of redox enzymes is an emerging
route for sustainable chemical synthesis. Among redox enzymes, the
family of Old Yellow Enzyme (OYE) dependent on the nicotinamide
adenine dinucleotide cofactor (NADH) catalyzes the stereoselective
reduction of α,β-unsaturated hydrocarbons. Here, we report OYE-
catalyzed asymmetric hydrogenation through light-driven
regeneration of NADH and its analogues (mNADHs) by N-doped
carbon nanodots (N-CDs), a zero-dimensional photocatalyst. Our
spectroscopic and photoelectrochemical analyses verified the
transfer of photo-induced electrons from N-CDs to an
organometallic electron mediator (M) for highly regioselective+
regeneration of cofactors. Light triggered the reduction of NAD
+
and mNAD s with the cooperation of N-CDs and M, and the
reduction behaviors of cofactors were dependent on their own
reduction peak potentials. The regenerated cofactors subsequently
delivered hydrides to OYE for stereoselective conversions of a
broad range of substrates with excellent biocatalytic efficiencies.
Ene reductases from the Old Yellow Enzyme (OYE) family
catalyze the asymmetric reduction of activated C=C bonds in α,β-
[
1]
unsaturated compounds. The oxidoreductase contains a flavin
mononucleotide (FMN) prosthetic group to which a hydride is
transferred from a reduced nicotinamide adenine dinucleotide
cofactor [NAD(P)H]. Subsequently, the substrate is trans-
hydrogenated by accepting a hydride from the reduced FMN and a
proton from a Tyr-residue of the OYE. Despite the potential of
OYE-catalyzed asymmetric reduction, the provision of
stoichiometric NAD(P)H makes the process economically not
Scheme 1. A schematic illustration of the photoenergy conversion into chemical
energy by integrating photocatalysis and biocatalysis. Photoexcited electrons
generated by N-doped carbon nanodots reduce the oxidized rhodium-based
+
complex (Mox) that regioselectively converts mNAD into mNADH. Subsequently,
[
2]
the enzymatically active mNADH returns to its oxidized form after donating a
hydride to a prosthetic group (i.e., flavin mononucleotide, FMN) in the old yellow
enzyme. The reduced FMN transfers a hydride onto Cβ of a substrate while a
Tyr-residue gives a proton onto Cα, resulting in the trans-specific reduction of
an activated C=C bond. EWG: electron-withdrawing group.
feasible due to the high price of the cofactor. This issue has
+
prompted studies on in situ conversion of NAD(P) to its reduced
form (or vice versa) using different methods (e.g., enzymatic,
chemical, electrochemical, photochemical, and photoelectro-
[
3]
[4]
the field of redox biocatalysis because of their low price, better
chemical). According to the literature, the use of hydride-
transfer mediators can facilitate regioselective regeneration of
NAD(P)H by avoiding the formation of enzymatically inactive NAD
dimers. Furthermore, the mediators can function as a diffusing
communicator between photoactive materials and redox enzymes.
As a substitute of the natural cofactor, synthetic nicotinamide
cofactor analogues (mNADHs) have sparked a renewed interest in
[
5]
chemical stability, and even improved biocatalytic efficiency.
Several studies have demonstrated applications of the analogues as
electron donors to drive various redox enzymes (e.g.,
monooxygenase, P450 BM-3, enoate reductase, malate
[
5]
dehydrogenase, D-lactate dehydrogenase, and malic enzyme).
Here, we report photoactivation of OYEs through light-driven
regeneration of mNADHs using carbon nanodots (CDs) as a
+
photosensitizer, as depicted in Scheme 1. Three different mNAD s
[
]
J. Kim, Dr. S. H. Lee, D. S. Choi, Prof. Dr. C. B. Park
Department of Materials Science and Engineering
Korea Advanced Institute of Science and Technology (KAIST)
have been synthesized for this study: 1-benzyl-3-carbamoyl-
+
+
pyridinium (mNH ), 1-butyl-3-carbamoylpyridinium (mBu ), and
2
+
1
-benzyl-3-carboxypyridinium ion (mCOOH ). According to the
335 Science Road, Daejeon 305-701 (Republic of Korea)
[
5-6]
literature,
these analogues possess one or two substituted
E-mail: parkcb@kaist.ac.kr
functional groups positioned on C3 and/or N1 atoms that give rise to
different kinetic parameters for the reductive reactions catalyzed by
Dr. C. E. Paul
Laboratory of Organic Chemistry, Wageningen University & Research
Stippeneng 4, 6708 WE Wageningen (The Netherlands)
E-mail: caroline.paul@wur.nl
OYEs.
We
employed
triethanolamine
(TEOA)
and
2
+
Cp*Rh(bpy)H O (Cp* = C Me , bpy = 2,2’-bipyridine) (M) as an
2
5
5
electron donor and an electron mediator, respectively, for
regioselective regeneration of mNADHs. Semiconducting materials
such as quantum dots have been used for enzyme-mediated sensing,
Dr. F. Tieves, Prof. Dr. F. Hollmann
Department of Biotechnology, Delft University of Technology
Van der Maasweg 9, 2629 HZ Delft (The Netherlands)
[
7]
photobiocatalysis, and bioenergetic applications. Nano-sized CDs
are zero-dimensional carbonaceous semiconducting nanomaterials
that exhibit UV-Vis absorptivity, good photostability,
biocompatibility, water solubility, and tunable fluorescence
[
] This work was supported by the National Research Foundation (NRF)
via the Creative Research Initiative Center (Grant number: NRF-2015
R1A3A2066191), Republic of Korea, for CBP and the Netherlands
Organisation for Scientific Research by a VICI grant (Grant number:
[8]
emissions, making them a prospective alternative to molecular
724.014.003) for FH and a VENI grant (Grant number: 722.015.011)
dyes and semiconductor quantum dots for photocatalytic
for CEP.
1
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Angewandte Chemie International Edition
10.1002/anie.201804409
Figure 1. (a) Comparison of changes in the cathodic peak current of 500 μM M
(ΔIM,p) in the presence of 400 μM cofactor. (b) Comparison of different reduction
peak potentials of cofactors (Ecofactor,p). The electrochemical analysis was carried
-
1
out in a phosphate buffer (100 mM, pH 7.5) at a scan rate of 50 mV s . Working
electrode: a polished glassy carbon electrode.
[
9]
[10]
applications, such as CO reduction, H evolution,
and water
2
2
[
11]
splitting . Recently, CDs have been applied to photoenzymatic
synthesis through their coupling with redox enzymes. For example,
positively charged CDs were hybridized electrostatically with
fumarate reductase for light-driven hydrogenation of fumarate to
succinate via direct transfer of photoexcited electrons.
We synthesized nitrogen-doped CDs (N-CDs) by one-pot
hydrothermal treatment (200 ºC, h) of citric acid and
ethylenediamine as a carbonaceous precursor and a nitrogen doping
[
12]
Figure 2. (a) N-CD-sensitized photobioenzymatic reduction of (a) 2-methyl-2-
cyclohexen-1-one (7 mM) and (b) trans-cinnamaldehyde (5 mM) by TsOYE.
5
-
1
Reaction conditions: N-CDs (0.10 mg mL ), M (25 μM), substrate, cofactor (2
mM), TsOYE (9 μM), and CaCl (5 mM) in a TEOA buffer (500 mM, pH 7.5) at
5 ºC. Light intensity: 200 mW cm . Light source: a xenon lamp equipped with
a 324 nm cut-on optical filter. TOF was determined after (a) 30 min and (b) 15
min of reaction. TTN and ee were determined after (a) 180 min and (b) 75 min
of reaction.
[
13]
2
agent, respectively,
and characterized them using multiple
-2
4
analytical tools (see Figures S1-S6 in the Supporting Information).
According to the UV-Vis spectrum in Figure S7a, N-CDs exhibited
UV absorption with tailing toward the visible light region. The
corresponding Tauc plot in Figure S7b indicates that the optical
bandgap of N-CDs is approximately 2.74 eV. Figure S8 shows the
onset of reduction potential, from which the corresponding LUMO
each cofactor using cyclic voltammetric analysis. The degree of M
mediation in the reduction of a cofactor was estimated by measuring
the increase in the reduction peak current of M (IM,p). According to
+
was calculated to be -0.83 V (vs. Ag/AgCl). A HOMO of 1.91 V (vs. Figures 1a and S15, the positive ΔIM,p was in the order of NAD >
+
+
+
Ag/AgCl) was estimated based on the optical bandgap value. This
electronic configuration suggests photoactivated N-CDs possess the
thermodynamic driving force to reduce M (redox potential of M: -
mNH
2
> mBu > mCOOH . From a thermodynamic point of view,
we ascribe the order to the different reduction peak potentials
+
+
(Ecofactor,p) of oxidized cofactors in the order of NAD > mNH
>
2
[
14]
+
+
0
.76 V vs. Ag/AgCl ). To verify the transfer of photoexcited
mBu > mCOOH (Figures 1b and S16) because the driving force
of M to reduce a cofactor becomes higher with a lower negative
electrons from N-CDs to M, we obtained PL spectra of N-CDs with
M in varying concentrations. According to Figure S9a, oxidative
fluorescence quenching of N-CDs by M was observed, and the
E
cofactor,p. On the molecular level, nicotinamide-based cofactors (i.e.,
+
+
+
NAD , mNH , mBu ) contain the common amide functionality on
2
corresponding Stern-Volmer plot (Figure S9b) displayed
a
C3 atom that is well known for the coordination to the Rh metal
center of M. However, compared to NAD , different substituents of
+
quadratic function of M. It suggests that the quenching of N-CDs
resulted from the transfer of photoexcited electrons to M and the
formation of an electrostatic complex between a negatively charged
N-CD and a positively charged M. Furthermore, we obtained
transient photocurrent responses of N-CDs to procure additional
evidence of N-CD-sensitized reduction of M (Figure S10), which
shows that the photoexcited electrons of N-CDs are delivered to
cofactor analogues on N1 atom exhibit lower electron-withdrawing
[
15]
+
+
abilities in the order of ribosyl (of NAD ) > 1-benzyl (of mNH )
2
+
> 1-butyl (of mBu ), making the pyridine ring less electrophilic and
less liable to accept electrons, resulting in higher negative Ecofactor,p
+
+
than that of NAD . On the other hand, mCOOH possesses the
negatively charged carboxylic group on C3 atom (at pH 7.5) that can
interrupt the desirable Rh-O-carbonyl coordination for hydride
transfer due to the ionic interactions between the carboxyl group and
+
mNH2 via M upon light irradiation.
We investigated light-driven regeneration of mNADHs by N-
[
16]
CDs under illumination (λcut-on: 324 nm) in the presence of M and
the metal center of M.
We further investigated the effect of
+
+
TEOA. The reduction of mNAD s to mNADHs was detected with
protonated carboxylic group of mCOOH on the catalytic efficiency
of M (Figure S17).
different yields (in the order of NADH > mNH H > mBuH >
2
mCOOHH). Detailed regeneration performances (e.g., total turnover
number (TTN), turnover frequency (TOF)) are listed in Table S1.
TEOA was selected as a sacrificial electron donor because its
Next, we studied trans-hydrogenation of conjugated C=C
bonds by implementing the photochemical recycling of cofactors
into the biocatalysis driven by an OYE from Thermus scotoductus
(TsOYE). Figure S18 shows a progressive reduction of 2-methyl-2-
cyclohexen-1-one with different production yields (in the order of
[
7b]
oxidation potential (ca. 0.86 V vs. Ag/AgCl)
is higher than the
HOMO of N-CDs, and its suitability was confirmed by the reductive
fluorescence quenching of N-CDs (Figure S11). Control
experiments in the absence of each component (i.e., N-CDs, light,
M, cofactor, and TEOA) resulted in no formation of NADH or
mNADHs (Figure S12). This result indicates that light promotes the
electrons of N-CDs to higher energy states that reduce M for
mNADH regeneration, while the holes created by excited electrons
are filled at the expense of TEOA oxidation. We also examined the
dependency of the initial rate of cofactor regeneration on the
intensity of incident light (Figure S13) and the concentration of N-
CDs, M, cofactor, and TEOA, respectively (Figure S14).
mNH
H > NADH > mBuH > mCOOHH) under illumination (λcut-on
:
2
324 nm) for 180 min. As shown in Figure 2a, mNH
H regeneration
2
-
1
displayed the highest TOFTsOYE of 511.8 h and TTNTsOYE of 670.1
among four different cofactors. The absence of cofactor (i.e., M
alone) caused a failure in the TsOYE-driven reduction of C=C
bonds. The result indicates that NADH and mNADHs, not M, are
the diffusing hydride donors to the prosthetic FMN in TsOYE. We
also observed that the photobiocatalytic performance increased with
the concentration of N-CDs, indicating that a higher regeneration
rate of mNH H enhances the rate and the production yield of OYE-
2
-
1
We explored the differential catalytic efficiency of M toward
driven trans-hydrogenation (Figure S19); TOFTsOYE of 576.3 h and
2
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Angewandte Chemie International Edition
10.1002/anie.201804409
Table 1. Comparison of photobiocatalytic efficiencies for asymmetric reduction
of activated C=C bonds in α,β-unsaturated compounds.
design of CDs exhibiting enhanced efficiencies of charge separation
and migration, and the structural modification of the hydride-
[
14]
OYE
type
TOFOYE
[h ]
Yield
[%]
transfer mediator to tune its redox potential.
Overall, the light-
Photocatalytic system
N-CD-sensitized
-1
TTNOYE
driven approach to regenerate better-than-nature cofactor analogues
is a promising strategy for efficient activation of oxidoreductases
using light energy.
regeneration of mNH
2
H
TsOYE
TsOYE
576.3
118.0
838.9
>99
67
[
a]
(this study)
Keywords: asymmetric catalysis • alkene hydrogenation • carbon
nanodot • NADH analogues • photobiocatalysis
Eosin Y-sensitized
direct activation of
295.0
[
17]
OYE
[
b]
[
Ru(bpz)
2
dClbpy]Cl
2
-
TOYE
121.5
100.4
500.0
>99
89
sensitized regeneration
[1] C. K. Winkler, G. Tasnádi, D. Clay, M. Hall, K. Faber, J. Biotechnol.
2012, 162, 381-389.
[
18]
[b]
of methyl viologen
CdSe-sensitized
PETNR
445.0
[
2] a) J. H. Kim, D. H. Nam, C. B. Park, Curr. Opin. Biotechnol. 2014, 28,
-9; b) S. H. Lee, J. H. Kim, C. B. Park, Chem. Eur. J. 2013, 19,
[
b]
[b]
[b]
1
regeneration of methyl
YqjM
23.3
82.2
8.0
12
[
19]
viologen
Au-TiO -sensitized
regeneration of free
4392-4406.
[
3] a) F. Hollmann, I. W. C. E. Arends, K. Buehler, ChemCatChem 2010,
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Okamoto, V. Köhler, C. E. Paul, F. Hollmann, T. R. Ward, ACS Catal.
2
[
b]
[b]
[b]
TsOYE
411.2
62
[
20]
FMN
[
a]
-1
[b]
With 0.20 mg mL N-CDs (Figure S19). Approximate estimation based on
data provided by the corresponding reference.
2
016, 6, 3553-3557.
+
[
4] L. Gorton, E. Domínguez, Electrochemistry of NAD(P) /NAD(P)H, in
Encyclopedia of Electrochemistry, Wiley-VCH Weinheim, 2007, pp.
-
1
TTNTsOYE of 838.9 were achieved by using 0.20 mg mL N-CDs.
These values are much higher compared to other systems reported
for photoactivation of OYEs (Table 1). We attribute the inverted
order to the better kinetic parameters of mNADHs. According to the
6
7-143.
[5] C. E. Paul, I. W. C. E. Arends, F. Hollmann, ACS Catal. 2014, 4, 788-
797.
[
6]
literature, the catalytic efficiency for the reductive half-reaction
kred/K ) of TsOYE with mNH H (533 mM s ) is around three
[6] T. Knaus, C. E. Paul, C. W. Levy, S. de Vries, F. G. Mutti, F.
Hollmann, N. S. Scrutton, J. Am. Chem. Soc. 2016, 138, 1033-1039.
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J. Parak, F. Lisdat, Langmuir 2010, 26, 1395-1400; c) N. Sabir, N.
Khan, J. Völkner, F. Widdascheck, P. del Pino, G. Witte, M. Riedel, F.
Lisdat, M. Konrad, W. J. Parak, Small 2015, 11, 5844-5850; d) S. H.
Lee, D. S. Choi, S. K. Kuk, C. B. Park, Angew. Chem. Int. Ed. 2018,
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D. H. Nam, J. Ryu, S. H. Ku, C. B. Park, Biosens. Bioelectron. 2011,
-
1
-1
(
D
2
-
1
-1
times higher than with NADH (163 mM s ). In addition, the lower
biocatalytic performance could be caused by chemical binding of
NADH to N-CDs that possibly prohibits NADH from diffusing
inside the catalytic site of TsOYE (Figure S20). Furthermore, we
examined the general applicability of the N-CD-sensitized
photobiocatalytic platform to the conversion of another α,β-
unsaturated compound (i.e., trans-cinnamaldehyde) by TsOYE. In
agreement with the conversion of 2-methyl-2-cyclohexen-1-one, the
regeneration platform prompted TsOYE activity toward the
reduction of the unsaturated aldehyde with varying yields in the
2
9
7
6, 1860-1865; h) D. H. Nam, S. H. Lee, C. B. Park, Small 2010, 6,
22-926; i) K. K. Sakimoto, A. B. Wong, P. Yang, Science 2016, 351,
4-77.
order of mNH H > NADH > mBuH > mCOOHH (Figure 2b).
2
Compared to the reaction of 2-methyl-2-cyclohexen-1-one, the
lower catalytic performance is ascribed to the inhibitory effect of the
[
8] G. A. M. Hutton, B. C. M. Martindale, E. Reisner, Chem. Soc. Rev.
2017, 46, 6111-6123.
[
17]
product 3-phenylpropionaldehyde on TsOYE. Taken together, N-
CD-sensitized regeneration of NADH and its analogues can be
applied to different unsaturated compounds and best results were
[9] L. Cao, S. Sahu, P. Anilkumar, C. E. Bunker, J. Xu, K. A. S. Fernando,
P. Wang, E. A. Guliants, K. N. Tackett, Y.-P. Sun, J. Am. Chem. Soc.
2
011, 133, 4754-4757.
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Joliat, C. Bachmann, R. Alberto, E. Reisner, Angew. Chem. Int. Ed.
obtained with mNH H compared to the natural cofactor and other
2
analogues.
In summary, we have demonstrated OYE-catalyzed
asymmetric hydrogenation by N-CDs via light-driven regeneration
2
016, 55, 9402-9406; c) B. C. M. Martindale, G. A. M. Hutton, C. A.
Caputo, S. Prantl, R. Godin, J. R. Durrant, E. Reisner, Angew. Chem.
Int. Ed. 2017, 56, 6459-6463.
of NADH and its analogues (i.e., mNH H, mBuH, and mCOOHH).
2
Through spectroscopic and (photo)electrochemical analyses, we
elucidated the capability of N-CDs to deliver photoexcited electrons
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Lee, J. Zhong, Z. Kang, Science 2015, 347, 970-974.
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W. J. Lockwood, J. N. Butt, E. Reisner, J. Am. Chem. Soc. 2016, 138,
+
to M and different photochemical reduction behaviors of NAD and
+
mNAD s. Initial rates and yields of regeneration were in the order of
1
6722-16730.
NADH > mNH H > mBuH > mCOOHH, which we ascribe to
2
[
[
13] J. Bian, C. Huang, L. Wang, T. Hung, W. A. Daoud, R. Zhang, ACS
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different reduction peak potentials of cofactors. The coupling of
photochemical regeneration of mNADHs with a TsOYE-driven
reaction allowed for the efficient synthesis of (R)-2-
methylcyclohexanone (~94% ee). Furthermore,
a
broader
[15] H. C. Lo, O. Buriez, J. B. Kerr, R. H. Fish, Angew. Chem. Int. Ed.
1999, 38, 1429-1432.
applicability of the photobiocatalytic system was demonstrated by
employing different substrates (e.g., 2-methyl-2-cyclohexen-1-one,
trans-cinnamaldehyde). The photochemical regeneration platform of
[
[
[
16] S. H. Lee, H. J. Lee, K. Won, C. B. Park, Chem. Eur. J. 2012, 18,
490-5495.
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Scrutton, Catal. Sci. Technol. 2016, 6, 169-177.
5
mNH H enabled excellent catalytic activities of TsOYE over the
2
substrates. Compared to other cofactors, mNH H’s excellent
2
performance is on account of the higher kred/KD of TsOYE with
mNH H that compensates for the lower efficiency of mNH H
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667-670.
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2
2
regeneration. Although our photobiocatalytic system recorded a
higher enzymatic efficiency than other platforms for light-induced
activation of OYEs, admittedly it still falls short of the
productivities obtained with classical cofactor regeneration
[
21] H. S. Toogood, T. Knaus, N. S. Scrutton, ChemCatChem 2014, 6,
[
21]
951-954.
systems.
Nevertheless, the limitations identified in this
contribution are the basis for further improvements, such as the
3
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Angewandte Chemie International Edition
10.1002/anie.201804409
Photobiocatalysis
Jinhyun Kim, Sahng Ha Lee,
Florian Tieves, Da Som Choi,
Frank Hollmann, Caroline E.
Paul*, and Chan Beum Park*
Page No. – Page No.
Biocatalytic C=C Bond
Reduction through Carbon
Nanodot-Sensitized
Regeneration of NADH
Analogues
We report the activation of Old Yellow Enzyme (OYE) through light-driven regeneration of
nicotinamide adenine dinucleotide cofactor (NADH) and its analogues (mNADHs) by using N-doped
2
carbon nanodots as a photosensitizer. Among cofactors, the photochemically regenerated mNH H
enabled the best catalytic activity of TsOYE. This work suggests that photochemical regeneration of
better-than-nature mNADHs can provide an efficient platform for enhancing biocatalytic efficiencies
using light energy.
4
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