Coupling Molecular Photocatalysis to Enzymatic Conversion

Article abstract of DOI:10.1002/cctc.201701232

A hetero-binuclear dyad that contains a ruthenium polypyridyl moiety bound through an aromatic bridging ligand to an organometallic catalytic center has been used for the light-driven reduction of the N-benzyl-3-carbamoylpyridinium cation, NAD⁺

Full text of DOI:10.1002/cctc.201701232

Accepted Article
Title: Coupling Molecular Photocatalysis to Enzymatic Conversion
Authors: Sven Rau, Alexander Mengele, Gerd Seibold, and Bernhard
Eikmanns
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Coupling Molecular Photocatalysis to Enzymatic Conversion
Alexander K. Mengele,[a] Gerd M. Seibold,[b] Bernhard J. Eikmanns[b] and Sven Rau*[a]
Abstract: A heterobinuclear dyad containing a ruthenium polypyridyl
moiety bound via an aromatic bridging ligand to an organometallic
catalytic center has been used for the light driven reduction of BNA+,
NAD+ and NADP+ yielding the two electron reduced analog. Direct
coupling with enzymatic conversion could be proven by means of
UV-vis spectroscopy as well as liquid chromatography, showing
cofactor recycling and enzymatic conversion with a turnover number
of 350 per photocatalyst. First insights into the complex behavior of
the catalytic system under irradiation points towards multiple
prerequisites on the molecular as well as on the macroscopic level in
order to generate highly efficient semiartificial photobiocatalytic
systems for future energy storage applications.
shown that even biomimetic cofactor analogs not bearing the
naturally occurring ribose containing N-substituents can be
recognized by certain enzymes enabling less expensive and
even more effective enzymatic stereoselective reactions.[15,18-20]
In addition these Rh catalysts can also play the role of an
efficient redox mediator in electrochemical NAD(P)H
regeneration systems by lowering the required potential,
simultaneously inhibiting unselective side reactions arising from
cofactor radical chemistry.[21-23]
To date photocatalytic reduction of nicotinamide cofactors is
mainly tackled using multicomponent systems.[24] Interestingly,
an intermolecular system, where the formation of a bimodular
aggregate by reversible coordination of Eosin
Y
to
[(bpy)Rh(Cp*)(H2O)]2+ (bpy = 2,2´-biypyridine) was proposed,
reached very high turnover frequencies (TOFs) by facilitating the
charge transfer onto the catalytic center.[25] In addition a polymer
compound linked to a similar rhodium catalyst has been shown
to drive enzymatic substrate conversion upon irradiation with
visible light.[26]
Introduction
The storage of light energy in chemical bonds is one of the most
promising approaches to the global energy shortage by
supplying mankind with renewable green fuels.[1] Among a
variety of different methods, photoredox catalysis using
multimetallic architectures is a promising concept to achieve this
goal.[2] To date a variety of such systems has been reported
As other studies proved oligonuclear photocatalysts performing
superior in comparison to intermolecular systems as well,[27,28]
we were interested in efficient nicotinamide cofactor recycling
using a structurally well-defined bimetallic dyad, enabling fast
photoinduced electron transfers to the [(N,N)Rh(Cp*)X]n+
catalytic center. In addition, decreasing the chance of forming a
highly reactive photoreduced chromophoric subunit by
eliminating diffusion controlled redox processes between the
chromophore and the catalyst might furthermore diminish the
unproductive radical chemistry of nicotinamide cofactors upon
one electron reduction. This process is known to generate
biologically inactive dimers, which could impede efficient
photobiocatalytic systems.[29,30] Therefore the well-known
mononuclear Ru complex [(tbbpy)2Ru(tpphz)]2+ (Rutpphz;
tbbpy = 4,4´-di-tert-butyl-2,2´-bipyridine, tpphz = tetrapyrido[3,2-
a:2´,3´-c:3´´,2´´-h:2´´´,3´´´-j] phenazine) was chosen as
chromophoric subunit, since it has been under extensive
investigation in terms of photocatalytic hydrogen production as
well as intramolecular sub-ns electron transfers using Pd or Pt
moieties as the catalytic centers.[31,32] Thus we sought to
examine the light driven reduction of artificial as well as
biologically utilizable NAD-like cofactors employing the
mainly focusing on proton[3] and CO2 reduction as well as on
[4]
water oxidation.[5] On the other side, coupling of light harvesting
systems with biocatalysts appears highly relevant as well.[6] It
allows the light induced powering of mild and selective
enzymatic transformations of potentially rather inert or
structurally complex substrates. Besides the direct light driven
regeneration of reduced flavins,[7-9] the reduction of oxidized
nicotinamide cofactors is of particular interest as well, since an
effectively working catalyst could provide the reducing
equivalents to an even larger number of oxidoreductases,
simultaneously saving resources by directly recycling the
consumed biological hydride donor.
For this purpose [(N,N)Rh(Cp*)X]n+ compounds with
Cp* = pentamethylcyclopentadienyl, N,N = N,N-chelating ligand
and X = Cl, H2O have been shown to be highly active in
selectively reducing meta functionalized pyridinium ions to the
1,4-dihydro form using either formate[10-12] or phosphite[13] as
reducing agent. Applying NAD(P)+ as the substrate for the
selective [(N,N)Rh(Cp*)X]n+ mediated generation of the
previously
[(tbbpy)2Ru(tpphz)Rh(Cp*)Cl]Cl(PF6)2
described
hydrogen
evolving
photocatalyst
(RutpphzRhCp*),[33]
biologically active reduced cofactor,
systems for the efficient reductive enzymatic synthesis of chiral
compounds has been described.[14-17] Moreover it has been
a variety of coupled
whose molecular structure is depicted in scheme 1.
Results and Discussion
[a]
[b]
M.Sc. A. K. Mengele, Prof. Dr. S. Rau
Institute of Inorganic Chemistry I, Materials and Catalysis
Ulm University
Albert-Einstein-Allee 11, 89081 Ulm, Germany
E-mail: sven.rau@uni-ulm.de
Dr. G. M. Seibold, Prof. Dr. B. J. Eikmanns
Institute of Microbiology and Biotechnology
Ulm University
In order to test the photocatalytic activity of RutpphzRhCp* for
nicotinamide cofactor reduction, initial experiments were
performed using BNA+ (N-benzyl-3-carbamoylpyridinium cation)
as convenient structural analog for the biologically active
compounds. Irradiation ( = 470 nm) of a solution containing
RutpphzRhCp*, BNA+, triethylamine (TEA) and NaH2PO4 in
degassed H2O:MeCN (1:1, v:v) under an argon atmosphere led
Albert-Einstein-Allee 11, 89081 Ulm, Germany
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presence of the m-carbamoyl pyridiniumion moiety itself.[11]
Additionally, in contrast to the previously reported
heterobinuclear complex [(tbbpy)2Ru(tpphz)PdCl2](PF6)2,[31a]
photocatalysis using RutpphzRhCp* is not inhibited by the
presence of excess chloride ions, proved by an experiment in
which BNA+ was reduced with a similar TOF in absence as well
in presence of an 5000 times excess of NaCl (Fig S4).
Furthermore on-off experiments revealed that the catalytically
active species is highly reactive with respect to BNA+ (Fig. 2).
After switching off the light source, no further increase in the
absorbance at 355 nm was detected, indicating that in presence
of a suitable substrate such as BNA+, no accumulation of the
catalytically active species is possible, which would react in the
dark with the cofactor analog. Therefore formation of the
catalytically active species seems to be the rate determining
step of the overall process.
Scheme 1. Molecular structures of the heterobinuclear photocatalyst
RutpphzRhCp* as well as the oxidized (BNA+) and the 1,4-reduced (BNAH)
nicotinamide cofactor analogs.
to the spectral changes shown in Fig 1. The raising band at
355 nm is characteristic for the formation of a reduced species
derived from BNA+.[24a] After 3.5 h a conversion of nearly 80%
was obtained, calculated by the increase of absorbance at
Surprisingly control reactions performed with Rutpphz instead of
RutpphzRhCp* led to similar spectral changes as in Fig 1. By
irradiation of a typical catalytic mixture described above, rising
bands at around 350 nm in presence of BNA+, NAD+ or NADP+
(Fig S5-S7) were observed. Although operating slower than
RutpphzRhCp*, we first believed that the photoreducible tpphz
ligand in Rutpphz may play a role as hydride mediator to
produce the 1,4-reduced product out of the three substrates. To
test this hypothesis, we performed a typical catalytic run without
adding BNA+ (see Fig S8). The loss of the two sharp bands at
around 365 nm and 385 nm during illumination clearly indicated
the photoreduction of the tpphz ligand in presence of TEA as
electron donor, as these two bands have been ascribed to →*
and n→* transitions of the fully aromatic system.[33]
355 nm
relative
to
the
initial
spectrum
(355nm(BNAH) = 7240 L mol-1 cm-1, taken from literature[24a]).
This correlates to a TON (turnover number) of 39 after 3.5 h with
a maximum TOF (turnover frequency) of almost 15 h-1.
Moreover an induction phase is observed (Fig. S1) and the
catalyst´s TOF reaches its maximum after 90 minutes with a
subsequent slight slope. This could be interpreted as formation
of the active catalyst at the first minutes of irradiation and a
substrate limitation of the catalysis at the end of the process.
The rising band at 650 nm, which leads to a color change from
orange to green during catalysis, was previously ascribed to the
reduction of the tpphz bridging ligand upon irradiation.[33]
Furthermore neither in the dark nor in the absence of
RutpphzRhCp* accumulation of reduced BNA species was
observed. Similar changes as in Fig 1 can be found using NAD+
and NADP+ as substrates under otherwise identical conditions
(Fig S2, S3). The fundamental ability of RutpphzRhCp* to
photocatalytically produce the reduced cofactor hence does not
depend on the group attached to the nitrogen but rather on the
To validate if the tpphz ligand in Rutpphz plays a crucial role in
transferring reducing equivalents to BNA+, [Ru(tbbpy)3](PF6)2
was tested under similar photocatalytic conditions as described
above. As can be seen in Fig 3, a rising band at around 350 nm
Figure 2. On-off-experiment for the photocatalytic reduction of BNA+
(c = 0,5 mM) using RutpphzRhCp* (c = 10 M) in
a
solution of
MeCN:H2O = 1:1 (v:v) containing TEA (c = 0.1 M) and NaH2PO4 (c = 0.1 M);
UV-vis spectra at different points in time and corresponding changes in the
absorbance at 355 nm. The blue arrows indicate that the light source (one
blue LED stick,  = 470 nm, 50 ± 2 mW/cm-2) is switched on, the black arrows
that it is switched off.
Figure 1. UV-vis spectral changes during irradiation of a catalytic mixture
containing RutpphzRhCp* (10 µM), BNA+ (0.5 mM), TEA (0.1 M), NaH2PO4
(0.1 M) in degassed MeCN:H2O = 1:1 (v:v) under an argon atmosphere with
blue light ( = 470 nm, 50 ± 2 mW cm-2).
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Since it is known that the 1,4-dihydro forms (BNAH, NADH,
NADPH) as well as the different possible dimeric structures
((BNA)2, (NAD)2, (NADP)2), obtainable by single electron
transfer onto the substrate and successive radical coupling,
show similar absorption profiles,[36] discrimination of the
photoproducts by this method is not possible.[29] Therefore an
enzymatic assay was used to test if the mononuclear complexes
Rutpphz and [Ru(tbbpy)3](PF6)2 as well as RutpphzRhCp* were
able to produce the biologically relevant two electron 1,4-
reduced form of the nicotinamide cofactors, since only this
species would be enzymatically consumable.
For this purpose a mixture of Rutpphz, [Ru(tbbpy)3](PF6)2 or
RutpphzRhCp* (c = 10 M), NAD+ (c = 0.96 mM), sodium
pyruvate
(c = 10 mM),
MgCl2
(c = 8.5 mM),
TEOA
(triethanolamine, c = 0.2 M) in MeCN:H2O (1:99, v:v, pH = 8.7)
and in presence of lactate dehydrogenase (LDH, 5 units/mL)
under an argon atmosphere was irradiated with one LED-stick
( = 470 nm, 50±2 mW/cm2) from the bottom side of standard
quartz cuvettes that were used as reaction vessels. Based on
the fast hydrolysis of the Rh-Cl bond and the determined pKa
value of the rhodium bound aquo ligand in similar systems,[37]
the resting state of the photocatalyst might be described best
with a RhCp*(OH) moiety at the catalytic center. Although the
higher  donating ability of the hydroxo ligand compared to an
aquo ligand might influence the intramolecular electron transfer
kinetics, RutpphzRhCp* behaves as expected for an efficient
catalyst, that produces the biologically consumable reduced
cofactor. As can be seen in Fig 4, almost no accumulation of
reduced NAD species can be observed in the presence of LDH
(diagram a)), indicated by negligible changes of the recorded
UV-vis spectra upon irradiation of the sample, whereas in
absence of LDH a rise of the 340 nm product absorbance is
observable (diagram b)). HPLC analysis after 17 h showed, that
in presence of LDH, 3.5 mM of lactate were formed. This
correlates to a TON of 350 and an average TOF of 20 h-1 with
respect to the photocatalyst RutpphzRhCp* (Fig S11).
Figure 3. a) UV-vis spectral changes during 4 hours of irradiation of a solution
containing [Ru(tbbpy)3](PF6)2 (c = 10 M), BNA+ (c = 0.5 mM), TEA (c = 0.1 M)
and NaH2PO4 (c = 0.1 M) in degassed MeCN:H2O = 1:1 (v:v) under an argon
atmosphere with blue light ( = 470 nm, 50 ± 2 mW cm-2). Diagram b) shows
the changes upon irradiation of a solution described above without BNA+ being
present.
occurred in presence of BNA+ (diagram a)), whereas in its
absence (diagram b)) only the photodegradation of
[Ru(tbbpy)3](PF6)2 was observed, indicated by the lowered
absorbance of the MLCT band at 450 nm with increasing
irradiation time.
Although the homoleptic complex is able to generate reduced
BNA species too, the efficiency of this process compared to
Rutpphz is lower. Despite the photolability of [Ru(tbbpy)3](PF6)2,
this result respresents a surprising detail of our studies, since
Rutpphz would feature due to its low lying tpphz based LUMO a
smaller driving force for electron transfer onto a substrate.[31a,34]
An attractive explanation for this behavior could be the
overcompensation of the lower reduction potential of Rutpphz
by the formation of a - interactions mediated supramolecular
aggregate with BNA+ via the planar tpphz ligand and the
aromatic systems of the cofactor analog.
As NAD+ itself was used in a concentration of 0.96 mM, cofactor
recycling was obtained. In the course of irradiation each NAD+
molecule was reduced on average more than 3.5 times. For the
reduction of oxidized nicotinamides, the active catalyst has been
proposed to be a Rh-H[11,12] or as previously suggested a
reduced Cp* moiety, formed after proton transfer from the
rhodium center to a carbon atom of the former aromatic
ligand.[38,39]
However, in the absence of LDH no lactate was detected,
indicating that the catalytically active species is not able to
reduce the keto group of pyruvate in a significant amount
itself.[40] Since no stereo information is existing in
RutpphzRhCp* proximate to the catalytic center, we
furthermore would have expected the formation of a racemic
lactate mixture.[41]
This is demonstrated by concentration dependent 1H-NMR
spectroscopy experiments. With an increasing amount of BNA+
predominantly the tpphz based signals of Rutpphz undergo a
shift (Fig. S9). This is in line with similar results obtained by the
--stacking between pyrene and Rutpphz.[35]
On the other hand [Ru(tbbpy)3](PF6)2 does not exhibit similarly
effective interactions with BNA+, indicated by constant 1H-NMR
spectroscopic shifts of the tbbpy signals upon increasing
concentrations of the cofactor analog (Fig. S10). This fortifies
the above discussed hypothesis for the superior electron
transfers onto BNA+ using Rutpphz via formation of a -
interactions mediated aggregate.
However, the results obtained from the UV-vis spectroscopic
investigations of the catalytic processes using RutpphzRhCp*,
Rutpphz and [Ru(tbbpy)3](PF6)2 as photoredox active molecules
revealed, that the fundamental photocatalytic activity of the three
complexes with respect to BNA+ may simply be related to the
photochemistry of ruthenium polypyridyl complexes in presence
of a sacrificial electron donor such as the used TEA.
Similarly no lactate was found after 17 h when the same sample
was stored in the dark or RutpphzRhCp* was absent, showing
that the overall photobiocatalytic process is driven by visible light
induced photoredox catalysis of the heterobinuclear complex,
providing enzymatically consumable reducing equivalents for
LDH.
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In order to strengthen this hypothesis, 1H-NMR experiments
using Rutpphz or [Ru(tbbpy)3](PF6)2 as phocatalysts, TEA as
sacrificial electron donor and BNA+ as terminal electron acceptor
showed, that upon irradiation with visible light ( = 470 nm) the
peaks assigned to BNA+ diminish and new signals appear
(Fig S15 and S16). Although this again highlights the ability of
ruthenium polypyridyl complexes to photochemically convert
BNA+ under appropriate reaction conditions, comparison with the
1H-NMR signal set of freshly synthesized BNAH were not in
accordance with the new peaks emerging under visible light
irradiation.
These findings together with the enzymatic tests and the results
obtained by following photocatalysis using UV-vis spectroscopy
have severe impacts on the molecular design of systems for
photocatalytic NADH production and coupled enzymatic
transformations using photosensitizers with high reduction
potentials such as ruthenium polypyridyl complexes. As
Rutpphz and [Ru(tbbpy)3](PF6)2 are able to photoreduce the
oxidized nicotinamide cofactors and its artificial analogs itself,
multicomponent systems composed of separated redox
chromophores and [(N,N)Rh(Cp*)X]n+ like catalysts have to be
designed carefully in order to enable fast electron transfers from
the excited/reduced chromophore onto the catalyst, thus
avoiding the accumulation of bioinactive dimeric cofactors.
Multimetallic or supramolecular architectures in which the
electron transfers are not limited by diffusion due to a well-
defined preorganisation of chromophore and catalyst are
therefore highly relevant alternatives to multicomponent systems.
In the latter, by virtue of the independent photochemistry of the
long-lived excited/reduced state of the chromophore with
nicotinamide cofactors, the need of intermolecular electron
transfers would exhibit a severe source of inefficiency inherent
to the system.
In order to detect possible side products in the photobiocatalytic
processes, we first performed thermal catalysis experiments
using RutpphzRhCp* as catalyst, NaHCO2 as hydride donor
and BNA+ as hydride acceptor to exclude negative influences of
the ligand environment around the rhodium center on the well-
known and highly selective hydride transfer of the
[(N,N)Rh(Cp*)X]n+ complexes for the generation of the 1,4-
reduced product.[10-12] As can be seen in Fig 5, the rising band at
355 nm again indicates the formation of a reduced BNA species.
A TON of 13 after 515 min and a TOF of 1.5 h-1 could be
calculated, which is significantly lower with respect to the
photobiocatalytic system described above. This again highlights
the potential of light driven cofactor reduction, as here the
formation of the active catalyst is much less dependent on the
temperature. Moreover, compared to recently published
literature,[19] the TOF of our formate driven system is reduced by
a factor of 4. As a higher thermal energy content will have a
dramatic effect on the overall BNA+ reduction by facilitating the
rate determining step, i.e. generation of the catalytically active
Rh species via -hydride elimination from the metal bound
formate,[12] the decreased TOF in our case can be ascribed to
the lower applied temperature.
Figure 4. UV-vis spectral changes during the first 3 hours of irradiation of a
catalytic mixture using one LED stick ( = 470 nm, 50 ± 2 mW/cm-2): a) The
mixture contains RutpphzRhCp* (c = 10 M), NAD+ (c = 0.96 mM), sodium
pyruvate (c = 10 mM), TEOA (c = 0.2 M), MgCl2 (c = 8.5 mM) and LDH
(5 U/ml) in MeCN:H2O = 1:99 (v:v, pH = 8.7); b) The mixture contains all the
above mentioned compounds without LDH; c) Increase in the absorbance at
340 nm with respect to the value before starting the illumination of the catalytic
mixtures described above.
The enzymatic test for Rutpphz as well as [Ru(tbbpy)3](PF6)2
revealed that neither in presence nor in absence of LDH lactate
was produced (Fig S12). Consistent with this finding the rise of
the 340 nm band was nearly the same for both mononuclear
complexes, irrespective of the presence of LDH (Fig S13 and
S14). We therefore conclude, that these two complexes
generate only biologically inactive reduced NAD species,
putatively a mixture of regioisomeric (NAD)2 dimers.[42] Similar
findings have already been published, highlighting the reactivity
of oxidized nicotinamide cofactors and its analogs towards one
electron reduction in presence of suitable chromophores.[29,30]
A
selective hydride transfer of the photoreduced tpphz ligand in
Rutpphz to the 4-position of the nicotinamide cofactor can
therefore also be excluded. We conclude additionally, that the
same dimer formation is true for the experiments described
above, using BNA+ as substrate and the mononuclear
complexes Rutpphz and [Ru(tbbpy)3](PF6)2 as photocatalysts.
As in Fig 5 the fine structured bands of the fully aromatic tpphz
ligand at 350 and 380 nm remain visible until they diminish
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in presence of TEA and BNAH as mutual competing electron
donors and BNA+ as terminal electron/hydride acceptor. As can
be seen in Fig 6 (the whole spectrum is shown in Fig S19) the
signal, attributed to the two hydrogen atoms located para to the
nitrogen atom in the heterocycle of BNAH at 2.97 ppm,
decreases with increasing irradiation time relative to the quintett
of MeCN-d2 at 1.94 ppm. This shows that also in the case of
RutpphzRhCp* consumption of BNAH occurs, generating a
mixture of different (BNA)2 dimers.
However, as the enzymatic assay described above proved that
the biologically active 1,4-dihydro NADH was successfully
generated photochemically using RutpphzRhCp*, the reduced
cofactor was consumed enzymatically faster than by the excited
binuclear complex.
These results lead to a second important design parameter of
semiartificial photobioctalytic systems using NAD(P)+ as
photocatalytically recyclable hydride mediator, which is that the
enzymatic transformation has to be significantly faster than the
unproductive consumption of the generated NAD(P)H by the
excited or photooxidized photosensitizer. This could be achieved
on the molecular scale by choosing a quickly operating enzyme
for redox biocatalysis and on a macroscopic level by a precise
control over the mass flow in the reaction vessel or via the
immobilization of multinuclear photocatalysts on semiconductor
electrode surfaces. The latter would inhibit the quenching of the
photoexcited chromophores by the photocatalytically generated
reduced nicotinamide cofactors due to steric reasons as well as
by a fast regeneration of the oxidized chromophore with an
electron from the valence band of the semiconductor.[46,47]
Moreover by immobilizing enzymes on microparticles,[48] this
approach would also allow spatial separation of photo- and
biocatalyst, which are prone to mutual inhibition.[49,50]
Figure 5. UV-vis spectral changes during the thermal BNAH production by a
mixture containing RutpphzRhCp* (c = 10 M), BNA+ (c = 1 mM), NaHCO2
(c = 50 mM) under argon atmosphere, using degassed solvents in a ratio of
MeCN:H2O = 1:9 (v:v) at room temperature.
under the increasing product absorbance, hydride transfer
reactions from the initially formed rhodium hydride onto the
tpphz bridging ligand with possible concomitant unselective
hydride transfers onto BNA+ are highly unlikely. Control
experiments without RutpphzRhCp* did not result in the
formation of the 355 nm product band. In order to study the
product distribution of this thermal process, 1H-NMR
experiments under similar conditions were performed (Fig S17).
Over time new peaks emerge exactly at these positions, where
the independently synthesized 1,4-reduced BNAH exhibits its
proton resonances. Therefore the well-known selective hydride
transfer property of the Rh(Cp*) catalytic center is preserved in
RutpphzRhCp*.
For a closer look into the product distribution under irradiation
1
using RutpphzRhCp* as catalyst, H-NMR investigations under
catalytic conditions using TEA as electron donor and BNA+ as
terminal electron acceptor were performed. Surprisingly, similar
to the experiments performed with the mononuclear complexes
Rutpphz and [Ru(tbbpy)3](PF6)2, no BNAH could be detected
(Fig S18). These findings were in strong contrast to the results
obtained by performing the enzymatic assays described above,
since only the 1,4-dihydro form of NADH can be used by LDH.
A possible explanation for the accumulation of reduced BNA
species different to that of the 1,4-dihydro form BNAH in the
case of RutpphzRhCp*, could be the consumption of formed
BNAH by an one electron oxidation process involving the
photoexcited ruthenium chromophore. BNAH is frequently used
as potent sacrificial electron donor in photoredox catalytic
systems due to its higher reduction potential than common
aliphatic amines.[43] In presence of a base, fast deprotonation of
the very acidic formed BNAH radical cation occurs, generating a
neutral radical species which then couples with a second BNA
radical to yield dimeric BNA species (BNA)2.[44,45]
Figure 6. 1H-NMR spectroscopic changes between 1.4 and 3.2 ppm of a
solution containing RutpphzRhCp* (c = 100 M), TEA (c = 0.1 M), NaH2PO4
(c = 0.1 M), BNA+ (c = 10 mM) and BNAH (c = 30 mM) under argon
atmosphere using a degassed solvent mixture of MeCN-d3:D2O = 1:1 (v:v).
Spectrum 1) was recorded before, spectrum 2) after 3, spectrum 3) after 22
and spectrum 4) after 42 hours of irradiation with one LED stick (45-
52 mW cm-2) at room temperature. The intensive peak at 3.05 ppm is
attributed to the methylene group of TEA.
To elucidate if this fast consumption process of generated BNAH
by excited chromophores also occurs for RutpphzRhCp*, 1H-
NMR experiments were performed using the binuclear complex
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Based on the performed experiments described above,
a
Hence semiartificial photobiocatalytic systems have to be
designed with great care as the continuous accumulation of
CFdim represents an irreversible loss pathway that leads
ultimately, due to the ever dwindling CFox amount, as it is also
observable in Fig S19, even in presence of a great excess of a
sacrificial electron donor with lower reduction potential than
CFred, to an unefficient enzymatically substrate conversion.
A further possible side product during the photobiocatalytic
process is H2, which cannot be detected using standard UV-vis
rational reaction scheme can be drawn. As can be seen in
scheme 2, part a), the binuclear photocatalyst RutpphzRhCp* is
able to use TEA under irradiation with visible light as sacrificial
electron donor in order to generate the bioactive reduced
cofactor (CFred) from its oxidized analog (CFox). In presence of
an efficiently working enzyme, CFred gets immediately
reconverted to CFox which can then be reused from
RutpphzRhCp* as hydride acceptor. These coupled processes
allow the photocatalytic recycling of nicotinamide cofactors and
the use of substochiometric cofactor amounts for enzymatic
substrate conversions. In absence of an enzyme, the initially
produced CFred gets no longer reconverted to CFox and by virtue
of its function as potent sacrificial electron donor CFred
undergoes an one electron oxidation with RutpphzRhCp*,
yielding the radical cationic species, which couples after
1
or H-NMR spectroscopy. Therefore the photocatalytic hydrogen
production under conditions for the initial BNA+ photoreduction
experiments described above was studied via gas
chromatography
(MeCN:H2O = 1:1,
v:v;
c(TEA) =0.1 M,
c(NaH2PO4) = 0.1 M; c(RutpphzRhCp*) = 10 M). After 100 h a
TON of only 3 was achieved. In presence of 100 equivalents
BNA+, a delayed onset of hydrogen production as well as a
reduced TON of 0.4 was observed. Moreover within the time
span of the photobioctalysis experiments described above, no
hydrogen was detected at all, indicating that under the applied
conditions BNA+ represents the strongly favored substrate
compared to H+.
deprotonation with
a second neutral nicotinamide cofactor
radical (CFrad) to one of the regioisomeric dimeric species
[42]
CFdim
.
As can be seen in part b) of scheme 2, Rutpphz as well as
[Ru(tbbpy)3](PF6)2 are not able to generate CFred due to the lack
of an appropriate catalytic center that guides the stored reducing
equivalents selectively to the 4-position of the heterocycle.
Therefore the mononuclear compounds only yield CFrad under
Conclusions
irradiation, which couple to the enzymatically unusable CFdim
.
Although the dimeric (BNA)2 species exhibits an even higher
reduction potential than BNAH, previous reports suggest that the
(BNA)2 radical cationic species, generated by photooxidation
with a ruthenium polypyridyl chromophore, decompose only very
slowly.[51] Therefore electron back transfer occurs very efficiently
and (BNA)2 represents the final product of photocatalysis as
shown in scheme 2.
In conclusion we presented with RutpphzRhCp* for the first time
a
well-defined binuclear architecture, able to reduce
nicotinamide cofactors into the biologically active forms under
irradiation with visible light. Herein the rate determining step of
the process is the light driven generation of the catalytically
active species. Moreover RutpphzRhCp* was able to power the
LDH catalyzed transformation of pyruvate to lactate in a proof of
concept system with a total TON of 350 per photocatalyst,
thereby using substochiometric amounts of NAD+ under
implementation of an internal cofactor recycling system.
Furthermore, comparing studies indicated that RutpphzRhCp*
exhibits under the applied conditions a much higher activity for
nicotinamide cofactor reduction than hydrogen evolution,
possibly suggesting unequal mechanistic pathways with respect
to the formation of the two different products.
First insights into the intertwined photocatalytic reaction
pathways using mono- and bimetallic ruthenium polypyridyl
complexes in presence of nicotinamide cofactor analogs point
towards design criteria for semiartificial photobiocatalytic
systems. These arise from the fact that multicomponent systems
with separated photo and catalytic centers exhibit a potential
source of error by design. Within the elongated lifetime of the
highly reactive excited or photoreduced chromophore due to the
collision induced electron transfer from the photo- to the
[(N,N)Rh(Cp*)X]n+ catalytic center, an one electron reduction of
the oxidized nicotinamide cofactors may occur, leading finally to
the end of productive photobiocatalysis by continuous
accumulation of dimeric species. Hence the combination of
oligonuclear photocatalysts with fast operating enzymes could
be a promising approach for the future design of efficient
photobiocatalytic systems.
Scheme 2. Possible reaction pathways under visible light irradiation using
RutpphzRhCp* (part a)) and Rutpphz (part b), same pathway is proposed for
[Ru(tbbpy)3](PF6)2); CFox = oxidized nicotinamide cofactor, CFred = reduced
nicotinamide cofactor (1,4-dihydro form), CFrad = radical nicotinamide cofactor
species, CFdim = dimeric nicotinamide cofactor species, S = substrate for
biocatalysis, P = product of the photobiocatalytic process.
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A heterobinuclear photocatalyst has
been found to drive an enzymatic
reduction of pyruvate to lactate by
continuous generation of NADH under
visible light irradiation.
Alexander K. Mengele, Gerd M. Seibold,
Bernhard J. Eikmanns, Sven Rau*
Page No. – Page No.
Coupling Molecular Photocatalysis to
Enzymatic Conversion
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