A Heterogeneous Recyclable Rhodium-based Catalyst for the Reduction of Pyridine Dinucleotides and Flavins

Article abstract of DOI:10.1002/cctc.201901726

Reduced pyridine nucleotides and flavins are important enzyme cofactors that require continuous regeneration for biotechnological development of the corresponding enzymes. This can be achieved with the assistance of a dehydrogenase system or by reduction with formate catalysed by a soluble organometallic {[Cp*Rh(bpy)(H₂O)]²⁺} (Cp=pentamethylcyclopentadienyl; bpy=bipyridine) complex. Here, we report that this Rh complex, once immobilized on bypiridine-periodic mesoporous organosilica, displays catalytic activity for flavin (including FAD, FMN and riboflavin) and NAD(P)⁺ reduction by formate. The recyclability of this solid catalyst makes it possible to achieve up to 20 cycles of FAD reduction without activity loss. This recyclable heterogeneous catalyst can also be used to assist a complex NADH-, FAD- and O₂-dependent monooxygenase system, allowing several cycles of transformation of a phenol into the corresponding catechol.

Full text of DOI:10.1002/cctc.201901726

Accepted Article
Title: A heterogeneous recyclable Rhodium-based catalyst for the
reduction of pyridine dinucleotides and flavins
Authors: YIFAN DENG, Mateusz Odziomek, OLIVIER BACK, Clément
Sanchez, VICTOR MOUGEL, and Marc Fontecave
This manuscript has been accepted after peer review and appears as an
Accepted Article online prior to editing, proofing, and formal publication
of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: ChemCatChem 10.1002/cctc.201901726
Link to VoR: http://dx.doi.org/10.1002/cctc.201901726
A Journal of
www.chemcatchem.org

ChemCatChem
10.1002/cctc.201901726
FULL PAPER
A heterogeneous recyclable Rhodium-based catalyst for the
reduction of pyridine dinucleotides and flavins
[
a]
[b]
[b]
[c]
[a], [d]
Yifan Deng , Mateusz Odziomek, Clement Sanchez, Olivier Back , Victor Mougel
Fontecave *[a]
and Marc
Abstract: Reduced pyridine nucleotides and flavins are important
enzyme cofactors that require continuous regeneration for
biotechnological development of the corresponding enzymes. This
can be achieved with the assistance of a dehydrogenase system or
by reduction with formate catalysed by a soluble organometallic
amounts of NAD(P)H with respect to reaction products are
consumed during the reaction. This severely limits the industrial
application of such biocatalytic processes for economic reasons
because these reduced pyridine nucleotides are very expensive
reactants. Furthermore, the accumulation of large concentrations
2+
{
[Cp*Rh(bpy)(H
2
O)] } (Cp* = pentamethylcyclopentadienyl; bpy =
of the corresponding oxidized forms of pyridine nucleotides, NAD+
bipyridine) complex. Here, we report that this Rh complex, once
immobilized on bypiridine-periodic mesoporous organosilica, displays
catalytic activity for flavin (including FAD, FMN and riboflavin) and
+
and NADP , during the reaction in some cases might lead to a
[2]
+
significant inhibition of the enzyme . Since NAD(P) is much less
expensive, a solution resides in using an effective and broadly
applicable method for in situ regeneration of NAD(P)H from the
+
NAD(P) reduction by formate. The recyclability of this solid catalyst
makes it possible to achieve up to 20 cycles of FAD reduction without
activity loss. This recyclable heterogeneous catalyst can also be used
+
oxidized NAD(P) form.
2
to assist a complex NADH-, FAD- and O -dependent monooxygenase
The most frequently used procedure is based on an
enzyme-dependent regeneration reaction, in which NAD(P)+ is
continuously reduced into the active NAD(P)H form in situ thanks
to the action of an alcohol dehydrogenase or a formate
system, allowing several cycles of transformation of a phenol into the
corresponding catechol.
Introduction
[
3]
dehydrogenase, both commercially available . With such a
regeneration coupled system, the biocatalytic reaction of interest
stoichiometrically consumes only cheap reductants, namely
ethanol or formate, respectively. However, because this strategy
implies the cooperation of several enzymes that need to be
adequately coupled, a challenging issue, a purely chemical
system might be preferable.
The reduced forms of pyridine dinucleotides, nicotinamide
adenine dinucleotide (NADH) and nicotinamide adenine
dinucleotide phosphate (NADPH), are essential biological hydride
donors in enzymatic reactions. They are thus used as cofactors
for reductases and dehydrogenases, some of which are
developed as effective biocatalysts in organic synthesis with high
[1]
regio-, chemo- and stereo-selectivity . However, stoichiometric
So far the only catalyst that proved efficient for the reduction
of
NAD(P)+
into
NAD(P)H
is
the
organometallic
bipyridyl
[
a] Laboratoire de Chimie des Processus Biologiques, Collège de France,
Sorbonne Université, CNRS UMR 8229, PSL Research University, 11
place Marcelin Berthelot, 75005 Paris, France.
pentamethylcyclopentadienyl
rhodium(III)
2+
{
2
[Cp*Rh(bpy)(H O)] } complex, named OC (for Organometallic
[
4]
Complex) in the following. First reported in 1987 , it was proved
active in aqueous solutions over a broad range of reaction
conditions. It can thus catalyze the reduction of NAD and NADP+
equally well and was thus used for a number of chemoenzymatic
[
b] Sorbonne Université, CNRS, Collège de France, PSL Research
University, Laboratoire Chimie de la Matière Condensée de Paris,
LCMCP, 4 Place Jussieu, F-75005, Paris, France
+
[
c] Solvay, Research & Innovation Center of Lyon, 85 avenue des frères
Perret, 69190 Saint-Fons, France.
[
5]
reductions, hydroxylations, epoxidations and halogenations .
[
d] Present Address: Department of Chemistry and Applied Biosciences,
Laboratory of Inorganic Chemistry, Swiss Federal Institute of
Technology, Zürich, Switzerland
Also, with respect to the source of reducing equivalents,
[Cp*Rh(bpy)(H
2
O)]2+ exhibits high versatility. It can be combined
with tertiary amines such as triethanolamine (TEOA) in a
photochemical regeneration system [5d] or with a cathode serving
Supporting information for this article is given via a link at the end of the
document
This article is protected by copyright. All rights reserved.

ChemCatChem
10.1002/cctc.201901726
FULL PAPER
[
6]
as an electrochemical source of electrons , while the most widely
used reducing agent is formate.
transformation, we used it for fueling an aromatic hydroxylation
enzyme reaction. During preparation of this manuscript, a similar
material was reported as a heterogeneous catalyst to assist a
NADPH-dependent alcohol dehydrogenase for asymmetric
Interestingly, [Cp*Rh(bpy)(H
reduction of oxidized forms of flavins, flavin adenine dinucleotide
FAD) and flavin monunucleotide (FMN), also important biological
O)]2+ also catalyzes the
2
hydrogenation of ketones[11]
.
(
[
5]
cofactors, by formate efficiently . This provides an opportunity to
develop chemoenzymatic oxidation reactions, in which a flavin-
dependent monoxygenase, requiring a reduced flavin cofactor for
Results and Discussion
Preparation and characterization of the catalyst.
oxygen activation, would be coupled to
a
reduced flavin
such as
regeneration
Cp*Rh(bpy)(H
chemical
system,
Immobilization of a RhCp* moiety on Bpy-PMO was
2
+
[
2
O)] /formate, in the following named OC/FA. In
2 2
achieved by incubating the neutral dimer [Cp*RhCl ] precursor
the absence of such a regeneration system, more expensive and
complex scenarios have to be set up : the reduced flavin, an
expensive and unstable compound, is either used
stoichiometrically or produced at the expense of stoichiometric
amounts of NAD(P)H, thanks to the addition of another enzyme,
a NAD(P)H :flavin oxidoreductase (also named flavin reductase).
Nice examples of the utilization of the OC/FA system for fueling
such a two-component monooxygenase system, composed of a
flavin reductase component and a hydroxylase component, have
been reported in the case of epoxidation[7] and aromatic
hydroxylation, more recently by us [8], reactions.
with a suspension of commercially available Bpy-PMO in
methanol during 4 hours at room temperature (scheme 1). After
centrifugation and several washing steps with methanol, the
powder was dried and submitted to elemental analysis. In order
to achieve the best loading conditions, the Rh precursor was
added to the reaction mixture at concentrations corresponding to
bpy:Rh molar ratio values of 1:0.01 ; 1:0.1 ; 1:0.2 and 1:1.1. As
shown from the results in Table S1, almost all Rh atoms were
immobilized on Bpy-PMO up to the 1: 0.2 ratio, leading indeed to
a maximal bpy:Rh ratio of 1:0.2 in the isolated powder. The same
Rh loading of 1:0.2 was found in the last sample prepared using
an initial 1:1.1 ratio. In the following we exclusively used the 1:0.1
sample which has an actual Rh loading of 1 :0.094 (0.296 mmol
Rh/g) and named it OC@Bpy-PMO.
Unfortunately, the OC/FA system suffers from the fact that
it is based on a rhodium, an expensive and rare metal, catalyst
and that it is homogeneous, thus ruling out the possibility to
separate, recover and recycle the catalyst. Therefore, the
development of a heterogeneous form of the [Cp*Rh(bpy)(H
2
O)]2+
complex is highly desirable. Since OC carries a bipyridine ligand,
we were highly interested in following Inagaki and Copéret
strategy, consisting in the fixation of metal ions, such as iridium or
ruthenium, to bypiridine-periodic mesoporous organosilica (called
Bpy-PMO in the following), in which the bipyridine ligands are
[
9]
exposed on the pore surface . These materials have the
advantage to display large pores, allowing easy diffusion of
substrate and product required for catalysis. Following this
strategy, we have recently demonstrated that Mn@Bpy-PMO
materials are excellent catalysts for the photoreduction of CO
2
∗
O)]2+
Scheme 1. Synthesis of OC@Bpy-PMO in which the OC ([Cp Rh(Bpy)(H
2
)
into CO and formate[10]. We here report the preparation of a Rh
complex is immobilized onto solid bypiridine-periodic mesoporous organosilica
Bpy-PMO).
catalyst immobilized on Bpy-PMO (named OC@Bpy-PMO in the
(
following) in which the [Cp*Rh(bpy)(H
2
O)]2+ coordination is
preserved. This solid catalyst proved active and recyclable in the
+
reduction of NAD(P) and FAD/FMN by formate. As an illustration
of its potential as a NADH regeneration system for biocatalytic
This article is protected by copyright. All rights reserved.

ChemCatChem
10.1002/cctc.201901726
FULL PAPER
+
Here we report detailed analytical data in order to
(a) NAD concentration, using 5 mg of OC@Bpy-PMO and 500
+
characterize the synthesized material and confirm the structure
shown in Scheme 1. First, the Transmission Electron Microscopy
revealed crystallographic planes in both OC@Bpy-PMO and Bpy-
PMO (Figure S1a,b) indicating crystallinity of both materials,
which was further confirmed by X-Ray Diffraction with patterns
that did not change upon metallation (Figure S2). Similarly, the
porous structure is essentially preserved after the immobilization
of metal complex (Figure S1c,d). Second, BET measurements
showed that the surface area decreased 3-fold upon metalation,
from 648 to 208 m2g-1 (Figure S3), which stays in agreement with
previous report[11a]. Third, 13C NMR spectrum of OC@Bpy-PMO
mM formate; (b) OC@Bpy-PMO mass using 50 µM NAD and 500
mM formate ; (c) formate concentration using 5 mg OC@Bpy-
+
PMO and 50 µM NAD . These data show that OC@Bpy-PMO is
+
a catalyst for NAD reduction to NADH : using 5 mg of OC@Bpy-
PMO and 500 mM formate, 500 µM NAD+ is almost totally
reduced within one hour, with an initial rate of around 17 µMmin-
1
+
. Under these conditions NADP is equally reduced into NADPH
(Figure S5). No reduction occurred in the absence of OC@Bpy-
PMO. Finally, no induction phase was observed during reduction
of NAD+ showing that OC@Bpy-PMO is the actual catalyst
(Figure S6).
*
nicely confirms the presence of the Cp ligands (9,1 and 98,6
For comparison with a homogeneous system we used the
ppm) on Rh and the coordination presented in Scheme 1 (Figure
∗
2+
[8]
). 29Si and H NMR spectra are shown in Figure S4. The former
1
1
2
previously reported [Cp Rh(bpy)(H O)] /formate system and
confirms the stability of PMO during immobilization of Rh by the
lack of cleavage of Si-C bonds upon metallation (no evolution of
the results are shown in Figure S7. Using a concentration of 100
µM of the soluble Rh complex led to reduction of 500 µM NAD+
n
Q units). The latter further supports the presence of Rh complex
-1
with the highest initial rate of about 8 µMmin . Based on the
by the appearance of peaks corresponding to methyl groups.
amount of Rh in OC@Bpy-PMO (0.296 mmol/g) we end up with
initial TOF values of 1.36 h-1 and 4.8 h-1 for the heterogeneous
and the homogeneous catalyst respectively. This shows that the
heterogeneization of the Rh complex has a limited effect on its
activity.
Recyclability tests were carried out as follows. In a first run
a solution containing 5 mg of OC@Bpy-PMO in suspension with
5
00 mM formate and 500 µM NAD+ was stirred and NADH
formation was monitored spectrophotometrically. After one hour
+
reaction, when NAD was almost totally reduced, the solution was
filtered to recover the catalyst and the latter was suspended again
+
in a fresh solution of formate and NAD for a second catalytic run.
The procedure was then repeated 8 times. As shown in Figure 2d
the catalyst was still fully active after 5 cycles, after which it started
to lose some activity (20% loss after 8 cycles), partly due to some
loss of material during filtration. These data thus nicely show that
OC@Bpy-PMO is a recyclable catalyst. The experiment shown in
Figure S8 shows that one can run as many as 15 cycles without
losing activity when using larger amounts of catalyst. After 30
cycles only half of the activity is lost, in perfect agreement with the
decrease of Rh content in the material (about 50% of the initial Rh
amount being leached), as shown by elemental analysis of the
recovered material. This shows that the remaining Rh sites have
retained full activity.
Figure 1. 13C NMR spectra of Bpy-PMO (black) and OC@Bpy-PMO (red). The
peaks are assigned according to the structure.
+
Reduction of NAD by formate
Using 20-500 mM sodium formate as the reducing agent
and 0.5-10 mg OC@Bpy-PMO in suspension in 2 ml of 20 mM
+
Tris buffer, pH 7.5, 20 mM NaCl, NAD conversion into NADH was
monitored spectrophotometrically from the increase of the
-1
-1
absorption at 340 nm (ε= 6.22 mM cm ), characteristic of NADH.
In Figure 2, the initial reaction rate is reported as a function of :
This article is protected by copyright. All rights reserved.

ChemCatChem
10.1002/cctc.201901726
FULL PAPER
To limit catalyst loss during filtration and allow a continuous
2
conditions since the product of the reaction, reduced flavin FADH ,
flow operation, we packed OC@Bpy-PMO in a column. This was
obtained by mixing 15 mg of the OC@Bpy-PMO powder with 60
mg of silica, to limitate overpressure issues and filling a Restek
empty HPLC column (2.1×50 mm) which was then connected to
is highly sensitive to oxygen. The reaction was also monitored
spectrophotometrically from the decrease of the absorption at 450
nm, characteristic of FAD. The initial reaction rates increased with
increasing concentrations of FAD, OC@Bpy-PMO and formate
(Figure 3). This clearly showed that OC@Bpy-PMO was a very
good catalyst for the reduction of FAD by formate, with initial rates
up to 20 µM.min-1 using 2 mg OC@Bpy-PMO, 500 mM formate
and 50 µM FAD, in 2 ml 20 mM Tris pH 7.5, 20 mM NaCl, so that
+
a HPLC system. Pumping a solution containing 5 mM NAD and
500 mM formate in 20 mM Tris buffer, pH 7.5, 20 mM NaCl,
through the column (optimized flow rate : 0.1 mL/min) allowed
+
efficient reduction of NAD to NADH as shown from the presence
of about 2 mM NADH in the solution collected after the column
after 5 min reaction almost all FAD was found converted to FADH
Under these conditions, FMN can also be reduced into FMNH
2
2
.
(
Figure S9). This column could be used at least 10 times without
losing any activity, thus providing a way to prepare NADH
solutions free of catalyst from NAD+.
and riboflavin into reduced riboflavin (Figure S10). No reduction
occurred in the absence of OC@Bpy-PMO.
Recyclability tests were carried out using 100 µM FAD as
2
the substrate, 5 mg OC@Bpy-PMO and 500 mM NaHCO at room
temperature. After 5 minutes reaction the catalyst was isolated
from the reaction mixture and re-suspended in a fresh solution of
formate and FAD for a second catalytic run. The procedure was
then repeated 20 times, without any loss of activity. This
establishes the remarkable stability and recyclability of the solid
catalytic material.
Figure 2. Reduction of NAD+ by OC@Bpy-PMO using formate as reductant.
Initial rate of NADH formation are reported as a function of: a) concentration of
+
NAD : 20, 50, 100, 250 and 500 µM with 500 mM formate and 5 mg OC@Bpy-
PMO; b) mass of OC@Bpy-PMO: 0.4, 1, 2, 5, 10 mg with 50 µM NAD+ and 500
mM formate; c) concentration of formate: 20, 50, 150, 250 and 500 mM with 50
+
µM NAD and 5 mg OC@Bpy-PMO. d) catalyst recycling: 5 mg OC@Bpy-PMO
was suspended in 2 mL of 20 mM Tris pH 7.5, 20 mM NaCl containing 500 µM
NAD+ and 500 mM formate at room temperature for 1h; after each cycle NADH
was measured and the powder isolated and reused in the following cycle carried
out with a fresh assay mixture.
Reduction of FAD
Figure 3. Reduction of FAD by OC@Bpy-PMO using formate as the reductant.
2
Initial rate of FADH formation is reported as a function of: a) concentration of
The same study was carried out now using FAD, flavin
adenine dinucleotide, as a substrate, however under anaerobic
FAD: 20, 50, 100 and 250 µM; b) mass of OC@Bpy-PMO: 0.2, 0.5, 1, 2 mg; c)
concentration of formate: 20, 50, 150, 250 and 500 mM. d) Catalyst recycling: 5
This article is protected by copyright. All rights reserved.

ChemCatChem
10.1002/cctc.201901726
FULL PAPER
mg OC@Bpy-PMO was suspended in 2 mL of 20 mM Tris pH 7.5, 20 mM NaCl
containing 500 µM NAD+ and 500 mM formate at room temperature for 5 min;
after each cycle FAD was measured and the powder isolated and reused in the
following cycle carried out with a fresh assay mixture.
trihydroxyphenylacetate THPA (Figure 4b). We verified that only
tiny amounts of Rh (0.2% of total Rh determined by elemental
analysis) were recovered in solution after catalysis and that the
activity was exclusively due to the solid OC@Bpy-PMO material.
Indeed the hydroxylation reaction was fully stopped after 15 min
when the solid was removed from the assay mixture by filtration
Application to NADH- and FAD-dependent biocatalytic
aromatic hydroxylation
(
Figure S11).
Unfortunately, reuse of the catalyst, after isolation from the
We recently reported an enzymatic aromatic hydroxylation
system converting a variety of phenols into catechols, based on
the two-component flavin-dependent 4-hydroxyphenylacetate
monooxygenase from Escherichia coli [8]. This system consists of
two enzymes: (i) a flavin reductase, named Fre, which catalyzes
first catalytic assay mixture, for further cycles appeared ineffective
(Figure S12). We named this spent catalyst OC@Bpy-PMO* in
the following. While still incompletely understood, the inactivation
occurring during this first enzyme assay is related, at least partly,
to the loss of catalytic activity of OC@Bpy-PMO* specifically for
FAD reduction (Figure S13). However, unexpectedly, OC@Bpy-
the reduction of FAD into FADH
named HpaB, which uses O to convert the reduced flavin FADH
into the corresponding flavin hydroperoxide for subsequent
hydroxylation of hydroxyphenylacetate (HPA) into
2
by NADH; (ii) an hydroxylase,
2
2
+
PMO* was found to be still fully active for NAD reduction (Figure
S14) and thus could be used in a second HPA hydroxylation
enzymatic cycle, however using Fre and NAD+ to allow FAD
reduction (Figure 5). Following this procedure, interestingly, one
could use the same catalyst for 4 catalytic cycles of complete HPA
hydroxylation without loss of activity, after which the solid catalyst
became less active; however, after 6 cycles still about 50% of the
activity was retained (Figure 5). No enzyme reaction occurred in
the absence of OC@Bpy-PMO.
dihydroxyphenylacetate (DHPA) (scheme 2). Because the system
consumes stoichiometric amounts of NADH, an expensive
2
reagent, we developed a simple assay in which FADH was
continuously regenerated though reduction of FAD by formate
catalyzed by the homogeneous organometallic complex
∗
2+
{
2
[Cp Rh(bpy)(H O)] } complex (OC), thus excluding NADH and
Fre. However the OC cannot be recovered and recycled easily.
This enzyme system provides an excellent model for going a step
further using OC@Bpy-PMO as a solid catalyst for FADH
regeneration.
2
Figure 4. Time dependent enzymatic conversion of HPA into DHPA by
HpaB/OC@Bpy-PMO. Conditions: 1 mM HPA, 10 µM HpaB, 50 µM FAD, 5 mg
OC@Bpy-PMO and 500 mM formate in 500 µL of 20 mM Tris pH 7.5, 20 mM
NaCl at 35 °C. a) HPA (circles) and DHPA (squares) quantities, determined after
separation on HPLC, as a function of time, from 50 µL of assay mixture. b)
Chromatograms showing HPA (peak at 11 min) into DHPA (peak at 7 min) and
then into THPA (peak at 3 min), monitoring absorbance wavelength at 225 nm.
Scheme 2. Hydroxylation reaction realized by enzymatic system HpaB/Fre, in
consuming NADH to convert HPA into DHPA.
Figure 4a shows that indeed OC@Bpy-PMO (5 mg) can be
2
used as an effective FADH -regeneration catalyst in combination
with the HpaB enzyme, FAD and formate to fully hydroxylate 1
mM HPA into DHPA, within 30 min reaction (TON = 0.33). Further
incubation
led
to
the
oxidation
of
DHPA
into
This article is protected by copyright. All rights reserved.

ChemCatChem
10.1002/cctc.201901726
FULL PAPER
monooxygenase, catalyzing the oxidation of phenols into
+
catechols, via its ability to generate NADH from NAD . Also within
this highly complex system, OC@Bpy-PMO could be cycled
several times with limited loss of activity. This is the first catalyst
displaying this property. While remarkably stable and reusable,
OC@Bpy-PMO nevertheless displays limited activity. There is
thus still a need to find new and efficient solid catalysts for the
reduction of such important biological cofactors as NAD+ and
flavins.
Figure 5. Catalyst recycling for HPA (circles) hydroxylation into DHPA (squares).
The first cycle was run using 1 mM HPA, 10 µM HpaB, 50 µM FAD, 5 mg
OC@Bpy-PMO and 500 mM formate in 500 µL solution of 20 mM Tris pH 7.5,
Experimental Section
Materials
20 mM NaCl at 35 °C. The following cycles were run using 1 mM HPA, 10 µM
+
HpaB, 2 µM FAD, 0.5 µM Fre, 500 µM NAD , 5 mg OC@Bpy-PMO and 500 mM
formate in 500 µL solution of 20 mM Tris pH 7.5, 20 mM NaCl at 35 °C. At the
end of each assay cycle, the solid was isolated and re-suspended in a fresh
reaction mixture for the next cycle. HPA (circles) and DHPA (squares) quantities
from 50 µL of assay mixture were measured after separation on HPLC.
+
+
NADH, NADPH, NAD , NADP , FAD, FMN and Riboflavin were
purchased from Sigma-Aldrich, Bpy-PMO from TCI Europe N.V.,
[
2 2
Cp*RhCl ] from Sigma-Aldrich. HpaB from Escherichia coli was purified
according to a recently reported procedure[8]
.
Synthesis of Rh@Bpy-PMO catalyst
Conclusions
50 mg Bpy-PMO is suspended in 12 mL CH
3
OH, and then 5.9 mg of
The development of NAD(P)H-dependent enzymes,
[
Cp*RhCl complex is added (with the Bipyridine : Rh molar ratio 1 : 0.1).
2 2
]
reductases,
dehydrogenases
or
monooxygenases,
as
The mixture was stirred for 4 hours at room temperature. The solution was
then centrifuged at 10000 rpm for 5 min to afford the yellow solid OC@Bpy-
biocatalysts has been limited by the cost of NAD(P)H which, in all
these reactions, is consumed stoichiometrically with respect to
product formation. While NAD(P)H-regenerating systems,
PMO after separation from the CH
3
OH solution. The solid was then
washed with CH OH until adsorbed Rh dimer stops diffusing out from the
3
solid (as monitored by UV-Vis spectroscopy). The quantity of bound
Rhodium was determined by elemental analysis (Table S1). We
synthesized different solids using different initial bpy:Rh ratios (molar ratio,
+
converting NAD(P) back to NAD(P)H, provide a partial solution
to that problem, up to now all of them were homogeneous
systems (enzymes or molecular catalysts) which are not
recoverable and recyclable.
1:1.1, 1:0.2, 1:0.1 and 1:0.01).
Characterization of Bpy-PMO and of OC@Bpy-PMO
Here, for the first time, we describe a solid material based
on a molecular Rh catalyst heterogenized on bypiridine-periodic
mesoporous organosilica, OC@Bpy-PMO, which displays
catalytic activity for the reduction of pyridine nucleotides, NAD(P)+,
and oxidized flavins, FAD and FMN, by formate. This material is
also a durable catalyst as it can be easily recovered and recycled,
allowing a large number of cycles before complete inactivation.
Solid state 1H, 29Si and 13C NMR were performed on Bruker Advance III
4
00 or 700 spectrometers with triple resonance 4 mm CP MAS probe, with
13
1
29
rotation 14 kHz ( C, H) and 10 kHz ( Si). Nitrogen gas adsorption and
desorption isotherms were recorded using Micrometrics ASAP 2010. XRD
analysis was carried out using Panalytical X’pert pro diffractometer
equipped with a Co anode (λKα = 1.79031 Å). TEM analysis was
performed on JOEL JEM 2010 UHR operating at 200 kV. Powder XRD
patterns were recorded on a Bruker D8 advance diffractometer operating
at the Cu−Kα wavelength.
+
For example after 8 cycles of 0.5 mM NAD reduction using 5 mg
+
of the same batch of catalyst, 80% of the activity was retained
while no catalyst deactivation could be observed after 20 cycles
of 0.1 mM FAD reduction. Finally, this heterogeneous catalyst can
be exploited in a biocatalytic reaction requiring NADH. We indeed
showed that the OC@Bpy-PMO/formate system can efficiently
assist a complex NADH-dependent two-component aromatic
Tests using OC@Bpy-PMO for NAD reduction by formate
The solid OC@Bpy-PMO is used as a catalyst and formate was used
+
as a reductant to reduce NAD . OC@Bpy-PMO (1:0.1 bpy:Rh ratio) was
+
used in these tests. The concentration of NAD , the amount of catalyst and
the concentration of formate were modulated .The formation of NADH was
monitored by UV-vis spectroscopy at 340 nm.
This article is protected by copyright. All rights reserved.

ChemCatChem
10.1002/cctc.201901726
FULL PAPER
As for the recycling experiments, the reaction was carried out in 2 or
the absorption at 225 nm. The conversion from peak area to quantity was
based on calibration curves established with standard samples.
4
mL solution (20 mM Tris pH 7.5, 20 mM NaCl) which contained 500 µM
+
NAD , 5 or 40 mg OC@Bpy-PMO and 500 mM formate at room
temperature for 1 h. Recycling tests were performed by filtration of
OC@Bpy-PMO using a micro-filter funnel under negative pressure from
the reaction mixture, washing it with 1 mL buffer solution before adding the
resulting solid to a fresh reaction mixture to start a new 1 h cycle.
+
Enzymatic tests using formate and OC@Bpy-PMO as NAD reductant
In the second system, the Flavin is reduced by NADH thanks to a
Flavin reductase, Fre from E. coli. Here NADH is constantly regenerated
from NAD+ by reduction by formate catalyzed by the heterogeneous
catalyst Rh@Bpy-PMO.
+
For continuous reduction of NAD , a Restek empty HPLC column
(2.1×50 mm) was filled with a mixture of 15 mg OC@Bpy-PMO powder
We established a protocol implying a pretreatment step in which 5 mg
OC@Bpy-PMO was first incubated during 1 h with 1 mM HPA, 10 µM
HpaB, 50 µM FAD and 500 mM formate in 500 µL solution (20 mM Tris pH
and 60 mg of silica, and then connected to a HPLC system. The two mobile
phases were A: 20 mM Tris buffer, pH 7.5, 20 mM NaCl and B: solution
+
containing 5 mM NAD and 500 mM formate in 20 mM tris buffer, pH 7.5,
7.5, 20 mM NaCl) at 35 °C. In this first step, HPA was fully converted into
20 mM NaCl. Conditions: flow rate: 0.1 mL/min; gradient: mobile phase A
DHPA. The solid was then recovered by filtration using a micro-filter funnel
and used in the following cycles. From then the reaction was realized in a
500 µL solution (20 mM Tris pH 7.5, 20 mM NaCl) which contains 1 mM
for 1 min then switch to B for 21 min and finally switch back to A.
Tests using OC@Bpy-PMO for FAD reduction by formate
+
HPA, 1 µM HpaB, 2 µM FAD, 0.5 µM Fre, 500 µM NAD , 5 mg Rh@Bpy-
The solid OC@Bpy-PMO was used as a catalyst and formate was
used as a reductant to reduce FAD. OC@Bpy-PMO (1:0.1 bpy:Rh ratio)
was used in these tests. The concentration of FAD, the amount of catalyst
and the concentration of formate were modulated. The formation of FAD
was monitored in a glove box, because of the air sensitivity of the reduced
flavin, by UV-vis spectroscopy following the decay of the band at 450 nm,
characteristic of the oxidized FAD.
PMO and 500 mM formate at 35 °C. The reaction was initiated by adding
the mixture of HpaB and Fre and quenched by filtration and addition of 1%
(v/v) of TCA. The solution samples at the end of each 1 h cycle were
analyzed by HPLC using the methods described above.
Acknowledgements
We thank Xia Wang for her contribution to catalyst synthesis and
for helpful discussions. We thank Cristina Coelho for NMR
analysis and Patricia Beaunier for TEM analysis.
As for the recycling experiments, the reaction was carried out in a 2
mL solution (20 mM Tris pH 7.5, 20 mM NaCl) which contained 100 µM
FAD, 5 mg OC@Bpy-PMO and 500 mM formate at room temperature for
5
min. Recycling tests were performed by filtration of OC@Bpy-PMO using
Keywords: recyclable catalyst • NAD(P)H • flavins •
hydroxylation• cofactor regeneration
a micro-filter funnel under negative pressure from the reaction mixture,
washing it with 1 mL buffer solution before adding the resulting solid to a
fresh reaction mixture to start a new cycle
[
[
1]
2]
A. Schmid, J. S. Dordick, B. Hauer, A. Kiener, M. Wubbolts, B. Witholt,
Nature 2001, 409, 258–268.
Enzymatic tests using formate and OC@Bpy-PMO as flavin reductant
V. Nivière, F. Fieschi, J.-L. Décout, M. Fontecave, J. Biol. Chem. 1999,
2
74, 18252–18260.
The reaction was performed in 500 µL solution (20 mM Tris pH 7.5, 20
mM NaCl) which contains 1 mM HPA, 10 µM HpaB, 50 µM FAD, 5 mg
OC@Bpy-PMO and 500 mM formate at room temperature or 35 °C. The
reaction was started by adding HpaB and quenched by filtration and then
addition of 1% (v/v) TCA. The recycling of OC@Bpy-PMO was carried out
by filtration using a micro-filter funnel followed by a washing step with 500
µL buffer and then the solid was added to a fresh solution to start a new
cycle. The samples were centrifuged at 13000 rpm for 5 min. Before
injection into the HPLC, the pH of the sample was adjusted to ca. 4 and 50
µL aliquots were injected in the HPLC by an auto-sampler. An Agilent 1200
Infinity HPLC system was used to analyze the samples. A column Agilent
Eclipse XDB-C18, 5 µm, 4.6×150 mm was used, the two mobile phases
[
[
[
3]
4]
5]
W. A. Van Der Donk, H. Zhao, Curr. Opin. Biotech. 2003, 14, 421–426.
U. Kölle, M. Grützel, Angew. Chem. Int. Ed. 1987, 26, 567–570.
a) F. Hollmann, B. Witholt, A. Schmid, J. Mol. Catal. B: Enzym. 2003,
19–20, 167–176; b) F. Hollmann, I. W. C. E. Arends, K. Buehler,
ChemCatChem 2010, 2, 762–782; c) T. Quinto, V. Köhler, T. R. Ward,
Top Catal 2014, 57, 321–331; d) A. K. Mengele, S. Rau, Inorganics
2
017, 5.
L. Zhang, N. Vilà, G. Kohring, A. Walcarius, M. Etienne, ACS Catal.
017, 7, 4386–4394.
[
6]
7]
2
[
F. Hollmann, P. Lin, B. Witholt, A. Schmid, J. Am. Chem. Soc. 2003,
125, 8209–8217.
[8]
Y. Deng, B. Faivre, O. Back, M. Lombard, L. Pecqueur, M. Fontecave,
ChemBioChem 2019, DOI 10.1002/cbic.201900277.
2 4
being A: 10 mM KH PO pH 2.6 and B: acetonitrile, with a flow rate of 1
[9]
a) Y. Maegawa, S. Inagaki, Dalton Trans. 2015, 44, 13007–13016; b)
N. Ishito, H. Kobayashi, K. Nakajima, Y. Maegawa, S. Inagaki, K. Hara,
mL/min. The typical gradient to analyze HPA and DHPA was 5-15% B
within 15 min. The reaction substrates and products were monitored from
This article is protected by copyright. All rights reserved.

ChemCatChem
10.1002/cctc.201901726
FULL PAPER
A. Fukuoka, Chem. Eur. J. 2015, 21, 15564–15569; c) X. Liu, Y.
Maegawa, Y. Goto, K. Hara, S. Inagaki, Angew. Chem. Int. Ed. 2016,
5, 7943–7947; d) W. R. Grüning, A. J. Rossini, A. Zagdoun, D. Gajan,
A. Lesage, L. Emsley, C. Copéret, Phys. Chem. Chem. Phys. 2013, 15,
3270–13274; e) W. R. Grüning, G. Siddiqi, O. V. Safonova, C.
5
1
Copéret, Adv. Synth. Catal. 2014, 356, 673–679.
[
[
10]
11]
X. Wang, I. Thiel, A. Fedorov, C. Copéret, V. Mougel, M. Fontecave,
Chem. Sci. 2017, 8, 8204–8213.
a) K. Matsui, Y. Maegawa, M. Waki, S. Inagaki, Y. Yamamoto, Catal.
Sci. Technol. 2018, 8, 534–539; b) T. Himiyama, M. Waki, Y. Maegawa,
S. Inagaki, Angew. Chem. Int. Ed. 2019, 58, 9150–9154.
This article is protected by copyright. All rights reserved.

ChemCatChem
10.1002/cctc.201901726
FULL PAPER
Table of contents
A Rh-based complex immobilized on bypiridine-periodic mesoporous organosilica(OC@Bpy-PMO) is a recyclable catalyst for flavin
+
and NAD(P) reduction by formate. The heterogeneous catalyst can assist a complex NADH-, FAD- and O
2
-dependent monooxygenase
enzyme system.
This article is protected by copyright. All rights reserved.