Enzyme-catalysed enantioselective oxidation of alcohols by air exploiting fast electrochemical nicotinamide cycling in electrode nanopores

Article abstract of DOI:10.1039/c9gc01534e

Enantioselective conversion of alcohols to ketones using air as the oxidant is achieved with high rates and efficiency using an indium tin oxide (ITO) electrode in which an alcohol dehydrogenase and a photosynthetic NADPH recycling enzyme are confined within nanopores. The massive catalytic enhancement arising from nanoconfinement is exploited in an air-driven electrochemical cell, which requires no complicating control features yet allows continuous monitoring of the reaction via the current that flows between anode (ITO: Organic chemistry) and cathode (Pt: O₂ from air).

Full text of DOI:10.1039/c9gc01534e

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Enzyme-catalysed enantioselective oxidation of
alcohols by air exploiting fast electrochemical
nicotinamide cycling in electrode nanopores†
Cite this: DOI: 10.1039/c9gc01534e
Received 8th May 2019,
Accepted 15th July 2019
a
Lei Wan, a Rachel S. Heath,b Bhavin Siritanaratkul, a Clare F. Megarity,
Adam J. Sills,a Matthew P. Thompson,‡b Nicholas J. Turner *b and
DOI: 10.1039/c9gc01534e
a
Fraser A. Armstrong
*
rsc.li/greenchem
Enantioselective conversion of alcohols to ketones using air as the small reactants and products enter and leave the pores.
oxidant is achieved with high rates and efficiency using an indium Importantly, the whole process can be controlled electrochemi-
tin oxide (ITO) electrode in which an alcohol dehydrogenase and a cally and monitored in real time through the electrical current
photosynthetic NADPH recycling enzyme are confined within nano- that flows via FNR through the rapidly-recycling NADP(H).16–18
pores. The massive catalytic enhancement arising from nanoconfi- An attractive option for oxidation reactions is to connect (FNR
nement is exploited in an air-driven electrochemical cell, which + E2)@ITO/support to an O2-reducing Pt cathode that is sup-
requires no complicating control features yet allows continuous plied with a stream of air, thus rendering aerial oxidation con-
monitoring of the reaction via the current that flows between anode tinuously quantifiable. Here, we describe a simple, eco-friendly
(ITO: organic chemistry) and cathode (Pt: O2 from air).
system for the enantioselective oxidation of an alcohol to a
ketone. The current ‘green’ options for performing such reac-
tions on an industrial scale are either chemical, using hydro-
gen peroxide, O2 or N-oxyl reagents, along with a transition-
metal catalyst,19–21 or enzymatic, using O2 with laccases or
alcohol oxidases.22,23 The nano-confined enzyme-electro-
chemical strategy could compete favourably with those options
once details, including scalability, have been worked out.
Fig. 1 shows the two-compartment cell in schematic form
along with a cartoon depicting the nanoconfinement effect:
actual assembly of the cell is shown in ESI, Fig. S1.† The
(FNR + E2)@ITO/Ti electrode is located in the main compart-
ment. During operation, a gentle stream of compressed air is
introduced to the smaller compartment, separated from the
main compartment by a Nafion 115 proton-exchange mem-
brane (PEM), which houses a Pt-electrode formed from plati-
nised carbon paper (PCP: details given in ESI†). The reaction
rate is monitored continuously by recording the current that
flows, the accumulated charge reporting on the progress at any
point in time.
The scope of this system is demonstrated using, as E2, a
recombinant alcohol dehydrogenase from Thermoanaerobacter
ethanolicus 39E (TeSADH W110A), which has already been
applied in hydrogen-borrowing cascades for synthesising
amines, starting from either alcohols or alkanes.4,24 The
enzyme favours oxidation of the (S)-enantiomer of secondary
alcohols;25–27 some of which are highly valued as precursors or
building blocks for pharmaceuticals.25,28 In a TeSADH-W110A-
catalysed oxidation of a racemic mixture of chiral alcohol into
the corresponding ketone, the (S)-enantiomer is transformed
As Biology’s hydride carriers, nicotinamide cofactors NAD(P)(H)
are involved in numerous enzyme-catalysed processes: an
increasing number of these are being exploited by the pharma-
ceutical industry,1 and a wide range of options have emerged
to deal with the challenges of rapid cofactor recycling.2–15
A
potentially powerful new approach uses principles that govern
rates and efficiency in living cells and exploits the observation
that the photosynthetic enzyme ferredoxin-NADP+-reductase
(FNR) and a NADP(H)-linked oxidoreductase (E2) are taken up
collectively into the nanopores of an indium tin oxide (ITO)
layer that is produced by depositing commercially available
nanoparticles (diameter ca. 50 nm) onto a suitable conductive
support, typically titanium foil or graphite.16 In the resulting
electrode, abbreviated as (FNR + E2)@ITO/support, each of the
two trapped enzyme partners are concentrated at a local level
and the inter-site diffusion distance of NADP(H) is decreased
to a very small length scale (e.g. <0.1 μm). The nanoconfine-
ment of two interdependent catalysts massively enhances the
rate and efficiency of biocatalytic transformations occurring as
aDepartment of Chemistry, University of Oxford, South Parks Road, Oxford,
OX1 3QR, UK. E-mail: fraser.armstrong@chem.ox.ac.uk
bSchool of Chemistry, University of Manchester, Manchester Institute of
Biotechnology, 131 Princess Street, Manchester M1 7DN, UK.
E-mail: nicholas.turner@manchester.ac.uk
†Electronic supplementary information (ESI) available. See DOI: 10.1039/
c9gc01534e
‡Current address: EnginZyme AB, Teknikringen 38 A, 114 28 Stockholm,
Sweden.
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Fig. 2 Left. Cyclic voltammograms (CVs) for NADP+/NADPH inter-
conversion coupled to the ADH-catalysed oxidation of 4-phenyl-2-
butanol recorded at an FNR@ITO/ITO-glass electrode (1.0 cm2, [FNR] =
1.25 mM, 3 μL, 30 seconds incubation); cycle numbers are indicated, the
‘0’ cycle being recorded before introducing alcohol dehydrogenase.
Right. A cyclic voltammogram for O2 reduction at a platinised carbon
paper (PCP) electrode (2.4 cm2). The CVs were performed at scan rates
of 1 mV s−1 for the FNR@ITO/ITO electrode and 5 mV s−1 for the PCP
electrode. Experimental conditions: Cell volume = 8 ml, 50 mM TAPS
buffer (pH 9.0), 25 °C, [ADH] = 0.13 μM, [NADPH] = 50 μM, [(rac)-4-
phenyl-2-butanol ((rac)-4P2B)] = 10 mM. SHE = Standard Hydrogen
Electrode, RHE = Reversible Hydrogen Electrode (corrected for pH). The
Fig. 1 Sketches showing the setup of the two-compartment air-driven
enzyme electrochemical cell and the basis for the nanoconfinement
effect underpinning the action of the (FNR + E2)@ITO/support electrode
for organic synthesis. A Nafion 115 proton exchange membrane (PEM) sep-
arates the main compartment (5 mL) from the cathode compartment
(1 mL). The current flowing between the anode (ITO: organic chemistry)
and cathode (Pt: O2 from air) yields the reaction rate.
air flow rate for PCP-catalysed O2 reduction was set at 30 scc min−1
.
more rapidly, thereby concentrating the (R)-enantiomer, i.e. a
kinetic resolution.28 Conversely, the reverse reaction favours
production of the (S)-alcohol.
Two ways of forming the (FNR + E2)@ITO/support electrode various times after introducing ADH. The initial cyclic voltam-
were used for the experiments. In the first case, FNR is taken mogram (scan 0, blue trace) corresponds to the quasi-revers-
up alone by dropcasting a concentrated solution onto the elec- ible electrochemistry of NADPH. Upon introducing ADH to the
trode surface and incubating for approximately 30 s before cell solution (final concentration after initial stirring
=
rinsing with purified water (Milli-Q) or buffer and placing in 0.13 μM) the oxidation current increases steadily as ADH
the electrochemical cell. A small aliquot of a stock solution of enters the ITO nanopores to form catalytically proficient nano-
TeSADH W110A (hereafter referred to as ADH) is then injected zones with FNR.16 The right side of Fig. 2 shows catalytic
into the cell solution containing all other components: this reduction (negative current) of O2 in air, achieved at an electro-
initiates electrocatalysis, which develops with time as ADH chemically deposited PCP cathode. A useful driving potential
binds and its ratio relative to FNR in the ITO pores increases. of 0.7–0.8 V is clearly available for the aerial oxidation of
In the second case, both enzymes are dropcast as a concen- alcohol (ΔE in Fig. 2).
trated mixture (various FNR/ADH ratios being used) onto the
Next, experiments were performed under hydrodynamic
electrode surface and incubated for approximately 3 minutes: control to compare the system’s overall activity and response
the electrode is then rinsed repeatedly with purified water towards product inhibition at different pH values. In each
(Milli-Q) or buffer, before being used in experiments. The case, a (FNR + ADH)@ITO/PGE rotating disc electrode was pre-
second method allows rapid formation of a (FNR + ADH) pared by dropcasting a concentrated mixture of FNR (705 μM)
@ITO/support electrode free of unbound enzyme and control and ADH (42 μM) onto the ITO layer for approximately 3 min,
over the FNR/ADH ratio to which ITO was originally exposed. taking care to ensure that the film did not dry. The electrode
In all cases the loading of FNR could be determined from the was then rinsed thoroughly with purified water (Milli-Q) before
amplitude of non-turnover signals obtained in the absence of connecting and placing in the electrochemical cell. The upper
NADP(H).17
cyclic voltammogram in Fig. 3A shows the oxidation of 5 mM
All experimental details are provided in ESI.† Fig. 2 shows (S)-4P2B at pH 9.0 carried out with 50 μM NADP(H) present in
the electrochemical characteristics of the anode and cathode solution (a 1 : 1 mixture of NADP+:NADPH was used in this
of the enzyme electrochemical cell used for air-driven organic case). Upon adding the product (4-phenyl-2-butanone) to a
synthesis. The left side shows cyclic voltammograms (CVs) final concentration of 5 mM, the oxidation current dropped
obtained with
a stationary FNR@ITO/ITO-glass electrode sharply and remained suppressed at a level of approximately
([FNR] = 1.25 mM, 3 μL, 30 seconds incubation) in an aqueous 50% of that recorded before its introduction: continuing the
cell solution containing NADPH (50 μM) and a racemic cycle, a large current due to ketone reduction was observed.
mixture (10 mM) of 4-phenyl-2-butanol (4P2B), recorded at Fig. 3B shows the same experiment carried out at pH 8.0, the
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Fig. 4 Timecourses (chronoamperograms, CA) showing oxidations of
(rac)-, (S)- and (R)-4P2B in the air-driven electrochemical cell.
Experimental conditions: Buffer = 0.2 M TAPS, pH = 9.0, 25 °C, [ADH]
(after introduction to solution) = 0.44 μM, cell volume, 1 mL for the
PCP-containing compartment and 5 mL for the main compartment,
active surface of (FNR + ADH)@ITO/Ti electrode = 7.2 cm2 ([FNR] =
425 μM, 24 μL, 30 seconds incubation), air flow rate for the PCP-con-
taining compartment = 2.6 scc min−1, [(rac)-4P2B] = 11.6 mM, [(R)-4P2B] =
9.9 mM, [(S)-4P2B] = 10.5 mM. The concentrations of NADP+ and types
of enantiomers used for each plot are shown. Detailed results for these
four experiments are shown in entries A, B, D, and E of Table 1.
obtain products for analysis. Additional insight was also
obtained by dropcasting FNR first then allowing ADH to
enter the pores from dilute solution. Fig. 4 shows oxidations of
(rac)-, (S)- and (R)-4P2B, monitored as a function of time after
Fig. 3 Cyclic voltammograms to measure the electrochemical oxi-
dation of 5 mM (S)-4P2B achieved at a (FNR + ADH)@ITO/graphite rotating
disc electrode. The experiments compare the reversibility, catalytic bias
and product (ketone) inhibition obtained at pH 9 (A) and pH 8 (B). An
aliquot of 4-phenyl-2-butanone was injected (giving 5 mM final concen-
tration) in each case during the scan (shown in red and enlarged in the
inset). The subsequent cycle following ketone injection is shown in blue.
Experimental conditions: 50 mM TAPS + 50 mM MOPS + 50 mM CHES
buffer (pH 9.0), [NADP+] = 25 μM, [NADPH] = 25 μM, 20 °C, scan rate =
3 mV s−1, electrode rotation speed = 500 rpm.
introducing ADH to the stirred electrochemical cell,
a
FNR@ITO/Ti electrode having been pre-prepared by dropcast-
ing an aliquot of FNR solution ([FNR] = 425 μM, 24 μL,
30 seconds incubation) onto a freshly prepared ITO layer. Each
experiment was initiated by injecting a 25 μL aliquot of ADH
into the main compartment to give a final concentration of
0.44 μM,29 while the cell was previously charged with the reac-
tant and NADP+ at the levels indicated. The alcohol concen-
tration in these experiments was limited by its solubility in
water (<20 mM). The traces contain four pieces of information:
upper voltammogram again being recorded before adding (1) the current at any particular time is proportional to an
ketone. Comparing the two sets of conditions, it is clear how overall empirical catalytic turnover rate k′ (mole per s per unit
the catalytic bias shifts in favour of alcohol oxidation as the area of electrode) via the relationship i = 2k′FA, where F is
pH is increased, and the effect of ketone as a product inhibitor Faraday’s constant and A is the surface area of the electrode.
is particularly marked at pH 8. The enantioselectivity for (2) The slope of the initial increase in current gives the rate at
(S)- vs. (R)-4P2B was also measured under ADH-limited con- which optimised catalytic zones comprised of nanoconfined
ditions using an electrode prepared by dropcasting a concen- FNR and ADH become assembled in the ITO layer. (3) The
trated FNR-rich ([FNR] : [ADH] = 22 : 1) mixture: a >95% selecti- total area (integral) under each trace gives the charge passed,
vity for the (S)-isomer was confirmed by comparing the S vs. R which is related to the total chemical yield via the equation
Ð
catalytic currents measured over the course of several hours.
idt ¼ 2F ꢀ total yield (segments of the integral may be
Results are shown in ESI (Fig. S2†). The enantiomeric ratio sampled to show the progress at any point in time). (4) The
E of TeSADH W110A, defined as (kScat/KmS )/(kcRat/KmR ), which current eventually decreases, which can be due to several
reflects the enzyme’s intrinsic kinetic enantioselectivity, was factors, i.e. the reaction approaches completion, becomes
determined independently by solution kinetics (Fig. S3†) using increasingly product-inhibited or there is a degradation in the
fixed concentrations of NADP+ and a known concentration of number or performance of active nanozones. Results of experi-
ADH. The E value was calculated to be 45.8.
ments performed over a range of reactants and NADP+ concen-
1
Air-driven alcohol electrooxidation runs were carried out at trations, as analysed by coulometry, H NMR and GC-FID are
pH 9.0 and 25 °C using a much larger electrode (7.2 cm2) to provided in Tables 1, S1 and Fig. S4–S12.† Runs A–C were
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Table 1 Compilation of data for the TeSADH-W110A-catalyzed oxidation of different enantiomers of 4-phenyl-2-butanol using an FNR@ITO/Ti
electrode in an air-driven electrochemical cell
Conversionb/%
Charge passed
TTNd (approx.)
Charge passed
Reactant
Reactant
amounta/mM
NADP+/μM
NMR
GC
eec (R)/%
NMR
GC
A
B
C
11.6
11.6
11.6
5
20
50
38.7
48.4
50.4
39.7
48.5
55.4
38.3
48.5
53.0
85 (R)
99 (R)
96 (R)
898
279
117
922
281
129
889
282
123
D
10.5
20
71.1
68.3
60.7
372
360
319
E
9.9
20
27.3
36.9
33.8
135
182
167
Experimental conditions: FNR@ITO/Ti (7.2 cm2), PCP electrode (2.4 cm2), temperature 25 °C, pH = 9.0, 0.2 M TAPS, [ADH] (after being intro-
duced to solution) = 0.44 μM, solution volume in main compartment = 5 ml, solution volume in PCP-containing compartment = 1 mL, gas flow
rate in PCP-containing compartment = 2.6 scc min−1
.
a Concentrations of reactants determined by 1H NMR. b Defined as [product]/[reactant] ×
CR ꢁ CS
100%. The product concentration has been normalised for volume change (details shown in Table S1†). c Defined as ¼ j
j ꢀ 100.
CR þ CS
d Defined as [product]/[NADP+].
carried out with a racemic mixture, while D and E were carried Significantly, when (R)-4P2B is the sole substrate, a much
out using S and R forms. longer time is taken to reach an optimum level, suggesting
Taking the experiment with ([NADP+] = 20 μM and [(rac)- that the much lower natural activity for this isomer – as
4P2B)] = 11.6 (mM) (red trace in Fig. (4) as an exemplary case, reflected in the enantiomeric ratio E determined from the
the oxidation current increases for approximately 40 minutes solution kinetics, can be compensated for as a greater number
after the injection of ADH, before peaking and eventually of ADH molecules become incorporated into catalytic nano-
decreasing to a low level after 15 h. The charge passed after zones in the ITO layer that already contain FNR. Obvious
15 hours was equivalent to 48.4% conversion, while product comparison can be made with the experiment described
analyses by 1H NMR and GC-FID each gave values of 48.5% above conducted using dropcasting with a fixed high FNR/
(Table 1). These values are encouraging considering that some ADH ratio (Fig. S2†) where a very high kinetic preference for
losses of reactant and product occur due to crossover (leakage S vs. R alcohol is maintained throughout.
into the Pt-electrode compartment) and volatility (the cell not
To elucidate further, the factors controlling the catalytic
being tightly sealed). Upon lowering the concentration of activity, experiments were also carried out to determine the
NADP+ to 5 μM, the experiment with (rac)-4P2B still resulted in response to specific interventions such as injections of further
a peak current of 0.2 mA, i.e. approximately 70% of the value reactant, product, cofactor and enzymes. A timecourse of the
obtained using 20 μM NADP+. As expected, the total turnover reaction monitored over 18 h is shown in Fig. 5, electrocataly-
number (TTN, defined as [ketone]/[NADP+], is highest for the sis at the FNR@ITO/Ti electrode having been initiated by intro-
lower NADP+ concentration, and must ultimately in these ducing 0.19 μM ADH to the cell solution. When the current
experiments be limited by the low amount of alcohol available had decreased to approximately 25% of its starting value after
for conversion. The (R) enantiomer is recovered at enantio- 6 h, a further amount of the reactant (rac)-4P2B was intro-
meric excess (ee) levels of 85, 99 and 96% respectively for the duced (the extra amounting to 2.5 mM in concentration),
runs with 5, 20 and 50 μM NADP+. The results are as expected resulting in an immediate increase in current. The next inter-
if S-4P2B is oxidised much more rapidly than R-4P2B, and the vention was the introduction of the ketone product (again,
analogous experiment carried out with the favoured enantio- giving a 2.5 mM final concentration increase) which caused
mer (S)-4P2B, alone (Fig. 4, blue trace), revealed that a higher the current to drop immediately by a third. Subsequent inter-
rate and greater yield are achieved. Unlike the solution assays ventions involved additions of TeSADH W110A, FNR, NADP+,
which involve known, fixed concentrations of ADH, the electro- and second injections of alcohol and ketone. Whereas
chemical system involves unknown active amounts of ADH in addition of more alcohol dehydrogenase to augment the orig-
the electrode pores. It is therefore not possible to calculate the inal 0.19 μM level resulted in an increase in current, addition
kinetic constants needed to give a meaningful enantiomeric of FNR caused no change and the injection of NADP+ to give a
ratio. Accordingly, the experiment carried out with pure total concentration of 100 μM, doubling the original amount,
(R)-4P2B showed that significant conversion still occurred, increased the current by less than 10%. Subsequent additions
albeit with
a
lower catalytic rate and decreased yield. of ketone and alcohol gave a decrease and increase in current,
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FNR being easy to produce and stable. The enzyme-ITO layer
(<3 μm) is easily removed and replaced between runs, while
the titanium and platinum electrodes are re-used continually.
The system uses a low concentration of NADP+ (<20 µM, vs.
0.5 mM for typical analytical scale biotransformations). The
results suggest that enantioselectivity will be optimised by
ensuring that the FNR/ADH ratio is high. Obviously, the cell is
easily adapted to driving reversible reactions in either direction,
as all that is required to reduce ketones is to replace air by H2.18
Both TTN and yields are expected to increase greatly by imple-
menting a two-phase system in which a much higher amount of
alcohol is presented as an organic phase into which the ketone
can also partition, thus also limiting product inhibition.
Real-time monitoring (via the current response) allows con-
tinuous probing of the system with additions of reactants, pro-
ducts or enzymes to determine their effects on the reaction.
One could envisage changing other factors such as tempera-
ture and pH during the timecourse of the reaction and moni-
toring their effects. Overall therefore, a single timecourse/
experiment with interventions could quickly yield all the
necessary information regarding the conditions required to
optimise a particular reaction, and this capability could be
implemented in batch processes.
Fig. 5 Time course showing how the system responds to different
interventions. Buffer = 0.2 M TAPS, pH = 9.0, 25 °C, initial [(rac)-4P2B] =
2.5 mM in the main compartment, initial [NADP+] = 50 μM, [ADH] (after
introduction to solution) = 0.19 μM, air flow rate = 10.0 scc min−1, cell
solution = 9 mL for main compartment and 1.2 mL for PCP-containing
compartment, active surface area of (FNR + ADH)@ITO/Ti anode =
3.6 cm2 ([FNR] = 425 μM, 12 μL, 30 seconds incubation), active surface
area of PCP cathode = 2.4 cm2. Argon was bubbled through the main
compartment throughout. The inset shows an enlarged view of the time
period during which several interventions were made. Inside this figure,
each injection of (rac)-4P2B and 4-phenyl-2-butanone raises the cell
concentration by 2.5 mM, injection of [NADP+] raises its concentration
by 50 μM, the concentration of ADH is raised by 0.1 μM, the concen-
tration of FNR is raised by 0.57 μM.
Conflicts of interest
respectively, proportional to those observed earlier. The results
show that the decrease in activity arises mainly from product
inhibition, which greatly exceeds losses due to deterioration of
the (FNR + ADH)@ITO/Ti electrode system or any instability of
NADP+. The fact that the ultimate position of equilibrium with
the air-driven experiments lies very much in favour of the
ketone product rules out the possibility that the reaction is
approaching its thermodynamic end point.
There are no conflicts to declare.
Acknowledgements
This project was supported in Oxford by the Biological and
Biotechnological Research Council (Follow-on Fund, Grant BB/
P023797/1) and SCG Chemicals (Thailand), and in Manchester
by an ERC Advanced Grant awarded to N. J. T (Grant number
742987) and the CoEBio3 Affiliates Program. Lei Wan is grate-
ful to the China Scholarship Council for funding.
Conclusions
The experiments and results we have described suggest a new
and simple approach to conduct enzyme-catalysed selective
oxidations of organic compounds – one that provides real-time
control and monitoring as well as significant scope for scaling
up to meet levels for special chemicals. The high catalytic rate
due to the intense local concentration of two NADP(H) enzyme
partners within electrode nanopores is a key factor. Although
the catalysis is electrochemical in kind, the cell requires only
anode and cathode, and reactor design is simplified. The
reduction of O2 in air to H2O at a platinum cathode is clean
and well established, and provides a large, steady overpotential
to drive the efficient, near-reversible organic oxidation at the
anode. No non-aqueous solvents are needed apart from the
acetone that is used in rapid electrophoretic preparation of the
ITO layer. Scalability should be feasible as ITO is inexpensive
and non-toxic and nanoparticle powders are already produced
commercially for manufacturing electronic displays. The
enzymes are required in only small amounts, recombinant
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