Hybrid enzymatic and organic electrocatalytic cascade for the complete oxidation of glycerol

Article abstract of DOI:10.1021/ja5098379

We demonstrate the complete electrochemical oxidation of the biofuel glycerol to CO₂ using a hybrid enzymatic and small-molecule catalytic system. Combining an enzyme, oxalate oxidase, and an organic oxidation catalyst, 4-amino-TEMPO, we are able to electrochemically oxidize glycerol at a carbon electrode, while collecting up to as many as 16 electrons per molecule of fuel. Additionally, we investigate the anomalous electrocatalytic properties that allow 4-amino-TEMPO to be active under the acidic conditions that are required for oxalate oxidase to function.

Full text of DOI:10.1021/ja5098379

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Hybrid Enzymatic and Organic Electrocatalytic Cascade for the
Complete Oxidation of Glycerol
David P. Hickey, Matthew S. McCammant, Fabien Giroud, Matthew S. Sigman,* and Shelley D. Minteer*
Department of Chemistry, University of Utah, 315 South 1400 East, Salt Lake City, Utah 84112, United States
S
* Supporting Information
with both the conditions of enzymatic oxidation as well as the
ABSTRACT: We demonstrate the complete electro-
chemical oxidation of the biofuel glycerol to CO2 using a
hybrid enzymatic and small-molecule catalytic system.
Combining an enzyme, oxalate oxidase, and an organic
oxidation catalyst, 4-amino-TEMPO, we are able to
electrochemically oxidize glycerol at a carbon electrode,
while collecting up to as many as 16 electrons per
molecule of fuel. Additionally, we investigate the
anomalous electrocatalytic properties that allow 4-amino-
TEMPO to be active under the acidic conditions that are
required for oxalate oxidase to function.
enzyme itself. In view of the numerous examples of alcohol
oxidation catalysts, we hypothesized TEMPO as a possible
solution in that it has an extensive history as an effective alcohol
oxidation catalyst and does not have the substrate specificity
limitations enzymes face.3 Furthermore, unlike many enzymatic
catalysts, TEMPO is capable of catalyzing the oxidation of
multiple oxygen-containing functional groups, such as those
encountered in biofuel oxidation processes.
The mechanism of a TEMPO catalyzed oxidation can
proceed through initial oxidation of the native form of
TEMPO, which contains a stable nitroxyl radical (I) to form
the catalytically active oxoammonium ion (II), as shown in
Scheme 1.4 This can be accomplished either electrochemically
nzymatic biofuel cells utilize an enzyme to electrochemi-
Ecally catalyze the anodic oxidation of a fuel or the cathodic
reduction of an oxidant. While enzymes are often limited in the
extent to which they can oxidize a fuel due to high substrate
specificity, they typically exhibit significantly higher catalytic
rates per active site than their precious metal counterparts. In
addition, enzymes are capable of operating under ambient
temperatures and in mild aqueous environments. Recent
research efforts have focused on the use of multi-enzyme
cascades to facilitate deep oxidation of a biofuel at a bioanode.1
This strategy maximizes the energy density extracted per
molecule of fuel; however, there are several oxidative
transformations for which a suitable biocatalyst is not yet
known. Thus, there remains a need to design green anodic
systems capable of complete oxidation of a fuel. Consequently,
we considered the possibility of merging the advantages of
enzymatic oxidation with a small-molecule catalyst to achieve
complete oxidation of a biofuel. Herein, we present a study
illustrating the potential of this approach by combining a simple
oxidation catalyst (TEMPO) with the enzyme oxalate oxidase
to completely convert glycerol to CO2, while collecting up to
16 electrons per glycerol molecule.
Oxalate oxidase (OxO) has previously been shown to
catalyze the oxidation of simple carboxylic acids such as
mesoxalic acid and oxalic acid.2 However, this enzyme is not
capable of recognizing and effectively oxidizing a high-energy
density and simple alcoholic biofuel such as glycerol. Thus, to
achieve complete oxidation of glycerol, another enzyme or a
small molecule catalyst would need to be utilized. In
considering this multicatalytic cascade strategy in a biofuel
cell setting, a promiscuous catalyst with the ability to oxidize
various alcohol starting materials and intermediates formed
throughout the conversion under electrocatalytic conditions is
desired. Additionally, the catalyst would need to be compatible
Scheme 1. Redox Cycle Highlighting the Nitroxyl Radical
(I), Oxoammonium Ion (II), and Hydroxylamine (III)
Oxidation States of TEMPO
or using a stoichiometric chemical oxidant. Recently,
Liebminger et al. reported such a system wherein TEMPO
catalyzes the oxidation of glycerol to mesoxalic acid using a
copper-containing oxidase to enzymatically regenerate the
active TEMPO catalyst with molecular oxygen as the
stoichiometric oxidant.5 However, there are no examples to
the best of our knowledge of coupling the redox capability of
both TEMPO and an enzyme in the oxidative processing of a
biofuel.
Success using this hybrid approach would require TEMPO
catalyzing the first five oxidative steps in the proposed glycerol
cascade (1 → 6, Scheme 2). The initial step in the cascade
results in the oxidation of glycerol (1) to glyceraldehyde (2).
While multiple subsequent oxidative pathways are possible, the
net result is the formation of mesoxalic acid (6). A combination
of OxO and TEMPO can then transform mesoxalic acid to
glyoxalic acid (7), oxalic acid (8), and finally CO2. Clearly the
main issue is compatibility in terms of catalytic activity of both
systems as TEMPO has not been explored extensively at both
Received: September 23, 2014
© XXXX American Chemical Society
A
dx.doi.org/10.1021/ja5098379 | J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

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significant increase in catalytic current density (Jmax) over
experiments performed without triethylamine (Figure S3). The
absence of an increase in Jmax suggests that the increased rate of
catalysis for TEMPO-NH2 is due to the presence of the
adjacent amine functional group, although its exact role in the
oxidation is unclear at this time.
Scheme 2. Proposed Electrocatalytic Oxidation Cascade of
Glycerol by TEMPO-R and Oxalate Oxidase
Much of the previous work detailing the electrochemical
oxidation of alcohols using TEMPO was either performed
under alkaline conditions or required a stoichiometric amount
of base to facilitate regeneration of the TEMPO nitroxyl radical
species.7 This presents a challenge as the basic conditions
required for catalytic TEMPO oxidation are mutually exclusive
to the acidic functional pH range of OxO. The Mn2+/Mn3+
active site of OxO is accessible through a narrow channel
containing a negative surface charge near the opening at pH 7,
which limits substrate access through a proposed gating
mechanism.8 In Figure 2, we depict the overlaid pH profiles
versus catalytic activity of OxO with TEMPO for oxalic acid
and glycerol, respectively.
the pH and aqueous conditions required for effective oxidation
using OxO.
To initiate our investigation several commercially available
derivatives of TEMPO were evaluated using cyclic voltammetry
(CV) at pH 7.0 to determine their electrocatalytic activity
toward glycerol. The results from the catalytic screening are
summarized in Figure 1. Both 9-azabicyclo[3.3.1]nonane-N-
◆
○
Figure 2. Overlaid pH profiles of oxalate oxidase ( ), TEMPO ( ),
●
and TEMPO-NH2 ( ). Dotted lines highlight the overlapping pH
range between OxO and TEMPO-NH2.
While UV−vis assays found that the activity of OxO was
unaffected by the presence of TEMPO (Figure S6), the
incompatibility of these two catalysts in a single system is clear
from the lack of overlap in their respective pH profiles. Based
on the intriguing properties of TEMPO-NH2 described above,
we also evaluated its pH profile. Unmodified TEMPO is
electrocatalytically active from alkaline conditions to pH 6.0,
while TEMPO-NH2 maintains measurable catalytic activity as
low as pH 4.0. This result identifies the expanded pH tolerance
of TEMPO-NH2, which allows for electrocatalytic activity
under acidic conditions that are otherwise required for OxO
catalysis.
The electrocatalytic performance of unmodified TEMPO,
shown in Figure 2, correlates to the pKa of the hydroxylamine
intermediate (formed during the TEMPO oxidation se-
quence).9 The reduced activity at low pH suggests that the
coupled deprotonation/oxidation of TEMPO hydroxylamine
(III, shown in Scheme 1) can no longer be facilitated and
prevents completion of the catalytic cycle. The ability of
TEMPO-NH2 to catalyze oxidation processes at a pH lower
than its pKa indicates that the amine functional group is
presumably capable of lowering the energy required for
deprotonation/oxidation of the TEMPO-NH2 hydroxylamine
intermediate. A comparison of the effects of pH on oxidation
Figure 1. Electrocatalytic screening of TEMPO compounds with
glycerol. Catalytic activity was determined by comparison of cyclic
voltammograms in the presence and absence of 0.1 M glycerol using
40 mM Robinson buffer, pH = 7.0, at 25 °C.
oxyl (ABNO) and 2-azaadamantane-N-oxyl (AZADO) exhibit a
5-fold catalytic rate increase over unmodified TEMPO.6 This is
likely the result of reduced steric hindrance from a more rigid
structure surrounding the nitroxyl radical within ABNO and
AZADO as has been observed previously.3a The highest
catalytic rate among the TEMPO compounds evaluated was
achieved by 4-amino-TEMPO (TEMPO-NH2), which ex-
hibited an 8-fold increase in catalytic rate over unmodified
TEMPO. To the best of our knowledge, the enhanced
electrocatalytic activity of TEMPO-NH2 over unmodified
TEMPO in aqueous environments has not previously been
reported. Similar experiments performed using unmodified
TEMPO with 2 equiv of triethylamine did not result in any
B
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Figure 3. Catalytic CVs of 5 mM TEMPO-NH2 in the absence (dashed) and presence (solid) of 100 mM substrate: (left to right, top row) glycerol,
glyceraldehyde, glyceric acid, (second row) tartronic acid, mesoxalic acid, glyoxalic acid. Experiments were performed using a 3 mm glassy carbon
electrode with 50 mM phosphate buffer, pH 5.2, at 5 mV s−1 and 25 °C.
potential shows that the protonation state of the amine on
TEMPO-NH2 significantly impacts its oxidation potential from
0.73 V at pH 4.0 to 0.45 V at pH 10.5 (Figure S4). The
oxidation potential of unmodified TEMPO is virtually
unaffected by pH. This shift in oxidation potential observed
for TEMPO-NH2 with pH could suggest a conformational
component in which the amine/ammonium species is in close
enough proximity to the nitroxyl radical to alter its electronic
properties through Coulombic repulsion.10 The exact nature of
this interaction as it relates to the expanded catalytic pH range
of TEMPO-NH2 is under further investigation.
The electrocatalytic properties of TEMPO-NH2 with several
intermediates of the proposed glycerol cascade were charac-
terized using CV to ensure that each step of the cascade could
be accomplished. The resulting CVs confirm the ability of
TEMPO-NH2 to catalyze the oxidation of glycerol (1),
glyceraldehyde (2), glyceric acid (3b), tartronic acid (5), and
glyoxalic acid (7) (Figure 3). Solutions of TEMPO-NH2 were
capable of generating >800 μA cm−2 in the presence of either
100 mM glycerol, glyceraldehyde, or glyoxalic acid at pH 5.2
and 25 °C. Not surprisingly, the highest rates of electrocatalytic
oxidation were observed with substrates containing a primary
alcohol or an aldehyde. Conversely, the lowest catalytic rates
were observed for substrates in which only a secondary alcohol
was available for oxidation, such as tartronic acid. This is
consistent with previously reported results suggesting that the
hindered nature of a secondary alcohol causes a decreased
oxidation rate using TEMPO.8a,11 Despite the modest rate of
tartronic acid oxidation with TEMPO-NH2, the observed
catalytic current density of 200 μA cm−2 is significantly higher
than that of tartronic acid with unmodified TEMPO. The
catalytic CV of TEMPO in the presence of 100 mM tartronic
acid indicates that TEMPO is virtually unreactive even at pH
7.0 (Figure S5).
pH 5.2. The oxidation of glycerol proceeded with TEMPO-
NH2 and OxO while generating current densities as high as 1.2
mA cm−2. The progress of the 22 h glycerol oxidation cascade
was monitored by HPLC, and the resulting analysis provided
evidence for the conversion of glycerol (1) to glyceric acid (3),
tartronic acid (5), mesoxalic acid (6), and glyoxylic acid (7)
(Figure S8). Product analysis also indicated that neither
glyceraldehyde nor oxalic acid is formed within detectable
concentrations after 22 h. These results suggest that the steady-
state concentration of both intermediates is below the
detectable limit due to the relatively high activity of both
TEMPO-NH2 for glyceraldehyde (0.02 μmol min−1, see Figure
3) and OxO for oxalic acid (0.90 μmol min−1, see Figure 2). In
addition to HPLC analysis, an isotopic labeling study was
carried out using 13C-labeled glycerol. In order to capture the
13CO2 generated by the reaction cascade, a small canister of
NaOH was suspended above the bulk electrolysis solution. At
completion, the contents of the canister were dissolved in D2O
and analyzed by 13C NMR. From the resulting 13C NMR
spectrum shown in Figure 4, a significant peak corresponding
to 13C-enriched CO32− at ca. 165 ppm was observed. While it is
difficult to quantify the catalytic turnover frequency for this
system due to diffusional kinetics, we do observe a Coulombic
yield of 90.6 C. This strongly suggests that TEMPO-NH2 and
OxO are operating as catalysts in the complete oxidation of
glycerol.
In summary, we have shown that TEMPO-NH2 and OxO
can be combined in a single electrochemical cell to catalyze the
complete oxidation of glycerol to CO2. This process completes
within 22 h when performed at 25 °C, while collecting a charge
of 90.6 C. Furthermore, catalytic current densities as high as
875 μA cm−2 were observed for individual steps of the oxidative
glycerol cascade. Ongoing research is focused on characterizing
the precise nature of the anomalous electrocatalytic activity of
TEMPO-NH2 as well as the immobilization of TEMPO-NH2
onto the surface of an electrode in the presence of OxO to
improve the performance of this hybrid system.
The complete electrochemical oxidation of glycerol by
TEMPO-NH2/OxO was carried out by bulk electrolysis of a
solution containing both the enzymatic and organic catalysts at
C
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Figure 4. 13C NMR spectrum of (a) a control sample of NaOH
exposed to ambient conditions for 22 h, and (b) 13CO2 trapped in the
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ASSOCIATED CONTENT
* Supporting Information
■
S
Detailed experimental conditions, electrochemical controls,
catalytic CVs for both various TEMPO catalysts and substrates,
HPLC chromatograms, and additional figures. This material is
available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Authors
minteer@chem.utah.edu
sigman@chem.utah.edu
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
The authors would like to thank the Army Research Office
MURI (#W911NF1410263) grant for funding.
■
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