ACS Catalysis
Research Article
the Supporting Information for more details). The conversion
proved highly dependent on the substrate and GOase variant
employed, reinforcing the unique specificity conferred by
enzymatic catalysis.
mediated electrochemical activation of GOase for high
substrate conversion.
These results collectively show that upon (re)activation of
GOase under suitable electrochemical conditions, bioelectro-
catalytic oxidation can be applied to a variety of benzyl
alcohols, cinnamyl alcohol, and aliphatic poly-ols, demonstrat-
ing its synthetic value for industrial biocatalytic processes.
Though the GOase M3‑5 or GALO-105 variants demonstrated
relatively broad substrate tolerance toward a series of
substituted benzyl alcohols, the successful application to
aliphatic poly-ols or other alcohols will require further careful
identification of the enzyme variant most suitable for each
individual substrate for the best performance.
The electro-oxidation of multiple benzyl alcohol derivatives
to aromatic aldehydes by either GOase M3‑5 or GALO-105
resulted in the high conversion of the starting material (Table
4, entries 1−5). Specifically, >99% conversion was observed for
the oxidation of 4-cyanobenzyl alcohol and 3,5-dinitrobenzyl
alcohol (entry 3 and 5). Following organic work-up, 4-
cyanobenzaldehyde and 3,5-dinitrobenzaldehyde were isolated
at 82 and 66% isolated yields, respectively. The yield for
benzaldehyde was only 37% (entry 1), and prolonged reaction
led to an even lower yield (15%) despite full conversion
(99%), likely because benzaldehyde underwent evaporation
due to the vigorous bubbling of air through the reaction
mixture. Benzoic acid as the overoxidation product was not
observed on 1H NMR spectrum. Oxidation of 4-methox-
ybenzyl alcohol afforded slightly lower conversion (92%) and
CONCLUSIONS
■
In summary, we have developed a novel electrochemical
activation method for GOase as an attractive alternative to the
expensive HRP activator in the GOase-catalyzed oxidation
reaction used for the preparation of islatravir. Electrochemistry
is an efficient tool that does not introduce additional protein
burden as in the case of HRP. GOase activation was
determined to be necessary for maintaining the catalytically
active Cu(II) tyrosyl radical oxidation state GOaseox in the
catalytic redox cycle to ensure high substrate conversion.
We observed the activation process of GALO-104 using
spectroscopic techniques. The formation of GOasesemi upon
mixing Cu and apo-enzyme and the generation of GOaseox
upon addition of Na2S2O8 into GOasesemi were both evidenced
by UV−vis spectroscopy. The activation of GOase by an
electrochemically generated mediator was then demonstrated
using cyclic voltammetry, which allowed us to interrogate the
PCET mechanism of GOase activation. The electron-transfer
rates between GOase and various mediators at different pH
values were determined using cyclic voltammetry in the
presence of 2-ethynylglycerol. The logarithm of electron-
transfer rate ln k increases not only with the redox potential of
the mediator but also with pH. The weak dependence on the
redox potential of the mediator and pH suggests that the
oxidation of GOase by redox mediators under anaerobic
conditions at pH 7−9 likely occurs by a concerted PCET
process.
1
72% yield by H NMR spectroscopy (entry 2). Even though
the oxidation of benzyl alcohols bearing two electron-
withdrawing nitro substituents was achieved within a much
shorter time (entry 5, ca. 1 h), para-substituted benzyl alcohols
did not show a strong correlation between substituent
electronic properties and the reaction rate, in good accordance
with the reported mechanism of a free radical transition state
with no charge buildup on the α-carbon of the alcohols.7
Furthermore, the reaction was not limited to benzyl alcohol
substrates: notably, the bioelectrocatalytic oxidation of
cinnamyl alcohol was also achieved, with 10 v/v% DMSO
added to improve solubility, providing cinnamaldehyde in 68%
yield (entry 6, 88% conversion).
The bioelectrocatalytic oxidation of aliphatic poly-ols was
also studied (Table 5). The constant current was used as
overoxidation was not observed with these substrates. Methyl
α-D-galactopyranoside was smoothly converted into the
corresponding aldehyde in 74% yield using GOase M1 under
the bioelectrocatalytic conditions. Electro-oxidation of dihy-
droxyacetone and xylitol using GALO-105 each resulted in full
conversion, affording 2-oxomalonaldehyde and xylose in 68
and 76% yield, respectively. These results are exciting, since
xylitol was previously identified as a poor substrate for Cu-
dependent alcohol oxidases CgrAlcOx from Colletotrichum
graminicola and CglAlcOx from C. gloeosporioides. Glycerol has
been reported as a poor substrate for FgGOase,54,72,73 but it
was accepted as a substrate for electro-oxidation using
GOaseM1, affording glyceraldehyde in 37% yield. Interestingly,
the electro-oxidation of 2,2-difluoro-1,3-propanediol selectively
yielded mono-oxidation or double-oxidation products, depend-
ing on the choice of the GOase variant utilized. When GOase
F2 was used, 2,2-difluoro-3-hydroxypropanal was preferentially
formed in 38% yield as the mono-oxidation product, and the
di-oxidation product, difluoromalonaldehyde, was formed in
only 6% yield. Compared with GOase F2, GALO-105 showed
higher reactivity for oxidizing both alcohol moieties, yielding
difluoromalonaldehyde cleanly in 64% yield. No hydro-
Applying this mediated electrochemical GOase activation in
a synthetic setting enabled the development of a bioelec-
trocatalytic aerobic oxidation of alcohols. Both O2 and the
mediator are necessary to achieve high conversions of the
alcohol substrates, because O2 serves to rapidly regenerate the
GOaseox from GOasered via a facile PCET process, and the
mediator is responsible for reactivating the catalytically inactive
GOasesemi via 1e− anodic oxidation to form GOaseox and
(re)enter the catalytic cycle. The mediator is necessary as an
electron relay between GOase and electrodes due to the high
kinetic barrier of direct electron transfer. The conversion and
selectivity of oxidation were found to be sensitive to the
operational voltage, but not correlated with the redox potential
of mediators, likely because the mediator-activated GOase
underwent rapid turnover via reactions with O2 and alcohol
until the deactivation occurred again. We showed that constant
electrochemical generation of the oxidized mediator at low
concentrations was far superior to using a larger amount under
nonelectrolytic conditions, because it circumvents the
inhibition or degradation of the enzyme by the mediator.
Compared with the GOase-mediated oxidation using either
HRP or a nonenzymatic manganese(III) activator, the
1
defluorination product was observed by H NMR spectrosco-
py. Difluoromalonaldehyde could represent a valuable
fluorinated synthon; however, there is only one prior report
on the synthesis of difluoromalonaldehyde, likely due to its
instability under the previous workup conditions.74 Without
electrolysis, the conversions for all above-mentioned oxidation
reactions were much lower, again showing the necessity of
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ACS Catal. 2021, 11, 7270−7280