Catalytic Promiscuity of Galactose Oxidase: A Mild Synthesis of Nitriles from Alcohols, Air, and Ammonia

Article abstract of DOI:10.1002/anie.201809411

We report an unprecedented catalytically promiscuous activity of the copper-dependent enzyme galactose oxidase. The enzyme catalyses the one-pot conversion of alcohols into the related nitriles under mild reaction conditions in ammonium buffer, consuming ammonia as the source of nitrogen and dioxygen (from air at atmospheric pressure) as the only oxidant. Thus, this green method does not require either cyanide salts, toxic metals, or undesired oxidants in stoichiometric amounts. The substrate scope of the reaction includes benzyl and cinnamyl alcohols as well as 4- and 3-pyridylmethanol, giving access to valuable chemical compounds. The oxidation proceeds through oxidation from alcohol to aldehyde, in situ imine formation, and final direct oxidation to nitrile.

Full text of DOI:10.1002/anie.201809411

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Accepted Article
Title: Catalytic Promiscuity of Galactose Oxidase: A Mild Synthesis of
Nitriles from Alcohols, Air and Ammonia
Authors: Jan Vilím, Tanja Knaus, and Francesco Mutti
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COMMUNICATION
Catalytic Promiscuity of Galactose Oxidase: A Mild Synthesis of
Nitriles from Alcohols, Air and Ammonia
[
a]
[a]
[a]
Jan Vilím , Tanja Knaus , and Francesco G. Mutti*
Dedication. ((optional))
Abstract: We present an unprecedented catalytic promiscuous
activity of the copper-dependent enzyme, galactose oxidase. The
enzyme catalyses the one-pot conversion of alcohols into the related
nitriles under mild reaction conditions in ammonium buffer, consuming
ammonia as the source of nitrogen and dioxygen (from air at
atmospheric pressure) as the only oxidant. Thus, this green method
does not require either cyanide salts, or toxic metals, or undesired
oxidants in stoichiometric amounts. The substrate scope of the
reaction includes benzyl and cinnamyl alcohols as well as 4- and 3-
pyridylmethanol, giving access to valuable chemical compounds. The
oxidation proceeds via oxidation from alcohol to aldehyde, in situ
imine formation and final direct oxidation to nitrile.
of alcohols to nitriles have been published, making use of Cu(II)
at high temperature or Fe(III)/TEMPO in MeCN.[
18]
Biocatalytic approaches enable synthesis of nitriles under mild
reaction conditions. Those methods include the use of aldoxime
dehydratases,[ hydroxynitrile lyases (i.e. addition of cyanide to
carbonyl compounds),[ halohydrin dehalogenases (i.e. ring-
19]
20]
[
21]
opening of epoxides by cyanide),
and amine oxidases in
[
22]
combination with cyanide salt. Other enzyme families such as
nitrile synthetase,[
23]
β-cyano-L-alanine synthase[24] and
cytochromes [ have limited synthetic applicability. However,
there is no report about a one-enzyme conversion of alcohols into
nitriles.
25]
Surprisingly, during the oxidation of benzyl alcohol (1a, 10 mM) to
benzaldehyde (1b) in ammonium formate buffer (600 mM, pH 9)
Catalytic enzyme promiscuity is the ability of an enzyme to
catalyse chemical reactions that are different from the natural
one.[1] Even after two decades of intensive investigations, new
notable cases of catalytic enzyme promiscuity have been recently
catalysed by purified Strep-tagged galactose oxidase (GOx, 25
[26]
µM) from Fusarium sp. M3-5
,
we noticed the unexpected
formation of just 1.2% of benzonitrile (1c) that sparked our interest
Scheme 1).
(
[
2]
revealed and applied in chemical synthesis
as well as in
[
3]
synthetic biology. Herein, we report an unprecedented catalytic
promiscuity of a galactose oxidase, which is the conversion of
selected alcohols into nitriles.
General methods for the synthesis of nitriles include dehydration
[
4]
[5]
of amides, formal acid-nitrile exchange, Sandmeyer and
[
6]
Rosenmund-von Braun reactions, transition-metal catalyzed
[
7]
[8]
cyanation, electrophilic cyanide transfer and radical-type
[
9]
Scheme 1. Conversion of alcohols to nitriles catalysed by single galactose
oxidase (GOx).
cleavage reactions. However, these methods generally require
toxic cyanide and heat. Cyanide-free routes to nitriles are possible
starting from aldehydes (using azide, hydroxylamine or
With the aim of increasing benzonitrile formation, we considered
that GOx (a Cu-dependent enzyme) requires the addition of
[
10]
ammonium salts as nitrogen source) , or amines (in presence of
[
11]
metal catalysts or catalytic TEMPO or stoichiometric oxidants),
2+
exogenous Cu to promote the stabilisation of its holo-form for
[
12]
[13]
[14]
or azides,
or pre-formed oximes,
or organic halides,
or
[
27]
biocatalytic reactions in vitro. The influence of the concentration
arenes.[15] Benzonitriles are also produced on industrial scale
from toluene by ammoxidation using heterogeneous catalysts,
ammonia and dioxygen (450 °C, 2 bar).[16] The direct conversion
of alcohols into nitriles attracts interest, but it requires a metal
and/or an organic catalyst in presence of supra-stoichiometric
amounts of an organic oxidant and ammonium species.[
However, replacing chemical oxidants with dioxygen would
increase significantly the atom-efficiency and the environmental
footprint of the reaction. A few systems for the aerobic conversion
2+
of added Cu towards the activity of GOx M3-5 for the oxidation of
alcohols to aldehydes has been determined previously in
phosphate buffer.[ However, the use of phosphate buffer poses
the issue of precipitation of nearly insoluble copper phosphate.
Thus, we evaluated the influence of the concentration of Cu2+ ions
as CuSO ) for the natural oxidation reaction of alcohol 1a to
aldehyde 1b in Tris-HCl buffer (100 mM, pH 8). Fig 1A shows that
the conversion of 1a (10 mM) into 1b, measured after 40 min, rose
progressively at increasing ratio between Cu and purified GOx
2.5 µM). The highest yield was observed at a molar ratio of
Cu /GOx ca. 60:1 (for details, SI 4.1). Switching from Tris-HCl to
HCOONH buffer resulted in a similar trend albeit nitrile 1c was
formed along with 1b. Thus, a 50:1 molar ratio of Cu /GOx was
used for the continuation of our study. Then, we investigated the
influence of the pH towards the formation of 1c by performing a
26f]
[
28]
17]
(
4
2+
(
2+
4
[
a]
J. Vilím, Dr. T. Knaus, Ass. Prof. Dr. F. G. Mutti
Van’t Hoff Institute for Molecular Sciences
HIMS-Biocat, University of Amsterdam
Science Park 904, 1098 XH, Amsterdam (The Netherlands)
E-mail: f.mutti@uva.nl
2+
2+
set of experiments at 30 °C with 1a (10 mM), GOx (20 µM), Cu
Supporting information for this article is given via a link at the end of
the document.((Please delete this text if not appropriate))
(1 mM) and catalase (17 µM). The pH was varied from 8 to 10 in
HCOONH buffer (600 mM). Interestingly, data regarding the
4
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COMMUNICATION
costly and time-consuming purification steps are avoided [26f] and,
possibly, higher GOx activity may be retained. Indeed,
optimisation of the reaction conditions for the conversion of 1a to
1
c using CFE permitted to increase the TON up to ca. 3300 (Fig.
2
B), which is a value already suitable for a large scale
application.[ Yields of 1c were in line with the experiments using
29]
purified GOx (Fig. 2A). In particular, the highest TON of ca. 3300
(1.28 µM GOx as CFE) correlated to 42% yield of 1c, whereas the
highest yield of 65% (2.55 μM GOx as CFE) correlated to a TON
of ca. 2600. Using a 2.55 µM GOx loading as CFE, the
Figure 1. Optimization of reaction conditions and determination of
substrate scope. A) Influence of copper B) Influence of pH C) Optimal
amount of catalase D) Concentration of ammonium species E) Influence of
temperature F) Supplementation of oxygen.
catalytic activity of GOx above pH 8 (in any type of buffer) were
not available in literature, while the beneficial effect of the addition
of catalase was documented.[26f] In fact, GOx produces H
2 2
O
during the catalytic cycle that may diminish, at certain
concentrations, the enzyme activity. Under the reaction conditions
reported above, the formation on nitrile versus pH showed a bell-
shape with a maximum yield at pH 9 (Figure 1B). A second set of
experiments aimed at minimising the amount of catalase for the
transformation of 1a (10 mM) to 1c at pH 9. Fig. 1C shows that
the addition of catalase affected positively the reaction albeit a
-
1
minimal concentration of 0.83 µM (equal to 0.05 mg ml ) was
sufficient. The evaluation of the influence of the concentration of
ammonium species and of temperature on the yield of 1c shows
+
maxima in the range of 400-600 mM of NH
3
/NH
4
and at 30 °C
(Fig. 1D and 1E). After the optimisation of the reaction parameters,
we investigated the influence of air and pure dioxygen (even
under pressure) on the progress of the reaction, as dioxygen is
the oxidant in the GOx catalytic cycle.[
26c, 26f, 27a, 27c]
. Interestingly,
2 2
the supplementation of O as pressurised air or pure O increased
slightly the yield of 1c (Fig. 1F). However, a large-scale
biocatalytic conversion of alcohols to nitriles operating under
pressure would have the disadvantage of consuming energy for
pressurisation of the system. Thus, further optimisation was
conducted using air at atmospheric pressure.
The work with highly purified GOx was crucial for demonstrating
the promiscuous formation of nitriles from alcohols (Figure S3).
Nonetheless, the chemical turnover (TON) for the reaction with
purified GOx reached a maximum value of ca. 230 that is
insufficient for a synthetic application with oxidoreductases.[29]
Figure 2. Synthesis of nitriles from alcohols using CFE. Analytical yield of 1c
+
(A) and TON (B) at varied concentration of NH
scope (C). For experimental details, see SI.
3
/NH
4
and GOx. Substrate
Hence, we tested GOx as E. coli cell free extract (CFE) because
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COMMUNICATION
GOx
Albumine
Yield (%)
(µM)
promiscuous biocatalytic conversion of alcohols into nitriles was
tested with a variety of substrates (10 mM) pre-dissolved in
Entry
Substrate
pH
(μM)
1
2
3
4
5
6
7
8
9
1b
1b[a]
1b
9
9
9
9
9
9
7
9
7
9
20
20
-
-
-
47
33
0
-
1
DMSO (2%, v v ). The reactions were run under the optimised
+
conditions (NH
3
/NH
4
400 mM, pH 9, catalase 0.83 µM, 30 °C).
-
With the exception of cyclohexylmethanol (16a), 2-
pyridylmethanol (19a), 2-phenylethanol (20a) and 3-phenyl-1-
propanol (21a), all the other alcohols were converted into nitriles
1b[a]
-
-
<0.5
0
1b
-
20
20
-
1b[a]
1d
-
<0.5
0
(for yields, TONs: Fig. 2C, SI 4.8).
20
20
20
20
Interestingly, within homologous series, benzyl alcohols
a
1d
-
0
containing electron-withdrawing substituents in ortho position
were converted with higher yields (4c, 7c, 10c) compared to the
para- (2c, 5c, 8c) and especially the meta-substituted ones (3c,
1d[b]
1d[b]
-
0
10
-
0
6
c, 9c). The effect was reversed with the electron-donating methyl
Table 1. Study on the formation of nitrile from 1b or 1d using GOx or Albumine.
If not stated otherwise, Cu2 (50 eq.) was added. For details, see, SI. [a]: H
+
2 2
O
substituent, as ortho-methyl benzyl alcohol was less converted
+
(
10 mM) was added. [b]: Cu2 was omitted.
(
(
13c) than para-methyl (11c) and meta-methyl benzyl alcohol
12c). The highest yield was 70% for the conversion of 2-
formation by reaction with ammonia and subsequent promiscuous
oxidation to nitrile via hydride abstraction, or 2) imine formation,
subsequent promiscuous hydroxylation to oxime and final
dehydration to nitrile. Thus, benzaldehyde oxime (1d) was
incubated with GOx under various conditions (Table 1 entries 7-
10; for details Table S11) but dehydration to nitrile was never
fluorobenzyl alcohol (4a) into 2-fluorobenzyl nitrile (4c). Cinnamyl
alcohol (22a) was also accepted, leading to 10% yield into the
related nitrile 22c. Moreover, 4-pyridyl methanol and 3-pyridyl
methanol were also transformed into corresponding nitriles (17c,
1
8c) with 10% and 55% yield, respectively. Besides the formation
of the nitrile products, variable amounts of carboxylic acids (1-14e, observed. Consequently, nitrile 1c is formed by direct oxidation of
17-18e, 22e) were detected in agreement with the findings
the imine intermediate.
reported in a concomitant publication focused on oxidation of
alcohols to carboxylic acids catalysed by GOx.[ We point out
that nitriles and carboxylic acids can be separated easily by
extracting the former directly from the reaction buffer (pH 9) and,
in case, the latter after acidification. In many cases, both nitriles
and carboxylic acids are valuable compounds (e.g. oxidation of
In conclusion, we have discovered a new promiscuous activity of
the galactose oxidase that is the one-pot synthesis of benzyl,
pyridyl and cinnamyl nitriles from the related alcohols using only
ammonia as source of nitrogen and dioxygen as innocuous
30]
oxidant. Compared to recently reported approaches used to
transform alcohols or aldehydes into nitriles, [
7a, 10a, 10e, 17]
the GOx-
1
8a to vitamins B3: 18c and 18e). However, interestingly, the yield
catalysed reaction has significant advantages such as mild
reaction conditions in aqueous medium, simple operational set-
up and elevated atom-economy. Moreover, utilization of GOx in
form of CFE increased the TON to synthetically applicable levels
and avoided any purification steps. This promiscuous activity of
GOx has already notable applications as cinnamonitrile is an
important synthetic aroma,[32] whereas benzonitriles constitute the
active core of the large majority of nitrile-containing
pharmaceuticals. Moreover, 3-cyanopyridine is a precursor to
vitamin B3, to which it can be converted by established enzymatic
methods.[34] Future research will focus on searching for other
promiscuous copper-dependent alcohol oxidases, which are
active on structurally different alcohols, in order to enable even
broader application of this new biocatalytic reaction.
of nitrile (and the chemoselectivity of the reaction) were somehow
dependent on the scale of the reaction. For instance, a
preparative scale synthesis was performed with 4a (151 mg, 1.2
mmol) under the optimised reaction conditions using CFE. After
2
4 h, the reaction afforded >99% analytical yield into nitrile 4c
(exactly quantified with internal standard). After extraction and
solvent evaporation, nitrile 4c was isolated in 75% yield and pure
form (no further purification step was required). Conversely, the
biocatalytic conversion of 4a in analytical scale (Fig. 2C) produced
[
33]
7
0% analytical yield of nitrile 4c and 5% of carboxylic acid 4e. We
attribute the discrepancy to different aeration and agitation
between analytical scale and preparative scale reactions.
Regarding the mechanism for the formation of nitrile from alcohol,
we further proved the promiscuous activity of GOx by exploring a
possible non-enzymatic or non-specific conversion of the
aldehyde 1b into nitrile 1c. In fact, there are literature reports
, Cu2+ or formate may contribute to the
Acknowledgements
describing that H
conversion of 1b to 1c (and derivatives thereof), but in presence
2 2
O
[
17a,
of additional reagents and under particular reaction conditions.
This project received funding from the European Research
Council (ERC) under the European Union’s Horizon 2020
research and innovation programme (grant agreement No.
638271).
31]
A series of reactions (Table 1) revealed that nitrile 1c is indeed
produced from aldehyde 1b only in presence of GOx (entry 1,
7% yield). Partial loss of GOx activity was observed when H
was also added into the mixture (entry 2, 33% yield), confirming
4
2 2
O
[
26f]
the detrimental effect of H
O
2 2
in high concentration.
Several
Keywords: biocatalysis • enzyme promiscuity • alcohol oxidation
2+
control experiments including albumin and/or Cu and/or H
2
O
O
2
•
nitriles • copper oxidases
afforded just traces of 1c (<0.5%) only in presence of H
2
2
(entries 4 and 6). In the same way, formation of 1c from 1a was
observed only in presence of GOx. Finally, the transformation of
aldehyde to nitrile may occur via two possible paths: 1) imine
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Angewandte Chemie International Edition
10.1002/anie.201809411
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Unprecedented catalytic promiscuity
of copper dependent alcohol oxidase
allows one-pot transformation of
selected alcohols into nitriles under
mild, cyanide-free conditions using
ammonia as nitrogen donor and
dioxygen as oxidant. This reaction
provides a valuable addition to
enzymatic methods for nitrile
J. Vilím, T. Knaus, F. G. Mutti*
Page No. – Page No.
Catalytic Promiscuity of Galactose
Oxidase: A Mild Synthesis of Nitriles
from Alcohols, Air and Ammonia
synthesis, since direct enzymatic
transformation of alcohol to nitriles
has been unknown so far.
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