The green and effective oxidation of alcohols to carboxylic acids with molecular oxygen via biocatalytic reaction

Article abstract of DOI:10.1016/j.tetlet.2007.12.032

A clean and effective alcohol oxidizing system using three enzymes has been developed. Regeneration of NAD⁺ by NADH oxidase with molecular oxygen enabled to oxidize alcohols to carboxylic acids in good yield under mild conditions (25 °C, 1 atm).

Full text of DOI:10.1016/j.tetlet.2007.12.032

Available online at www.sciencedirect.com
Tetrahedron Letters 49 (2008) 1217–1219
The green and effective oxidation of alcohols to carboxylic
acids with molecular oxygen via biocatalytic reaction
Jun-ichiro Hirano, Kenji Miyamoto, Hiromichi Ohta *
Keio University, Department of Biosciences and Informatics 3-14-1 Hiyoshi, Yokohama 223-8522, Japan
Received 27 October 2007; revised 6 December 2007; accepted 7 December 2007
Available online 14 December 2007
Abstract
A clean and effective alcohol oxidizing system using three enzymes has been developed. Regeneration of NAD+ by NADH oxidase
with molecular oxygen enabled to oxidize alcohols to carboxylic acids in good yield under mild conditions (25 °C, 1 atm).
Ó 2007 Published by Elsevier Ltd.
Keywords: Alcohol oxidation; Molecular oxygen; Biocatalyst; Green chemistry; Regeneration of cofactor
Oxidation of alcohols is one of the most important and
essential processes in organic chemistry. Many synthetic
methods have been reported so far. However, hitherto
known methods have some drawbacks as the industrial
processes from the environmental point of view, because
they require a stoichiometric amount of toxic metal ions,
harmful organic solvents, or a large amount of energy con-
sumption with high pressures and/or high temperature.
Therefore, development of the catalytic process of alcohol
oxidation under mild conditions is essential for green and
sustainable chemistry. Recently, several catalytic methods
have been reported, such as aerobic oxidation using palla-
dium complexes,1 ruthenium complex,2 and platinum cata-
lyst.3 However, the metal-independent oxidations of
alcohols are very few. Thus we tried to construct a biocata-
lytic system for alcohol oxidation. Much attention has been
paid to biocatalysts for these years as environmentally
friendly and sustainable catalysts, because they are biosyn-
thesized from recyclable biomaterials and are biodegrad-
able. Some biocatalytic methods for the oxidation of
alcohol using whole cells and isolated enzyme were
reported.4 As the representative examples, the enantioselec-
tive oxidation of 2-phenylpropanol by Acetobacter aceti5
and the oxidation of primary alcohols to aldehyde by
Gluconobacter oxydans6 have been reported. In these cases,
intact cells were used as the oxidizing reagents. On the
other hand, the enantioselective oxidation of amines7,8
and secondary alcohols using enzymes9 have also been
reported. However, the enzymatic oxidation of primary
alcohols to carboxylic acids, which can be applicable to a
broad range of alcohols, has not been studied enough.
Recently, we have isolated Brevibacterium sp. KU 1309
from soil, which grows on the medium containing 2-phenyl-
ethanol as the sole carbon source. This microorganism
oxidized various primary and secondary alcohols to the
corresponding carboxylic acids and ketones, respectively.10
NAD+ dependent 2-phenylethanol dehydrogenase (PEDH)
was purified from this strain and characterized.11 This
enzyme is presumed to be responsible for the abovemen-
tioned oxidation by the intact cells, because it exhibited
similar activity to a wide range of aromatic and aliphatic
alcohols. In addition, NAD+ dependent phenylacetalde-
hyde dehydrogenase (PADH) was also purified from the
same microorganism.12 PEDH and PADH are expected
to be useful enzymes because of their exceptionally broad
substrate specificities. For this type of enzymatic oxidation
to proceed effectively, the cooperation of an NAD+-regener-
ating system is essential. NADH oxidase was reported as
*
Corresponding author. Tel.: +81 45 566 1703; fax: +81 45 566 1551.
E-mail address: hohta@bio.keio.ac.jp (H. Ohta).
0040-4039/$ - see front matter Ó 2007 Published by Elsevier Ltd.
doi:10.1016/j.tetlet.2007.12.032

1218
J. Hirano et al. / Tetrahedron Letters 49 (2008) 1217–1219
effective regeneration system of NAD+.13 Thus, we
employed the NADH oxidase (NOX) isolated from Lacto-
bacillus brevis.14 NOX from L. brevis catalyzes oxidation of
NADH to NAD+ with molecular oxygen and produces
H2O. The family of NOX is divided into two types accord-
ing to the reduction of oxygen such as H2O-producing or
H2O2-producing. H2O-producing NOX is suitable for
isolated enzymatic reaction, because high concentration
of H2O2 is harmful for enzymes. Herein, we would like to
report that we have accomplished a green and highly effec-
tive oxidation system of alcohols using the above three
enzymes (Scheme 1). 2-Phenylethanol was oxidized to
phenylacetaldehyde by PEDH, which in turn was further
oxidized to phenylacetate by PADH. The resulting reduced
form cofactor NADH was oxidized with molecular oxygen
to regenerate NAD+ by the aid of NOX. PEDH and
PADH were used as partially purified enzyme from Brevi-
bacterium sp. KU1309. NOX was isolated from recom-
binant Escherichia coli BL21 (DE3) overexpressing the
NOX transformed by plasmid containing nox gene under
the control of T7 promoter (see Supplementary data).
Thus, we achieved the oxidation of alcohols (5–10 mM)
forming H2O as the only by-product under normal pres-
sure (1 atm) and at room temperature (25 °C).
Table 1
The total effect of pH and NAD+ concentration on the combined three
enzymatic reactions
PEDH, PADH, NOX
Ph
CH2OH
Ph CO2H
NAD+
1
2
buffer
25 ºC, 12 hr
10 mM
Entry
pH
[NAD+] (mM)
Yield of 2a (%)
1
2
3
4
5
6
7
a
8
9
10
9
1
1
1
62
86
12
5
0
9
9
9
0.1
0.5
10.0
34
84
5
The details of reaction condition and determination of yields were
described in the part of Supplementary data.
and the cost of the procedure. The conversion of 2-phenyl-
ethanol to phenylacetate requires 2 equiv of NAD+.
However, oxidation reaction proceeded smoothly and
quantitatively when only 5 mol % of NAD+ was used in
the presence of NOX, because molecular oxygen could be
utilized as the final electron acceptor (Table 1). On the
other hand, alcohol was not oxidized in the absence of
NOX. Neither was phenylacetate detected after incubation
of phenylacetaldehyde under the same conditions, but
without enzymes. In the absence of PADH, the oxidation
of alcohol did not proceed. Thus, it was confirmed that
the oxidation of the aldehyde was an enzyme-catalyzed
reaction. On the contrary, the oxidation reaction did not
proceed in the presence of high concentration of NAD+
(10 mM). Under high concentration of NAD+, the absor-
bance at 340 nm, which corresponds to NADH, did not
increase regardless of the presence of 2-phenylethanol
and PEDH. Thus we considered that PEDH was inhibited
by a large amount of NAD+. In this way, the efficiency of
the NAD+ regenerating system is the key to achieve the
oxidation of alcohols in high yields.
Generally, the rate of enzymatic reaction depends on the
pH of the medium, because each enzyme exhibits its own
charge distribution. These pH dependencies are especially
important in the case of biotransformation using plural
enzymes. Accordingly, pH profiles of PEDH, PADH, and
NOX were examined first (described in detail in Supple-
mentary data). PEDH and PADH prefer basic conditions.
On the other hand, although NOX showed its highest
activity at pH 6, it also exhibited a maximum activity of
about 30% at pH 9. Consequently, the pH range in which
the three enzymes showed their activities at the same time
was revealed to be between 8 and 10. Thus, in all three
enzymatic reactions were performed at once in this pH
range and the results are summarized in Table 1. When
the excess amount of NOX was added to the system, the
reaction proceeded most effectively at pH 9. Because
NOX was easily obtained in large amount from E. coli
BL21 (DE3) transformed by the plasmid vector containing
nox gene, it was not difficult to use a relatively large
amount of this enzyme.
Utilizing this reaction system, various primary alcohols
were oxidized to the corresponding carboxylic acids in
good yields (Table 2), which are generally prone to be
further degraded via b-oxidation pathway in the case of
whole-cell reaction. Although the yields of the products
were less than 100% in some cases, other by-products
were not detected in the reaction mixture. The yields of
The concentration of NAD+ is an extremely important
factor from the standpoint of the efficiency of reaction
Brevibacterium sp.KU1309
PEDH
Brevibacterium sp.KU1309
PADH
Ph
CH2OH
H2O
Ph CO2H
Ph CHO
NADH + H+
NADH + H+
NAD+
NAD+
1/2 O2 H2O
1/2 O2
L. brevis ATCC14869
NOX
L. brevis ATCC14869
NOX
Scheme 1. Three enzymatic oxidation reactions of alcohol to carboxylic acid via molecular oxygen.

J. Hirano et al. / Tetrahedron Letters 49 (2008) 1217–1219
1219
Table 2
enable this oxidizing system to be applied to the industrial
process.
Enzymatic oxidation of alcohols coupled with NADH oxidase
PEDH, PADH, NOX
R CH2OH
R CO2H
Acknowledgment
NAD+ (1mM)
buffer
25 ºC, 12 hr
This research was partially supported by the Ministry of
Education, Culture, Sports, Science and Technology,
Grant-in-Aid for the 21st century COE Program entitled
‘Understanding and Control of Life’s Function via Systems
Biology’, Keio University.
Entry Substratea
Product
Yield (%)
86
CH2OH
CH2OH
CO2H
CO2H
1
2
74
87
35
Supplementary data
CH2OH
CO2H
Supplementary data (preparation of PEDH, PADH,
and NOX and general experimental procedure) associated
with this article can be found, in the online version, at
doi:10.1016/j.tetlet.2007.12.032.
3
4
O
O
CO2H
CH2OH
References and notes
CH2OH
CH2OH
CO2H
CO2H
5
86
60
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In summary, we developed a novel approach to the
oxidation of alcohol using biocatalyst. This reaction
proceeds at room temperature (25 °C) and under the atmo-
spheric pressures (1 atm). Using an NAD+ regenerating
system, the required amount of the expensive cofactor
could be greatly reduced. Because the final electron
acceptor is molecular oxygen and the only by-product is
water, this system can be considered as green and sustain-
able. The preparation of PEDH and PADH in large scale
utilizing genetic and protein technology is expected to
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