A mild and highly efficient laccase-mediator system for aerobic oxidation of alcohols

Article abstract of DOI:10.1039/c3gc42124d

With the aid of the highly active nitroxyl radical AZADO (2-azaadamantane N-oxyl), a simple method for the aerobic catalytic oxidation of alcohols is presented. The oxidations could typically proceed under practical ambient conditions (room temperature, air atmosphere, no moisture effect, metal-free, etc.) with a broad generality of the alcohol substrates, and especially for the oxidation of complex and highly functionalized alcohols. An ionic mechanism is proposed for the present system.

Full text of DOI:10.1039/c3gc42124d

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A mild and highly efficient laccase-mediator
system for aerobic oxidation of alcohols†
Cite this: Green Chem., 2014, 16,
1131
Chenjie Zhu,a,b Zhi Zhang,a,b Weiwei Ding,a,b Jingjing Xie,a,b Yong Chen,a,b
Received 13th October 2013,
Accepted 2nd December 2013
Jinglan Wu,a,b Xiaochun Chena,b and Hanjie Ying*a,b
DOI: 10.1039/c3gc42124d
www.rsc.org/greenchem
With the aid of the highly active nitroxyl radical AZADO (2-azaada- oxidoreductase, EC 1.10.3.2), a blue multi-copper oxidase, has
mantane N-oxyl), a simple method for the aerobic catalytic oxi- drawn considerable attention in white biotechnology. As a
dation of alcohols is presented. The oxidations could typically result of their nontoxic nature, high stability, and lack of sub-
proceed under practical ambient conditions (room temperature, strate inhibition, laccases are nowadays ideal candidates in the
air atmosphere, no moisture effect, metal-free, etc.) with a broad fields of waste detoxification, textile dye transformation, bio-
generality of the alcohol substrates, and especially for the oxi- sensors, food industry, and pulp bleaching.5 The demand for
dation of complex and highly functionalized alcohols. An ionic green chemical processes has also inspired interest in the
mechanism is proposed for the present system.
application of this enzyme to address modern synthetic
organic chemistry challenges. Due to their broad substrate
scope, laccases find increasing significance in a variety of
organic synthetic methodologies and the manufacture of syn-
thetic building blocks for fine chemistry.6
The selective oxidation of alcohols to the corresponding carbo-
nyl compounds is a fundamental transformation both in labo-
ratory synthesis and industrial production.1 Numerous
oxidizing reagents in stoichiometric amounts have been tra-
ditionally employed to accomplish this transformation with
considerable drawbacks such as the use of expensive reagents,
volatile organic solvents, and discharge of environmentally
pernicious wastes. Recent growing environmental concerns
have spurred research activities directed toward the develop-
ment of greener oxidation methods in which the use of mole-
cular oxygen as the terminal oxidant has drawn considerable
attention.2
On the other hand, enzyme-catalyzed transformations have
emerged as an elegant synthetic methodology that allows the
development of eco-friendly processes within the basic prin-
ciples of green chemistry.3 The development of enzyme-cata-
lyzed oxidations is highly attractive because of their great
potential to remove pollutants and catalyse a great variety of
redox processes with no hazardous side effects.4 Furthermore,
most enzymatic oxidations usually use aerial oxygen as the
terminal oxidant and can be performed under very mild reac-
tion conditions. In this context, laccase (benzenediol : oxygen
Despite the synthetic importance of laccases, their broader
application has been restricted due to the low redox potential
(typically about 0.5–0.8 mV vs. the normal hydrogen elec-
trode).6a For the reactions where the substrate to be oxidized has
a redox potential higher than laccase, or the substrate is too
large to penetrate into the enzyme active site, the presence of a
low-molecular weight chemical mediator is required to facili-
tate oxidative reactions.7 A mediator acts as a sort of ‘electron
shuttle’; once it is oxidised by laccase, it diffuses away from
the enzymatic pocket and in turn oxidises any substrate that,
due to its size, could not directly enter the enzymatic pocket.8
The structures and abbreviated names of representative artifi-
cial laccase mediators are given in Fig. 1. By using these so-
called ‘chemical mediators’, the redox potential of laccases
can be extended which allows the oxidation of a wide range of
non-phenolic substrates, such as sugars,9 ethers,10 alkenes,11
amides,12 aromatic methyl groups,13 polycyclic aromatic hydro-
carbons,14 lipids,15 and alcohols.16 Despite all the associated
advantages of using these mediators, there are still several
drawbacks from a practical point of view. These are: (i) low cata-
lytic efficiency, excess amounts of mediator (typically 30 mol%
on substrate) are needed, with some even needing more
than 1 equiv.; (ii) in some cases, laccase is inactivated by the
mediator radicals, or the latter can be transformed into in-
active compounds with no more mediating capability (e.g. gen-
eration of benzotriazol from HBT by losing the hydroxyl group);
(iii) some of the mediators can generate toxic derivatives; and
aCollege of Biotechnology and Pharmaceutical Engineering, Nanjing University of
Technology, Xin mofan Road 5, Nanjing 210009, China.
E-mail: yinghanjie@njut.edu.cn, zhucj@njut.edu.cn; Fax: +86 25 58139389;
Tel: +86 25 86990001
bNational Engineering Technique Research Center for Biotechnology, Nanjing, China
†Electronic supplementary information (ESI) available: General experimental
procedures and compound characterization data including 1H and 13C NMR. See
DOI: 10.1039/c3gc42124d
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Table 1 Optimization studies for the aerobic oxidation of 1a
Entry
Catalyst
Equiv.
Additive
Yieldb (%)
1
2
3
4
TEMPO
AZADO
Nor-AZADO
ABNO
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.1
0.05
0.05
—
—
—
—
—
CTAB
SDS
Tween-20
Rhamnolipid R1
PhCF3
PhCF3
PhCF3
PhCF3
7
63
60
46
29
66
52
67
55
87 (85)
85
80
82
5
ABOO
6c
7d
8e
9f
10g
11
12
13h
AZADO
AZADO
AZADO
AZADO
AZADO
AZADO
AZADO
AZADO
Fig. 1 Structures of representative artificial laccase mediators.
a Reactions were performed by using 1 (1 mmol), laccase (40 U), a
nitroxyl radical in acetate buffer (pH 4.5) under an O2 atmosphere at
room temperature for 12 h unless otherwise noted. b Yield was
determined by 1H NMR using 1,1,2,2-tetrachloroethane as an internal
standard. c CTAB (cetrimonium bromide, 0.2 equiv.) was used.
d SDS (sodium dodecylsulfate, 0.2 equiv.) was used. e Tween-20
(sorbitan monooleate ethoxylate, 0.1 g) was used. f Rhamnolipid R1
(3-(3-[(6-deoxy-alpha-L-mannopyranosyl)oxy]decanoyl oxy) decanoic
acid, 0.1 g) was used. g PhCF3 (1 mL) was used as a co-solvent; isolated
yield is shown in parentheses. h This reaction was run using air as an
oxidant for 24 h.
(iv) long reaction times are usually required. In view of the
aforementioned drawbacks of existing routes and the desire
for aerobic oxidation of multifunctionalized complex mole-
cules, the development of a new and highly efficient laccase-
mediator system is particularly attractive.
In continuation of our efforts to explore green approaches
for synthetic chemistry,17 we report herein a new and highly
efficient laccase-mediator system for the oxidation of alcohols
using O2 as a green and economic oxidant under mild reaction
conditions.
Initial experiments were carried out using 1,2:4,5-di-O-iso- comparable catalytic activity to AZADO and serves as another
propylidene-β-D-fructopyranose 1 as a model substrate; the good catalyst to enhance the reaction remarkably. However,
corresponding product ketone 2 (1,2:4,5-di-O-isopropylidene- the catalytic activity of ABNO (9-azabicy-clo[3.3.1]nonane
β-D-erythro-2,3-hexadiulo-2,6-pyranose, Shi’s catalyst) is
a
N-oxyl) and ABOO (8-azabicyclo[3.2.1]octane N-oxyl) is much
useful reagent, which is often used as a precursor for tran- lower than that of AZADO, probably due to the stability of the
sition metal free catalytic asymmetric olefin epoxidation.18 Cata- corresponding cations.23b,24 Encouraged by these results and
lytic oxidation of 1 has proved to be particularly difficult, with in order to search for the optimum reaction conditions, we
numerous methods such as the TPAP-O2 system,19 the screened a variety of parameters of this reaction, such as a
NHPI-O2 system,20 and the Pd(bathophenanthroline)-(AcO)2-O2 different source of laccase, the type and pH of buffer and
system,21 which were successfully applied to oxidation of alco- a broad range of co-solvents (for more details see Table S2 in
hols, failing to convert 1 to 2 due to steric reasons. In the the ESI†).
laccase catalysed aerobic oxidation of alcohols, TEMPO and its
It is well known that in aqueous-phase reactions, addition
derivatives proved to be the most effective.22 However, using of surfactants can improve the reactions by solubilization or
TEMPO as the catalyst, the yield of 2 was less than 10% the micelle formation effect. To improve the solubility of the
(Table 1, entry 1). The low catalytic efficiency of TEMPO as well reactant and catalyst in the buffer, we examined a variety of
as recent significant advances in designing the structure of surfactants, including cationic, anionic, nonionic, and biologi-
nitroxyl radicals23 prompted us to reconsider the TEMPO cata- cal surfactants (Table 1, entries 6–9). However, all of them
lyst and provided an opportunity of using a more reactive were unsatisfactory although CTAB and Tween-20 showed a
mediator for laccase. We were very pleased to find that in the slight improvement. Further investigation of the co-solvent on
case of using AZADO (2-azaadamantane N-oxyl) as a catalyst, this reaction showed a strong solvent dependence effect.
the desired product was obtained in 63% yield (Table 1, entry Earlier studies revealed that laccases are often deactivated by
2). In the control experiments, there was no product formation organic solvents.25 A broad range of co-solvents were tested for
in the absence of laccase, AZADO, or when using heat-killed this reaction (see Table S2 in the ESI†); among them, PhCF3
laccase (see Table S1 in the ESI†). With the aim of finding an proved to be the most efficient co-solvent, giving the highest
appropriate mediator for laccase, we evaluated the catalytic yield of 2 (Table 1, entry 10). A further study showed that a co-
performance of other nitroxyl radicals (Table 1, entries 3–5). solvent was not necessary for the reaction; for most reactants,
Among them, Nor-AZADO (9-azanoradamantane N-oxyl) shows the use of a buffer as the solvent was effective enough to
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complete the oxidation. With regard to the AZADO dosage, it centre in laccase from Trametes versicolor has a redox potential
was possible to decrease the amount of AZADO to as low as of ca. 790 mV versus NHE.22b,26 Consequently, laccase from
0.05 equiv. without a significant loss in catalytic efficiency Trametes versicolor has sufficient oxidation potential to convert
(Table 1, entry 12). It is noteworthy that the reaction can also TEMPO or AZADO into their corresponding oxidised state. The
proceed smoothly even using air as an oxidant, albeit a longer different catalytic reactivity between TEMPO and AZADO
reaction time was required (Table 1, entry 13).
seems to present another rule, that is, the lower the redox
With the aim to develop and define the scope and limit- potential, the easier the oxidation into the corresponding
ation of the present method, this laccase–AZADO catalytic oxi- oxoammonium species by laccase and the higher catalytic
dation system was then extended for the oxidation of a wide activity. To check this point, the experiments on the reaction
range of alcohols, including benzylic, allylic, heterocyclic, ali- rate were carried out using TEMPO, AZADO, 1-Me-AZADO and
cyclic, and aliphatic alcohols (Method A in Table 2). Using a 1,3-dimethyl-AZADO as catalysts. As shown in Fig. 2, the cataly-
similar strategy, the laccase–TEMPO system was also applied tic activities of these nitroxyl radicals are in the order of
for the purpose of comparison (Method B in Table 2).
AZADO > 1-Me-AZADO > 1,3-dimethyl-AZADO > TEMPO.
As shown in Table 2, most alcohols underwent oxidation to However, the Eo′ values (vs. Ag/Ag+) of these nitroxyl radicals
afford the corresponding carbonyl products in excellent yields. are in the order of TEMPO (294 mV) > AZADO (236 mV) >
The present laccase–AZADO system afforded aldehydes from 1-Me-AZADO (186 mV) > 1,3-dimethyl-AZADO (136 mV).23a No
primary alcohols and ketones from secondary alcohols. Excel- obvious correlation between the two orders is found. Thus, we
lent chemoselectivity was observed under the present system. believe that another factor that affects catalytic activity is due
For the oxidation of primary alcohol, no noticeable over-oxi- to the steric hindrance effect, compared to TEMPO or 1,3-
dation of aldehyde to carboxylic acid was detected. As the func- dimethyl-AZADO, in which the oxygen-centered unpaired elec-
tional groups, several oxidation-sensitive alkenes, electron-rich tron is sterically protected by the surrounding four or two
aromatic, heteroaromatic rings, halogens, and alkyne groups methyl groups; the α-hydrogen of AZADO furnishes a less hin-
were also tolerated during the reactions. It is noteworthy that dered reaction center, thereby enabling smooth contact
2,6-dichlorobenzyl alcohol and 2,4,6-trimethylbenzyl alcohol, between the active species due to the reduced steric hindrance
which often resist oxidation using a laccase-mediator system effect; this influence is more obvious in the oxidation of steri-
due to the steric hindrance effect,16b were also readily oxidized cally hindered alcohols (Table 2, entries 1–4, 12, 13).
to the corresponding aldehydes in high yields (Table 2, entries
In contrast to the redox potential, the energy level, or the
12 and 13). However, the oxidation of 4-hydroxybenzyl alcohol chemical structures of the mediator, little attention has been
and 4-aminobenzyl alcohol was not successful, which may be paid to the kinetic features of the redox mediator during oxi-
due to laccase oxidizing the –OH or –NH2 group by inducing dative regeneration or electron self-exchange reaction among
dimerisation and oligomerization through the intermediacy of mediator molecules. These differences between AZADO and
resonance-stabilized radicals (Table 2, entries 21 and 22). Oxi- TEMPO may be the other important factors that affect the cata-
dation of benzyl mercaptan was also not successful, and no lytic activity. Recently, the electrochemical and spectroscopic
oxidation to benzaldehyde was detected, in spite of its struc- properties of these nitroxyl radicals were measured by the
tural analogy with benzyl alcohol (Table 2, entry 23). The free Nishide group.27 According to their report, the electron self-
thiol group (–SH) has been shown to be a potent inhibitor of exchange reaction rate constant (kex) of AZADO is about 3.3 ×
laccases, presumably via co-ordination of the thiol to the 108 M−1 s−1, which is 10 times higher than that of TEMPO
copper atoms in the enzyme active site.7c Even so, the present (2.9 × 107 M−1 −1) (Scheme 1). In addition, the heterogeneous
s
laccase–AZADO system still has enough activity to oxidate electron-transfer rate constant (k0) of AZADO and TEMPO is
thioether-containing molecules, and thioanisaldehyde was 1.6 and 0.29 cm s−1, which is an important parameter indicating
produced from the corresponding alcohol with a high yield how fast the regeneration reaction of the mediator cation is.
(94%) (Table 2, entry 24); this is a very selective reaction with The significantly faster regeneration and self-exchange reac-
no sulphoxide, sulphone, or Dakin-type (phenols) by-products tion rate of AZADO are expected to be responsible for its excel-
being produced.
lent catalytic activity through smooth contact and easier
To assess the feasibility of using this method on a prepara- oxidation by laccase.
tive scale, the multigram-scale catalytic system was then exam-
The mechanistic details of the laccase-mediator catalyzed
ined for the oxidation of menthol on a 100 mmol scale. As aerobic oxidation system are still unclear.28 However, they are
expected, the reaction proceeded smoothly, similar to the generally believed to involve one electron oxidation of the
smaller-scale case, and the desired menthone was obtained in mediator by the oxidized form of laccase (cupric of T1 site in
an 80% isolated yield (Table 2, entry 3).
laccase), followed by the reaction of the oxidized mediator
The different catalytic activities between the laccase–AZADO with the substrate, either via a radical hydrogen-atom transfer
and laccase–TEMPO systems observed in Table 2 cannot be route (HAT) which is suggested for the laccase–HBT, laccase–
explained simply on the basis of the redox potential of the NHPI, and laccase–VA system, or via the electron transfer route
nitroxyl radicals. TEMPO and AZADO produced electrochemi- (ET) which is suggested for the laccase–ABTS system, or via the
cally reversible responses with redox potentials at 294 mV and ionic oxidation route which is suggested for the laccase–
236 mV versus Ag/AgCl, respectively.23h However, the T1 copper TEMPO system. To shed light on the reaction pathway of the
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Table 2 Green and selective oxidation of alcohols
Entry
1c
Alcohols
Methoda
Yieldb (%)/time
Entry
13
Alcohols
Method
Yield (%)/time
A
B
85/12 h
7/12 hd
A
B
82/24 h (87/6 hg)
45/24 h
2
A
B
92/12 h
61/24 h
14
A
B
92/6 h
90/12 h
3c
4c
A
B
84/12 h (80/24 he)
5/36 hd
15
16
A
B
79/6 h
71/12 h
A
B
90/12 h
23/24 h
A
B
85/6 h
92/6 h
5
6
A
B
72/12 h
35/24 h
17
18
A
B
86/12 h
80/12 h
A
B
92/8 h
73/12 h
A
B
94/6 h
87/6 h
7c
8
A
B
90/8 h
67/12 h
19c
20
A
B
93/6 h
90/6 h
A
B
88/8 h
65/12 h
A
B
98/4 h
96/4 h
h
9
A
B
76/12 h
47/24 h
21
22
23
A
B
—
i
10
11f
A
B
93/8 h
85/12 h
A
B
—
A
B
89/12 h
72/12 h
A
B
—/8 hj
—/8 h
12c
A
B
78/24 h
22/24 h
24
A
B
94/6 h
88/8 h
a Method A: laccase–AZADO system. Method B: laccase–TEMPO system. b Isolated yields unless otherwise noted. c The reaction was carried out in
the presence of PhCF3 as a co-solvent. d Yield was determined by 1H NMR using 1,1,2,2-tetrachloroethane as an internal standard. e Yield of large-
scale synthesis on a 100 mmol scale. f Yield was determined by GC-MS. g 20 mol% AZADO was used. h Formation of black tar was observed in this
reaction. i The reaction mixture was too complex to identify. j Unchanged substrate is recovered (91%).
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A further experiment was conducted using 5-hexen-1-ol as a
probe substrate; oxidation of 5-hexen-1-ol with the laccase–
AZADO system produced only 5-hexen-1-al. In case of the HAT
route, removal of α-H to the hydroxy group is known to afford
cyclopentyl alcohol through an intramolecular radical rearran-
gement.29a,30 The absence of a rearrangement product con-
firms that the present laccase–AZADO system also does not
follow the HAT route.
We determined the intramolecular kinetic isotope effect in
the oxidation of a suitably synthesized α-monodeutero-p-
methylbenzyl alcohol31 using Sheldon’s method.32 The kinetic
isotope effect (kH/kD value) for the laccase–AZADO catalysed
aerobic oxidation of this alcohol at room temperature was
1
determined to be 2.16 by H NMR. This value is much lower
than those obtained for the laccase–NHA system (6.2), the
laccase–HBT system (6.4), and the laccase–VA system (6.4),7a
which are expected for the HAT route. However, this value is in
the range found for the stoichiometric oxidation of benzyl
alcohols by the oxoammonium ions (1.7–3.1)33 and compares
exceptionally well with the isotope effect observed with the
laccase–TEMPO system (2.32).22b As a further proof, the AZADO
oxoammonium chloride AZADO+Cl− was synthesized ex situ23k
and used as a stoichiometric oxidant to oxidise α-monodeu-
tero-p-methylbenzyl alcohol. The KIE value for AZADO+Cl− oxi-
dation of this alcohol was determined to be 2.11, which is
much closer to that obtained from the laccase–AZADO system
(2.16). The similar kinetic isotope effects strongly suggest that
oxoammonium cation AZADO+ was generated during the reac-
tion; the oxoammonium ion oxidizes alcohol via a ionic route
which is similar to the proposed mechanism for the laccase–
TEMPO system.28
Fig. 2 Comparison of the catalytic activity of AZADO, 1-Me-AZADO,
1,3-dimethyl-AZADO, and TEMPO.
Scheme 1 One-electron transfer redox reaction of nitroxyl radicals.
On the basis of all of the aforementioned results, an ionic
oxidation mechanism is suggested for the present laccase–
AZADO system (Scheme 3). In this route, one electron oxi-
dation of AZADO affords the oxoammonium cation (I); a
nucleophilic attack of the lone-pair of alcohol onto the oxo-
ammonium cation takes place to form an adduct (II); deprotona-
tion of the adduct either intramolecularly (from N–O−) or
intermolecularly (from the base in the buffer, i.e. B)34 gives the
carbonyl product and the hydroxylamine (III). Laccase oxidises
the hydroxylamine to regenerate AZADO, and further oxidation
leads to the oxoammonium cation. Beyond that, the oxoammo-
nium cation (I) also can be restored through acid-induced dis-
proportionation of AZADO.23f The laccase is finally re-oxidised
Scheme 2 Oxidation with the laccase–AZADO system.
present laccase–AZADO system, we conducted the following by oxygen, thereby completing this green catalytic cycle.
experiments (Scheme 2). We first treated 2,2-dimethyl-1-
phenyl-1-propanol 3 with the present laccase–AZADO system.
Under the ET route, oxidation of 3 will produce benzaldehyde
4 and a tert-butyl radical by Cα–Cβ bond cleavage. Conversely,
under the HAT route, 3 undergoes cleavage of the Cα–H
benzylic bond and produces ketone 5. This clear-cut behaviour
makes 3 a useful model substrate, enabling it to assess the oxi-
dation mechanism from product analysis.28b,e,29 Results have
shown that ketone 5 was the only product produced from the
laccase–AZADO system, thereby suggesting that the reaction
Scheme 3 Suggested mechanism for the laccase–AZADO catalyzed
pathway does not involve the ET route.
aerobic oxidation.
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Infrared spectra were recorded as a KBr pellet on a Perkin-
Elmer 1600 series FT-IR spectrophotometer.
General experimental procedure for green oxidation of alco-
hols using the laccase–AZADO system (Table 2): To a stirred
solution of alcohol (1 mmol), AZADO (0.1 mmol) in acetate
buffer (0.2 M, pH 4.5, 5 mL), laccase from Trametes versicolor
(4 mg, 10 U mg−1) was then added. In case of some solid water
insoluble substrates, PhCF3 (1 mL) was added. The solution
was stirred at room temperature under an oxygen atmosphere
(balloon) for several hours while checking the reaction pro-
gress by using gas or thin-layer chromatography. After com-
pletion, the mixture was extracted with diethyl ether (3 ×
5 mL). The organic phase was concentrated under vacuum and
the crude product was purified by column chromatography
Scheme 4 Synthesis of wasabidienone B1 and wasbidienone B0.
One of the applications of the present green laccase–AZADO
system is to synthesize 1-(4-(benzyloxy)-2,6-dimethoxy-3,5-
dimethylphenyl)-2-methylbutan-1-one (7). Oxidation of 1-(4-
(benzyloxy)-2,6-dimethoxy-3,5-dimethylphenyl)-2-methylbutan-
1-ol (6) has proved difficult due to the steric hindrance effect.
The Quideau group reported the successful oxidation of 6 to 7
using stabilized IBX (3 equiv.) in the THF–DMSO solution.35
However, this method involves the use of expensive and excess
amounts of the oxidant. In the case of using the laccase–AZADO
(hexane–EtOAc
= 10 : 1) to provide the analytically pure
product, which was characterized by 1H NMR, 13C NMR and
GC-MS. The ESI† provides details of these measurements.
system, desired product
7 was obtained in 76% yield
(Scheme 4). 7 is a precursor to synthesize natural products
wasabidienone B1 and wasbidienone B0. These fungal poly-
ketides were isolated in the 1980s by Soga and co-workers from a
potato culture of Phoma wasabiae Yokogi, a fungus responsible
for the blackleg disease causing widespread destruction
among cruciferous crops such as rape, cabbage, and wasabi
(Japanese horseradish).36
In conclusion, a new and highly efficient mediator of
laccase has been comparatively evaluated. With the aid of the
highly active nitroxyl radical AZADO, a simple green method
for the aerobic catalytic oxidation of alcohols is presented. The
advantages of this catalytic oxidation system are summarized
as follows: (1) avoids the use of a transition metal catalyst; (2)
high atom economy and environmental consciousness, with
the only by-product being H2O; and (3) mild reaction con-
ditions (room temperature, air atmosphere, and no moisture
effect). Moreover, the present non-transition metal catalytic
system also provides an easy scale-up and separation protocol.
Acknowledgements
The work was supported by the National Science Fund for
Distinguished Youth Scholars (grant no. 21025625); Program
for Changjiang Scholars and Innovative Research Team in
University (grant no. IRT1066); National High-Tech Research and
Development Plan of China (863 Program, 2012AA021201);
The National Basic Research Program of China (973 Program,
2013CB733602); The Major Research plan of the National
Natural Science Foundation of China (grant no. 21390204);
The Natural Science foundation of Jiangsu Province (grant no.
BK2011031) and the Priority Academic Program from Develop-
ment of Jiangsu Higher Education Institutions.
Notes and references
1 For books and reviews, see: (a) G. Tojo and M. Fernández,
Oxidation of Alcohols to Aldehydes and Ketones, Springer,
Berlin, 2006; (b) J. E. Bäckvall, Modern Oxidation Methods,
Wiley-VCH, New York, 2004; (c) M. F. Schlecht, in Compre-
hensive Organic Synthesis, ed. B. M. Trost, I. Fleming
and S. V. Ley, Pergamon, Oxford, 1991, vol. 7, ch. 2,
pp. 251–327.
2 For reviews and accounts: (a) R. A. Sheldon, I. W. C. E. Arends,
G.-J. ten Brink and A. Dijksman, Acc. Chem. Res., 2002, 35,
774–781; (b) R. A. Sheldon and I. W. C. E. Arends, J. Mol.
Catal. A: Chem., 2006, 251, 200–214; (c) R. A. Sheldon and
I. W. C. E. Arends, Adv. Synth. Catal., 2004, 346, 1051–1071;
(d) M. J. Schultz and M. S. Sigman, Tetrahedron, 2006, 62,
8227–8241.
3 For books and reviews, see: (a) C.-H. Wong and
G. M. Whitesides, Enzymes in Synthetic Organic Chemistry,
Pergamon, Oxford, 1994; (b) K. Faber, Biotransformation in
Organic Chemistry, Springer, Berlin, 6th edn, 2011;
(c) K. Drauz, H. Gröger and O. May, Enzyme Catalysis in
Organic Synthesis, Wiley-VCH, New York, 3rd edn, 2012;
Experimental
Laccases from Trametes versicolor, Rhus vernicifera, and Agari-
cus bisporus were purchased from Sigma-Aldrich as a light
brown lyophilized powder and used without modification.
Nor-AZADO, ABNO and ABOO were synthesized according to
the reported procedures.23b,c Other reagents were ACS reagent
grade and used without further purification. NMR spectra were
recorded on a Bruker Ascend 400 MHz NMR spectrometer at
400 MHz (1H NMR) and 100 MHz (13C NMR). Chemical shifts
are reported in parts per million (ppm). H and 13C chemical
1
shifts are referenced relative to the tetramethylsilane. GC-MS
were recorded on a Thermo Trace DSQ GC-MS spectrometer
using a TRB-5MS (30 m × 0.25 mm × 0.25 mm) column.
ESI-MS were recorded on a Thermal Finnigan TSQ Quantum
ultra AM spectrometer using a TRB-5MS (30 m × 0.25 mm ×
0.25 mm) column. Melting points were determined in an open
capillary tube with a Mel-temp II melting point apparatus.
1136 | Green Chem., 2014, 16, 1131–1138
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(d) R. N. Patel, Stereoselective Biocatalysis, Academic Press, 12 (a) A. Coniglio, C. Galli, P. Gentilli and R. Vadal, J. Mol.
San Diego, 1998; (e) A. S. Bommarius and B. R. Riebel, Bio-
catalysis, Wiley-VCH, Weinheim, 2004; (f) R. N. Patel, Bio-
Catal. B: Enzym., 2008, 50, 40–49; (b) A. Wells, M. Teria and
T. Eve, Biochem. Soc. Trans., 2006, 34, 304–308.
catalysis in the Pharmaceutical and Biotechnology Industries, 13 (a) A. Potthast, T. Rosenau, C.-L. Chen and J. S. Gratzl,
CRC Press, Boca Raton, 2007; (g) K. M. Koeller and
C.-H. Wong, Nature, 2001, 409, 232–240; (h) U. T. Bornscheuer
J. Org. Chem., 1995, 60, 4320–4321; (b) E. Fritz-Langhals
and B. Kunath, Tetrahedron Lett., 1998, 39, 5955–5956.
and R. J. Kazlauskas, Angew. Chem., Int. Ed., 2004, 43, 14 (a) S. Camarero, A. I. Canas, P. Nousiainen, E. Record,
6032–6040.
A. Lomascolo, M. J. Martinez and A. T. Martinez, Environ.
Sci. Technol., 2008, 42, 6703–6709; (b) A. I. Canas, M. Alcalde,
F. Plou, M. J. Martinez, A. T. Martinez and S. Camarero,
Environ. Sci. Technol., 2007, 41, 2964–2971.
4 For books and reviews, see: (a) R. D. Schmid and
V. B. Urlacher, Modern Biooxidations: Enzymes, Reactions
and Applications, Wiley-VCH, Weinheim, 2007; (b) F. Hollmann,
I. W. C. E. Arends, K. Buehler, A. Schallmey and B. Bühler, 15 S. Molina, J. Rencoret, J. C. del Rio, A. Lomascolo,
Green Chem., 2011, 13, 226–265; (c) D. Monti, G. Ottolina,
G. Carrea and S. Riva, Chem. Rev., 2011, 111, 4111–4140;
E. Record, A. T. Martinez and A. Gutierrez, Appl. Microbiol.
Biotechnol., 2008, 80, 211–222.
(d) J. B. van Beilen, W. A. Duetz, A. Schmid and B. Witholt, 16 (a) M. Fabbrini, C. Galli, P. Gentilli and D. Macchitella,
Trends Biotechnol., 2003, 21, 170–177; (e) Z. Li, J. B. van
Beilen, W. A. Duetz, A. Schmid, A. de Raadt, H. Griengl
and B. Witholt, Curr. Opin. Chem. Biol., 2002, 6, 136–
144.
Tetrahedron Lett., 2001, 42, 7551–7553; (b) A. Potthast,
T. Rosenau, C. L. Chen and J. S. Gratzl, J. Mol. Catal. A:
Chem., 1996, 108, 5–9; (c) A. Barrilli, F. Belinghieri,
D. Passarella, G. Lesma, S. Riva, A. Silvani and B. Danieli,
Tetrahedron: Asymmetry, 2004, 15, 2921–2925.
5 For reviews, see: (a) S. R. Couto and J. L. T. Herrera, Bio-
technol. Adv., 2006, 24, 500–513; (b) P. Widsten and 17 (a) C. Zhu and Y. Wei, ChemSusChem, 2011, 4, 1082–1086;
A. Kandelbauer, Enzyme Microb. Technol., 2008, 42,
293–307; (c) T. Kudanga, G. S. Nyanhongo, G. M. Guebitz
and S. Burton, Enzyme Microb. Technol., 2011, 48, 195–
208; (d) M. Fernández-Fernández, M. Á. Sanromán and
D. Moldes, Biotechnol. Adv., 2013, 31, 1808–1825.
6 For reviews, see: (a) S. Witayakran and A. J. Ragauskas, Adv.
Synth. Catal., 2009, 351, 1187–1209; (b) A. Mikolasch and
F. Schauer, Appl. Microbiol. Biotechnol., 2009, 82, 605–624;
(c) A. Kunamneni, S. Camarero, C. García-Burgos, F. J. Plou,
(b) C. Zhu, A. Yoshimura, P. Solntsev, L. Ji, Y. Wei,
V. N. Nemykin and V. V. Zhdankin, Chem. Commun., 2012,
48, 10108–10110; (c) C. Zhu and Y. Wei, Adv. Synth. Catal.,
2012, 354, 313–320; (d) C. Zhu, A. Yoshimura, L. Ji, Y. Wei,
V. N. Nemykin and V. V. Zhdankin, Org. Lett., 2012, 14,
3170–3173; (e) C. Zhu, A. Yoshimura, Y. Wei, V. N. Nemykin
and V. V. Zhdankin, Tetrahedron Lett., 2012, 53, 1438–1444;
(f) C. Zhu, L. Ji and Y. Wei, Catal. Commun., 2010, 11,
1017–1020.
A. Ballesteros and M. Alcalde, Microbiol. Cell Fact., 2008, 7, 18 (a) Y. Tu, Z.-X. Wang and Y. Shi, J. Am. Chem. Soc., 1996,
32; (d) S. Riva, Trends Biotechnol., 2006, 24, 219–226;
(e) S. G. Burton, Curr. Org. Chem., 2003, 7, 1317–1331.
7 (a) C. Galli and P. Gentili, J. Phys. Org. Chem., 2004, 17,
973–977; (b) O. V. Morozova, G. P. Shumakovich,
118, 9806–9807; (b) Z.-X. Wang, Y. Tu, M. Frohn,
J.-R. Zhang and Y. Shi, J. Am. Chem. Soc., 1997, 119, 11224–
11235; (c) L. Shu and Y. Shi, Tetrahedron, 2001, 57,
5213–5218.
S. V. Shleev and Y. I. Yaropolov, Appl. Biochem. Microbiol., 19 (a) S. V. Ley, J. Norman, W. P. Griffith and S. P. Marsden,
2007, 43, 523–535; (c) A. Wells, M. Teria and T. Eve,
Biochem. Soc. Trans., 2006, 34, 304–308; (d) P. Baiocco,
Synthesis, 1994, 639–666; (b) R. A. Sheldon, I. W. C. E. Arends
and A. Dijksman, Catal. Today, 2000, 57, 157–166.
A. M. Barreca, M. Fabbrini, C. Galli and P. Gentili, Org. 20 Y. Ishii, S. Sakaguchi and T. Iwahama, Adv. Synth. Catal.,
Biomol. Chem., 2003, 1, 191–197; (e) K. Li, F. Xu and 2001, 343, 393–427.
K.-E. L. Eriksson, Appl. Environ. Microbiol., 1999, 65, 21 G.-J. ten Brink, I. W. C. E. Arends and R. A. Sheldon,
2654–2660. Science, 2000, 287, 1636–1639.
8 F. d’Acunzo, C. Galli and B. Masci, Eur. J. Biochem., 2002, 22 (a) M. Fabbrini, C. Galli and P. Gentili, J. Mol. Catal. B:
269, 5330–5335.
Enzym., 2002, 16, 231–240; (b) I. W. C. E. Arends, Y.-X. Li,
R. Ausan and R. A. Sheldon, Tetrahedron, 2006, 62, 6659–
6665; (c) P. Astolfi, P. Brandi, C. Galli, P. Gentilli,
M. F. Gerini, L. Greci and O. Lanzalunga, New J. Chem.,
2005, 29, 1308–1317.
9 (a) M. Marzorati, B. Danieli, D. Haltrich and S. Riva, Green
Chem., 2005, 7, 310–315; (b) D. Monti, A. Candido,
M. M. C. Silva, V. Kren, S. Riva and B. Danieli, Adv. Synth.
Catal., 2005, 347, 1168–1174; (c) L. Baratto, A. Candido,
M. Marzorati, F. Sagui, S. Riva and B. Danieli, J. Mol. Catal. 23 (a) M. Shibuya, M. Tomizawa, I. Suzuki and Y. Iwabuchi,
B: Enzym., 2006, 39, 3–8.
10 F. d’Acunzo, P. Baiocco and C. Galli, New J. Chem., 2003,
27, 329–332.
11 (a) C.-L. Chen, A. Potthast, T. Rosenau, J. Gratzl,
A. G. Kirkman, D. Nagai and T. Miyakoshi, J. Mol. Catal. B:
Enzym., 2000, 8, 213–219; (b) M.-L. Niku-Paavola and
L. Viikari, J. Mol. Catal. B: Enzym., 2000, 10, 435–444.
J. Am. Chem. Soc., 2006, 128, 8412–8413; (b) M. Shibuya,
M. Tomizawa, Y. Sasano and Y. Iwabuchi, J. Org. Chem.,
2009, 74, 4619–4622; (c) M. Hayashi, Y. Sasano,
S. Nagasawa, M. Shibuya and Y. Iwabuchi, Chem. Pharm.
Bull., 2011, 59, 1570–1573; (d) M. Shibuya, Y. Sasano,
M. Tomizawa, T. Hamada, M. Kozawa, N. Nagahama and
Y. Iwabuchi, Synthesis, 2011, 3418–3425; (e) M. Shibuya,
This journal is © The Royal Society of Chemistry 2014
Green Chem., 2014, 16, 1131–1138 | 1137

View Article Online
Communication
Green Chemistry
Y. Osada, Y. Sasano, M. Tomizawa and Y. Iwabuchi, J. Am.
Chem. Soc., 2011, 133, 6497–6500; (f) M. Hayashi,
M. Shibuya and Y. Iwabuchi, J. Org. Chem., 2012, 77, 3005–
197; (f) A. Marjasvaara, M. Torvinen and P. Vainiotalo,
J. Mass Spectrom., 2004, 39, 1139–1146; (g) G. Cantarella,
C. Galli and P. Gentili, New J. Chem., 2004, 28, 366–372.
3009; (g) M. Shibuya, T. Sato, M. Tomizawa and Y. Iwabuchi, 29 (a) E. Baciocchi, S. Belvedere, M. Bietti and O. Lanzalunga,
Chem. Commun., 2009, 1739–1741; (h) M. Shibuya, F. Pichierri,
M. Tomizawa, S. Nagasawa, I. Suzuki and Y. Iwabuchi,
Tetrahedron Lett., 2012, 53, 2070–2073; (i) B. Graetz,
S. Rychnovsky, W.-H. Leu, P. Farmer and R. Lin, Tetra-
Eur. J. Org. Chem., 1998, 299–302; (b) E. Baciocchi,
S. Belvedere and M. Bietti, Tetrahedron Lett., 1998, 39,
4711–4714; (c) E. Baciocchi, M. Mattioli, R. Romano and
R. Ruzziconi, J. Org. Chem., 1991, 56, 7154–7160.
hedron: Asymmetry, 2005, 16, 3584–3598; ( j) Y. Demizu, 30 D. Griller and K. U. Ingold, Acc. Chem. Res., 1980, 13, 317–323.
H. Shiigi, T. Oda, Y. Matsumura and O. Onomura, Tetrahe- 31 (a) A. D. N. Vaz and M. J. Coon, Biochemistry, 1994, 33,
dron Lett., 2008, 49, 48–52; (k) M. Hayashi, M. Shibuya and
Y. Iwabuchi, Org. Lett., 2012, 14, 154–157.
6442–6449; (b) H. L. Holland, F. M. Brown and M. Conn,
J. Chem. Soc., Perkin Trans. 2, 1990, 1651–1655.
24 Y. Kuang, Y. Nabae, T. Hayakawa and M. Kakimoto, Green 32 (a) A. Dijksman, A. Marino-González, A. M. Payeras,
Chem., 2011, 13, 1659–1663.
25 (a) O. Milstein, B. Nicklas and A. Hüttermann, Appl. Micro-
biol. Biotechnol., 1989, 31, 70–74; (b) J. Rodakiewicz-Nowak,
I. W. C. E. Arends and R. A. Sheldon, J. Am. Chem. Soc.,
2001, 123, 6826–6833; (b) A. Dijksman, I. W. C. E. Arends
and R. A. Sheldon, Org. Biomol. Chem., 2003, 1, 3231–3237.
Top. Catal., 2000, 11/12, 419–434; (c) J. M. M. Verkade, 33 (a) M. F. Semmelhack, C. R. Schmid and D. A. Cortés, Tetra-
L. J. C. van Hemert, P. J. L. M. Quaedflieg, H. E. Schoemaker,
M. Schürmann, F. L. van Delft and F. P. J. T. Rutjes, Adv.
Synth. Catal., 2007, 349, 1332–1336.
hedron Lett., 1986, 27, 1119–1122; (b) V. A. Golubev,
V. N. Borislavskii and A. L. Aleksandrov, Izv. Akad. Nauk.
SSSR, Ser. Khim., 1977, 2025–2034.
26 K. Piontek, M. Antorini and T. Choinowski, J. Biol. Chem., 34 (a) Z. Ma and J. M. Bobbitt, J. Org. Chem., 1991, 56, 6110–
2002, 277, 37663–37669.
27 F. Kato, A. Kikuchi, T. Okuyama, K. Oyaizu and H. Nishide,
Angew. Chem., Int. Ed., 2012, 51, 10177–10180.
28 (a) F. d’Acunzo, P. Baiocco, M. Fabbrini, C. Galli and
P. Gentilli, Eur. J. Org. Chem., 2002, 4195–4201; (b) M. Fabbrini,
6114; (b) A. E. J. de Nooy, A. C. Besemer and H. van
Bekkum, Synthesis, 1996, 1153–1174.
35 L. Pouysegu, M. Marguerit, J. Gagnepain, G. Lyvinec,
A. J. Eatherton and S. Quideau, Org. Lett., 2008, 10, 5211–
5214.
C. Galli and P. Gentili, J. Mol. Catal. B: Enzym., 2002, 18, 36 (a) O. Soga, H. Iwamoto, S. Date, T. Watanabe, K. Tanaka,
169–171; (c) G. Cantarella, C. Galli and P. Gentilli, J. Mol.
Catal. B: Enzym., 2003, 22, 135–144; (d) C. Crestini,
L. Jurasek and D. S. Argyropoulos, Chem.–Eur. J., 2003, 9,
5371–5378; (e) P. Baiocco, A. M. Barreca, M. Fabbrini,
C. Galli and P. Gentili, Org. Biomol. Chem., 2003, 1, 191–
K. Hata, A. Takuwa and M. Nakayama, Chem. Lett., 1984,
339–340; (b) O. Soga, H. Iwamoto, A. Takuwa, H. Nozaki,
J. Kuramoto and M. Nakayama, Agric. Biol. Chem., 1987, 51,
283–284; (c) J. O. Omolo, H. Anke, S. Chhabra and
O. Sterner, J. Nat. Prod., 2000, 63, 975–977.
1138 | Green Chem., 2014, 16, 1131–1138
This journal is © The Royal Society of Chemistry 2014