A lipase-glucose oxidase system for the efficient oxidation of: N -heteroaromatic compounds and tertiary amines

Article abstract of DOI:10.1039/c6gc00780e

In this work, a lipase-glucose oxidase system has been designed and proven to be an efficient system for the oxidation of N-heteroaromatic compounds and tertiary amines. This dual-enzyme system not only displays environmental friendliness, but also demonstrates its huge potential in industrial applications.

Full text of DOI:10.1039/c6gc00780e

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A lipase-glucose oxidase system for the efficient oxidation of N-
heteroaromatic compounds and tertiary amines
Received 00th January 20xx,
Accepted 00th January 20xx
a
a
b
a,*
a,*
Fengjuan Yang, Xiaowen Zhang, Fengxi Li, Zhi Wang, and Lei Wang
DOI: 10.1039/x0xx00000x
www.rsc.org/
In this work, a lipase-glucose oxidase system has been designed for the efficient oxidation of N-heteroaromatic compounds
and proved to be an efficient system for the oxidation of N- and tertiary amines (Scheme 1). In this system, glucose oxidase,
heteroaromatic compounds and tertiary amines. This dual-enzyme an inexpensive enzyme, was adaped for the in situ generation
system not only displays environmental friendliness, but also of H
2
O
2
to avoid the enzyme inactivation. And then the
demonstrates the huge potential of industrial applications.
2 2
generated H O was used for the lipase-catalyzed synthesis of
peracids, which could catalyze the oxidation of N-
heteroaromatic compounds and tertiary amines. This “feed-
on-demand” reaction system is a green and efficient system.
Furthermore, this is the first report of a dual-enzyme system
contained GOX and lipase.
Aliphatic and heterocyclic N-oxides (amine N-oxides) are
important components for their wide usefulness in inorganic
and organic chemistry [1-3]. They can be used as protecting
groups, auxiliary agents, oxidants, catalysts, surrogates for
heterocyclic boronic acids and ligands in metal complexes [4-8].
A variety of catalysts have been developed to prepare these
amine N-oxides, such as Caro's acid (H SO ), dioxiranes,
Glucose oxidase
Glucose
2 2
H O
in situ
2
5
in situ Ethyl acetate/Lipase
peracids, activated molecular oxygen/hydrogen peroxide/tert-
butylhydroperoxide and magnesium monophthalate [9-15].
However, most of them are expensive, inefficient and toxic.
Thus, mining an environmentally clean synthetic method is
essential.
Peracid
N
or
NR
3
N
O
oRr
3
H
2
N
O
in situ Oxidation
1
2
Scheme
1 Lipase-glucose oxidase system for the oxidation of N-heteroaromatic
Lipase (EC 3.1.1.3) could catalyze the generation of
peracids through a perhydrolysis of carboxylic acids or esters
in the presence of hydrogen peroxide (H O ). And the formed
compounds and tertiary amines
2
2
Pyridine was used as a model substrate for the oxidation to
the corresponding N-oxide in the lipase-glucose oxidase
system. Lipase B from Candida antarctica (CalB) and glucose
oxidase from A. niger were reported to be the efficient
peroxyacids have been successfully utilized in the epoxidation
of alkenes and Baeyer-Villiger reactions [16-20]. In our
unpublished research, we found that this method can also be
applied for the epoxidation of N-heteroaromatic compounds.
2 2
catalysts for the in situ generation of peracids and H O ,
However, the activated oxygen species such as H O2 or
2
respectively [23-26]. Therefore, we selected these two
enzymes as the catalysts in this work. Initially, the different
hydroxyl radicals could easily induce the enzyme inactivation
and decrease the final oxidation yield. It’s well known that
glucose oxidase (GOX, EC 1.1.3.4) can generate H O in a mild
feeding modes of H
investigated and the results were shown in Table 1. Either
stepwise or continuous addition of H combined with CalB in
this oxidation led to the lower production of the pyridine N-
oxide (entry 1, 2). The in situ generation of H by GOX could
improve the efficiency of H , avoid the inactivation of lipase,
2 2
O in the oxidation of pyridine were
2
2
reaction condition with glucose as its substrate. Water and
gluconolactone are its side products which is non-toxic and
environment friendly [21-22].
2
O
2
2
O
2
Herein, we designed a novel lipase-glucose oxidase system
2 2
O
and then higher yield could be obtained (entry 3). It should be
noted that in the dual-enzyme catalyzed oxidation of pyridine,
the highest yield could be obtained (entry 4) when the oxygen
(
1 mL/min) was bubbled into the reaction mixture because the
activity of GOX would be increased in the oxygen. So, oxygen
was used to improve the efficiency of this dual-enzyme system
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for further investigations. And also, the result of the oxidation could not be improved with further increasing the concentration
catalyzed by chemical catalyst (entry 5) or the blank of enzymes. Therefore, 30 U/mL of CalB and 42 U/mL of GOX
experiment (entry 6) demonstrated that the lipase-glucose turned out to be sufficient in this dual-enzyme catalyzed
oxidase system is mild, efficient and worthy of further study.
oxidation.
2 2
Table 1. The effect of feeding modes of H O in the oxidation of pyridine
Entry
Catalyst
CalB+ H (Stepwise)
CalB+ H
Yield
48.2±1.2
71.3±2.1
89.1±1.6
97.6±1.8
53.9±2.7
0
a
100
1
2
3
4
5
6
2 2
O
a
2
O
2
(Continuous)
90
b
CalB+GOX ( air)
8
7
6
5
4
3
0
0
0
0
0
0
b
CalB+GOX (oxygen)
c
m-CPBA
2 2
H O
a)
Reaction conditions: pyridine (1 mmol), Ethyl acetate (EA, 3 mL), CalB (30
b)
U/mL),
2 2
H O (10% aqueous solution, 1.2 mmol), room temperature, 1h;
pyridine (1 mmol), glucose (1.2 mmol), Phosphate buffer (PB, pH 7.0), Ethyl
c)
acetate (EA, 2 mL), CalB (30 U/mL), GOX (42 U/mL), room temperature, 1h;
catalyzed by 3-chloroperbenzoic acid (m-CPBA, 1.2 mmol).
10
15
20
25
30
35
40
The concentration of CalB (U/mL)
2 2
The production and consumption of H O is a key factor for
Fig. 2. The effect of the concentration of CalB in the oxidation of pyridine. Reaction
conditions: pyridine (1 mmol), glucose (1.2 mmol), reaction media (3 mL, PB/EA=1/2),
oxygen (1mL/min), room temperature, GOX:CalB = 1.4:1, 1h.
the dual-enzyme system. Therefore, an optimum operational
ratio of GOX and CalB is important in this reaction system. We
fixed the concentration of CalB at 30 U/mL and investigated
the effects of the ratio of GOX and CalB. The results indicated
Reaction medium may seriously affect the catalytic
performance and stability of enzyme [27]. It’s well known that
phosphate buffer is the common solvent for GOX catalyzed
reactions [28]. And ethyl acetate can be used as substrate and
reaction medium for the lipase-mediated oxidation according
to the previous reports [20, 24]. Accordingly, we adapted a bi-
phase reaction medium (PB and EA) for this dual-enzyme
reaction. The effect of ratio of PB to EA was investigated when
the total reaction volume was fixed to 3 mL. As shown in
Figure 3, the yield of N-oxide exhibited a bell shaped curve
with the ratio PB to EA changing, and the optimal ratio was
2 2
that the initial formation of H O appeared to be the rate-
limiting step for this dual-enzyme catalyzed oxidation. As
shown in Fig. 1, the yield of pyridine N-oxide increased as the
ratio of GOX and CalB was increased (1:1-1.4:1). Further
increasing the GOX concentration from 1.5:1 to 2.0:1 resulted
in gradual decline in the yield of N-oxide. This was possibly due
2 2
to that the accumulation of H O in the reaction system could
inactivate CalB. We therefore concluded that in this system,
the optimal ratio of GOX and CalB was 1.4:1.
1
:2. The possible explanation may be that the ratio of PB and
EA could affect the rate of generation of H O , and then
2
2
1
10
00
influence the final yield of N-oxide.
1
9
8
7
6
5
4
3
2
0
0
0
0
0
0
0
0
1
00
9
8
7
6
5
4
3
0
0
0
0
0
0
0
1.0
1.2
1.4
1.6
1.8
2.0
the ratio of GOX and CalB (U/U)
Fig. 1. The effect of the ratio of GOX and CalB in the oxidation of pyridine. Reaction
conditions: pyridine (1 mmol), glucose (1.2 mmol), reaction media (3 mL, PB / EA = 1/2),
CalB (30 U/mL), oxygen (1mL/min), room temperature, 1h.
2:1
1:1
1:2
1:3
1:4
The ratio of phosphate buffer to ethyl acetate
The effect of the concentration of enzyme on the yield was
investigated when the volume of reaction media was fixed at 3
mL and the ratio of two enzymes (GOX : CalB) was kept
constant (1.4:1). It was found that higher concentration of
enzymes could enhance the yield of N-oxide. But the yield
Fig. 3. The effect of reaction media in the oxidation of pyridine. Reaction conditions:
pyridine (1 mmol), glucose (1.2 mmol), reaction media (3 mL), CalB (30 U/mL), GOX (42
U/mL), oxygen (1mL/min), room temperature, 1h.
2
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The time course of the enzyme mediated synthesis of Table 2. The oxidation of varity N-heteroaromatic compounds and tertiary amines
pyridine N-oxide was illustrated under two type feeding modes
2 2
of H O , respectively. As displayed in Fig. 4, the initial reaction
Pyridine derivatives (1a-1l)
rate of the continuous feeding was higher than that of the in
situ generation mode. After 0.4 h, the oxidation carried out in
the dual-enzyme system is significantly faster and affords
higher yields than the reaction using the continuous feeding
mode of H
reached its equilibrium in approximately 1 h with a yield of
7.6% of pyridine N-oxide. This interesting observation
indicates that the in situ generation of H can obviously
2 2
O . The oxidation in the dual-enzyme system
9
2 2
O
avoid the enzyme inactivation and increase the final oxidation
yield.
N-heteroaromatic compounds (1m-1p) and tertiary amines (1q-1t)
1
00
80
60
40
20
0
in situ generation
continuous feeding
0.2
0.4
0.6
0.8
1.0
1.2
1.4
Time (h)
a)
Fig. 4. The time course of the synthesis of pyridine N-oxide. Reaction conditions:
pyridine (1 mmol), glucose (1.2 mmol), reaction media (3 mL, PB/EA = 1/2), CalB (30
b)
U/mL), GOX (42 U/mL), oxygen (1mL/min), room temperature, 1h; pyridine (1 mmol),
Reaction conditions: N-heteroaromatic compound or tertiary amine (1 mmol),
glucose (1.2 mmol), reaction media (3 mL, PB/EA=1/2), CalB (30 U/mL), GOX (42
U/mL), oxygen (1mL/min), room temperature, 1h.
EA (3 mL), CalB (30 U/mL), H
h.
2 2
O (10% aqueous solution, 1.2 mmol), room temperature,
1
Under the optimal reaction conditions, we scaled up the
dual-enzyme system to 100-fold (pyridine (100 mmol), glucose
120 mmol), ethyl acetate (200 mL), phosphate buffer (100
mL), CalB (9000 U), GOX (12600U)). The yield of pyridine N-
Conclusions
(
In this work, we reported a novel dual-enzyme system,
oxide was up to 98.7% after 1h. This result demonstrated that which can be applied for the oxidation of N-heteroaromatic
this green method has high potential for industrial production. compounds and tertiary amines effectively. Furthermore, this
To evaluate the scope and limitation of this method, a wide green method can be easily scaled up, and so seems to be a
variety of N-heteroaromatic compounds and tertiary amines very exciting and promising method which could contribute to
were used in this oxidation (Table 2). It could be observed the development of new synthetic approache and green
from Table 2 that pyridines containing electron donating technology. However, the reusability of catalyst should be
groups (1a-1i) were more active than pyridines bearing considered in this dual-enzyme system. It's well known that
electron withdrawing groups (1j-1l). And steric effects of the immobilization is a useful tool to improve the reusability and
substituents also affected the reaction, because ortho- stability of enzyme in modern biotechnology [29-30]. In order
substituted pyridines resulted in the lower yields. Other N- to decrease the cost of this dual-enzyme system, a study
heteroaromatic compounds (1m-1p) and tertiary amines (1q- adopting the technique of co-immobilization is underway and
1t
) can also be oxidized to their corresponding N-oxides in will be reported in due course.
good to excellent yields. All the above results indicated that
this dual-enzyme system can work well with different N-
heteroaromatic compounds and tertiary amines in this
oxidation.
Acknowledgements
We gratefully acknowledge the National Natural Science
Foundation of China (no. 21172093), the Natural Science
Foundation of Jilin Province of China (no. 20140101141JC) and
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Graduate Innovation Fund of Jilin University for the financial
support (no. 2015023).
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