Michael addition of pyrimidine derivatives with acrylates catalyzed by lipase TL im from Thermomyces lanuginosus in a continuous-flow microreactor

Article abstract of DOI:10.1039/c3ra47470d

Lipase-catalyzed Michael addition of pyrimidine derivatives to acrylates in a continuous-flow microreactor is described. The influence of the structure of the Michael acceptor and the corresponding donor on the enzymatic addition was also investigated. The important features of this method include mild reaction conditions, short reaction times (30 min) and high yields.

Full text of DOI:10.1039/c3ra47470d

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Michael addition of pyrimidine derivatives with
acrylates catalyzed by lipase TL IM from
Thermomyces lanuginosus in a continuous-flow
microreactor†
Cite this: RSC Adv., 2014, 4, 7770
Received 10th December 2013
Accepted 13th January 2014
a
a
ab
Li-Hua Du,* Hui-Min Ling and Xi-Ping Luo
DOI: 10.1039/c3ra47470d
www.rsc.org/advances
Lipase-catalyzed Michael addition of pyrimidine derivatives to acry- reaction times. Our interest in microreactors prompted us to
lates in a continuous-flow microreactor is described. The influence of ask if a Michael addition of pyrimidine derivatives to acrylates
the structure of the Michael acceptor and the corresponding donor on catalyzed by lipase TL IM from Thermomyces lanuginosus would
the enzymatic addition was also investigated. The important features work when performed in a continuous-ow microreactor. We
of this method include mild reaction conditions, short reaction times report here, for the rst time, the production of lipase-catalyzed
(
30 min) and high yields.
Michael additions of pyrimidine derivatives (uracil, 5-uoro-
uracil and thymine) to acrylates (methyl acrylate, acrylonitrile,
butyl acrylate and methyl methacrylate) in a continuous-ow
microreactor (Scheme 1). The aim of this paper is to investigate,
under a continuous-ow microreactor, the effect of the donor
structure (uracil, 5-uorouracil and thymine) and the acceptor
Microreactors are a relatively new technology for performing
safer, more efficient, and more selective reactions.
improved performance is attributed to more rapid heat transfer
and mixing as a result of the increased surface-area to volume
ratio. The advantages of microreactors have been substantiated
by a growing number of examples over the past decade.
In recent years, microreactors containing immobilized enzymes
have attracted considerable attention, as a consequence of
1
–4
The
(methyl acrylate, acrylonitrile, butyl acrylate and methyl meth-
2
acrylate) on the reaction yield. These reactions were catalyzed by
Lipozyme TL IM from Thermomyces lanuginosus using DMSO as
a solvent.
The equipment conguration that was used for the enzy-
matic Michael addition of pyrimidine derivatives with acrylates
starting from 5-uorouracil and methyl acrylate is described in
Fig. 1. Harvard Apparatus PHD 2000 syringe pumps were used
to deliver reagents from syringes to the reactor. On the syringe
pump, a 10 mL syringe with the 5-uorouracil solution and a
5–13
14–23
their many potential industrial applications.
Important
advantages over conventional batch reactions for biocatalytic
reactions include high throughput, reduced reaction time, and
high conversion efficiency.
The Michael addition reaction is among the most funda-
mental types of reactions in organic synthesis. Michael addi-
tions are generally promoted by harsh bases or strong acids,
which could lead to environmentally hazardous residues and
undesirable by-products.
types of catalysts have been developed. The most common
catalysts reported for Michael additions are transition metals or
10 mL syringe with methyl acrylate in DMSO were mounted.
Lipozyme TL IM was lled in silica gel tubing (inner diameter
ID ¼ 2.0 mm, length ¼ 1 m). The temperature of this reaction
was controlled by a water bath by immersion of tubing in water
and control the temperature of water. Streams 1 and 2 were
24–29
To avoid these problems, various
30
lanthanide catalysts. Enzymes provide a alternative to these
inorganic catalysts. Some natural lipases
31–33
34–36
and proteases
have been applied in Michael-type addition reactions to form
carbon–nitrogen and carbon–sulfur bonds. However, most of
these methods led to lower conversion yields and longer
a
College of Pharmaceutical Science, ZheJiang University of Technology, Hangzhou,
3
10014, China. E-mail: orgdlh@gmail.com; orgdlh@zjut.edu.cn; Fax: +86 571
8320903
8
b
Department of Environmental Science and Technology, ZheJiang A&F University,
Hangzhou, 311300, China
Scheme 1 Michael addition of pyrimidine derivatives with acrylates
catalyzed by lipase TL IM from Thermomyces lanuginosus in a
continuous-flow microreactor.
†
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7770 | RSC Adv., 2014, 4, 7770–7773
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Fig. 1 Microreactor setup for the continuous-flow Michael addition
reaction of pyrimidine derivatives with acrylates catalyzed by Lipozyme
TL IM from Thermomyces lanuginosus.
ꢀ1
mixed together at a ow rate of 10.4 mL min in a Y-mixer at 50
ꢁ
ꢀ1
C and the resulting stream (20.8 mL min ) was connected to a
sample vial which was used to collect the nal mixture.
The structure of the Michael acceptor and donor can affect
the results of the enzymatic Michael reaction. Fig. 2 summarizes
the donor structure effect on the Michael addition in micro-
reactors. These results indicate that 5-uorouracil was the most
reactive substrate. In fact, conversion yields were approximately
Fig. 3 Acceptor structure effect on the Michael addition performance
with 5-fluorouracil carried out in microreactors using from Thermo-
myces lanuginosus lipase.
investigated in ratios from 1 : 1 to 1 : 7 for pyrimidine–acrylate.
Fig. 4 shows the strong effect of this parameter on the enzymatic
Michael addition reaction; the best result can be obtained with
the ratio of donor–acceptor ¼ 1 : 5.
96, 88 and 80%, respectively, for 5-uorouracil, uracil and
thymine. The high conversion yield obtained with 5-uorouracil
can be explained as an effect of an electron-withdrawing group
on the donor structure. An electron-withdrawing group on the
donor will increase its reactivity, and an electron-donating group
will have the opposite effect. Under the same condition, the
Michael reaction of uracil and methyl acrylate was more rapid
than that using thymine as the donor, while using 5-uorouracil
as the donor lowered the reaction rate.
We have also investigated the acceptor structure effect on
Michael additions and found the longer alcohol chain, the
lower the yield. Using 5-uorouracil as the donor, the decrease
of yields was detected with the increase of the alcohol chain.
The steric effect of the side chain in the acceptor also affects its
reactivity. The yield of adduct detected by HPLC was less than
Furthermore, the inuence of the reaction time/ow rate on
0
the conversion of methyl 3-(1 -uracil)propionate was also studied.
0
Fig. 5 shows that the best conversion of methyl 3-(1 -uracil)
propionate was observed at a residence time of 30 minutes and a
ꢀ
1
ow rate of 20.8 mL min
.
Finally, to explore the scope and limitations of this new high-
speed Michael addition of pyrimidine derivatives to acrylates in
a continuous-ow microreactor, three pyrimidine derivatives,
5
(
-uorouracil (1a), uracil (1b), thymine (1c), and four acrylates
2a–d) were subjected to the general reaction conditions, using
both a single-mode shaker reactor and a continuous
ow/microreactor processing. For the shaker experiments,

1
(
0% in the reaction of 5-uorouracil and methyl methacrylate
Fig. 3).
Following the standard procedure given above, the effect of
reaction times needed to be about 24 h or more to obtain
ideal conversion (Method A). Using lipase-catalyzed Michael
addition of pyrimidine derivatives with acrylates under
continuous-ow conditions, 12 adducts have synthesized
in parallel in a single experiment at the same ow rate
molar ratio (donor–acceptor) on the enzymatic Michael reaction
of pyrimidine derivatives with acrylates in a microreactor was
Fig. 2 Donor structure effect on the conversion of the methyl acrylate
reaction with N-substituted pyrimidine derivatives carried out in Fig. 4 The influence of Michael donor–acceptor on the enzymatic
microreactors using Thermomyces lanuginosus lipase.
Michael addition reaction in a microreactor.
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unprecedented efficiency using a ow microreactor approach.
The large surface-area-to-volume ratios of the catalyst, Lipozyme®
TL IM adsorbed on silica particles is the key to the success of this
protocol. The adsorbed catalyst permits the substrate pyrimidine
derivatives and acrylates to make efficient contact and react within
the microreactor environment. The salient features of this
ꢁ
method include mild reaction conditions (50 C), short reac-
tion times (30 min) and high yields that make our method-
ology a valuable contribution to the eld of N-substituted
pyrimidine derivatives synthesis. The method of enzymatic
synthesis in a microreactor environment described here may
have general applications to synthetic organic chemistry
by enzymatic catalysis in the future. Michael additions of
imidazole, purine, amine and other nitrogen nucleophiles to
a,b-ethylenic compounds catalyzed by lipase TL IM from
Thermomyces lanuginosus in a continuous-ow microreactor
are in progress.
The authors would like to thank National Natural Science
Foundation of China (no. 21306172) and the Natural Science
Foundation of Zhejiang Province (LY13B020010 and
LY13C160008), the Science and Technology Research Program
of Zhejiang Province (2008C12084) as well as the Natural
Science Foundation of Zhejiang University of Technology
Fig. 5 The influence of reaction time on the conversion of methyl 3-
0
(
1 -uracil)propionate in microreactor.
(Method B). The results were better with ow/microreactor pro-
cessing than with the single-mode shaker (Table 1, entry 1–12).
Importantly, applying continuous ow/microreactor processing,
resulted in a conversion of to N-substituted pyrimidine deriva-
tives of 80% or more. This allows us to reduce the reaction time
and simplify the purication of products.
In conclusion, we have demonstrated that Michael addition
of pyrimidine derivatives with acrylates can be carried out with
(116004029) for nancial support.
Notes and references
Table 1 Shaker and continuous flow synthesis of pyrimidine deriva-
tives to acrylates catalyzed by Lipozyme TL IM from Thermomyces
lanuginosus
1
2
3
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70
75
65
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71
96
92
80
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10
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85
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68
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ꢀ1
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