Probing the effects of microwave irradiation on enzyme-catalysed organic transformations: The case of lipase-catalysed transesterification reactions

Article abstract of DOI:10.1039/b617544a

The lipase-catalysed transesterification reaction of methyl acetoacetate in toluene as a solvent has been studied using carefully controlled conditions. Results suggest that microwave heating does not have a noticeable effect on reaction rate or product conversion. This journal is The Royal Society of Chemistry.

Full text of DOI:10.1039/b617544a

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www.rsc.org/obc | Organic & Biomolecular Chemistry
Probing the effects of microwave irradiation on enzyme-catalysed organic
transformations: the case of lipase-catalysed transesterification reactions†
Nicholas E. Leadbeater,* Lauren M. Stencel and Eric C. Wood
Received 1st December 2006, Accepted 26th January 2007
First published as an Advance Article on the web 16th February 2007
DOI: 10.1039/b617544a
The lipase-catalysed transesterification reaction of methyl acetoacetate in toluene as a solvent has been
studied using carefully controlled conditions. Results suggest that microwave heating does not have a
noticeable effect on reaction rate or product conversion.
Introduction
Results and discussion
Microwave heating is a valuable tool for synthetic chemists. It
is possible to improve product yields and enhance the rate of
reactions as well as being a safe and convenient method for heating
reaction mixtures to elevated temperatures.1,2 It is possible to
perform reactions even at modest reaction temperatures and still
see great improvements in rate and yield. Sealed reaction vessels
are only one option, standard reflux and open vessel chemistry can
benefit from microwave irradiation. As the range of techniques for
microwave heating has expanded, so have the areas in which it can
have a profound impact. One case is its application to biologically
relevant processes such as the synthesis of peptides,3 peptoids,4
oligopeptides5 and carbohydrates6 and in the field of proteomics.7
Also included in this broad area is the application of microwave
heating to enzyme-catalyzed organic synthesis.8 Over the last
few years there have been a number of reports suggesting that
enzyme activity can be enhanced by using controlled microwave
irradiation and suggesting that these effects are non-thermal in
origin. Much of this work has been focused around the use
of lipases: researchers stating that reaction rates and product
yields obtained in esterification and transesterification reactions
catalysed by lipases are significantly higher when using microwave
heating.9–13 Also, enzymatic stability is reportedly higher under
microwave heating than under conventional thermal heating.14
Very recently, a paper by some of the authors of these previous
reports10,12 was published. It suggests that identical initial rate and
conversion yield were obtained under microwave and conventional
heating for the transesterification reaction between ethyl butyrate
and butanol in a solvent-free system but that the kinetics of
inactivation of the lipase are influenced differently depending on
the heating method used.15 This has prompted us to report recent
findings from our laboratory directed around the investigation
of microwave-promoted lipase-catalysed transesterification reac-
tions.
Since the transesterification reaction between methyl acetoacetate
and primary alcohols had been the subject of several previous
investigations, we decided to start our study using this as a
model.13 As the enzyme, we used lipase from Candida antartica.
Our initial objective was to compare the rate of reaction using
microwave and conventional heating. To achieve this, we knew that
it was key to perform the reactions under as closely comparable
reaction conditions as possible. For microwave experiments we
used a scientific monomode apparatus, equipped with a fiber-
optic temperature measurement device. Reactions were performed
in a round-bottomed flask with the temperature probe inserted
into the reaction mixture by means of a sapphire thermal well.
Conventional experiments were performed in the same vessel using
the same temperature probe but were placed into a pre-heated oil
bath. In both cases, the reaction mixture was stirred throughout.
We focused on the reaction of methyl acetoacetate and n-propanol
using toluene as a solvent. Toluene was chosen both because it
has been used previously but also because it is not particularly
microwave absorbent, a characteristic that would be useful in later
experiments.
Reaction mixtures were heated to the desired temperature in the
absence of the enzyme and only when this was stabilized did we
add the lipase (the reaction did not occur in the absence of the
enzyme). We performed the reaction at 50 ◦C and at 70 ◦C for
2.5 h. Plots of product conversion vs. time are shown in Figs. 1
and 2. The plots show that there is negligible difference in rate
when using microwave and conventional heating.
When running the reaction at 70 ◦C, the microwave apparatus
holds the reaction mixture at temperature using intermittent pulses
of 25 W power. This is a relatively low irradiation power and
thus we next wanted to determine the effect, if any, of varying
microwave power delivered from the magnetron while keeping the
bulk temperature of the reaction mixture the same. To achieve
this we applied controlled cooling in conjunction with microwave
heating. Data are shown in Table 1. By irradiating a reaction
mixture with microwaves while simultaneously cooling the outer
vessel walls with compressed air or cryogenic fluid, it is possible
to irradiate the sample with significant microwave power while
holding it at a set bulk temperature during the whole period of the
Department of Chemistry, University of Connecticut, 55 North Ea-
gleville Road, Storrs, CT 06269-3060, USA. E-mail: nicholas.leadbeater@
uconn.edu; Fax: +1 860 486 2981; Tel: +1 860 486 5076
† Electronic supplementary information (ESI) available: Power and tem-
perature vs. time profiles for the microwave-promoted reactions. See DOI:
10.1039/b617544a
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efficiency. Thus, both to address this and act as a control, we
first performed the reaction conventionally using a glass tube. The
◦
reaction mixture was equilibrated at 50 C in an oil bath before
adding the enzyme and running the reaction for 1 h. A product
conversion of 31% was obtained (Table 1, entry 1), compared to
49% when using a round-bottomed flask.
We next performed the reaction in the microwave with no
external cooling. The reaction was performed using the CEM
Coolmate apparatus. This comprises of the same monomode
apparatus as used previously but equipped with a jacketed
vessel around which microwave-transparent cryogenic fluid can
be passed.18,19 Due to the dimensions of the vessel (∼0.6 cm i.d.),
it was hard to ensure complete delivery of the enzyme while the
equipment was running. As a result, rather than equilibrate
the reagents at the desired temperature of 50 ◦C and then add
the enzyme, we took a different strategy. We pre-equilibrated a
◦
toluene solution of propanol and the enzyme at 50 C and then
added the methyl acetoacetate. After 1 h microwave irradiation of
the complete reaction mixture a product conversion of 32% was
obtained (Table 1, entry 2); this being almost identical to that from
the control reaction using oil-bath heating.
Fig. 1 Lipase catalysed transesterification between methyl acetoacetate
and n-propanol at 50 ◦C.
We performed the reaction using air cooling in conjunction
with microwave heating. To do this, we used a 10 mL glass tube
as the reaction vessel. Significantly more microwave power was
applied during the course of the reaction as compared to the
case with no cooling but a similar product conversion (28%) was
obtained (Table 1, entry 3). We finally ran the reaction with cooling
using cryogenic fluid. In this case it was possible to introduce the
maximum power possible with the apparatus (300 W) but with no
increase in product conversion (30%, Table 1, entry 4).
Our results suggest that the microwave power applied does
not have a noticeable effect on product conversion in the lipase-
catalysed transesterification reaction of methyl acetoacetate in
toluene as a solvent. Also, because the enzyme was added
before the equilibration stage, it is also apparent that microwave
irradiation does not have a noticeable effect on the inactivation of
the lipase during this period.
Kerep and Ritter recently reported the results of studies on the
microwave-assisted lipase-catalysed ring-opening polymerization
of e-caprolactone in boiling solvents.20 They found that in the case
of boiling toluene or benzene, the microwave reaction proceeded
slower compared to oil bath heating. On the other hand, using
boiling diethyl ether as solvent, the product yield was about
10% higher when using microwave heating as opposed to oil
bath heating. We believe that these results can be interpreted
in terms simply of the temperature at which the reactions are
performed. When refluxing toluene or benzene using significant
microwave energy input, instantaneous local temperatures could
be significantly higher than the boiling point and this could lead
to rapid denaturing of the lipase. Using conventional heating,
the lifetime could be somewhat extended and thus more product
could be formed before the enzyme denatures. With diethyl ether,
because the boiling temperature is lower, denaturing of the enzyme
is less of a concern. The fact that the authors obtained a higher
product yield in diethyl ether using microwave heating made
us want to probe the effect of moving from toluene to diethyl
ether on our reaction. Thus, we performed the lipase catalysed
transesterification reaction between methyl acetoacetate and 1-
propanol in boiling diethyl ether using both conventional and
Fig. 2 Lipase catalysed transesterification between methyl acetoacetate
and n-propanol at 70 ◦C.
Table 1 Effect of microwave power on the lipase-catalysed transesterifi-
cation reaction of methyl acetoacetate in toluene as a solvent at 50 ◦Ca
Entry
Reaction conditions
Product conversion/%
1
2
3
4
Conventional heating
31
32
28
30
Microwave heating, no cooling
Microwave heating, air cooling
Microwave heating, cryogenic
cooling
a Microwave power defined as that delivered from the magnetron.
reaction.16,17 We performed reactions using compressed air cooling
and cryogenic fluid as coolant. In both cases it was necessary to
move from working in a round-bottom flask to a glass tube. We
foresaw the change in vessel as having an effect on the product
conversion due to differences in scale, dimensions and stirring
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microwave heating. When using microwave heating we obtained
a 38% yield of the desired product after a reaction time of 1 h.
Performing the identical experiment using conventional heating
resulted in a 36% product yield.
Transesterification reaction with no cooling using a glass tube.
In a dedicated jacketed glass tube were placed propanol (0.75 mL,
0.601 g, 10 mmol), lipase (53 mg) and toluene (4 mL). The
temperature probe was inserted into the reaction mixture and the
open vessel placed into the microwave cavity. Initial microwave
irradiation of up to 300 W was used, the temperature being ramped
from rt to 50 ◦C. Once this temperature was reached and held
steadily, methyl acetoacetate (1.08 mL, 1.16 g, 10 mmol) was added
and the reaction mixture held at 50 ◦C for 1 h. At the end of
Conclusions
While more studies are clearly required in order to be able to
determine the effects of microwave irradiation across a broad
range of enzyme-catalysed transformations, we find no differences
between conventional and microwave heating in the reaction
studied here. We have used carefully controlled comparisons to
probe the effects both of reaction temperature and microwave
power delivered by the magnetron.
1
the reaction, the H-NMR spectrum of the product mixture was
recorded and the product conversion determined.
Transesterification reaction with air cooling using a glass tube.
In a 10 mL glass tube were placed propanol (0.75 mL, 0.601 g,
10 mmol), lipase (53 mg) and toluene (4 mL). The temperature
probe was inserted into the reaction mixture and the open vessel
placed into the microwave cavity. Initial microwave irradiation of
up ◦to 300 W was used, the temperature being ramped from rt to
50 C with compressed air (40 psi) being passed constantly over
the outer vessel walls. Once this temperature was reached and
held steadily, methyl acetoacetate (1.08 mL, 1.16 g, 10 mmol) was
added and the reaction mixture held at 50 ◦C for 1 h. At the end of
Experimental
General
All reagents were obtained from commercial suppliers and used
without further purification. Lipase acrylic resin from Candida
antartica was used. The lipase was kept in a sealed dry environment
and while it is hygroscopic no evidence of macroscopic water
uptake was noted during the course of the study. 1H- and 13C-NMR
spectra were recorded at 293 K on a 400 MHz spectrometer.
1
the reaction, the H-NMR spectrum of the product mixture was
recorded and the product conversion determined.
Transesterification reaction with cryogenic fluid cooling using
a glass tube. In a dedicated jacketed glass tube were placed
propanol (0.75 mL, 0.601 g, 10 mmol), lipase (53 mg) and toluene
(4 mL). The temperature probe was inserted into the reaction
mixture and the open vessel placed into the microwave cavity.
Initial microwave irradiation of up to 300 W was used, the
Description and use of the microwave apparatus
Microwave reactions were conducted using a commercially avail-
able monomode microwave unit (CEM Discover). The machine
consists of a continuous focused microwave power delivery system
with operator selectable power output from 0–300 W. Reactions
were performed either in round-bottom flasks or glass tubes. The
temperature of the contents of the vessel was monitored using a
calibrated fiber-optic probe inserted into the reaction vessel by
means of a sapphire immersion well. In all cases, the contents
of the vessel were stirred by means of a rotating magnetic plate
located below the floor of the microwave cavity and a Teflon-coated
magnetic stir bar in the vessel. Temperature, pressure and power
profiles were monitored using commercially available software
provided by the microwave manufacturer. For reactions performed
using cryogenic cooling, a specially designed jacketed reaction
vessel was used through which a microwave transparent fluid was
passed continuously by means of a pump (CEM Coolmate).
◦
temperature being ramped from rt to 50 C with cryogenic fluid
being passed constantly over the outer vessel walls at a rate such
as to maximise microwave input. Once the target temperature was
reached and held steadily, methyl acetoacetate (1.08 mL, 1.16 g,
10 mmol) was added and the reaction mixture held at 50 ◦C for 1 h.
At the end of the reaction, the 1H-NMR spectrum of the product
mixture was recorded and the product conversion determined.
Representative example of a transesterification reaction using a
round-bottom flask using diethyl ether as a solvent. In a 50 mL
round-bottom flask were placed propanol (1.5 mL, 1.202 g,
20 mmol), methyl acetoacetate (2.15 mL, 2.32 g, 10 mmol) and
diethyl ether (16 mL). The temperature probe was inserted into the
reaction mixture and the open vessel placed into the microwave
cavity. Initial microwave irradiation of 100 W was used, the
temperature being ramped from rt to 37 ◦C. Once this temperature
was reached and held steadily, lipase (105 mg) was added and the
reaction mixture held at 50 ◦C for 1 h. Samples were removed over
time, diluted in CDCl3, the 1H-NMR spectrum recorded and the
product conversion determined.
General experimental procedures
Representative example of a transesterification reaction using
a round-bottom flask. In a 50 mL round-bottom flask were
placed propanol (1.5 mL, 1.202 g, 20 mmol), methyl acetoacetate
(2.15 mL, 2.32 g, 10 mmol) and toluene (8 mL). The temperature
probe was inserted into the reaction mixture and the vessel placed
into the microwave cavity. Initial microwave irradiation◦of 35 W
was used, the temperature being ramped from rt to 50 C. Once
this temperature was reached and held steadily, l◦ipase (106 mg)
was added and the reaction mixture held at 50 C for 2.5 h.21
Samples were removed over time, diluted in CDCl3, the 1H-NMR
spectrum recorded and the product conversion determined.
Acknowledgements
CEM Microwave Technology is thanked for provision of mi-
crowave apparatus. Funding from the University of Connecticut
is acknowledged.
References
1 A number of books on microwave-promoted synthesis have been
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published recently: (a) A. Loupy, ed., Microwaves in Organic Synthesis,
Wiley-VCH, Weinheim, 2006; (b) C. O. Kappe and A. Stadler, Mi-
crowaves in Organic and Medicinal Chemistry, Wiley-VCH, Weinhiem,
2005; (c) P. Lidstro¨m and J. P. Tierney, eds., Microwave-Assisted Organic
Synthesis, Blackwell, Oxford, 2005; (d) A. Loupy, ed., Microwaves
in Organic Synthesis, Wiley-VCH, Weinheim, 2002; (e) B. L. Hayes,
Microwave Synthesis: Chemistry at the Speed of Light, CEM Publishing,
Matthews NC, 2002.
2 For recent reviews see: (a) C. O. Kappe, Angew. Chem., Int. Ed., 2004,
43, 6250; (b) M. Larhed, C. Moberg and A. Hallberg, Acc. Chem.
Res., 2002, 35, 717; (c) A. Lew, A., P. O. Krutzik, M. E. Hart and A. R.
Chamberlain, J. Comb. Chem., 2002, 4, 95; (d) P. Lidstro¨m, J. P. Tierney,
B. Wathey and J. Westman, Tetrahedron, 2001, 57, 9225.
A. Enada and G. L. Myers, Rapid Commun. Mass Spectrom., 2005,
19, 2865; (e) H.-F. Juan, S.-C. Chang, H.-C. Huang and S.-T. Chen,
Proteomics, 2005, 5, 840.
8 For a review see: I. Roy and M. N. Gupta, Curr. Sci., 2003, 85,
1685.
9 J.-R. Carillo-Munoz, D. Bouvet, E. Guibe-Jampel, A. Loupy and A.
Petit, J. Org. Chem., 1996, 61, 7746.
10 M.-C. Parker, T. Besson, S. Lamare and M.-D. Legoy, Tetrahedron
Lett., 1996, 37, 8383.
11 G. Lin and W.-Y. Lin, Tetrahedron Lett., 1998, 39, 4333.
12 M. Vacek, M. Zarevu´cka, Z. Wimmer, K. Stra´nsky, K. Demnerova´ and
M.-D. Legoy, Biotechnol. Lett., 2000, 22, 1565.
13 G. D. Yadav and P. S. Lathi, J. Mol. Catal. A: Chem., 2004, 223, 51.
14 B. Re´jasse, T. Besson, M.-D. Legoy and S. Lamare, Org. Biomol. Chem.,
2004, 2, 1086.
3 J. M. Collins and M. J. Collins, in Microwaves in Organic Synthesis, ed.
A. Loupy, Wiley-VCH, Weinheim, 2006.
4 (a) H. J. Olivos, P. G. Alluri, M. M. Reddy, D. Salony and T. Kodadek,
Org. Lett., 2002, 4, 4057; (b) B. C. Gorske, S. A. Jewell, E. J. Guerard
and H. E. Blackwell, Org. Lett., 2005, 7, 1521.
15 B. Re´jasse, T. Besson, M.-D. Legoy and S. Lamare, Org. Biomol. Chem.,
2006, 4, 3703.
16 N. E. Leadbeater, S. J. Pillsbury, E. Shanahan and V. A. Williams,
Tetrahedron, 2005, 61, 3565.
17 R. K. Arvela and N. E. Leadbeater, Org. Lett., 2005, 7, 2101.
18 For more detailed information see: http://cem.com/synthesis/
subam.asp.
19 For a previous report of the use of this apparatus see: B. K. Singh, P.
Appukkuttan, S. Claerhout, V. S. Parmar and E. Van der Eycken, Org.
Lett., 2006, 8, 1863.
5 (a) T. Matsushita, H. Hinou, M. Kurogochi, H. Shimizu and S.-I.
Nishimura, Org. Lett., 2005, 7, 877; (b) T. Matsushita, H. Hinou, M.
Fumoto, M. Kurogochi, N. Fujitani, H. Shimizu and S.-I. Nishimura,
J. Org. Chem., 2006, 71, 3051.
6 M. Bejugam and S. L. Flitsch, Org. Lett., 2004, 6, 4001.
7 (a) A. K. Bose, Y. H. Ing, N. Lavlinskaia, C. Sareen, B. N. Pramanik,
P. L. Bartner, Y.-H. Liu and L. Heimark, J. Am. Soc. Mass Spectrom.,
2002, 13, 839; (b) B. N. Pramanik, U. A. Mirza, Y. H. Ing, Y.-H. Liu,
P. L. Bartner, P. C. Weber and A. K. Bose, Protein Sci., 2002, 11, 2676;
(c) W. Sun, S. Gao, L. Wang, Y. Chen, S. Wu, X. Wang, D. Zheng and
Y. Gao, Mol. Cell. Proteomics., 2006, 5, 769; (d) H. W. Vesper, L. Mi,
20 P. Kerep and H. Ritter, Macromol. Rapid Commun., 2006, 27, 707.
21 Once the desired target temperature is reached, the microwave power
is automatically controlled to maintain the reaction mixture at that
temperature.
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The Royal Society of Chemistry 2007
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