Kinetic resolution of rac-1-phenylethanol with immobilized lipases: A critical comparison of microwave and conventional heating protocols

Article abstract of DOI:10.1021/jo9010443

(Figure Presented) The lipase-catalyzed kinetic resolution of rac-1-phenylethanol with vinyl acetate as acyl donor and cyclohexane as solvent has been investigated applying both microwave dielectric heating and conventional thermal heating in order to probe the existence of nonthermal microwave effects. All transformations were conducted at 40°C in a dedicated reactor setup that allowed accurate internal reaction temperature measurements with use of fiber-optic probes. Quartz reaction vessels that allow higher levels of microwave power to be administered to the reaction mixture were used for all experiments. For all five studied immobilized lipases, the observed reactivities and enantioselectivities in microwave and oil bath experiments were identical and thus not related to the presence of the microwave field. The effect of magnetic stirring proved critical as too rapid stirring in some instances destroyed the enzyme support structure and led to altered reactivities and selectivities.

Full text of DOI:10.1021/jo9010443

pubs.acs.org/joc
Kinetic Resolution of rac-1-Phenylethanol with Immobilized Lipases: A
Critical Comparison of Microwave and Conventional Heating Protocols
†
Rodrigo Octavio M. A. de Souza, Octavio A. C. Antunes, Wolfgang Kroutil, and
†,z
‡
,§
C. Oliver Kappe*
†
Instituto de Quı ´m ica, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Bloco A 641, RJ 21941-909,
Brazil, Research Center Applied Biocatalysis, Institute of Chemistry, Karl-Franzens-University Graz,
‡
§
Heinrichstrasse 28, A-8010 Graz, Austria, and Christian Doppler Laboratory for Microwave Chemistry
(CDLMC) and Institute of Chemistry, Karl-Franzens-University Graz, Heinrichstrasse 28, A-8010 Graz,
Austria.
zDeceased, June 1, 2009.
oliver.kappe@uni-graz.at
Received May 19, 2009
The lipase-catalyzed kinetic resolution of rac-1-phenylethanol with vinyl acetate as acyl donor and
cyclohexane as solvent has been investigated applying both microwave dielectric heating and
conventional thermal heating in order to probe the existence of nonthermal microwave effects. All
transformations were conducted at 40 °C in a dedicated reactor setup that allowed accurate internal
reaction temperature measurements with use of fiber-optic probes. Quartz reaction vessels that allow
higher levels of microwave power to be administered to the reaction mixture were used for all
experiments. For all five studied immobilized lipases, the observed reactivities and enantioselec-
tivities in microwave and oil bath experiments were identical and thus not related to the presence of
the microwave field. The effect of magnetic stirring proved critical as too rapid stirring in some
instances destroyed the enzyme support structure and led to altered reactivities and selectivities.
Introduction
hydrolases EC 3.1.1.3) are among the most useful enzymes
2
applied in both aqueous and nonaqueous media. Lipases
Biocatalysis with enzymes is an attractive alternative to other
catalytic methods (for example metal- or organocatalysis) for
possess wide substrate specificity, have an excellent ability to
recognize chirality, and do not require labile cofactors. The
kinetic resolution of secondary alcohols or their carboxylates
either via esterification or hydrolysis has been extensively studied
with a variety of different lipases, where the enzyme discriminates
1
effecting chemical transformations. In general, high levels of
chemo-, regio-, and enantioselectivities can be achieved due to
1
highly selective recognition of the substrate by the enzyme.
Among the many enzyme classes used as biocatalysts for
synthetic organic transformations, lipases (triacyl glycerol
2
between the two enantiomers present in the racemic mixture.
(
2) (a) Gotor-Fernandez, V.; Rebolledo, F.; Gotor V. In Biocatalysis in the
Dedicated to the memory of Professor Octavio A. C. Antunes.
Pharmaceutical and Biotechnology Industry; Patel, R. N., Ed.; CRC Press:
Boca Raton, FL, 2007; pp 203-248. (b) Hidalgo, A.; Bornscheuer, U. T. In
Biocatalysis in the Pharmaceutical and Biotechnology Industry; Patel, R. N.,
Ed.; CRC Press: Boca Raton, FL, 2007; pp 159-179. (c) Ghanem, A. Tetra-
hedron 2007, 63, 1721. (d) Hasan, F.; Ali Shah, A.; Hameed, A. Enzyme Microb.
Technol. 2006, 39, 235. (e) Ghanem, A.; Aboul-Enein, H. Y. Chirality 2005
17, 1.
*
To whom the correspondence should be addressed. Phone: +43-316-
80-5352. Fax: +43-316-380-9840.
1) (a) Woodley, J. M. Trends Biotechnol. 2008, 26, 321. (b) Pollard, D. J.;
3
(
Woodley, J. M. Trends Biotechnol. 2007, 25, 66. (c) Breuer, M.; Ditrich, K.;
Habicher, T.; Hauer, B.; Kesseler, M.; St u€ rmer, R.; Zelinski, T. Angew.
Chem., Int. Ed. 2004, 43, 788.
DOI: 10.1021/jo9010443
r 2009 American Chemical Society
Published on Web 07/14/2009
J. Org. Chem. 2009, 74, 6157–6162 6157

de Souza et al.
JOCArticle
In the field of synthetic organic chemistry, microwave
dielectric heating for the past two decades has provided a
powerful method to enhance chemical processes and to
provide, in many cases, improved yields and cleaner reaction
have been invoked to rationalize the observed effects since
control experiments with conventional heating in a similar
temperature range often led to different reaction rates or
5-12
selectivities.
In fact, a 2008 publication has presented
3
profiles in significantly shorter reaction times. More re-
evidence that microwave irradiation can induce changes in
the tertiary structure/conformation of a hyperthermophilic
enzyme not related to a macroscopic temperature change,
which resulted in high biocatalytic hydrolysis rates at bulk
solution temperatures far below the thermal optimum of the
cently, microwave heating has also been successfully applied
in the biosciences field, including areas such as peptide
synthesis, protein digestion (proteomics), or DNA amplifi-
4
cation by polymerase chain reaction (PCR). As far as
1
0,15
synthetic enzymatic transformations are concerned, a signi-
ficant number of reports over the past few years have des-
cribed microwave-assisted, mostly lipase-catalyzed esterifi-
enzyme.
On the other hand, a recently conducted careful
analysis of lipase-catalyzed transesterification reactions led
to the conclusion that, in contrast to previous literature
reports, no differences between microwave heating and
5
-12
cations, transesterifications, or hydrolysis reactions.
In most instances, the published results suggest that micro-
wave irradiation can have an influence on enzyme stability
1
6
conventional heating existed.
Prompted by the ongoing and controversial debate on the
involvement of nonthermal microwave effects in biocatalytic
5
-7
and activity,
in addition to altering/enhancing reaction
5,11,12
5
-10
5
transformations, we herewith describe a critical comparison
rates and/or enantioselectivities.
these publications so-called nonthermal microwave effects
In the majority of
1
3,14
of microwave and conventionally heated kinetic resolutions
of a racemic secondary alcohol using immobilized lipases in
nonaqueous media. The kinetic resolution of a secondary
alcohol appears to be an ideal probe for investigating non-
thermal microwave effects in enzymatic transformations,
since apart from the standard parameter “conversion” that
is typically monitored in comparing conventional heating
and microwave heating experiments, the “enantioselectivity”
(
3) (a) For a recent review with >900 references and a tabular survey of
ca. 200 microwave chemistry review articles, books, and book chapters, see:
Kappe, C. O.; Dallinger, D. Mol. Diversity 2009, 13, 71.(b) Caddick, S;
Fitzmaurice, R. Tetrahedron 2009, 65, 3325 and references cited therein.
(
4) For a review, see: Collins, J. M.; Leadbeater, N. E. Org. Biomol. Chem.
007, 5, 1141.
5) For a review, see: R ꢀe jasse, B.; Lamare, S.; Legoy, M.-D.; Besson, T. J.
Enzyme Inhib. Med. Chem. 2007, 22, 518.
6) (a) R ꢀe jasse, B.; Besson, T.; Legoy, M.-D.; Lamare, S. Org. Biomol.
2
(
17
parameter can also be studied here. The enantioselectivity
(
Chem. 2006, 4, 3703. (b) R ꢀe jasse, B.; Lamare, S.; Legoy, M.-D.; Besson, T.
Org. Biomol. Chem. 2004, 2, 1086.
for a lipase-catalyzed kinetic resolution would be expected to
be rather sensitive to temperature and therefore a good probe
to distinguish between thermal and nonthermal microwave
effects. Previous investigations of lipase-catalyzed kinetic
resolutions of secondary alcohols with microwave irradia-
tion have in several cases demonstrated significant enhance-
ments in both rate and/or enantioselectivity compared to the
(7) (a) Zhao, H.; Baker, G. A.; Song, Z.; Olubajo, O.; Zanders, L.;
Campbell, S. M. J. Mol. Catal. B: Enzym. 2009, 57, 149.(b) Yadav, G. D.;
Borkar, I. V. Ind. Eng. Chem. Res. 2009, 48, published online October 9, 2008,
DOI: 10.1021/ie800591c.
(
8) (a) Huang, W.; Xia, Y.-M.; Gao, H.; Fang, Y.-J.; Wang, Y.; Fang, Y.
J. Mol. Catal. B: Enzym. 2005, 35, 113. (b) Yadav, G. D.; Lathi, P. S. J. Mol.
Catal. A: Chem. 2004, 223, 51. (c) Roy, I.; Gupta, M. N. Tetrahedron 2003,
5
9, 5431. (d) Parker, M.-C.; Besson, T.; Lamare, S.; Legoy, M.-D. Tetra-
hedron Lett. 1996, 37, 8383.
9) (a) Kidwai, M.; Mothsra, P.; Gupta, N.; Kumar, S. S.; Gupta, R.
11
results obtained applying conventional heating. To distin-
(
guish between thermal and nonthermal microwave effects in
these reactions we herewith present a critical evaluation of
lipase-catalyzed kinetic resolutions of a secondary alcohol
using accurate and fast responding internal fiber-optic tem-
Synth. Commun. 2009, 39, 1143. (b) Major, B.; Kelemen-Horv ꢀa th, I.;
Csan ꢀa di, Z.; B ꢀe lafi-Bak oꢀ , K.; Gubicza, L. Green Chem. 2009, 11, 614. (c)
Fang, Y.; Huang, W.; Xia, Y.-M. Process Biochem. (Oxford, U.K.) 2008, 43,
306. (d) Sarma, K.; Borthakur, N.; Goswami, A. Tetrahedron Lett. 2007, 48,
6776. (e) Yadav, G. D.; Sajgure, A. D. J. Chem. Technol. Biotechnol. 2007, 82,
964. (f) Yadav, G. D.; Lathi, P. S. Clean Technol. Environ. Policy 2007, 9, 281.
14,17,18
perature probes.
(
g) Yadav, G. D.; Borkar, I. AIChE J 2006, 52, 1235. (h) Kerep, P.; Ritter, H.
Macromol. Rapid Commun. 2006, 27, 707. (i) Yadav, G. D.; Lathi, P. S.
Enzyme Microb. Technol. 2006, 38, 814. (j) Yadav, G. D.; Lathi, P. S. Synth.
Commun. 2005, 35, 1699. (k) Mazumder, S.; Laskar, D. D.; Prajapati, D.;
Roy, M. K. Chem. Biodiversity 2004, 1, 925. (l) Bradoo, S.; Rathi, P.; Saxena,
R. K.; Gupta, R. J. Biochem. Biophys. Methods 2002, 51, 115. (m) Kidwai,
M.; Dave, B.; Bhushan, K. R.; Misra, P.; Saxena, R. K.; Gupta, R.;
Gulati, R.; Singh, M. Biocatal. Biotranform. 2002, 20, 377. (n) Vacek, M.;
Zarev uꢀ cka, M.; Wimmer, Z.; Str ꢀa nsk ꢀy , K.; Demnerov ꢀa , K.; Legoy, M.-D.
Biotechnol. Lett. 2000, 22, 1565. (o) Gelo-Pujic, M.; Guib ꢀe -Jampel, E.;
Loupy, A. Tetrahedron 1997, 53, 17247.
Results and Discussion
General Considerations. As a simple and representative
model system for our planned comparison studies between
microwave and conventionally heated kinetic resolutions
using secondary alcohols and lipases we have chosen rac-
1-phenylethanol (1) as the alcohol component and vinyl
acetate (2) as the acyl donor (Scheme 1). This system has
(
10) Young, D. D.; Nichols, J.; Kelly, R. M.; Deiters, A. J. Am. Chem.
Soc. 2008, 130, 10048.
11) (a) Bachu, P.; Gibson, J. S.; Sperry, J.; Brimble, M. A. Tetrahedron:
(
(15) By applying microwave dielectric heating, it may be expected that
certain movements of the biocatalyst required for catalysis (for example, the
opening of the active site of a lipase by the so-called lid-movement) can be
stimulated by selectively inducing changes in protein conformation by
microwave irradiation. See also: (a) De Pomarai, D. I.; Smith, B.; Dawe,
A.; North, K.; Smith, T.; Archer, D. B.; Duce, I. R.; Jones, D.; Candido, E. P.
M. FEBS Lett. 2003, 543, 93. (b) Porcelli, M.; Cacciapuoti, G.; Fusco, S.;
Massa, R.; d’Ambrosio, G.; Bertoldo, C.; De Rosa, M.; Zappia, V. FEBS
Lett. 1997, 402, 102. (c) La Cara, S. M. R.; D’Auria, S.; Massa, R.;
d’Ambrosio, G.; Franceschetti, G.; Rossi, M.; De Rosa, M. Bioelectromag-
netics 1999, 20, 172.
Asymmetry 2007, 18, 1618. (b) Yu, D.; Chen, P.; Wang, L.; Gu, Q.; Li, Y.;
Wang, Z.; Cao, S. Process Biochem. (Oxford, U.K.) 2007, 42, 1312. (c) Yu, D.;
Wang, Z.; Chen, P.; Jin, L.; Cheng, Y.; Zhou, J.; Cao, S. J. Mol. Catal. B:
Enzym. 2007, 48, 51. (d) Lin, G.; Lin, W.-Y. Tetrahedron Lett. 1998, 39, 4333.
(
e) Carrillo-Munoz, J.-R.; Bouvet, D.; Guib ꢀe -Jampel, E.; Loupy, A.; Petit, A.
J. Org. Chem. 1996, 61, 7746.
12) (a) Yadav, G. D.; Sajgure, A. D.; Dhoot, S. B. J. Chem. Technol.
(
Biotechnol. 2008, 83, 1145. (b) Lundell, K.; Kurki, T.; Lindroos, M.;
Kanerva, L. T. Adv. Synth. Catal. 2005, 347, 1110. (c) Pchelka, B. K.; Loupy,
A.; Plenkiewicz, J.; Blanco, L. Tetrahedron: Asymmetry 2000, 11, 2719.
(
13) For leading reviews, see: (a) Perreux, L.; Loupy, A. Tetrahedron
(16) (a) Leadbeater, N. E.; Stencel, L. M.; Wood, E. C. Org. Biomol.
Chem. 2007, 5, 1052. (b) See also: N u€ chter, M.; M u€ ller, U.; Ondruschka, B.;
Tied, A.; Lautenschl €a ger, W. Chem. Eng. Technol. 2003, 26, 12.
(17) Hosseini, M.; Stiasni, N.; Barbieri, V.; Kappe, C. O. J. Org. Chem.
2007, 72, 1417.
2
2
1
3
001, 57, 9199. (b) Perreux, L.; Loupy, A. In Microwaves in Organic Synthesis,
nd ed.; Loupy, A., Ed.; Wiley-VCH: Weinheim, Germany, 2006; Chapter 4, pp
34-218. (c) De La Hoz, A.; Diaz-Ortiz, A.; Moreno, A. Chem. Soc. Rev. 2005,
4, 164.
(
14) Herrero, M. A.; Kremsner, J. M.; Kappe, C. O. J. Org. Chem. 2008,
(18) Bacsa, B.; Horv ꢀa ti, K.; Bosze, S.; Andreae, F.; Kappe, C. O. J. Org.
Chem. 2008, 73, 7532.
7
3, 36.
6
158 J. Org. Chem. Vol. 74, No. 16, 2009

de Souza et al.
JOCArticle
of absolute configuration for alcohol (S)-1 was confirmed by
SCHEME 1
1
9,23
comparison with an authentic sample.
Several parameters were initially evaluated for the opti-
mization of the reaction conditions, including solvent,
reaction temperature, enzyme source, and acyl donor
concentration. Clearly, the proper choice of solvent is
important in lipase-catalyzed kinetic resolutions, as the
solvent can influence reaction rates and the enantioselecti-
2
4
vity for a given reaction. As expected, unpolar solvents
such as hexane or cyclohexane provided the highest activity
in kinetic resolutions of alcohol rac-1 (Scheme 1) in test
runs performed at 70 °C for 2 h, using 2 equiv of acyl donor 2,
been studied numerous times in the literature and the para-
meters effecting the kinetic resolution of this alcohol by
lipases are well understood. As far as the enzyme source
is concerned we have selected a variety of immobilized lipases
1
9
1
0% (w/w, based on substrate) enzyme loading, and a
(
rather than working with the free enzymes) in our screening
substrate concentration of 0.11 M (Figure S3, Supporting
Information). In a subsequent experiment, the effect of the
acyl donor concentration on the conversion was evaluated.
Utilizing Amano Lipase PS-C I in cyclohexane as a model
system and the conditions detailed above (70 °C, 2 h, 10% w/w
enzyme loading) the optimum concentration of the acyl
donor was found to be 2 equiv. Neither lower (1 equiv) nor
higher concentrations (3 and 5 equiv) of vinyl acetate led to
an improvement of the transesterification rate (Figure S4,
Supporting Information).
Of most interest for our planned comparison studies
between microwave and conventional heating was the influ-
ence of reaction temperature on enzyme activity (reac-
tion rate) and enantioselectivity. On the basis of the initial
optimization studies described above, a series of experiments
was performed with 10% (w/w) Novozym 435 and Amano
Lipase PS-C I in cyclohexane with 2 equiv of the acyl donor
in a temperature range of 25-70 °C. For Amano Lipase AK
results with 10% enzyme loading were unsatisfactory and
therefore 100% (w/w) enzyme loading was used. Table 1
summarizes the conversions and selectivities obtained after
campaigns since immobilization of the enzyme will generally
improve thermal stability and thus make the enzymes more
suitable for transformations at higher temperatures. Since it
is known that the properties of the immobilized enzyme
strongly depend on the microenvironment of the support
surface, a selection of lipases immobilized on different sup-
ports was chosen.
Initially, the following lipases were selected: Novozym
4
35 (candida antartica lipase B, CAL-B, immobilized on a
macroporous polyacrylate resin); Amano Lipase PS-C I
Pseudomonas cepacia lipase immobilized on a ceramic
(
support); and Amano Lipase AK (Pseudomonas fluorescens
lipase, PFL lipase). All of the above immobilized lipase
preparations can be considered as thermostable, having their
optimum range of activity between 55 and 80 °C, and
therefore appear well suited for the planned microwave
irradiation studies.
Reaction Optimization. We began our study with the
optimization of reaction conditions for the kinetic resolution
of rac-1-phenylethanol (1) catalyzed by the immobilized
lipases Novozym 435, Amano Lipase PS-C I, and Amano
Lipase AK using conventional heating. At this point, the
transesterification reactions (Scheme 1) were performed in
parallel directly in standard sealed HPLC/GC autosampler
vials equipped with small stir bars. The vials were fitted
inside the 20 wells (5 ꢀ 4 matrix) of a silicon carbide reaction
block, placed on a standard hot plate (Figure S1, Supporting
2
0
2
h of reaction time. The superiority of Novozym 435 over
the other enzymes in terms of reaction rate and selectivity
was clearly evident. Even at room temperature, high conver-
sions and excellent selectivities (E) were obtained. Increasing
the temperature to 70 °C did not significantly change con-
version or selectivities, confirming the good thermal stability
of this enzyme. In contrast, Amano Lipase PS-C I exhibited a
somewhat lower conversion in the range from 25 to 50 °C,
but showed perfect conversion at 60 and 70 °C. For Amano
Lipase AK only a moderate increase in the reactivity level
was obtained, going from 25 to 70 °C (Table 1).
In addition to the experiments described above, the recycl-
ability of the immobilized enzymes was investigated. For
both Novozym 435 and Amano Lipase PS-C I reusability of
the enzyme was excellent. After 10 cycles at 70 °C under the
conditions described in Table 1, both the recorded activity
and selectivity levels remained excellent, with only a minor
decrease seen after the eighth cycle (Figures S5 and S6,
Supporting Information).
2
1
Information). Because of the high thermal conductivity of
2
2
silicon carbide, rapid and homogeneous heating of the
reaction mixtures contained inside the HPLC/GC vials to
the selected target temperatures was generally achieved
within less than 2 min (Figure S2, Supporting Information).
The outcome of the kinetic resolutions was established by
directly monitoring conversion by GC-FID and enantio-
meric excess of the substrate (S)-1 and the product (R)-3 by
chiral HPLC. The enantiopreference for all lipases was
always as displayed in Scheme 1 and the correct assignment
(
19) (a) Wang, Y. H.; Wang, R.; Li, Q. S.; Zhang, Z. M.; Feng, Y. J. Mol.
Microwave versus Oil-Bath Heating. To accurately com-
pare the results obtained by direct microwave heating with
the outcome of a conventionally heated reaction we have
used a reactor system that allows us to perform both types
of transformations in the identical reaction vessel and to
Catal. B: Enzym. 2009, 56, 142. (b) Csajagi, C.; Szatzker, G.; Toke, E. R.;
Urge, L.; Darvas, F.; Poppe, L. Tetrahedron: Asymmetry 2008, 19, 237. (c)
Kourist, R.; de Maria, P. D.; Bornscheuer, U. T. ChemBioChem 2008, 9, 491.
(
2
9
d) Cheedrala, R. K.; Sachwani, R.; Krishna, P. R. Tetrahedron: Asymmetry
008, 19, 901. (e) Shah, S.; Gupta, M. N. Bioorg. Med. Chem. Lett. 2007, 17,
21. (f) Schofer, S. H.; Kaftzik, N.; Wasserscheid, P.; Kragl, U. Chem.
Commun. 2001, 425.
(
(
(
20) See the following database: http://www.brenda-enzymes.info.
21) Damm, M.; Kappe, C. O. J. Comb. Chem. 2009, 11, 460.
22) Kremsner, J. M.; Kappe, C. O. J. Org. Chem. 2006, 71, 4651 and
(23) Jing, Q.; Kazlauskas, R. J. Chirality 2008, 20, 724.
(24) Park, S.; Kazlauskas, R. J. J. Org. Chem. 2001, 66, 8395.
(25) Rakels, J. L. L.; Straathof, A. J. J.; Heijnen, J. J. Enzyme Microb.
Technol. 1993, 15, 1051.
references cited therein.
J. Org. Chem. Vol. 74, No. 16, 2009 6159

de Souza et al.
JOCArticle
a
TABLE 1. Temperature Dependence of the Kinetic Resolution of rac-1-Phenylethanol by Different Lipases in Cyclohexane (Scheme 1)
b
c
c
d
E
entry
enzyme
temp (°C)
conv (%)
ee
S
(%) (S-1)
ee
P
(%) (R-3)
1
2
3
4
5
6
7
8
9
Novozym 435
Amano PS-C I
Amano AK
Novozym 435
Amano PS-C I
Amano AK
Novozym 435
Amano PS-C I
Amano AK
Novozym 435
Amano PS-C I
Amano AK
25
25
25
40
40
40
50
50
50
60
60
60
70
70
70
51
31
7
98
51
97
97
53
95
99
55
98
98
97
97
97
97
97
99
98
97
98
99
97
98
97
97
98
99
97
99
97
98
>200
164
>200
>200
>200
>200
>200
114
>200
>200
>200
>200
>200
>200
>200
54
33
23
48
31
23
51
50
18
52
52
30
1
1
1
1
1
1
0
1
2
3
4
5
a
Novozym 435
Amano PS-C I
Amano AK
rac-1-Phenylethanol (1) (20 mg, 0.16 mmol), vinyl acetate (2) (27.5 mg, 0.32 mmol, 2 equiv), and 2 mg (10% w/w) of the corresponding immobilized
b
enzyme (100% for Amano AK) were reacted in cyclohexane (1.5 mL) for 2 h. For further details, see the Experimental Section. Based on GC analysis
c
HP-5MS column). Determined by chiral HPLC analysis (chiral column OD-H) employing heptane:2-propanol (95:5) as the mobile phase at 0.5 mL/
(
d
S P
min (254 nm). Selectivity was calculated as E = f(ee ,ee ) according to ref 25.
monitor the internal reaction temperature in both experi-
mg of 2, and 2 mg of Novozym 435) in a standard microwave
-4 28
1
ments directly with a fiber-optic probe device. Similar to
7
Pyrex vessel (tan δ = 10 ꢀ 10
)
and in a custom-made
of exactly the same
2
6
-4 28
the setup first described by Maes and co-workers we have
used a CEM Discover single mode microwave reactor
equipped with a fiber-optic probe for directly monitoring
the internal reaction temperature in a 10 mL sealed reaction
vessel made either out of Pyrex or fully microwave trans-
quartz vessel (tan δ = 0.6 ꢀ 10
)
geometry. In the case of the Pyrex vessel experiment, the
temperature of the reaction mixture measured by the fiber-
optic probe sensor was rapidly raised to 70 °C within 2 min.
In contrast, the same experiment in the quartz vessel required
45 min, and even using the full 300 W maximum nominal
magnetron output power of the microwave reactor, the
internal reaction temperature could not be raised above
70 °C (Figure S8, Supporting Information). Apparently,
the comparatively small amounts of polar reagents 1 and 2
contained in the reaction medium do not influence the over-
all microwave absorptivity of the reaction mixture enough to
allow efficient heating by microwave dielectric heating
1
7
parent quartz glass. This setup can be immersed either into
the cavity of the microwave reactor or into a preheated and
temperature equilibrated oil bath placed on a magnetic
stirrer (Figure S7, Supporting Information). In both cases,
the software of the microwave instrument is recording the
internal temperature and similar heating profiles can be
obtained (see below). This system has the advantage that
the same reaction vessel and the same method of temperature
measurement is used. In this way all parameters apart from
the mode of heating are identical and therefore a fair
comparison between microwave heating and thermal heating
can be made.
2
2,29
effects.
Heating of the reaction mixture in the Pyrex vial
thus undoubtedly occurs mostly by conductive heating via
the hot glass surface from the self-absorbing reaction vessel,
not unlike in a conventional oil bath experiment. In contrast,
the use of a quartz reaction vessel ensures that most of the
microwave irradiation will be able to directly interact with
the chemistry inside the reaction vessel, thereby allowing the
potential observation of nonthermal microwave effects.
On the basis of the observations made above, the compa-
rison experiments between oil bath and microwave heating
for the kinetic resolution of rac-1-phenylethanol (1) with
vinyl acetate (2) as acyl donor (Scheme 1) were therefore
performed in a quartz reaction vial with cyclohexane as
solvent at a fixed temperature of 40 °C measured internally
by a fiber-optic probe. The comparatively low reaction
temperature selected for these studies ensured that thermal
effects could be separated from nonthermal microwave
effects, since although the temperature was relatively low, a
high magnetron output power still had to be applied in all
microwave experiments in order to reach 40 °C (see above).
Our initial model system involved Novozym 435 as immo-
bilized lipase keeping the other reaction parameters essen-
tially as described in Table 1. To avoid weighing/pipetting
errors, stock solutions of rac-1-phenylethanol (1) and
Since the chosen optimum solvent for the lipase-catalyzed
kinetic resolution of alcohol rac-1 was microwave-trans-
2
7
parent cyclohexane (tan δ < 0.01), the use of quartz
reaction vessels for all microwave experiments was manda-
tory. On the basis of our previous experience with low
absorbing solvents under microwave conditions, we recog-
nized that the standard Pyrex reaction vessels are not truly
microwave transparent and will absorb a significant amount
of microwave energy, thereby acting as “passive heating
2
2
elements”. For the planned microwave-assisted kinetic
resolutions in cyclohexane this was readily demonstrated
by comparing the heating and power profiles of a typical
reaction mixture (1.5 mL of cyclohexane, 20 mg of rac-1, 27.5
(
26) Hostyn, S.; Maes, B. U. W.; Van Baelen, G.; Gulevskaya, A.;
Meyers, C.; Smits, K. Tetrahedron 2006, 62, 4676.
27) The ability of a specific solvent to convert microwave energy into
heat at a given frequency and temperature is determined by the so-called loss
(
0
0
0
tangent (tan δ) expressed as the following quotient, tan δ = ε /ε . A reaction
medium with a high tan δ at the standard operating frequency of a microwave
synthesis reactor (2.45 GHz) is required for good absorption and, conse-
quently, for efficient heating. Solvents used for microwave synthesis can be
classified as high (tan δ > 0.5), medium (tan δ 0.1-0.5), and low microwave
absorbing (tan δ < 0.1). See the following references for further details: (a)
Gabriel, C.; Gabriel, S.; Grant, E. H.; Halstead, B. S.; Mingos, D. M. P.
Chem. Soc. Rev. 1998, 27, 213. (b) Mingos, D. M. P.; Baghurst, D. R. Chem.
Soc. Rev. 1991, 20, 1.
(28) Kappe, C. O.; Dallinger, D.; Murphree, S. S. Practical Microwave
Synthesis for Organic Chemists;Strategies, Instruments, and Protocols;
Wiley-VCH: Weinheim, Germany, 2009.
(29) Kappe, C. O. Angew. Chem., Int. Ed. 2004, 43, 6250.
6
160 J. Org. Chem. Vol. 74, No. 16, 2009

de Souza et al.
JOCArticle
TABLE 2. Comparison of Microwave and Conventional Heating in the Kinetic Resolution of rac-1-Phenylethanol by Different Lipases in Cyclohexane
a
(
Scheme 1, 40 °C, 2 h)
b
c
d
d
e
E
entry
enzyme/loading (%)
heating method
conv (%)
ee
s
(%) (S-1)
ee
P
(%) (R-3)
1
2
3
4
5
6
7
8
9
Novozym 435/10
Novozym 435/10
Amano PS C I/10
Amano PS C I/10
Amano AK/100
MW (200 W)
oil bath
MW (200 W)
oil bath
MW (200 W)
oil bath
MW (200 W)
oil bath
50
50
35
33
41
38
23
25
11
14
97
97
51
53
56
51
33
30
25
25
98
99
97
98
97
96
84
83
79
81
>200
>200
109
168
115
81
Amano AK/100
Lipozyme TL IM/100
Lipozyme TL IM/100
Lipozyme RL IM/100
Lipozyme RL IM/100
15
14
10
12
MW (200 W)
oil bath
1
0
a
rac-1-Phenylethanol (1) (20 mg, 0.16 mmol), vinyl acetate (2) (27.5 g, 0.32 mmol, 2 equiv), and 10-100% (w/w) of the corresponding immobilized
enzyme were reacted in cyclohexane (1.5 mL) for 2 h. For further details, see the Experimental Section. All experiments have been performed three times
b
and the given values reflect median values. Both microwave and oil bath experiments were performed in the same 10 mL quartz reaction vessel with
c
d
magnetic stirring at 100 rpm (oil bath) or by using the setting “low” (MW), respectively. On the basis of GC analysis (HP-5MS column). Determined by
e
chiral HPLC analysis (Chiralcel OD-H) employing heptane:2-propanol (95:5) as the mobile phase at 0.5 mL/min (254 nm). Selectivity was calculated as
S P
E = f(ee ,ee ) according to ref 25.
FIGURE 1. Comparison of microwave and conventional heating for the kinetic resolution of rac-1-phenylethanol by Amano PS-C I lipase in
cyclohexane at 40 °C (Scheme 1, Table 2).
vinyl acetate (2) in cyclohexane were employed in both the
microwave and oil bath runs. In the microwave experiments
a maximum nominal magnetron output power of 200 W was
selected so that the heating profiles obtained in the oil bath
mimicked the heating rates achieved by dielectric effects in
the microwave reactor (Figure S9, Supporting Information).
Given the comparatively long reaction times of 2 h the initial
heating rate in these transformations was, however, less
important and similar results were achieved with 50 or
not match the stirring speed used in the oil bath experiments
3
1
(100 rpm). Adjusting the stirring speed in the microwave
instrument to “low”, the obtained activity and selectivity
much closer resembled the data seen with conventional
heating (Table 2, entries 1 and 2). This effect is clearly related
to the destruction of the immobilized enzyme support
(polyacrylate resin) by the frictional forces exerted by the
magnetic stir bar and the quartz vessel if too rapid stirring
is performed, and can be evidenced by investigating the
immobilized enzymes after treatment (Figure S10, Support-
3
0
1
00 W of initial magnetron power (data not shown).
In the experiments with Novozym 435 at 40 °C an appar-
3
2
ing Information).
ently significant nonthermal microwave effect was initially
observed, comparing the activities and selectivities achieved
in the oil bath with the activities/selectivities obtained under
the influence of microwave irradiation at the same tempera-
ture after 2 h. These experiments were repeated a number of
times and seemed to indicate that both the activity
To additionally support the notion that the stirring speed
has a major role on this enzymatic reaction we have per-
formed a control experiment at room temperature (no
microwave power applied) in the microwave reactor invol-
ving Novozym 435 and rac-1-phenylethanol (1) under other-
wise identical reaction conditions (Table 2, entry 1). In
agreement with the observations discussed above, there
was a significant effect of the stirring speed on the activi-
ties/selectivities obtained in these transformations, with
(conversion) and the selectivity of the enzyme in all cases
was significantly lower in the microwave experiment as
compared to the oil bath runs. After considerable experi-
mentation and variations of conditions we realized that the
stirring speed in the microwave reactor (set on “high”) did
(31) Three different stirring levels can be set on the CEM Discover
microwave reactor (low, medium, high). The correlation with actual stirring
speeds in rpm are not disclosed by the instrument vendor.
(32) Kvittingen, L.; Sjursnes, B.; Halling, P.; Anthonsen, T. Tetrahedron
1992, 48, 5259.
(30) Glasnov, T. N.; Findening, S.; Kappe, C. O. Chem.;Eur. J. 2009, 15,
001.
1
J. Org. Chem. Vol. 74, No. 16, 2009 6161

de Souza et al.
JOCArticle
higher stirring speeds leading to reduced activities/selecti-
vities (see Table S1 in the Supporting Information).
Because of the fact that Novozym 435 displays near
perfect selectivities for the investigated kinetic resolution
displayed in Scheme 1 over a wide temperature range
(for example Novozym 435) do not withstand intensive
stirring with a magnetic stir bar as already previously
described. Choosing a too high stirring speed will lead to a
destruction of the immobilized enzyme support by the fric-
tional forces exerted by the magnetic stir bar and the vessel.
These changes in the enzyme support structure may lead to
apparent altered reactivity/selectivity levels and can there-
fore mimic microwave effects if different stirring speeds in
the oil bath and microwave experiments were selected. We
would like to emphasize that performing meaningful com-
parison experiments between conventional and microwave
heated transformations must therefore not only carefully
consider accurate internal temperature measurements and
identical vessel geometries, but also the stirring speed.
(Table 1), this enzymatic system is apparently not well suited
for a study on nonthermal microwave effects. We have
therefore looked at Amano Lipase PS-C I where our initial
temperature screen has revealed a comparatively lower
selectivity at 40 °C bulk temperature in addition to a
temperature dependence on reactivity (Table 1). Under the
conditions used for the comparison of microwave and oil
bath heating in the 10 mL quartz tube, an excellent agree-
ment between the conversion and selectivity values obtained
via the two heating modes was obtained. All experiments
were repeated three times each with nearly identical results
being obtained in all cases (Table 2, entries 3 and 4). In
addition, the degree of conversion was monitored after
different time intervals and also here no significant difference
between microwave and oil bath heating to 40 °C was seen
Experimental Section
Microwave Irradiation Experiments. Microwave irradiation
experiments were performed with a single-mode Discover Sys-
1
7
tem from CEM Corporation, using either custom-made high
purity quartz or standard Pyrex vessels (capacity 10 mL). The
temperature profiles for microwave and oil bath experiments
were recorded with use of a fiber-optic probe protected by a
sapphire immersion well inserted directly into the reaction
mixture (Figure S7 in the Supporting Information).
(Figure 1). Interestingly, the stirring speed with Amano
Lipase PS-C I is less critical, as the ceramic support is
apparently more resistant to degradation. Even with use of
the 400 rpm stirring speed, the results were still the same as
with low stirring (data not shown)
Parallel Screening of Enzyme Reactivity and Selectivity for the
Kinetic Resolution of rac-1-Phenylethanol by Different Lipases
with Conventional Heating (Table 1). The 1.5 mL stock solution
of rac-1-phenylethanol (1) (20 mg, 0.16 mmol) and vinyl acetate
(2) (27.5 g, 0.32 mmol, 2 equiv) in cyclohexane was charged with
10-100% w/w of the immobilized lipases in standard sealed
HPLC/GC autosampler vials equipped with small stir bars. The
vials were fitted inside the 20 wells (5 ꢀ 4 matrix) of a silicon
Finally, we wanted to evaluate if microwave irradiation
could perhaps improve the performance of lipases that dis-
play very poor selectivity in kinetic resolutions. Apart from
Amano Lipase AK (see above) we have additionally per-
formed experiments with Lipozyme TL IM (Thermomyces
lanuginosus lipase immobilized on porous silica) and Lipo-
zyme RM IM (Mucor miehei lipase immobilized on anionic
resin). In all three cases, the observed conversions and
selectivities at 40 °C with conventional heating were extre-
mely low. However, no changes in both activity and selec-
tivity of the immobilized lipases under the influence of
microwave irradiation was observed.
2
1
carbide reaction block and placed on a standard hot plate
(
Figure S1, Supporting Information). The mixture was subse-
quently heated applying the appropriate temperature (Table 1).
Samples (0.1 mL) were taken from the reaction vessel to monitor
the conversion of the reaction (GC-FID). After completion of
the reaction, the enzyme was removed by filtration and washed
five times with cyclohexane and placed to dry under ambient
temperature. The crude reaction mixture was subjected to
HPLC analysis with use of a chiral column (Table 1).
Conclusions
Microwave and Conventional Heating in the Kinetic Resolution
of rac-1-Phenylethanol by Different Lipases in Cyclohexane
(Table 2). To a 1.5 mL stock solution of rac-1-phenylethanol
(1) (20 mg, 0.16 mmol) and vinyl acetate (2) (27.5 g, 0.32 mmol,
2 equiv) in cyclohexane were added the appropriate immobilized
lipases (10-100% w/w). The mixture was subsequently heated
with stirring in a 10 mL microwave process vial (Figure S7,
Supporting Information) for 2 h applying the appropriate mode
of heating and stirring (Table 2). Samples (0.1 mL) were taken
from the reaction vessel to monitor the conversion of the
reaction (GC-FID). After the reaction was completed, the
enzyme was removed by filtration, washed five times with
cyclohexane, and dried under ambient temperature. The crude
reaction mixture was subjected to HPLC analysis with use of a
chiral column (Table 2).
In summary, we have conducted a detailed investigation
on the existence of nonthermal microwave effects in the
kinetic resolution of a secondary alcohol with five different
immobilized lipases. For all the studied lipases and experi-
mental conditions we have shown that no difference between
heating in an oil bath or heating by microwave irradiation
exist. The observed reactivities and selectivities in all cases
were 9within experimental error9identical, clearly demon-
strating the absence of any nonthermal microwave effects in
these enzymatic transformations. This is despite the fact that
the reaction mixtures containing the enzymes were exposed
to significant amounts of microwave power for an extended
time period (100-200 W for 2 h). Of critical importance for
our work was the use of microwave transparent quartz
reaction vessels. Only by using quartz vessels did it prove
possible to deliver a substantial amount of microwave
irradiation to the reaction mixture. Utilizing standard Pyrex
glass, most of the applied microwave energy was consumed
by heating the vessel material and not the reaction mixture.
As an additional critical factor in these experimental
setups involving immobilized enzymes we have identified
the stirring speed. Apparently, some enzyme preparations
Acknowledgment. This work was supported by a grant
from the Christian Doppler Society (CDG). We thank the
following Brazilian science foundations for financial assis-
tance: CNPq, FAPERJ, and FINEP.
Supporting Information Available: Temperature/power
profiles and pictures of equipment used in this study. This
material is available free of charge via the Internet at http://
pubs.acs.org.
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162 J. Org. Chem. Vol. 74, No. 16, 2009