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Lipase-mediated epoxidation utilizing urea–hydrogen peroxide in ethyl

acetate{
Emanuel G. Ankudey,a Horacio F. Olivo*b and Tonya L. Peeplesa
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Received 7th April 2006, Accepted 18th July 2006

First published as an Advance Article on the web 8th August 2006
DOI: 10.1039/b604984b

A green method for alkene epoxidation based on the chemo-enzymatic perhydrolysis of

carboxylic acids and esters has been optimized using Novozyme 435, the immobilized form of
Candida antarctica lipase B, and the complex urea–hydrogen peroxide (UHP). UHP, an
anhydrous form of hydrogen peroxide, has the potential of releasing hydrogen peroxide in a
controlled manner and thus avoids the need to add the aqueous hydrogen peroxide slowly to the
reaction mixture. The absence of water in the reaction media was also beneficial, because it
minimized undesired reactions of the oxidized products. A minimum amount of enzyme was
necessary to show the catalytic effect. On recycling, the enzyme maintained its activity up to six
rounds of epoxidations. A range of alkenes was epoxidized by this method providing yields
ranging from 75 to 100 percent.

Introduction no optimization of reaction parameters has yet been reported.

Lipases (EC 3.1.1.3) catalyze the hydrolysis and synthesis of
fatty acid ester bonds in triglycerides.1 Organic chemists have
exploited the ability of lipases to accept a wide variety of
substrates and also non-natural acyl acceptors.2 Lipase-
mediated perhydrolysis of carboxylic acids in the presence of
aqueous hydrogen peroxide was initially described by
Björkling and co-workers in 1990.3 Hydrogen peroxide reacts
with the acyl enzyme complex, formed by a fatty acid and the
hydroxyl group of a serine aminoacid in the active site, to yield
a peroxycarboxylic acid. The peroxycarboxylic acid released
has been utilized as an in situ formed oxidant for the
epoxidation of alkenes (Scheme 1),3–6 in Baeyer–Villiger
reactions,7,8 and also in the oxidation of sulfanyl to sulfinyl
groups.3b Candida antarctica lipase-B was found to efficiently
catalyze the perhydrolysis of octanoic acid more effectively
among a variety of lipases. The gene of Candida antarctica
encoding for lipase-B has been cloned into a host micro-
organism, Aspergillus oryzae.9 The overexpressed enzyme has
been immobilized in a macroporous polyacrylic resin by Novo
Nordisk (Novozyme-435). Although several publications
describe the lipase-based epoxidation with Novozyme-435,3–8
Scheme 1
a
Department of Chemical and Biochemical Engineering, The University
of Iowa, Iowa City, IA 52242, USA
b
Division of Medicinal and Natural Products Chemistry The University
of Iowa, Iowa City, IA 52242, USA. E-mail: horacio-olivo@uiowa.edu;
Fax: +1 (319) 335-8766; Tel: +1 (319) 335-8849
{ Electronic supplementary information (ESI) available: 1H and 13C
NMR spectra for 1–12. See DOI: 10.1039/b604984b
As part of our program in environmentally beneficial catalysis,
we are interested in designing a ‘‘green’’ process to effectively
carry out the epoxidation reaction. We report herein, an
inexpensive, practical, safe and environmentally friendly
method to oxidize a variety of alkenes under extremely mild
conditions.
Results and discussion
One of the drawbacks in the original protocol reported by
Björkling and co-workers,3 is the necessity to gradually add
aqueous hydrogen peroxide to the reaction mixture over
several hours to avoid lipase deactivation and to obtain higher
conversions. To overcome this problem, we replaced the aq.
hydrogen peroxide with urea–hydrogen peroxide complex
(UHP) because of its potential to release the oxidant in a
controlled manner.10,11 Other advantages of utilizing this
anhydrous form of hydrogen peroxide include safer handling
and minimal undesired hydrolysis of the epoxide products in
the absence of water.
Effect of solvent
The lipase-mediated epoxidation of phenyl cyclohexene with a
catalytic amount of octanoic acid was studied in different
solvents employing UHP and 50% aq. hydrogen peroxide,
Table 1. In general, reactions were faster when using aq.
hydrogen peroxide than when using UHP. High conversions
were observed when using low partition coefficient solvents,12
except in the case of acetonitrile and diethyl ether. The lowest
conversions were observed when using hydrocarbon solvents
(entries 1–3) and diethyl ether (entry 8). The conversions in
aromatic solvents were higher than in non-aromatic hydro-
carbon solvents (entries 4 and 6). It is known that some esters
can also be used as substrates in the lipase-mediated
perhydrolysis; no octanoic acid was added when an ester was
used as solvent (entry 9). High conversions were observed

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Table 1 Epoxidation of phenylcyclohexene in different solvents Table 2 Optimization of amount of enzyme

% conversion % conversion aq. Entry Amount of enzyme/mg Time/h Conversion (%)
Entry Solvent Log P UHP added 50% H2O2 added
1 50 5 82
1 Hexane 3.5 14 39 2 40 5 86
2 Pentane 3.4 11 47 3 25 5 86
3 Cyclohexane 3.2 10 31 4 15 5 85
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4 Toluene 2.5 35 66 5 10 5 85
5 Chloroform 2.2 21 75 6 5 5 86
6 Benzene 2.0 42 84 7 2 5 81
7 Dichloromethane 1.4 45 97 8 1 5 61
8 Diethyl ether 0.8 22 11 9 No enzyme 65 0
9 Ethyl acetate* 0.7 82 91
10 Acetonitrile 20.3 42 27
a
Conditions: UHP (1 equiv.) or 50% aq H2O2 (1 equiv.), rt, Table 2. Interestingly, similar conversions were observed even
octanoic acid (cat), time = 5 h. * No octanoic acid was added. when the amount of enzyme was minimal. No appreciable
amount of epoxide was detected when the experiment was
conducted in the absence of lipase.
when ethyl acetate was employed with both oxidants. In this
case, the presence of acetic acid was observed at the end of the
Examination of enzyme recycle
reaction. We selected ethyl acetate to be the solvent of choice
because of its low boiling point, ability to dissolve many The re-use of Novozyme-435 was investigated to assess the
substrates, highest conversion, environmentally friendliness, economic potential of the process, Fig. 2. The lipase was
and non-toxicity. Interestingly, conversions using UHP in washed with acetonitrile–water (9 : 1) to remove urea, and
acetonitrile and diethyl ether were lower when using aq. washed with ethyl acetate after each cycle. The activity of the
hydrogen peroxide.3a lipase was retained in the first two cycles. Conversion
decreased to 81–85% after three cycles. The enzyme was
Oxidant recycled six times without appreciable loss of activity. The
ability to recycle the immobilized lipase is important to
Bjorkling et al. reported that exposure of the enzyme to high
implement a low cost process. In contrast, when aq. hydrogen
concentrations of aq. hydrogen peroxide resulted in complete
peroxide was employed, the activity of the enzyme was lost
deactivation of the lipase.3a Therefore, slow addition of aq.
after the second cycle.
hydrogen peroxide to the reaction media was shown to increase
the yields of peroxycarboxylic acids. We found that replace-
General procedure
ment of the aq. hydrogen peroxide for UHP was beneficial.
High concentrations of UHP had a positive effect on the A general procedure was applied to the oxidations of a variety
chemo-enzymatic reaction (Fig. 1). However, one equivalent of of olefins, Table 3. The reaction was carried out employing
UHP was enough to carry the reaction to completion. 1.1 equiv. of UHP, and a small amount of Novozyme-435
with a variety of olefins dissolved in ethyl acetate. Oxidation
Amount of enzyme of cyclic olefins furnished the corresponding epoxides in very
good and excellent yields (entries 1–6). Epoxidations of
The conversion of phenylcyclohexene to the epoxide was
norbornene and a-pinene (entries 5 and 6) were completely
carried out in ethyl acetate with different amounts of
stereoselective, furnishing the oxirane ring exclusively on the
Novozyme-435 and determined by 1H NMR spectroscopy,
less hindered side of the olefins in high yields.13 Oxidation of

Fig. 1 Amount of UHP. Conditions: phenylcyclohexene, 1 mmol;

ethyl acetate, 1.5 mL; Novozyme-435, 30 mg; time, 5 h. Fig. 2 Enzyme recycling.

924 | Green Chem., 2006, 8, 923–926 This journal is ß The Royal Society of Chemistry 2006
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Table 3 Epoxidation of different alkenes Conclusion

We presented a general and practical chemo-enzymatic
procedure for the oxidation of a variety of olefins which
minimizes the use of enzyme. The method employs an oxidant
which is safer to handle than aq. hydrogen peroxide, utilizes an
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environmentally friendly solvent and generates peracetic acid

in situ. The resulting epoxides were obtained in very good to
excellent yields using stoichiometric amounts of UHP and
catalytic amounts of Novozyme-435 in ethyl acetate. Studies
on the asymmetric version of this chemo-enzymatic procedure
are currently underway in our laboratory.

Yield
Entry Alkene Epoxide Time/h (%) Experimental
1 Cyclohexene 40 83 Effect of solvent
The experiments were performed with 1 mmol of the alkene,
1.1 mmol of UHP, 50 mg of Novozyme-435, and 3 ml of the
2 1-Methylcyclohexene 2 100
corresponding solvent. The reaction was shaken in a test tube
sealed with a cap in a shake-table at 27 uC and 250 rpm. The
reaction was stopped after 5 h and filtered through a cotton
3 1-Phenylcyclohexene 28 100
plug. The solvent was evaporated and the residue was analyzed
by 1H NMR to determine the ratio of alkene to epoxide. The
residue was dissolved in ethyl acetate and washed with water
4 Cyclooctene 11 100
and aq. sat. soln of NaHCO3 to remove urea and acid.

General procedure for the epoxidation of olefins

5 Norbornene 60 90 A solution of the olefin (1 mmol) in ethyl acetate (1.5 mL) was
added urea–hydrogen peroxide (1.1 mmol) and Novozyme-435
(50 mg). The mixture was shaken in a test tube closed with a
6 a-Pinene 5.5 95 cap in a shake-table at 250 rpm for the time as shown in
Table 4. The solution was filtered through a small piece of
cotton and the solid washed with more ethyl acetate. The
filtrate was washed with water, dried over Na2SO4, filtered and
7 1-Hexene 161 73 the solvent evaporated under reduced pressure. The products
were analyzed by 1H and 13C NMR.
8 1-Octene 46 85
Cyclohexene oxide, 1. 1H NMR (CDCl3): d 3.13 (2H, m),
2.00–1.91 (2H, m), 1.86–1.76 (2H, m), 1.49–1.36 (2H, m), 1.31–
9 Styrene 33 81 1.16 (2H, m); 13C NMR (CDCl3): d 52.3 (2CH), 24.6 (2CH2),
19.6 (2CH2).

10 a-Methylstyrene 46 90 1-Methylcyclohexene oxide, 2. 1H NMR (CDCl3): d 2.95

(1H, br s), 1.95–1.78 (3H, m), 1.66 (1H, m), 1.48–1.34 (2H, m),
1.30 (3H, d, J = 1.8 Hz), 1.32–1.10 (2H, m); 13C NMR
(CDCl3): d 59.7 (CH), 57.6 (C), 30.0 (CH), 24.9 (CH3), 24.1
11 b-Methylstyrene 46 86 (CH2), 20.2 (CH2), 19.8 (CH2).

Phenylcyclohexene oxide, 3. 1H NMR (CDCl3): d 7.40–7.21

(5H, m), 3.06 (1H, s), 2.27 (1H), 2.11 (1H), 1.98 (2H, m), 1.66–
12 Indene 50 77
1.24 (4H, m); 13C NMR (CDCl3): d 142.6 (C), 128.4 (2CH2),
127.3 (CH), 125.4 (2CH2), 62.0 (CH), 60.3 (C), 29.0 (CH2),
24.8 (CH2), 20.2 (CH2), 19.9 (CH2).

Cyclooctene oxide, 4. 1H NMR (CDCl3): d 2.93–2.87 (2H,

terminal olefins of lineal hydrocarbons is known to be slow m), 2.19–2.10 (2H, m), 1.68–1.39 (8H, m), 1.35–1.21 (2H, m);
(entries 7 and 8).14 Oxidation of olefins bonded to aromatics 13
C NMR (CDCl3): d 55.7 (2CH), 26.7 (2CH2), 26.4 (2CH2),
was also successful (entries 9–12). 25.7 (2CH2).

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exo-Norbornene oxide, 5. 1H NMR (CDCl3): d 3.06 (2H, s),
2.44 (2H, s), 1.48 (2H, m), 1.31 (1H, m), 1.21 (2H, m), 0.70
(1H, d, J = 9.9 Hz); 13C NMR (CDCl3): d 51.6 (2CH), 36.8
(2CH), 26.4 (CH2), 25.3 (2CH2).
a-Pinene oxide, 6. 1H NMR (CDCl3): d 3.07 (1H, m), 2.06–
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1.90 (4H, m), 1.73 (1H, m), 1.62 (1H, m), 1.35 (3H, s), 1.30
(3H, s), 0.94 (3H, s); 13C NMR (CDCl3): d 60.5 (C), 57.0 (CH),
45.2 (CH), 40.7 (C), 39.9 (CH), 27.8 (CH2), 26.8 (CH3), 26.0
(CH2), 22.5 (CH3), 20.3 (CH3).
1-Hexene oxide, 7. 1H NMR (CDCl3): d 2.91 (1H, m), 2.75
(1H, dd, J = 5.0, 4.0 Hz), 2.47 (1H, dd, J = 5.1, 2.7 Hz), 1.58–
1.26 (6H, m), 0.92 (3H, t, J = 6.7 Hz); 13C NMR (CDCl3): d
52.6 (CH), 47.3 (CH2), 32.4 (CH2), 28.3 (CH2), 22.7 (CH2),
14.2 (CH3).
1-Octene oxide, 8. 1H NMR (CDCl3): d 2.92 (1H, m), 2.76
(1H, t, J = 4.6 Hz), 2.48 (1H, dd, J = 5.1, 2.7 Hz), 1.58–1.27
(10H, m), 0.90 (3H, t, J = 7.0 Hz); 13C NMR (CDCl3): d 52.6
(CH), 47.3 (CH2), 32.7 (CH2), 31.9 (CH2), 29.3 (CH2), 26.1
(CH2), 22.7 (CH2), 14.2 (CH3).
Styrene oxide, 9. 1H NMR (CDCl3): d 7.39–7.26 (5H, m),
3.87 (1H, dd, J = 4.0, 2.6 Hz), 3.15 (1H, dd, J = 5.5, 4.1 Hz),
2.81 (1H, dd, J = 5.5, 2.6 Hz); 13C NMR (CDCl3): d 137.8 (C),
128.7 (2CH), 128.4 (CH), 125.7 (2CH), 52.6 (CH), 51.4 (CH2).
a-Methylstyrene oxide, 10. 1H NMR (CDCl3): d 7.38–7.23
(5H, m), 2.95 (1H, d, J = 5.5 Hz), 2.78 (1H, dq, J = 5.5, 0.8 Hz),
1.71 (3H, t, J = 0.7 Hz); 13C NMR (CDCl3): d 141.3 (C), 128.5
(2CH), 127.6 (CH), 125.4 (2CH), 57.1 (CH), 56.9 (C), 21.9
(CH3).
b-Methylstyrene oxide, 11. 1H NMR (CDCl3): d 7.34–7.21
(5H, m), 3.55 (1H, d, J = 2.0 Hz), 3.01 (1H, dq, J = 5.1, 2.0 Hz),
1.42 (3H, t, J = 5.1 Hz); 13C NMR (CDCl3): d 137.9 (C), 128.5
(2CH), 128.1 (CH), 125.6 (2CH), 59.6 (CH), 59.1 (CH), 18.0
(CH3).
Indene oxide, 12. 1H NMR (CDCl3): d 7.47 (1H, d, J =
7.2 Hz), 7.26–7.14 (3H, m), 4.23 (1H, d, J = 2.8 Hz), 4.09 (1H,
t, J = 2.8 Hz), 3.18 (1H, d, J = 18 Hz), 2.93 (1H, dd, J = 17.9,
2.9 Hz); 13C NMR (CDCl3): d 143.6 (C), 140.9 (C), 128.6
926 | Green Chem., 2006, 8, 923–926
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(CH), 126.2 (CH), 126.1 (CH), 125.2 (CH), 59.1 (CH), 57.7
(CH), 34.6 (CH2).
Acknowledgements
This research was supported with funds provided by the
Center for Environmentally Beneficial Catalysis under the
National Science Foundation Engineering Research Grant
(EEC-0310689).
References
1 A. Houde, A. Kademi and D. Leblanc, Appl. Biochem. Biotechnol.,
2004, 118, 155–170.
2 (a) U. T. Bornscheuer and R. J. Kazlauskas, Angew. Chem., Int.
Ed., 2004, 43, 6032–6040; (b) P. Bernhardt, K. Hult and
R. J. Kazlauskas, Angew. Chem., Int. Ed., 2005, 44, 2742–2746.
3 (a) F. Björkling, S. E. Godtfredsen and O. Kirk, J. Chem. Soc.,
Chem. Commun., 1990, 1301–1303; (b) F. Björkling, H. Frykman,
S. E. Godtfredsen and O. Kirk, Tetrahedron, 1992, 48, 4587–4592;
(c) O. Kirk, M. W. Christensen, T. Damhus and S. E. Godtfredsen,
Biocatalysis, 1994, 11, 65–77.
4 R. M. Lau, F. van Rantwijk, K. R. Seddon and R. A. Sheldon,
Org. Lett., 2000, 2, 4189–4191.
5 (a) S. Warwel and M. Rüsch gen. Klaas, J. Mol. Catal. B: Enzym.,
1995, 1, 29–35; (b) M. Rüsch gen. Klaas and S. Warwel, J. Mol.
Catal. A: Chem., 1997, 117, 311–319; (c) M. Rüsch gen. Klaas,
M. Kunz and S. Warwel, J. Mol. Catal. B: Enzym., 1999, 5–6,
283–289; (d) M. Rüsch gen. Klaas and S. Warwel, Org. Lett., 1999,
1, 1026–1026.
6 (a) V. Skouridou, H. Stamatis and F. N. Kolisis, Biocatal.
Biotransform., 2003, 21, 285–290; (b) V. Skouridou, H. Stamatis
and F. N. Kolisis, J. Mol. Catal. B: Enzym., 2003, 21, 67–69.
7 S. C. Lemoult, P. F. Richardson and S. M. Roberts, J. Chem. Soc.,
Perkin Trans. 1, 1995, 89–91.
8 B. K. Pchelka, M. Gelo-Pujic and E. Guibe-Jampel, J. Chem. Soc.,
Perkin Trans. 1, 1998, 2625–2627.
9 I. Hoegh, S. Patkar, T. Halkier and M. T. Hansen, Can. J. Bot.,
1995, 73, S869–S875.
10 M. S. Cooper, H. Heaney, A. J. Newbold and W. R. Sanderson,
Synlett, 1990, 533–535.
11 S. Taliansky, Synlett, 2005, 1962–1963.
12 The parameter P is defined as the partition coefficient of the given
solvent in an equimolar mixture of octanol and water.
13 1H NMR of the reaction crude for the oxidation of norbornene
and a-pinene oxide matched previously reported spectra. See the
electronic supplementary information.{ Also, for norbornene
oxide, see: M. Hamamoto, K. Nakayama, Y. Nishiyama and
Y. Ishii, J. Org. Chem., 1993, 58, 6421–6425. For a-pinene oxide,
see: R. Saladino, V. Neri, A. R. Pellicia and E. Mincione,
Tetrahedron, 2003, 59, 7403–7408.
14 W. Kirmse and B. Kornrumpf, Angew. Chem., Int. Ed. Engl., 1969,
8, 75–76.
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