Kinetic resolution of a drug precursor by Burkholderia cepacia lipase immobilized by different methodologies on superparamagnetic nanoparticles

Article abstract of DOI:10.1016/j.molcatb.2010.03.002

Burkholderia cepacia lipase was immobilized on superparamagnetic nanoparticles using three different methodologies (adsorption, chemisorption with carboxibenzaldehyde and chemisorption with glutaraldehyde) and employed in the kinetic resolution of a chiral drug precursor, (RS)-2-bromo-1-(phenyl) ethanol, via enantioselective acetylation reaction. An excellent improvement of lipase catalytical performance was observed. Free B. cepacia lipase gave the ester (S)-2 with poor E-value <30, and after its immobilization to magnetic nanoparticles the E-value was up to >200. The effect of several reaction parameters in the kinetic resolution was studied. The best results for kinetic resolution were obtained using vinyl acetate as acetyl donor and toluene as solvent, typically yielding the ester in high enantiomeric excess (>99%) and E-value (E > 200). Of the three tested immobilization methods, chemisorption with glutaraldehyde was the best one in terms of temperature stability and yield product.

Full text of DOI:10.1016/j.molcatb.2010.03.002

Journal of Molecular Catalysis B: Enzymatic 66 (2010) 55–62
Contents lists available at ScienceDirect
Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Kinetic resolution of a drug precursor by Burkholderia cepacia lipase immobilized
by different methodologies on superparamagnetic nanoparticles
Leandro H. Andradea,∗, Lya P. Rebeloa, Caterina G.C.M. Nettob, Henrique E. Tomab
a Laboratory of Fine Chemistry and Biocatalysis, Institute of Chemistry, University of São Paulo, CEP 05508-900, São Paulo, SP, Brazil
b Supramolecular NanotechLab, Institute of Chemistry, University of São Paulo, CEP 05508-900, São Paulo, SP, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history:
Burkholderia cepacia lipase was immobilized on superparamagnetic nanoparticles using three dif-
ferent methodologies (adsorption, chemisorption with carboxibenzaldehyde and chemisorption with
glutaraldehyde) and employed in the kinetic resolution of a chiral drug precursor, (RS)-2-bromo-1-
(phenyl)ethanol, via enantioselective acetylation reaction. An excellent improvement of lipase catalytical
performance was observed. Free B. cepacia lipase gave the ester (S)-2 with poor E-value <30, and after
its immobilization to magnetic nanoparticles the E-value was up to >200. The effect of several reaction
parameters in the kinetic resolution was studied. The best results for kinetic resolution were obtained
using vinyl acetate as acetyl donor and toluene as solvent, typically yielding the ester in high enan-
tiomeric excess (>99%) and E-value (E > 200). Of the three tested immobilization methods, chemisorption
with glutaraldehyde was the best one in terms of temperature stability and yield product.
© 2010 Elsevier B.V. All rights reserved.
Received 15 December 2009
Received in revised form 9 March 2010
Accepted 10 March 2010
Available online 20 March 2010
Keywords:
Lipase
Kinetic resolution
Superparamagnetic nanoparticles
Burkholderia cepacia lipase
Chiral drug
1. Introduction
of the biocatalyst without spending energy, by using permanent
magnets.
The use of lipases as catalysts is contributing to the rise of
the exciting and rapidly growing area of chiral organic synthe-
sis [1–10]. As a matter of fact, research in this area is pursuing
the discovery of new efficient enzymes, new target compounds,
and also of new convenient solid supports, capable of sustaining
the enzymatic activity in organic media with minimum loss. In
particular, the immobilization of enzymes on magnetic particles
is viewed as a very attractive strategy, for allowing their sepa-
ration and recovery from the reaction media by the simple use
of a magnet [11–15]. On the other hand, the reusability of the
immobilized enzymes represents an outstanding green-chemistry
approach, reducing the cost and amount of such expensive biocat-
alysts. It should be noticed that superparamagnetic nanoparticles
exhibit a single magnetic domain in their nanocrystalline struc-
tures, and this is responsible for their exceptional magnetization
properties in the presence of a magnetic field. A suitable superpara-
magnetic species is based on magnetite (␥-Fe3O4). Large surface
area, high mass transference and rapid response to external mag-
netic fields are remarkable characteristics ennobling this type of
nanometric support or carrier. In addition, enzyme immobiliza-
tion on superparamagnetic nanoparticles can permit the recovery
Recently, Burkholderia cepacia lipase (BCL, also known as Pseu-
domonas cepacia lipase) has been employed as biocatalyst in a
variety of synthetic applications, performing reactions, such as
hydrolysis [16–18], enantioselective acylation [19] and transes-
terification [20–23]. In view of the wide usefulness of P. cepacia
lipase, the possibility of exploring its recyclability using superpara-
magnetic nanoparticles may be quite rewarding. For this reason,
a detailed study focusing on the immobilization of this lipase
on superparamagnetic nanoparticles was carried out, aiming its
application in the kinetic resolution of the chiral drug precur-
sor, (RS)-2-bromo-1-(phenyl)ethanol. This compound is envisaged
as a common building block to the chiral compounds Fluoxetine,
Tomoxetine and Nisoxetine [24–26], which are among the most
important pharmaceuticals for the treatment of psychiatric disor-
ders (depression, anxiety, alcoholism) and also metabolic problems
(obesity and bulimia).
2. Results and discussion
Immobilization of enzymes at superparamagnetic nanoparti-
cles involves specific molecular interactions at the surface, and can
induce some structural reorganization, thus changing the behavior
of the catalytic sites. Therefore, a comparative study was carried out
in this work, by immobilizing the lipase according to three differ-
ent procedures. Initially, magnetite (Fe3O4) nanoparticles (MagNP)
were prepared by the co-precipitation method, mixing Fe3+ and
∗
Corresponding author.
E-mail addresses: leandroh@iq.usp.br (L.H. Andrade), henetoma@iq.usp.br
(H.E. Toma).
1381-1177/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.molcatb.2010.03.002

56
L.H. Andrade et al. / Journal of Molecular Catalysis B: Enzymatic 66 (2010) 55–62
species (Carboxy-APTS-MagNP). Thus, the lipase can be covalently
bound to the carboxy-substituted species via carbodiimide acti-
vation. This step involves the conversion of the carboxyl group
into an active ester by reacting with EDC (1-ethyl-3-(3-dimethyl
aminopropyl) carbodiimide hydrochloride), allowing the binding
of the lipase to afford the biocatalyst named as BCL-Carboxy-APTS-
MagNP.
Scheme 1. (i) Co-precipitation of Fe2+ and Fe3+ oxides in alkaline solution; (ii)
silanization of the Fe3O4 nanoparticles with APTS.
By using this methodology, it was possible to bind 0.25 mg of
protein to 20 mg of Carboxy-APTS-MagNP, quantified by Bradford
method.
In the third procedure, B. cepacia lipase was immobilized
onto the superparamagnetic nanoparticles using glutaraldehyde to
afford the biocatalyst named as BCL-Glu-APTS-MagNP (Scheme 3).
The role of glutaraldehyde is to react with the amino group of
APTS, forming an imine bond and leaving another terminal alde-
hyde group for reacting with the amino residues of the enzyme, as
shown in Scheme 3.
Fe2+ ions in NaOH solution, under an argon atmosphere. In order to
eliminate any possible interference from the iron-oxide/hydroxide
sites in the catalytic process, and to promote a better nanoparti-
cles protection against air oxidation, a silanization treatment was
applied, using ␥-aminopropyltriethoxysilane (APTS), as shown in
Scheme 1. The resulting black material exhibited strong magne-
tization in the presence of a magnetic field. Transmission electron
microscopy revealed the presence of nearly cubic particles, exhibit-
ing an average core size of 10 nm [27]. The silanization treatment
provides a stable silicate coating by forming strong Fe–O–Si and
cross-linked Si–O–Si bonds, leaving the ␥-aminopropyl groups
available for interacting with external species, protons, metal ions
and solvent molecules. At neutral or slightly acidic solutions, the
APTS amino groups are protonated (pKa = 9), yielding a net positive
charge responsible for the stabilization of the colloidal solutions.
The first immobilization procedure consisted in the direct
interaction of lipase with the APTS modified superparamagnetic
nanoparticles (APTS-MagNP). In spite of its rather simple nature,
the direct interaction of the enzyme with the functionalized
nanoparticles surface is driven by adsorption, proceeding accord-
ing to a Langmuir isotherm which depends on many parameters,
including the relative amounts of the interacting species, temper-
ature and time. In this work, by using the same concentration of
B. cepacia lipase (0.55 mg/mL) at 32 ◦C, the amount of APTS-MagNP
was varied from 10 to 30 mg. The extent of enzyme binding was
quantified using Bradford’s method [28] after 3 successive wash-
ings. A typical analysis indicated the presence of 0.21 mg of protein
bound to 20 mg of APTS-MagNP, a result consistent with a relatively
strong enzyme–nanoparticle interaction. It can involve hydrogen
bonding formation and electrostatic interactions, since at pH 7, B.
cepacia lipase has a negative charge (isoelectric point = 5.2) [29] and
the magnetic nanoparticles exhibit a positive charge due to the pro-
tonation of the aliphatic amino group [30]. In addition, there are
also many amide and aminoacid residues in this lipase available
for interacting with the proton.
A typical amount of lipase immobilized by this methodology
was 0.28 mg of protein to 20 mg of Glu-APTS-MagNP, quantified by
Bradford method.
2.1. Topographic analysis
B. cepacia lipase immobilized onto APTS-MagNPs, seems to form
extensive conglomerates when it was deposited at a flat mica sur-
face, as can be seen in the AFM topographic and phase contrast
imaging shown in Fig. 1. The conglomerate images are reproduced
in the magnetic force microscopy (Fig. 1C) confirming their mag-
netic nature. Similar images were observed for the immobilized
enzymes prepared by the several methods.
The conglomerate profiles are better seen in the 3D and cross-
diagonal scan images, shown in Fig. 2. They actually appear like
islands about 500 nm wide, composed by agglomerated nanoparti-
cles. However, it should be noticed that the typical AFM images of
APTS-MagNP exhibit a rather uniform distribution of nanoparticles
all over the flat mica surface (Fig. 3). Therefore, the formation of
such conglomerates, instead of a random distribution of the parti-
cles, indicates that the enzyme is facilitating their agglomeration
onto the flat mica surface, during the drying process.
2.2. FTIR vibrational analysis
Typical FTIR spectra of the magnetic nanoparticles (MagNP) and
their APTS modified forms (Fig. 4), as well as of the free B. cepacia
lipase and their immobilized forms were superimposed in Fig. 5,
for comparison purposes. The strong peaks at 585 and 632 cm−1
in the magnetic nanoparticles correspond to the ꢀ(Fe–O) vibra-
tional mode characteristic of bulk magnetite [31–34]. The silica
network is adsorbed on the magnetite surface by Fe–O–Si bonds,
and the corresponding infrared signals are usually overlapped with
In the second procedure, immobilization of B. cepacia lipase
on superparamagnetic nanoparticles was carried out chemically
through the formation of a covalent link, after the modification
of APTS-MagNP with 4-carboxybenzaldehyde (Scheme 2). In this
procedure, the amino group of APTS reacts with aldehyde, forming
an imine intermediate, which is then reduced with NaBH4, along
with the remaining aldehyde groups, to give a carboxy-substituted
Scheme 2. (i) 4-Carboxybenzaldehyde, 4 h; (ii) NaBH4, 1 h; (iii) EDC, 20 min; (iv) Burkholderia cepacia lipase, 1 h.
Scheme 3. (i) Glutaraldehyde; (ii) B. cepacia lipase.

L.H. Andrade et al. / Journal of Molecular Catalysis B: Enzymatic 66 (2010) 55–62
57
Fig. 1. Atomic force microscopy of lipase from B. cepacia immobilized via glutaraldehyde method on magnetic nanoparticles: (A) topography, (B) phase contrast and (C)
magnetic force microscopy.
Fig. 2. (A) 3D view of the atomic force microscopy of lipase from B. cepacia immobilized via glutaraldehyde method on magnetic nanoparticles and (B) a diagonal scan
showing the specific roughness.
Fig. 3. Atomic force microscopy of (A) topography and (B) phase contrast of a free lipase from B. cepacia film, and (C) topography of magnetic nanoparticles film (APTS-MagNP).
and 726 cm−1. The vibrational peaks around 1100–1000 cm−1 are
associated with the C–C and C–N composite vibrations of the pro-
tein chain.
When B. cepacia lipase is immobilized onto APTS-MagNP
(Fig. 5A) there is a strong overlap of the vibrational peaks of
the enzyme and nanoparticles. The amide I band appears at
1635 cm−1. The observed shift has been associated with an increase
in the inter- and intramolecular ␤-structure and a decrease in
the ␣-helix amount. This interchange of secondary structures can
sometimes be observed in other nanoparticles–enzyme systems
the Fe–O band of magnetite. The silane polymer onto the surface
of magnetite particles is responsible for the broad vibrational band
at 1100–900 cm−1, assigned to the Si–O–H and Si–O–Si groups. The
weak bands at 3417 and 1625 cm−1 are ascribed to the N–H stretch-
ing and H–N–H bending modes of the free/protonated amino group,
respectively [35,36]. Hydrogen-bonded silanols also absorb around
3200 and 3470 cm−1 [35,37].
The FTIR spectrum of B. cepacia lipase shows the characteristic
amide I [38] band at 1652 cm−1, while the amide II band appears
around 1568 cm−1. The amide III and IV bands are observed at 1285
Scheme 4. Kinetic resolution of (RS)-2-bromo-1-(phenyl)ethanol (1) by B. cepacia lipase-catalyzed acetylation.

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L.H. Andrade et al. / Journal of Molecular Catalysis B: Enzymatic 66 (2010) 55–62
Table 1
Kinetic resolution of (RS)-2-bromo-1-(phenyl)ethanol by free B. cepacia lipase-
catalyzed acetylation.a.
Entry
Solvent
Time (h)
ee (%)b
(R)-1
Conv. (%)c
Ed
(S)-2
1
2
3
4
TBME
TBME
Toluene
Toluene
5
24
5
2
3
10
20
30
45
70
78
5
6
12
20
<30
<30
<30
<30
24
a
General conditions: (RS)-2-bromo-1-(phenyl)ethanol (0.01 mmol), lipase
(16 mg), vinyl acetate (30 ␮L), solvent (1 mL), 32 ◦C, 800 rpm.
b
ee = enantiomeric excess was determined by chiral GC analysis.
Conv. = conversion = eeS/(eeS + eeP).
E = {ln[eeP(1 − eeS)]/(eeP + eeS)}/{ln[eeP(1 + eeS)]/(eeP + eeS)} [40,41].
c
d
studies, we prepared a lyophilized form of B. cepacia lipase from
this enzymatic solution to be used in the kinetic resolution of (RS)-
2-bromo-1-(phenyl)ethanol.
As one can see in Table 1, the reactions using free B. cepacia lipase
in different solvents and reaction time afforded the ester 2 with very
low E-value, low conversion and poor enantiomeric excess of ester.
Similar experiments were carried out using the enzyme immo-
bilized onto the magnetic nanoparticles, according to three
different procedures. Initially, we evaluated the influence of
different acyl donor nature, solvent, and reaction time for the enan-
tioselective acetylation of (RS)-1 catalyzed by lipase immobilized
onto magnetic nanoparticles using adsorption method (BCL-APTS-
MgNP). In this study, we employed vinyl acetate, isoprenyl acetate
and ethyl acetate, in two different solvents (tert-butyl methyl
ether = TBME and toluene). The results are shown in Table 2 (entries
1–6).
Among the acyl donor evaluated, ethyl acetate did not afford the
ester 2 in both solvents (Table 2, entries 3 and 6). However, vinyl
acetate showed to be a better acyl donor than isoprenyl acetate in
terms of conversion (Table 2, entries 1 and 4). When one compares
the reactions using toluene and TBME as solvent, a little difference
in conversion was observed (Table 2, entries 1 and 4; 2 and 5).
The E-values in all cases were higher than 200, which indicated
an excellent enantioselectivity. As shown in Table 1, KR of (RS)-1
catalyzed by free B. cepacia lipase gave the ester (S)-2 with poor
E-value, and now, by the simple immobilization of this lipase to
magnetic nanoparticles the E-value was up to >200, which indi-
cated a excellent improvement of the catalytical performance of
the B. cepacia lipase.
Fig. 4. Infrared spectra of (A) nude magnetic nanoparticles (MagNP), (B) silanized
magnetic nanoparticles (APTS-MagNP) and (C) magnetic nanoparticles functional-
ized with carboxybenzaldehyde (Carboxy-APTS-MagNP).
[39]. The characteristic amide II band is shifted to 1557 cm−1
.
The amide II position is sensitive to the environment around the
protein. Its shift to lower wavenumbers seems to reflect the immo-
bilization and possible structural changes. In addition, there is
strong enhancement of the vibrational peaks in the 1500–900 cm−1
region, involving the amide III, the skeletal C–C, C–N, and Si–O
vibrations. Although a detailed assignment is not possible at the
present time because of the complexity involved, the observed
changes in the vibrational spectra corroborate the existence of dif-
ferent binding modes of the enzyme to the magnetic nanoparticles.
2.3. Enzyme activity: kinetic resolution of
(RS)-2-bromo-1-(phenyl)ethanol
The lipase activity for each immobilization method was eval-
uated in the kinetic resolution (KR) of a chiral drug precursor
(RS)-2-bromo-1-(phenyl)ethanol (1) via enantioselective acetyla-
tion reaction (Scheme 4).
Typical results obtained for the free B. cepacia lipase, are shown
in Table 1, and will be taken as reference for comparison purposes
with respect to the catalytic activity of the immobilized enzyme
(Table 1). As we employed a lipase solution in the immobilization
In view of these results, vinyl acetate was selected as the acyl
donor to be employed on the evaluation of the influence of reaction
temperature and time in the enzymatic kinetic resolution of the
(RS)-2-bromo-1-(phenyl)ethanol (Table 2, entries 7–12). We car-
ried out the acetylation reaction at temperatures varying from 32
to 52 ◦C using different reaction times (24, 48, and 72 h). The highest
conversion was obtained with the reaction temperature main-
tained at 42 ◦C after 24 h (Table 2, entry 9). However, at the same
temperature, an increase in reaction time resulted a decreased of
conversion occurred probably due to a desorption process, typical
when the lipase is immobilized by adsorption method. In spite of
this, the adsorption method can be a good option to immobilize B.
cepacia lipase onto magnetic nanoparticles, since this protocol is
much easier to handle.
The activities of the B. cepacia lipase immobilized via glu-
taraldehyde and carboxybenzaldehyde methods on magnetic
nanoparticles were also determined in the KR of (RS)-2-bromo-1-
(phenyl)ethanol. The results are shown in Table 3.
Initially, we evaluated the acyl donor and solvent for KR of (RS)-
1 (Table 3, entries 1–6 and 13–18). When ethyl acetate was used as
acyl donor, the ester 2 was not obtained by any immobilized form
of B. cepacia lipase (Table 3, entries 3, 6, 15 and 18). Once again,
Fig. 5. Infrared spectra of (A) lipase from B. cepacia adsorbed on APTS-MagNP, (B)
lipase immobilized onto magnetic nanoparticles by carboxybenzaldehyde method
(Carboxy-APTS-MagNP), (C) lipase immobilized with glutaraldehyde and (D) free
lipase from B. cepacia.

L.H. Andrade et al. / Journal of Molecular Catalysis B: Enzymatic 66 (2010) 55–62
59
Table 2
KR of (RS)-2-bromo-1-(phenyl)ethanol via enantioselective acetylation catalyzed by B. cepacia lipase immobilized onto magnetic nanoparticles using adsorption method.a
.
Entry
T (◦C)
Time (h)
Acylant
Solvent
ee (%)b
(R)-1
Conv. (%)c
Ed
(S)-2
1
2
3
4
5
6
32
32
32
32
32
32
24
24
24
24
24
24
Vinyl acetate
Isoprenyl acetate
Ethyl acetate
Vinyl acetate
Isoprenyl acetate
Ethyl acetate
TBME
TBME
TBME
Toluene
Toluene
Toluene
11
3
–
15
6
–
>99
>99
–
>99
>99
–
10
4
–
13
5
–
>200
>200
–
>200
>200
–
7
8
32
32
48
72
Vinyl acetate
Vinyl acetate
Toluene
Toluene
23
21
>99
>99
19
17
>200
>200
9
10
42
42
24
48
Vinyl acetate
Vinyl acetate
Toluene
Toluene
51
16
>99
>99
34
14
>200
>200
11
12
52
52
24
48
Vinyl acetate
Vinyl acetate
Toluene
Toluene
14
10
>99
>99
12
9
>200
>200
(–) = no reaction.
a
General conditions: (RS)-2-bromo-1-(phenyl)ethanol (0.01 mmol), acylant (30 ␮L), solvent (1 mL), lipase (0.21 mg/20 mg APTS-MgNP), 800 rpm.
ee = enantiomeric excess was determined by chiral GC analysis.
Conv. = conversion = eeS/(eeS + eeP).
E = {ln[eeP(1 − eeS)]/(eeP + eeS)}/{ln[eeP(1 + eeS)]/(eeP + eeS)} [40,41].
b
c
d
vinyl acetate was the best acyl donor, and reactions using toluene
reagents and reaction steps, then, a sharp decrease in enzyme sta-
bility can occur due to these chemical treatments.
and TBME as solvent showed a little difference in conversion but
with excellent enantioselectivities (E-values >200, Table 3, entries
1, 4, 13, and 16).
2.4. Conclusion
KR of (RS)-1 catalyzed by B. cepacia lipase immobilized via glu-
taraldehyde and carboxybenzaldehyde methods was performed
at temperatures varying from 32 to 52 ◦C using different reaction
time (Table 3, entries 7–12 and 19–24). In all cases, the ee prod-
uct and E-values were excellent as observed with BCL immobilized
by adsorption method (Table 2). These results indicate that differ-
ent immobilization methods have no effect on the enzyme activity
in terms of enantioselectivity. In case of the lipase immobilization
by the carboxybenzaldehyde method (BCL-Carboxy-APTS-MagNP),
the increase in reaction time from 24 to 48 h did not make a
significant conversion improvement (Table 3, entries 1, 4, 7, and
8). In addition, the KR at 52 ◦C gave lower conversion than 42 ◦C
indicating a small decay of enzyme activity caused by tempera-
ture (Table 3, entries 9–12). However, a good conversion (24%)
was observed for enantioselective acetylation after 48 h at 42 ◦C
(Table 3, entry 10).
The highest conversions for KR of (RS)-1 were obtained with
the lipase immobilized by the glutaraldehyde method (up to 34%;
Table 3, entry 24). In addition, varying the temperature from 32
to 52 ◦C a linear increase in conversion was obtained (Table 3,
entries 19–24). In this case, we can mention that both immobilized
form of B. cepacia lipase via chemisorption showed very good effi-
ciency at high temperature after 48 h reaction. These results can
be attributed to the covalent binding of lipase to the magnetic
nanoparticles (␥-aminopropyltriethoxysilane modified nanopar-
ticles) which helps stabilizing the enzyme at high temperatures
and long reaction time [42,43]. On the other hand, these results
can also be affect by the protocol employed in the preparation of
modified magnetic nanoparticles. The best results were obtained
with B. cepacia lipase immobilized by the glutaraldehyde method,
in which the lipase was easily treated with previously activated
nanoparticles with glutaraldehyde. Meanwhile, in the preparation
of BCL-Carboxy-APTS-MagNP it was employed different chemical
B. cepacia lipase can be immobilized on magnetite by differ-
ent methods, exhibiting good compatibility with the supporting
material. An excellent improvement of lipase catalytical perfor-
mance, as determined in the KR of (RS)-2-bromo-1-phenylethanol,
was observed. Free B. cepacia lipase gave the ester (S)-2 with poor
E-value <30, and after its immobilization onto magnetic nanoparti-
cles the E-value was up to >200. Immobilization via glutaraldehyde
and carboxybenzaldehyde methods proved to be more effective for
transesterification reactions in high temperatures (up to 52 ◦C).
3. Experimental
3.1. General methods
Amano Lipase PS from B. cepacia was purchased from
Sigma–Aldrich. Solvents were purified by standard procedures.
Some reagents are commercially available and were used without
further purification. Thin-layer chromatography (TLC) was per-
formed using pre-coated plates (Aluminum foil, Silica Gel 60 F254
Merck, 0.25 mm). Silica gel (0.035–0.070 mm, Acros) was used for
column chromatography. GC analyses were performed in a Shi-
madzu GC-17A instrument with a FID detector, using hydrogen as
a carrier gas (100 kPa). The GC chiral column used was a Chirasil-
Dex CB ␤-cyclodextrin (25 m × 0.25 mm) for determination of the
enantiomeric excesses. Low Resolution Mass Spectra (LRMS) were
recorded on a Shimadzu GCMS P5050A (70 eV) spectrometer. Opti-
cal rotations were determined on a JASCO DIP-378 polarimeter.
Infrared spectra were recorded on a Perkin–Elmer 1750-FT-IR
spectrometer. NMR spectra were recorded on Varian Gemini-200
instrument. For NMR 1H (instrument operating at 200 MHz, ı val-
ues are referenced to (CH3)4Si (0 ppm) and for NMR 13C (instrument
operating at 50 MHz, ı values are referenced to CDCl3 (77.0 ppm).

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L.H. Andrade et al. / Journal of Molecular Catalysis B: Enzymatic 66 (2010) 55–62
Table 3
Kinetic resolution of (RS)-2-bromo-1-(phenyl)ethanol via enantioselective acetylation catalyzed by B. cepacia lipase immobilized onto APTS-MagNP using
carboxybenzaldehydea and glutaraldehydeb method.
Entry
T (◦C)
Time (h)
Acylant
Solvent
ee (%)c
(R)-1
Conv. (%)d
Ee
(S)-2
1a
2a
3a
4a
5a
6a
32
32
32
32
32
32
24
24
24
24
24
24
Vinyl acetate
Isoprenyl acetate
Ethyl acetate
Vinyl acetate
Isoprenyl acetate
Ethyl acetate
TBME
TBME
TBME
Toluene
Toluene
Toluene
15
34
–
11
7
>99
>99
–
>99
>99
–
16
12
–
10
6
>200
>200
–
>200
>200
–
–
–
7a
8a
32
32
48
72
Vinyl acetate
Vinyl acetate
Toluene
Toluene
17
16
>99
>99
15
14
>200
>200
9a
42
42
24
48
Vinyl acetate
Vinyl acetate
Toluene
Toluene
21
36
>99
>99
18
24
>200
>200
10a
11a
12a
52
52
24
48
Vinyl acetate
Vinyl acetate
Toluene
Toluene
20
26
>99
>99
17
21
>200
>200
13b
14b
15b
16b
17b
18b
32
32
32
32
32
32
24
24
24
24
24
24
Vinyl acetate
Isoprenyl acetate
Ethyl acetate
Vinyl acetate
Isoprenyl acetate
Ethyl acetate
TBME
TBME
TBME
Toluene
Toluene
Toluene
20
23
–
23
10
–
>99
>99
–
>99
>99
–
17
18
–
18
9
>200
>200
–
>200
>200
–
–
19b
20b
32
32
48
72
Vinyl acetate
Vinyl acetate
Toluene
Toluene
33
40
>99
>99
25
29
>200
>200
21b
22b
42
42
24
48
Vinyl acetate
Vinyl acetate
Toluene
Toluene
22
38
>99
>99
18
26
>200
>200
23b
24b
52
52
24
48
Vinyl acetate
Vinyl acetate
Toluene
Toluene
31
50
>99
>99
24
34
>200
>200
(–) = no reaction; general conditions: (RS)-2-bromo-1-(phenyl)ethanol (0.01 mmol), acylant (30 ␮L), solvent (1 mL), lipase (carboxybenzaldehyde method = 0.25 mg
lipase/20 mg APTS-MgNP, glutaraldehyde method = 0.28 mg lipase/20 mg APTS-MgNP), 800 rpm.
a
BCL immobilized by carboxybenzaldehyde method.
BCL immobilized by glutaraldehyde method.
ee = enantiomeric excess was determined by chiral GC analysis.
Conv. = conversion = eeS/(eeS + eeP).
b
c
d
e
E = {ln[eeP(1 − eeS)]/(eeP + eeS)}/{ln[eeP(1 + eeS)]/(eeP + eeS)} [40,41].
Chemical shifts are given in ppm and coupling constants are given
in Hertz (s = singlet, d = doublet, m = multiplet).
room temperature (25 ◦C) for 24 h. After this period, water (10 mL)
was added to the mixture. The resulting solution was extracted
with CH2Cl2 (3× 10 mL). The combined organic phases were dried
over anhydrous MgSO4 and then filtered. The organic solvent was
evaporated under vacuum and the residue was purified by silica
gel column chromatography, using a mixture of hexane and ethyl
acetate (9:1) as eluent to give the compound 2.
3.2. Synthesis of (RS)-2-bromo-1-(phenyl)ethanol (1)
In an one-necked round-bottomed flask, styrene (260 mg,
2.5 mmol) was dissolved in water (10 mL) and NBS (N-
bromosuccinimide, 470 mg, 2.5 mmol). This mixture was stirred
vigorously at room temperature until the solid NBS disappeared
(35 min). The resulting solution was extracted with CH2Cl2 (3×
10 mL). The combined organic phases were dried over anhydrous
MgSO4 and then filtered. The organic solvent was evaporated
under vacuum and the residue was purified by silica gel column
chromatography, using a mixture of hexane and ethyl acetate (4:1)
as eluent to give the compound 1.
Yield: 90%. IR (KBr) cm−1: 2937, 1604, 1447, 1374, 1219, 1084,
997, 967. 1H NMR (200 MHz, CDCl3): 7.37–7.34 (m, 5H), 6.01–5.94
(m, 1H), 3.65–3.59 (m, 2H), 2.13 (s, 3H). 13C NMR (50 MHz): 171.8,
137.6, 128.2, 129.3, 125.9, 73.6, 36.6, 21.0. MS, m/z (relative inten-
sity): 243 (0.45), 241 (1.22), 118 (4.8), 105 (100), 91 (2.84), 77 (41),
65 (1.98), 51 (15.79).
3.4. Synthesis of Fe3O4 nanoparticles
Yield: 65%. IR (KBr) cm−1: 3395, 3030, 2961, 1493, 1452, 1217,
1061, 991, 761. 1H NMR (200 MHz, CDCl3): 7.33–7.33 (m, 5H), 4.89
(dd, 1H, J = 3.4 Hz, 9 Hz), 3.67–3.48 (m, 2H), 2.46 (s, 1H). 13C NMR
(50 MHz): 140.3, 128.5, 128.3, 125.9, 73.7, 39.9 MS, m/z (relative
intensity): 200–202 (M+2) (3), 121 (1), 107 (100), 91 (6), 79 (51),
65 (4), 51 (15).
Superparamagnetic nanoparticles of magnetite were obtained
by co-precipitation method [44]. To protect the oxidation of the
nanoparticles, water (Milli-Q) was deoxygenated by a flow of N2
and the reaction was maintained under N2 atmosphere. A aqueous
solution of Fe2+ and Fe3+ mixed oxides (0.1 and 0.2 mol/L, respec-
tively) was slowly dropped into a solution of NaOH (0.5 M). The
reaction was maintained under mechanical stirrer (2000 rpm) for
30 minutes. After this period, the black suspension was washed
several times with water until reach pH 7.
3.3. Synthesis of (RS)-1-bromo-2-acetoxy-2-(phenyl)ethane (2)
In an one-necked round-bottomed flask, (RS)-2-bromo-
1-(phenyl)ethanol
1
(207 mg, 1.03 mmol) was dissolved in
The black suspension was washed with methanol (3× 10 mL)
with a help of an external magnetic field. This suspension was dis-
persed in 70 mL of toluene/methanol mixture (1:1) and heated at
95 ◦C under N2 atmosphere until the evaporation of 50% of the solu-
dichloromethane (2.5 mL) with triethylamine (0.42 mL, 3.1 mmol),
DMAP (4-N,N-dimethylaminopyridine; 6.32 mg, 0.052 mmol) and
acetic anhydride (0.3 mL, 3.1 mmol). The mixture was stirred at

L.H. Andrade et al. / Journal of Molecular Catalysis B: Enzymatic 66 (2010) 55–62
61
tion volume. After the evaporation, 35 mL of methanol was added
and the mixture was re-evaporated to one-half. This procedure was
repeated three more times and then, the magnetic nanoparticles
used for surface functionalization.
After the immobilization process, the magnetic nanoparticles
were retained by a magnet and, 100 ␮L of the supernatant solution
was collected and added 3 mL of Bradford reagent. After 3 min the
value of absorbance at 595 nm was obtained and compared with a
calibration curve to calculate the amount of immobilized lipase on
magnetic nanoparticles.
3.5. Functionalization of magnetic nanoparticles
3.5.1. Preparation of amine-functionalized magnetic
nanoparticles (APTS-MagNP)
3.7. General method for lipase immobilization
In a 2 mL microtube (eppendorff®) containing 20 mg of func-
tionalized magnetic nanoparticles were added 1 mL of enzymatic
solution (0.55 mg lipase/mL). The mixture was stirred at 32 ◦C and
800 rpm for 1 h. The immobilized lipase was recovered magneti-
cally and washed with a phosphate buffer solution (3× 100 ␮L, pH
10, 100 mM). The protein content in the supernatant was measured
by Bradford method [28].
The ␥-aminopropyltriethoxysilane was added to the magnetic
nanoparticles (0.2 mL/1 mg magnetic nanoparticles). The suspen-
sion was heated under N2 and refluxed at 110 ◦C through 12 h.
The suspension was washed with methanol (10× 10 mL) and
ethanol (10× 10 mL), and then the amine-functionalized magnetic
nanoparticles were dried under vacuum for 24 h.
In order to confirm the coating of the magnetite surface through
the silanization reaction, an FTIR spectrum of the APTES-magnetite
was obtained [27].
3.8. Enzymatic kinetic resolution of
(RS)-2-bromo-1-(phenyl)ethanol (1)
3.5.2. Preparation of carboxybenzaldehyde-functionalized
In a 2 mL microtube (eppendorff®) containing 20 mg of magnetic
nanoparticles with immobilized lipase by appropriate methodol-
ogy (see Tables 1–3), (RS)-2-bromo-1-(phenyl)ethanol (0.01 mmol)
and vinyl acetate (0.3 mmol) were dispersed in toluene or TBME and
stirred at appropriate temperature (see Tables 1–3) for appropriate
time (see tables) under 800 rpm.
magnetic nanoparticles (Carboxy-APTS-MagNP)
In a three-necked round-bottomed flask, were added amine-
functionalized magnetic nanoparticles (500 mg) and a solution of
TBME and ethanol (1:1, 500 mL). The solution was cooled to 0 ◦C
and carboxybenzaldehyde (6.5 g) was added with a solution of tert-
butyl methyl ether (TBME) and ethanol (1:1, 500 mL). The solution
was stirred for 4 h at 0 ◦C. After this period, the carboxy-imine-
functionalized magnetic nanoparticles were washed with TBME
(50 mL) and dried under vacuum for 24 h.
3.9. GC analysis for determination of the enantiomeric excess
Carboxy-imine-functionalized magnetic nanoparticles (20 mg),
NaBH4 (10 mg) and benzoic acid (24 mg) were ground for 1 h in an
agate mortar and a pestle at room temperature. Then, the particles
were washed with saturated aqueous solution of sodium bicarbon-
ate (1 mL) and TBME (1 mL), and dried under vacuum for 6 h to give
the carboxy-amino-functionalized magnetic nanoparticles.
In an Erlenmeyer flask containing 20 mg of this complex, were
added 5 mL of EDC solution [5% v/v; 1-ethyl-3-(3-dimethyl amino-
propyl) carbodiimide hydrochloride]. This mixture was sonicated
at 25 ◦C during 10 min. After this period, the magnetic nanoparti-
cles were washed with phosphate buffer solution (pH 7, 100 mM,
3× 10 mL) and then used for lipase immobilization.
The enantiomeric excesses of (RS)-2-bromo-1-(phenyl)ethanol
(1) and the product (RS)-1-bromo-2-acetoxy-2-(phenyl)ethane (2)
were analyzed by GC/FID in a chiral capillary column (Chirasil-
Dex CB-Varian). GC conditions: Injector 220 ◦C; detector: 220 ◦C;
pressure: 100 kPa. Column temperature: 110 ◦C, 1 ◦C/min up
to 135 ◦C. Retention times for (RS)-2-bromo-1-(phenyl)ethanol
(1): [(R)-1 = 19.2 min, (S)-1 = 20.0 min]. (RS)-1-bromo-2-acetoxy-2-
(phenyl)ethane (2): [(R)-2 = 14.2 min, (S)-2 = 15.2 min].
3.10. Absolute configuration
The absolute configuration of 2-bromo-1-(phenyl)ethanol and
1-bromo-2-acetoxy-2-(phenyl)ethane was determined by compar-
ison of the sign of the measured specific rotation with those of the
literature [45].
3.5.3. Preparation of glutaraldehyde-functionalized magnetic
nanoparticles (GLU-APTS-MagNP)
In a 2 mL microtube (eppendorff®) containing 20 mg of amine-
functionalized magnetic nanoparticles (APTS-MagNP) were added
25 ␮L of aqueous solution of glutaraldehyde (25% v/v). The mix-
ture was stirred at 25 ◦C and 800 rpm for 2 h. After this period,
the black material with strong magnetization was recovered. The
glutaraldehyde-functionalized magnetic nanoparticles were used
for the lipase immobilization.
Acknowledgements
The authors thank FAPESP and CNPq for financial support. The
authors acknowledge the support from FAPESP, CNPq, and PETRO-
BRAS, and Professor Pedro K. Kyohara (Institute of Physics, Univ. S.
Paulo) for obtaining TEM images of APTS-MagNP.
3.5.4. Preparation of a B. cepacia lipase aqueous solution
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