Enantioselective transesterification catalysis by Candida antarctica lipase immobilized on superparamagnetic nanoparticles

Article abstract of DOI:10.1016/j.tetasy.2009.08.022

Lipase B from Candida antarctica can be directly immobilized onto functionalized superparamagnetic nanoparticles, preserving its enzymatic activity in the enantioselective transesterification of secondary alcohols, with excellent results in terms of enantiomeric discrimination. The immobilized enzyme can be easily recovered with a magnet, allowing its reuse with negligible loss of activity.

Full text of DOI:10.1016/j.tetasy.2009.08.022

Tetrahedron: Asymmetry 20 (2009) 2299–2304
Contents lists available at ScienceDirect
Tetrahedron: Asymmetry
journal homepage: www.elsevier.com/locate/tetasy
Enantioselective transesterification catalysis by Candida antarctica lipase
immobilized on superparamagnetic nanoparticles
a,
Caterina G. C. M. Netto a, Leandro H. Andrade b, , Henrique E. Toma
*
*
a Supramolecular NanotechLab, Instituto de Quimica, Universidade de São Paulo, São Paulo—SP, Brazil
b Laboratory of Fine Chemistry and Biocatalysis, Instituto de Quimica, Universidade de São Paulo, 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:
Lipase B from Candida antarctica can be directly immobilized onto functionalized superparamagnetic
nanoparticles, preserving its enzymatic activity in the enantioselective transesterification of secondary
alcohols, with excellent results in terms of enantiomeric discrimination. The immobilized enzyme can
be easily recovered with a magnet, allowing its reuse with negligible loss of activity.
Ó 2009 Elsevier Ltd. All rights reserved.
Received 4 August 2009
Accepted 20 August 2009
Available online 26 September 2009
1. Introduction
2. Results and discussion
Green chemistry has intensified the interest toward new cata-
lytic processes, targeting different types of catalysts and enzymes,
and improved methods of immobilization.1 As essential biocata-
lysts, the enzymes are perfectly adapted to perform with high
activity and selectivity under relatively mild conditions. Among
the enzymes, lipases are well known for their versatility in organic
synthesis, biotechnology, medicinal chemistry, and the food indus-
try. A typical example is lipase B from Candida Antarctica (CALB), an
enzyme capable of performing a wide variety of reactions, such as
hydrolysis, transesterification, and aldol reactions.2 However, one
of the problems of using enzymes as catalysts is their serious lim-
itations in terms of recovery;1h,3 when enhancing the stability and
maintaining the activity of the biocatalyst, the choice of the en-
zyme support is of extreme importance.4,5
In this sense, superparamagnetic nanoparticles derived from
magnetite (iron oxide) which exhibit a large surface area, and high
mass transference, can be easily recovered by using an external
magnetic field.6 In addition to the environmental compatibilities,
their use as enzyme support provides an outstanding green chem-
istry approach, allowing the recovery and extending the useful life-
time of the biocatalyst.
The superparamagnetic nanoparticles of magnetite (MagNP)
were prepared by the co-precipitation method6,8 and treated with
c-aminopropyltriethoxysilane (APTS) to yield the APTS-functional-
ized magnetic nanoparticles, here designated as APTS-MagNP
(Scheme 1). The APTS treatment leads to a silicate coating which
helps stabilizing the magnetic nanoparticles against oxidation by
air. On the other hand, the amino residues are important for pre-
venting the nanoparticle aggregation at pH 7, and for promoting
the molecular interactions with the enzyme. The analytical compo-
sition of the samples employed in this work was determined as
Fe3O4(O3SiC3H8)0.29, and the transmission electron microscopy re-
vealed the presence of nearly cubic particles, exhibiting a core size
distribution from 5 to 10 nm.
Magnetite exhibits a cubic crystallographic cell of 0.83 nm,
encompassing eight Fe3O4 units.9 The specific nanoparticle mass
can be estimated from the number of crystallographic cells con-
tained in the corresponding nanoparticle volume, as given in Table 1.
Enzyme immobilization was carried out by the addition of CAL-B
to the APTS-functionalized magnetic nanoparticles. After work-up,
the immobilized lipase nanoparticles, designated as CAL-B/APTS-
MagNP were dried under vacuum for 2 h and stored at ꢀ7 °C. In
addition to the protein analysis based on the Bradford method, the
presence of the immobilized enzyme was investigated based on
the FTIR spectra of the magnetic nanoparticles, as shown in Figure 1.
The strong peak at 585, 632 cmꢀ1 in the magnetic nanoparticles
Herein we report a direct procedure for the immobilization of
lipase B from C. antarctica (CAL-B)7 onto functionalized magnetic
nanoparticles, and a detailed investigation of its catalytic activity
in the enantioselective transesterification of substituted secondary
alcohols.
corresponds to the m(Fe–O) vibrational peak characteristic of bulk
magnetite.10–13 The silica network is adsorbed on the magnetite sur-
face by Fe–O–Si bonds, while the corresponding infrared signals are
usually overlapped with the Fe–O band of magnetite. The silane
polymer on the surface of the magnetite particles is responsible for
the broad vibrational band at 1100–900 cmꢀ1, assigned to the
* Corresponding authors. Tel.: +55 11 3091 3887; fax: +55 11 3815 5579 (H.E.T.).
E-mail addresses: leandroh@iq.usp.br (L.H. Andrade), henetoma@iq.usp.br (H.E.
Toma).
0957-4166/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2009.08.022

2300
C. G. C. M. Netto et al. / Tetrahedron: Asymmetry 20 (2009) 2299–2304
Fe2+
2Fe3+
NH2
OH
OH
OH
O
O
Si
+
O
NH2
O
Si
O
O
SOH-
Scheme 1. Synthesis of APTS-functionalized magnetic nanoparticles.
Table 1
Calculated molecular weight (MW) of nanoparticles
Size (nm)
Volume (nm)3
Number of cells
Number of Fe3O4 units
MW for Fe3O4
MW of Fe3O4(APTS)0.29
5
6
125
216
211
366
1688
2928
389,928
676,368
455,611
790,303
7
8
9
10
343
512
729
1000
5812
868
1236
1695
4648
6944
9888
13,560
1,073,688
1,604,064
2,284,128
3,132,360
1,254,552
1,874,270
2,668,891
3,660,009
The binding of CAL-B lipase to APTS-MagNP was found to be
strong enough to resist successive washing processes, yielding
reproducible results. Presumably, the binding of the enzyme to
APTS-MagNP is driven by hydrogen bonding and electrostatic
interactions, since at pH 7, CAL-B has a negative charge (isoelectric
point = 6)18 and the magnetic nanoparticles exhibit a positive
charge due to the protonation of the aliphatic amines (pKa ꢁ9).19
In addition, there are many amide and aminoacid residues in
CAL-B available for interacting with the protonated amine groups
of APTS-MagNP.
Before the catalytic tests, the enzyme immobilization procedure
was optimized by varying the temperature, concentration, immo-
bilization time, and washing procedures. The amount of adsorbed
protein was determined by monitoring the total and the remaining
concentrations of the enzyme in the solution, using the Bradford
method.20
For the purpose of discussion, the immobilization of the CAL-B
enzyme on the magnetic nanoparticles (APTS-MagNP) can be rep-
resented by
Figure 1. FTIR spectra of (a) MagNP, (b) CAL-B/APTS-MagNP, (c) APTS-MagNP, and
(d) CAL-B in KBr pellets.
nCAL-B þ APTS-MagNP ꢀ APTS-MagNPðCAL-BÞn
where
Si–O–H and Si–O–Si groups. The weak bands at 3417 and 1625 cmꢀ1
are ascribed to the N–H stretching and H–N–H bending modes of the
free/protonated amino group, respectively.14,15 Hydrogen-bonded
½APTS-MagNPðCAL-BÞnꢂ
K ¼
ð1Þ
n
½CAL-Bꢂ ½APTS-MagNPꢂ
silanols also absorb at around 3200 and 3470 cmꢀ1 14,16
.
The FTIR spectrum of CAL-B (Fig. 1d) shows the characteristic
amide I17 band at 1645 cmꢀ1 , while the amide II band appears
around 1500 cmꢀ1. The amide III and IV bands are observed at
1328 cmꢀ1 and 610 cmꢀ1. The vibrational peaks around 1100–
1000 cmꢀ1 are associated with the C–C and C–N composite vibra-
tions of the protein chain.
When CAL-B is immobilized onto APTS-MagNP (Fig. 1b) there is
a strong overlap of the vibrational peaks of the enzyme (amide I)
and nanoparticles at 1645 cmꢀ1 (amide I), but the characteristic
amide I band is observed at 1500 cmꢀ1. In addition, there is a
strong enhancement of the vibrational peaks in the 1500–
900 cmꢀ1 region, involving the amide III, and 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
binding of the CAL-B enzyme to APTS-MagNP.
The kinetics involved were evaluated from the amount of the
absorbed protein as a function of time, as shown in Figure 2.
As can be seen in Figure 2, the binding process requires about
4 h for equilibration. This observation indicates that specific inter-
actions between the enzyme and the APTS-coated nanoparticles
should take place, rather than just a diffusion-controlled physical
adsorption process.
The extent of immobilization also depends on the amounts of
MagNP employed in the experiments. For a constant mass of
enzyme, there is a non-linear increase of the immobilized enzyme
versus the amount of MagNP, as shown in Figure 3.
Based on the data shown in Figure 3 and Table 1, one can eval-
uate the equilibrium constants for the interaction of CAL-B and
APTS-MagNP by inserting the known concentrations of the enzyme
([CAL-B]total), and of the immobilized species, [APTS-MagNP(CAL-
B)n] into the corresponding equations.

C. G. C. M. Netto et al. / Tetrahedron: Asymmetry 20 (2009) 2299–2304
2301
3
n ¼ 3 K ¼ 3x=f½CAL-Bꢂtotal ꢀ 3xg f½APTS-MagNPꢂtotal ꢀ xg
ð4Þ
Although the values of x are experimentally known, the concen-
tration of the magnetic nanoparticles [APTS-MagNP]total depends
upon the molecular weight assumed for the different sizes (Table
1), and this point should be taken into consideration in the calcu-
lations. For n = 1 and 2, the calculated values of K were typically
negative and can be discarded for the majority of the concentra-
tions of the APTS-MagNP employed, assuming a nanoparticle size
distribution from 5 to 10 nm. However, in the case of n = 3, the cal-
culated values of K were positive and constant within one order of
magnitude, as one can see in Table 2. The average constant (K1:3
)
was 1.41 ꢃ 1013 molꢀ3 dm9 at 32 °C.
The influence of the temperature on the immobilization yield
was also investigated. As shown in Figure 4, when the temperature
increases, the amount of adsorbed enzymes decreases. The calcula-
tion of the association constants at 37 and 42 °C led to K1:3
=
2.96 ꢃ 1012 and 8.53 ꢃ 1011 molꢀ3 dm9, respectively. The corre-
sponding thermodynamic parameters based on the Gibbs equation
were obtained from the linear plot of ln K versus 1/T (Fig. 4 inset)
Figure 2. Kinetics of immobilization of CAL-B (5.2 mg/mL) on APTS-MagNP (30 mg/
mL) at 32 °C.
as
D
H = ꢀ220 kJ molꢀ1 and
D
S = ꢀ480 J molꢀ1 Kꢀ1. It should be
mentioned that CAL-B undergoes 50% denaturation only at
53 °C,21 quite far from the temperatures employed in the immobi-
lization procedure. Therefore, the thermodynamic data are consis-
tent with relatively strong chemical interactions, presumably
involving hydrogen bonds between CAL-B and the magnetic nano-
particles, rather than a simple adsorption process.
After carrying out a controlled and reproducible binding of CAL-
B on the APTS-modified magnetic nanoparticles, a systematic study
was performed to evaluate their enantioselective behavior on the
transesterification reaction of secondary alcohols. In particular,
Figure 3. Relative amounts of adsorbed protein vs the mass of APTS-MagNP used in
the immobilization process.
Considering that
½APTS-MagNPðCAL-BÞnꢂ ¼ nx
½CAL-Bꢂfree ¼ ½CAL-Bꢂtotal ꢀ nx
½APTS-MagNPꢂfree ¼ ½APTS-MagNPꢂtotal ꢀ x
Equations can be expressed in the following ways, for
n ¼ 1 K ¼ x=f½CAL-Bꢂtotal ꢀ xgf½APTS-MagNPꢂtotal ꢀ xg
ð2Þ
ð3Þ
Figure 4. Effect of temperature on the enzyme binding to the magnetic nanopar-
ticles (30 mg) showing the linear ln K versus 1/T plot in the inset.
2
n ¼ 2 K ¼ 2x=f½CAL-Bꢂtotal ꢀ 2xg f½APTS-MagNPꢂtotal ꢀ xg
Table 2
Calculated association constants, K1:3 (molꢀ3 dm9) based on Eq. 4, assuming the [MagNP(CAL-B)3] composition (32 °C), for several amounts of MagNP and different nanoparticles
size
Size (nm)
Mass (mg)
30
5
10
20
40
50
60
5
6
7
8
9
2.03 ꢃ 1013
9.85 ꢃ 1012
2.39 ꢃ 1013
8.52 ꢃ 1013
2.53 ꢃ 1012
5.10 ꢃ 1012
1.04 ꢃ 1013
2.52 ꢃ 1013
1.73 ꢃ 1014
4.47 ꢃ 1012
8.63 ꢃ 1012
1.63 ꢃ 1013
3.26 ꢃ 1013
8.17 ꢃ 1013
2.28 ꢃ 1015
4.68 ꢃ 1012
8.12 ꢃ 1012
1.29 ꢃ 1013
1.92 ꢃ 1013
2.74 ꢃ 1013
3.76 ꢃ 1013
3.51 ꢃ 1012
6.55 ꢃ 1012
1.16 ꢃ 1013
2.04 ꢃ 1013
3.77 ꢃ 1013
8.23 ꢃ 1013
3.12 ꢃ 1012
5.75 ꢃ 1012
9.99 ꢃ 1012
1.71 ꢃ 1013
2.98 ꢃ 1013
5.69 ꢃ 1013
10

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C. G. C. M. Netto et al. / Tetrahedron: Asymmetry 20 (2009) 2299–2304
the enantioselective acetylation of alcohols derived from (R,S)-1-
phenylethanol mediated by CAL-B/APTS-MagNP was investigated.
In order to demonstrate the characteristic catalytic activity for
lipases, we first examined the performance of the CAL-B/APTS-
MagNP in the hydrolysis of para-nitrophenyl palmitate. In this
case, the observed activity was about 0.8 U/mg for immobilized
CAL-B, while that for free CAL-B was 0.4 U/mg. The activity of
CAL-B increased by a factor of 2 in the immobilized form.
The enantioselective activity of CAL-B/APTS-MagNP was then
evaluated in the transesterification reactions using (RS)-1-phe-
nyl-ethanol (para-nitro, para-methyl, para-bromo, para-chloro,
and para-methoxy derivatives) in the presence of vinyl acetate as
an acyl donor. All the reactions exclusively yielded the correspond-
ing (R)-1-phenylethyl acetate, as shown in Figure 5, demonstrating
the successful use of CAL-B/APTS/MagNP as catalyst in the kinetic
resolution of secondary alcohols via transesterification reaction.
As a matter of fact, it is known that the CAL-B enzyme is highly
selective for the (R)-enantiomer of the secondary alcohols because
there is a physical restriction in the active site, generating a stereo-
specific pocket to accommodate the methyl group of 1-phenyleth-
anol, in the (R)-configuration.22
ondary alcohols. More important, the immobilized enzyme can be
conveniently recovered with a magnet and used at least four times,
with negligible loss of activity.
4. Experimental
4.1. Materials
All commercially available chemicals were used without further
purification. Ferric chloride hexahydrate (FeCl3ꢄ6H2O, >98%) and
ferrous chloride (FeCl2, 98%) were obtained from Sigma–Aldrich.
Toluene (99.9%), monobasic sodium phosphate monohydrate
(NaH2PO4ꢄH2O, >99%), and dibasic sodium phosphate (HNa2PO4,
99%) were obtained from Merck. Sodium hydroxide (NaOH, 97%),
ethanol (99.8%), and methanol (99.9%) were obtained from Synth.
c-Aminopropyltriethoxisilane was obtained from Pierce. C. antarc-
tica Lipase B (CALB-L) was donated by Novozymes (Parana-Brazil).
Phenylethanols (RS)-1a–f were prepared by reduction of the corre-
sponding commercially available acetophenones (Sigma–Aldrich)
with NaBH4 in methanol. The acetates (RS)-2a–f were prepared
by acetylation of the precursor alcohols (RS)-1a–f by reacting with
acetic anhydride in dichloromethane, triethylamine, and 4-(N,N-
dimethylamino)pyridine as catalyst.
Table 3 summarizes the results obtained for all the reactions
tested. It is noteworthy that the (R)-enantiomer from the racemic
mixture of substituted 1-phenylethanols 1a–f was acetylated with
excellent enantioselectivity, >99%. The enantiomeric ratio E (E-va-
lue), given by Eq. 5,1 was quite high (E >300) for all the reactions
investigated (Table 1) reflecting the high specificity of the CAL-B
immobilized on the superparamagnetic nanoparticles.
4.2. Synthesis of superparamagnetic nanoparticles
Superparamagnetic nanoparticles of magnetite were obtained
by the co-precipitation method. A solution of Fe2+ and Fe3+ ions
(0.1 mol/L and 0.2 mol/L, respectively) was slowly dropped into a
solution of NaOH (0.5 mol/L). To protect the nanoparticles against
oxidation a flow of N2 was maintained during the reaction. The
reaction mixture was kept under mechanical stirring (2000 rpm)
for 30 min. After this period, the black suspension obtained was
washed several times with deoxygenated Milli-Q water until pH
7 was reached.
E ¼ lnfð1 ꢀ eesÞ=ð1 þ ees=eepÞg= lnfð1 þ eesÞ=ð1 þ ees=eepÞg
ð5Þ
where eep refers to the enantiomeric excess of the product and ees
refers to the enantiomeric excess of the substrate.
In order to evaluate the recycling potential of the CAL-B immo-
bilized on the supermagnetic nanoparticles, a series of repetitive
experiments were carried out for the transesterification reaction
using (R,S)-1-phenylethanol 1a as a substrate. After each process,
the catalytic particles were collected with a magnet, washed, and
used again in a new experiment. As shown in Figure 6, the immo-
bilized enzyme maintained its activity, exhibiting less than 5% de-
cay after four cycles. The E-value and the enantiomeric excess of
product 2a were the same after each cycle, that is, >300% and
>99%, respectively).
4.3. Nanoparticles surface modification
For the surface modification of the nanoparticles, an adaptation
of the Kim et al. method was used.22 After confining the particles
with the help of an external magnetic field, the black solid was
washed three times with 50 mL of analytical grade methanol. This
material was dispersed in 70 mL of toluene/methanol mixture
(1:1) and heated at 95 °C under N2 atmosphere until 50% of the
solution volume was evaporated. After the evaporation, 35 mL of
methanol was added and the mixture was re-evaporated to one-
half. This procedure was repeated until the residual water was
3. Conclusion
The adsorption of lipase B from C. antarctica on magnetic nanopar-
ticles provides an efficient strategy in enzymatic catalysis, preserving
its highly enantioselective activity in the transesterification of sec-
magnet
O
CAL-B
CAL-B
OH
O
OH
O
O
+
+
R
R
R
OH
O
(RS)-1a-f
(R)-1a-f
(S)-2a-f
1a R = H,
1b R = NO2
1c R = CH3O 1f R = Me
1d R = Cl
1e R = Br
Figure 5. Application of CAL-B/APTS/MagNP as a catalyst in the kinetic resolution of secondary alcohols via a transesterification reaction.

C. G. C. M. Netto et al. / Tetrahedron: Asymmetry 20 (2009) 2299–2304
2303
Table 3
Kinetic resolution of (RS)-1-phenylethanols 1a–f via transesterification reaction catalyzed by CAL-B supported on supermagnetic nanoparticlesa
Substrate
Vinyl acetate (mmol)
Time (h)
(S)-1a–f eec (%)
(R)-2a–f eec (%)
Conv.d (%)
Ee
OH
5
6
24
13
48
>99
>99
12
32
226
320
(RS)-1a
OH
50b
24
76
>99
43
458
(RS)-1a
OH
5
6
24
13
48
>99
>99
12
32
226
320
O2N
MeO
Cl
(RS)-1b
OH
5
6
24
19
55
>99
>99
16
36
239
346
(RS)-1c
OH
5
6
6
6
21
32
35
>99
>99
>99
17
25
26
264
319
320
(RS)-1d
OH
5
5
Br
(RS)-1e
OH
Me
(RS)-1f
a
b
c
Reaction conditions: substrate (1 mmol) 1a–f, methyl tert-butyl ether (1 mL); 32 °C, 30 mg of CAL-B/APTS-MagNP (this system contains 0.4 mg of the CAL-B).
10 mmol of (RS)-1-phenylethanol and 300 mg of CAL-B/APTS-MagNP (This system contains 4 mg of the CAL-B).
ee = Enantiomeric excess was determined by chiral GC analysis.
Conv. = Conversion = ees/ees + eep.
E = ln{(1 ꢀ ees)/(1 + ees/eep)}/ln{(1 + ees)/(1 + ees/eep)}.
d
e
thoroughly removed. Then, the
c
-aminopropyltriethoxysilane
shaker at 32 °C and 160 rpm for 2 h. After the immobilization time,
the magnetic nanoparticles were washed three times with phos-
phate buffer solution (pH 7, 3 ꢃ 0.5 mL), dried at vacuum for 2 h,
and then stored at ꢀ7 °C.
(0.2 mL/mg of the magnetic nanoparticle) was added to the mag-
netic nanoparticles. The suspension was heated under N2 and re-
fluxed at 110 °C through 12 h. After the surface modification the
suspension was confined using a magnet, and the solid was
washed 10 times with 10 mL of methanol and 10 mL of ethanol,
and then dried under vacuum for 24 h. Elementary Anal. Calcd
for Fe3O4(O3SiC3H8N)0.29: C, 3.4; H, 0.76; N, 1.32. Found: C, 3.8;
H, 0.83; N, 1.11.
The Bradford method20 was used for determining the amount of
the protein in the supernatant using Bovine Serum Albumin as
standard. By carrying out a mass balance, the amount of lipase
immobilized into the magnetic nanoparticles was evaluated. A vis-
ible-UV HP 8453-A spectrophotometer from Hewlett Packard was
employed for the determination of the protein content.
4.4. CAL-B immobilization
4.5. Nanoparticle characterization
Magnetic nanoparticles (30 mg) were dispersed in 1 mL of
phosphate buffer solution (pH 7.0, 0.1 M). Then, 600
B from C. antarctica (5.2 mg/mL, protein content) was added into
the suspension and the resulting mixture was stirred on an orbital
l
L of lipase
The characterization of the magnetic nanoparticles was carried
out as previously reported.8 Relevant vibrational information con-
firming the presence of the enzyme in the CAL-B/APTS-MagNPs

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C. G. C. M. Netto et al. / Tetrahedron: Asymmetry 20 (2009) 2299–2304
(RS)-1-(4-Chlorophenyl)ethyl acetate 2e: Isotherm at 130 °C,
(R)-enantiomer 5.31 min, and (S)-enantiomer 6.00 min.
(RS)-1-(4-Methylphenyl)ethyl acetate 2f: 93 °C up to 113 °C,
1 °C/min, (R)-enantiomer 10.43 min, and (S)-enantiomer 11.75 min.
4.8. Absolute configuration
The absolute configurations of all compounds were determined
by comparison of the sign of the measured specific rotation with
those in the literature.1
4.9. Enzymatic activity assay using the hydrolysis of para-
nitrophenyl palmitate (pNPP)
The activity assay was done by monitoring at 410 nm the
appearance of para-nitrophenol during the hydrolysis of pNPP as
substrate, in accordance with the procedure reported by Teng
et al.23 One unit of lipase activity is defined as the amount of lipase
which catalyzed the production of 1 lmol of the para-nitrophenol
per minute under the experimental conditions.
Figure 6. Recycling behavior of CAL-B immobilized on magnetite nanoparticles for
kinetic resolution of the (RS)-1-phenylethanol rac-1a: 30 mg of CAL-B/APTS-
MagNP; 1 mmol of rac-1a; 5 mmol of vinyl acetate; 6 h at 32 °C; aconversion
expressed as c = ees/ees + eep.
was obtained with Fourier transform infrared spectroscopy (FTIR-
8300, SHIMADZU) using KBr pellets. Each spectrum was collected
Acknowledgments
after accumulating 50 scans at a resolution of 2 cmꢀ1
.
The authors acknowledge the support from FAPESP, CNPq, and
PETROBRAS, and Professor Pedro K. Kyohara (Institute of Physics,
Univ. S. Paulo) for obtaining TEM images of APTS-MagNP.
4.6. General procedure for the kinetic resolution of the alcohols
(RS)-1a–f
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enzyme on magnetic nanoparticles (0.4 mg protein/30 mg mag-
netic nanoparticle) was added the appropriate alcohol 1a–f
(1 mmol). The reaction mixture was stirred on a rotary shaker
(32 °C, 160 rpm) for the appropriate time (Table 1). After this, the
mixture was filtered and the solvent was evaporated.
4.7. Evaluation of the enzymatic kinetic resolutions
After the reaction time given in Table 1, the samples were
analyzed by GC analysis using a chiral capillary column. The enan-
tiomeric excess of the compounds were determined by chromato-
graphic comparison with authentic samples of (RS)-alcohols 1a-e
and (RS)-acetates 2a–e synthesized chemically (see Section 4.1).
GC conditions (carrier gas-H2, 100 kPa): injector 220 °C, detec-
tor 220 °C, column temperature, and retention time, tR (min), for
each compound are indicated below.
(RS)-phenylethanol 1a: Isotherm at 107 °C, (R)-enantiomer
6.22 min, and (S)-enantiomer 6.87 min.
(RS)-1-(4-Nitrophenyl)ethanol 1b: Isotherm at 150 °C, (R)-
enantiomer 19.92 min, and (S)-enantiomer 20.30 min.
(RS)-1-(4-Methoxyphenyl)ethanol 1c: Isotherm at 119 °C, (R)-
enantiomer 6.22 min, and (S)-enantiomer 6.87 min.
(RS)-1-(4-Bromophenyl)ethanol 1d: Isotherm at 135 °C, (R)-
enantiomer 9.54 min, and (S)-enantiomer 10.52 min.
(RS)-1-(4-Chlorophenyl)ethanol 1e Isotherm at 130 °C, (R)-
enantiomer 7.33 min, and (S)-enantiomer 8.19 min.
(RS)-1-(4-Methylphenyl)ethanol 1f: Isotherm at 113 °C, (R)-
enantiomer 6.79 min, and (S)-enantiomer 7.71 min.
(RS)-1-Phenylethyl acetate 2a: 110 °C up to 103 °C, 1 °C/min,
(R)-enantiomer 4.35 min, and (S)-enantiomer 5.05 min.
(RS)-1-(4-Nitrophenyl)ethyl acetate 2b: Isotherm at 150 °C, (R)-
enantiomer 9.44 min, and (S)-enantiomer 10.23 min.
(RS)-1-(4-Methoxyphenyl)ethyl acetate 2c: Isotherm at 110 °C,
(R)-enantiomer 11.79 min, and (S)-enantiomer 13.47 min.
(RS)-1-(4-Bromophenyl)ethyl acetate 2d: Isotherm at 135 °C,
(R)-enantiomer 6.93 min, and (S)-enantiomer 7.75 min.
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