The influence of conventional heating and microwave irradiation on the resolution of (RS)-sec-butylamine catalyzed by free or immobilized lipases

Article abstract of DOI:10.1590/S0103-50532012005000033

The lipases CAL-B, PSL, PSL-C, PSL-D, and A. niger lipase, free or immobilized in starch (obtained from two types of yam, known in Brazil as cara? (Discorea alata L.) and inhame (Colocasia esculenta (L.) Schott) or gelatin films, were used in the acylation of (RS)-sec-butylamine with different acyl donors in various organic solvents applying conventional heating (CH) or microwave (MW) irradiation. In the case of free A. niger lipase, the conversion degrees were three times higher using MW irradiation when compared to conventional heating at 35 °C. Using free A. niger lipase, the (R)-amide was obtained with a conversion degree of 21percent, resulting in eep > 99percent and E-value (enantioselectivity value) > 200, in 1 min of reaction under MW irradiation. When the A. niger lipase was immobilized in yam starch films, the (R)-amide was obtained in moderate conversions of 8-25percent after 3 or 5 min of reaction under MW irradiation, but with higher selectivity (eep > 99percent and E > 200) in comparison with the free form (conversion degree of 45percent, eep 81percent and E value of 18).

Full text of DOI:10.1590/S0103-50532012005000033

J. Braz. Chem. Soc., Vol. 23, No. 9, 1688-1697, 2012.
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Article
A
The Influence of Conventional Heating and Microwave Irradiation on the
Resolution of (RS)-sec-Butylamine Catalyzed by Free or Immobilized Lipases
Cristiane Pilissão,a Patrícia de Oliveira Carvalhob and Maria da Graça Nascimento*,a
aDepartamento de Química, Universidade Federal de Santa Catarina,
88040-900 Florianópolis-SC, Brazil
bCurso de Farmácia, Universidade São Francisco, Av. S. Francisco, 218,
12916-900 Bragança Paulista-SP, Brazil
As lipases CAL-B, LPS, LPS-C, LPS-D e a de A. níger, livres ou imobilizadas em filmes de amido
de cará (Discorea alata L.), inhame (Colocasia esculenta (L.) Schott) ou de gelatina, foram utilizadas
na acilação da (RS)-sec-butilamina com diferentes doadores acila em vários solventes orgânicos sob
aquecimento convencional (CH) ou irradiação por microondas (MW).Ao utilizar a lipase de A. niger
livre, a conversão foi três vezes maior sob irradiação por MW em relação ao CH a 35 ºC. Usando
a lipase de A. niger livre, a (R)-amida foi obtida com 21% de conversão, resultando em valores de
eep > 99% e de E (enantiosseletividade) > 200, em 1 min de reação em MW. Ao utilizar a lipase de
A. niger imobilizada em filmes de amido de cará e inhame, em 3 ou 5 min de reação em MW, a amida
foi obtida com conversões moderadas, sendo de 8 a 25%, porém com maior seletividade (eep > 99%
e E > 200) em comparação à forma livre (45% de conversão, eep 81% e 18 para o valor de E).
The lipases CAL-B, PSL, PSL-C, PSL-D, and A. niger lipase, free or immobilized in starch
(obtained from two types of yam, known in Brazil as “cará” (Discorea alata L.) and “inhame”
(Colocasia esculenta (L.) Schott) or gelatin films, were used in the acylation of (RS)-sec-butylamine
with different acyl donors in various organic solvents applying conventional heating (CH) or
microwave (MW) irradiation. In the case of free A. niger lipase, the conversion degrees were three
times higher using MW irradiation when compared to conventional heating at 35 °C. Using free
A. niger lipase, the (R)-amide was obtained with a conversion degree of 21%, resulting in eep > 99%
and E-value (enantioselectivity value) > 200, in 1 min of reaction under MW irradiation. When
the A. niger lipase was immobilized in yam starch films, the (R)-amide was obtained in moderate
conversions of 8-25% after 3 or 5 min of reaction under MW irradiation, but with higher selectivity
(eep > 99% and E > 200) in comparison with the free form (conversion degree of 45%, eep 81% and
E value of 18).
Keywords: enzymatic resolution, lipase, immobilization, microwave irradiation
Introduction
the kinetic resolution of racemic alcohols, acids, esters
or amines as well as desymmetrization of prochiral
compounds and the efficient synthesis of chiral building
blocks, drugs and fragrances.4-6
The synthesis of optically pure amides is an area
of growing interest in synthetic chemistry. Amines and
their derived amides are important compounds in organic
synthesis because of the presence of these functional groups
in many pharmacologically active compounds such as
(S)-methyldopa and (S)-penicillin.6-10
Biocatalysts are an attractive alternative to conventional
methods for effecting asymmetric organic transformations,
offering unique characteristics when compared to
homogeneous or heterogeneous chemical catalysts.
Biocatalytic reactions are also generally safer and the
reaction conditions are mild.1-3
Lipases (triacylglycerol acyl hydrolases, EC 3.1.1.3)
are considered the most ubiquitous and valuable enzymes
for asymmetric synthesis. Lipase applications include
The classic amide synthesis method is the reaction of
carboxylic acids with amines at high temperature. Due to
the low activity of carboxylic acids, various methods for
*e-mail: maria.nascimento@ufsc.br

Vol. 23, No. 9, 2012
Pilissão et al.
1689
their activation have been reported in the literature. The
most common is the conversion of a carboxylic acid to a
more reactive functional group, such as an acyl chloride,
mixed anhydride, acyl azide or an active ester.10,11
In the last decade, microwave (MW) irradiation has
been used to simplify and improve the reaction conditions
for many classic organic reactions. Reactions performed
under MW irradiation proceed faster, are cleaner,
present much better yields and are more reproducible
than those performed under conventional conditions.12-17
Besides, in some enzymatic reactions the selectivity,
such as stereoselectivity or regioselectivity, are clearly
influenced.17-19
used repeatedly. Thus, they are easy to handle and can be
recovered from solution, which makes the process practical
and economically viable. Enzymes can be immobilized in
organic and/or inorganic material for their use in synthesis.
These materials include biopolymers, organogels, glass,
silica and nanoparticles.21-27
Yadav and Lathi28 reported that the initial activity for
the transesterification of methyl acetoacetate with various
alcohols in the presence of immobilized lipases, such as
novozym 435, lipozyme RM IM and lipozyme TL IM, was
around 2.2-4.6 times greater when using MW irradiation
than in CH reactions. The reaction followed the ping-pong
bi-bi mechanism.
Yu et al.18 observed that MW irradiation increased both
the activity and enantioselectivity of novozym 435, and
also the thermal stability and reusability in the resolution
of (RS)-2-octanol with vinyl acetate as the acyl donor. A
conversion of 50% was achieved in a 3 h reaction under
MW irradiation while around 12 h was required to obtain
the same conversion under conventional heating. Using
the optimum conditions and applying MW irradiation
(S)-2-octanol was obtained at 50.5% conversion with 99%
of enantiomeric excess in 2 h reaction.
Recently, the kinetic resolution of ( )-mandelonitrile
was studied using lipase from Candida antarctica under
conventional condition and MW irradiation in toluene. In
both conditions, the (S)-mandelonitrile acetate was obtained
with high selectivity (ee of 92-98%).19
In this study the influence of both temperature-
controllable microwave (MW) assisted irradiation and
conventional heating (CH) was evaluated in the enzymatic
resolution of (RS)-sec-butylamine using different
acyl donors (ethyl acetate, vinyl acetate and isopropyl
acetate). The reaction was catalyzed by four commercial
lipases, one from Pseudomonas cepacia (PSL) free or
immobilized in films produced from yam starch (obtained
from “cará” or “inhame”, referred herein to yam I and
yam II, respectively) or gelatin, and three commercial
samples: two of immobilized P. cepacia lipase (PSL-C and
PSL-D) and one of immobilized Candida antarctica lipase
(novozym 435 - CAL-B). The behavior of the native A.
niger lipase, free or immobilized in yam starch or gelatin
films, was also evaluated (Scheme 1).
However, most enzymes are deactivated by the fast
increase in temperature under MW irradiation.18 This
problem may be solved by the use of immobilized enzymes
which, in general, exhibit higher thermal stability than the
free form.20,21
Experimental
Materials and methods
Enzyme immobilization in different supports is an
important technique used to increase the productivity
of synthetic reactions in organic media. In general,
immobilized enzymes are more stable in relation to changes
in pH and temperature compared to free forms. Moreover,
they remain stable for months and the catalyst can be
The following lipases produced by Pseudomonas
cepacia were received from Amano Pharmaceutical
Co. (Nagoya, Japan): PSL (30,000 U g-1), (PSL-D)
(immobilized on diatomaceous earth, 500 U g-1), and
PSL-C (immobilized on diatomite and chemically-modified
ceramics, 600 U g-1). Lipase produced by Candida antarctica
O
NH2
HN
O
(S)-1
NH2
(R)-2
lipases
OR
O
microwave or
conventional heating
(RS)-sec-butylamine (1)
NH2
HN
,
(R)-1
(S)-2
R =
,
Scheme 1. Acylation of (RS)-sec-butylamine (1) with different acyl donors mediated by lipases using MW irradiation or conventional heating (CH).

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The Influence of Conventional Heating and MW Irradiation on the Resolution of (RS)-sec-Butylamine
J. Braz. Chem. Soc.
(CAL-B, novozym 435, 10,000 PLU g-1 immobilized on
a polyacrylate resin) was received from Novozymes Latin
America Ltda (Brazil) and lipase produced by Aspergillus
niger AC-54 (lipase activity 2.86 µmol min-1 mg-1 for the
hydrolysis of olive oil at pH 6.0 and 40 °C) was isolated and
purified as previously described.29,30 (RS)-sec-butylamine
(99%) and (R)-sec-butylamine (99%) were purchased
from Sigma-Aldrich. n-Hexane, n-heptane, cyclohexane,
toluene, acetonitrile, ethyl acetate, vinyl acetate and iso-
propenyl acetate were obtained from Vetec (Brazil), Fluka
and Sigma-Aldrich at the highest available purity (> 99%).
The starch was obtained from vegetable roots of yam I or
yam II, and was extracted as previously described for ginger
starch.27 The pharmaceutical-grade gelatin was obtained
from Sigma-Aldrich (300 Bloom).
stirring and cooled in an ice bath for 0.5 h. The amide
was purified by extraction with an aqueous solution of
5% hydrochloric acid (3 × 10 mL) and 5% of sodium
bicarbonate (3 × 10 mL) using ethyl ether (3 × 10 mL).
The organic phase was dried over anhydrous magnesium
sulfate (MgSO4) and the solvent was then evaporated. Pure
(R)-sec-butylacetamide was obtained with a specific optical
rotation [a ] = -8.87 (0.09 g mL-1, CHCl3). This value
presented the same sign reported for (R)-sec-butylamine;
[a] = -7.50 (c = neat).34 1H NMR (400 MHz, CDCl3) d 0.91
(3H, t, J 7.2 Hz, H5), 1.12 (2H, d, J 6.8 Hz, H4), 1.46 (2H,
q, H3), 1.98 (3H, s, H6), 3.89 (1H, sex, H2), 5.76 (1H, s,
H1); IR (KBr) nmax / cm-1: 3287 (NH), 2973-2932 (C-H
aliphatic), 1647 (C=O), 1558 (NH).35
General procedure for lipase-catalyzed resolution of
Immobilization of lipases in starch and gelatin films
(RS)-sec-butylamine (1)
The yam starch (two types) and gelatin films were
prepared by dissolving 1.0 g of starch (from yams I and II
separately) or gelatin in 25 mL of distilled water (pH 5.6) in
a 50 mL beaker. The mixture was kept under mild heating
(ca. 60 oC) and constant magnetic stirring until complete
dissolution of the polymers occurred. Glycerol (0.3 mL)
and the lipases (50 mg) were then added to the solution and
the mixture was stirred for another 20 min. The solution
was transferred to a Petri dish and heated in a sand bath at
40 °C for water evaporation. The films with the immobilized
enzymes were cut into small and regular pieces and kept
in organic solvent for later use.
The water content present in the films obtained from
the yam I and yam II starch or gelatin was determined
by Karl-Fisher titration. The films contained between
10-14% of water, an amount sufficient to maintain the
three-dimensional structure of the enzyme and consequently
its activity.31,32
In these reactions, 0.2 mL (2 mmol) of (RS)-sec-
butylamine (1) and different acyl donors (2-8 mmol), such
as ethyl acetate, vinyl acetate and iso-propenyl acetate, were
used. The lipases (50 mg), free or immobilized in the starch
or gelatin films and different organic solvents (7 or 10 mL)
(n-hexane, n-heptane, ciclohexane, toluene, chloroform,
dichloromethane and tert-butanol) were then added. The
reaction mixture was shaken in a rotary shaker (200 rpm)
and heated to 20-50 °C in a water bath (Tecnal TE-093) or in
a microwave oven (MW irradiation). The reactions carried
out under MW irradiation were performed in a CEM®
Discover benchmate microwave reactor with temperature
monitored by a built-in infrared sensor.A mixture of 0.2 mL
(2 mmol) of (RS)-sec-butylamine and 0.2 mL (2 mmol) of
ethyl acetate, 7 mL of the different organic solvents and
A. niger lipase (50 mg), free or immobilized, was placed
in a 10 mL glass MW tube with a magnetic stirrer. The vial
was then sealed with a septum and the reaction mixture
placed into the reactor, closed and subjected to a maximum
power of 50 W at 50 PSI pressure and 50 °C. The total
reaction time was the sum of five successive irradiation
pulses of 1 min, not considering the ramp time (1 min) for
each applied pulse.
The reaction progress and enantiomeric excesses values
were determined with a gas chromatograph (GC, Shimadzu-
14B) equipped with a chiral column (Rt-bDEXsm
30 m × 0.32 mm × 0.25 mm, Restek). H2 was used as the
carrier gas, the detector and injector were set at 230 °C, and
the column was ramped from 50 to 200 °C at 3 °C min-1.
The retention time of R-amide 2 was 11.62 min, and this
was compared with an enantiopure standard compound
using chiral gas chromatography. The retention time of
(S)-amide 2 was 11.30 min. The enantioselectivity values
The activities of the free or immobilized lipases were
determined by the hydrolysis of olive oil which is based
on the method proposed by Thomson et al.33 One unit of
lipolytic activity was defined as the amount of enzyme
that releases one micromole of fatty acid per min under
test conditions (pH 6.0, 40 °C in 1 h reaction) (results
presented in Table 1).
Preparation of (R)-sec-butylacetamide
Volumes of 2.50 mL (25 mmol) of (R)-sec-butylamine
and 4 mL (50 mmol) of pyridine in 50 mL of chloroform
were transferred to a 250 mL beaker. To this solution 5
mL of acetic anhydride (50 mmol) were added drop by
drop. The reaction medium was kept under mechanical

Vol. 23, No. 9, 2012
Pilissão et al.
1691
(E-values) were calculated from the enantiomeric excess of
the product (eep) and the conversion degree (c) according
to the method described by Chen et al.36 and Sih and Wu.37
The specific conditions of each study will be described
in the results and discussion for each experiment. Control
experiments were conducted without lipases under similar
reaction conditions, and no product was obtained.
When CAL-B was used as the biocatalyst the degree of
conversion into (R)- amide 2 was 54% with an eep value of
84%, resulting in an E-value of 30 in 0.5 h of reaction. The
selectivity of CAL-B for this reaction (expressed by the
E-value) can be considered as moderate (Table 1, entry 7).
When the native A. niger lipase was employed, free or
immobilized, the conversion degrees were in the range of
15-41%, resulting in eep and E-values of 81-89% and 10-32
in 1 h of reaction, respectively (Table 1, entries 8-11). In
2 h of reaction the (R)-amide 2 was obtained in conversion
degrees of 24-46% with eep and E-values of 82-84% and
13-21, respectively (data not presented in Table 1). As
previously mentioned, in general, using longer reaction
times, the amide 2 was formed in higher conversion degrees
but with lower eep and E-values.
As discussed above, these data show the influence
of using free or immobilized lipases in the resolution of
(RS)-sec-butylamine. Furthermore, using the free A. niger
lipase improved the results for the conversion degrees and
selectivity compared to the use of immobilized lipase in
1 h of reaction. Using CAL-B, the conversion degree was
higher (54%) than that obtained using the A. niger lipase
(15-41%) in a shorter reaction time, however the E-values
were similar (Table 1, entries 7 and 8).
Results and Discussion
Screening of lipases
In a first approach, five lipases from different sources
were screened in the acylation of (RS)-sec-butylamine (1)
with ethyl acetate in n-hexane at 35 ºC, under CH.
Lipases produced by C. antarctica (CAL-B), P. cepacia
(PSL, PSL-C and PSL-D) and the native A. niger free or
immobilized in yam starch or gelatin films were selected
for this study. The results are presented in Table 1.
The products were analyzed by chiral gas
chromatography and in most studies the (R)-amide 2
was the main product. These data followed the empirical
rule proposed by Kazluaskas and Weissfloch38 and are in
agreement with data reported in the literature which shows
that CAL-B and other lipases have a highly pronounced
catalytic preference for the R-enantiomer.
In general, the conversion degrees and E-values were
dependent on the lipase source and activity. When the
commercial lipases PSL-C and PSL-D, free or immobilized
in starch or gelatin films, were employed low values (0-5%)
for the conversion into the corresponding amide and low
E-values (< 2.0) were obtained in 6 h of reaction (Table 1,
entries 1-6).
These preliminary results showed that the A. niger
lipase presented good potential for use in the resolution
of (RS)-1. It is also important to consider the exploration
of non-commercial lipases obtained from the great
diversity of potential source species available in Brazil,
especially in asymmetric synthesis. This lipase has
previously been used for the resolution of ibuprofen29 and
(RS)-phenylethylamine.39 The potential of this biocatalyst
for industrial use has also been described recently.5 Thus,
Table 1. Acylation of (RS)-sec-butylamine (1) with ethyl acetate using different free and immobilized lipases under conventional heating
entry
1
lipase
LPS
lipase activity
10.72b
33.4b
time / h
c / %
5
eep / % (R)-2
E-valuea
6
6
25
-
1.7
-
2
PSL/yam II
PSL/yam I
PSL/gelatin
PSL-D
-
3
17.6b
6
5
14
-
1.3
-
4
18.40b
1.69c
6
-
5
6
-
-
-
6
PSL-C
2.35c
6
-
-
-
7
CAL-B
3.04c
0.5
1
54
41
15
37
30
84
89
81
85
89
30
32
10
20
24
8
A. niger
2.86b
9
A. niger/yam II
A. niger/yam I
A. niger/gelatin
10.8b
1
10
11
0.80b
1
4.20b
1
Reaction conditions: (RS)-1 (0.20 mL, 2 mmol), ethyl acetate (0.20 mL, 2 mmol), n-hexane (10 mL), enzyme (50 mg), 35 ºC;aenantiomeric ratio;bU g-1;
cµmol min-1 mg-1, c = conversion determined by GC-FID.

1692
The Influence of Conventional Heating and MW Irradiation on the Resolution of (RS)-sec-Butylamine
J. Braz. Chem. Soc.
the A. niger lipase was selected for use in the studies
that followed, which included the effect of acyl donor,
temperature, organic solvents and the use of immobilized
A. niger lipase under CH and MW irradiation.
which ranged from 1:1 to 1:5. The results are presented
in Figure 2.
Effect of type and amount of acyl donor
In the next study, the type and relative amount of acyl
donor were evaluated for the resolution of (RS)-1 using free
A. niger lipase as the biocatalyst. Firstly, for this study three
acyl donors (ethyl acetate, vinyl acetate and iso-propenyl
acetate) were screened. The results for the conversion
degrees and eep values are presented in Figure 1.
Figure 2. Influence of molar ratio on the acylation of (RS)-sec-butylamine
(1) with ethyl acetate using free lipase from A. niger. Reaction conditions:
(RS)-1 (0.20 mL, 2 mmol), ethyl acetate (2-8 mmol), n-hexane (10 mL),
enzyme (50 mg), 1h, 35 ºC, () conversion (%) and () eep (%).
The data show that the degrees of conversion to the
(R)-amide 2 increased from 41 to 73% as the molar ratio
increased from 1:1 to 1:3. However, a decrease in the eep
values from 89 to 76% was observed. Using a molar ratio
of 1:4 and 1:5, the conversion degrees (71 and 68%) and
eep values (77 and 73%) remained practically constant and
were similar to those obtained when a molar ratio of 1:3
was used.
The best result in relation to selectivity was achieved
when a molar ratio of amine:ethyl acetate 1:1 was used and
this value was therefore selected for use in the subsequent
studies to investigate the effects of temperature and organic
solvents under CH and MW irradiation.
Figure 1. Influence of acyl donor type on the resolution of (RS)-sec-
butylamine (1) catalyzed by free A. niger lipase. Reaction conditions:
(RS)-1 (0.20 mL, 2 mmol), ethyl acetate (0.20 mL, 2 mmol), n-hexane
(10 mL), enzyme (50 mg), 1 h, 35 ºC, () c (%), () eep (%),() E-value.
The results showed that when vinyl acetate or iso-
propenyl acetate were used the amide was formed with
conversions of 90 and 95%, resulting in eep values of 12
and 36% and E-values of 3.7 and 1.3, respectively, in 1 h
reaction.As observed, vinyl and iso-propenyl acetate were
good acyl donors and formed the racemic amide with high
conversion degrees. However, the E-values are low and
considered unacceptable for practical purposes, especially
in enzymatic resolution.3
These data are in agreement with those presented by
Bachu et al.12 These authors reported that the use of vinyl
acetate and iso-propenyl acetate is not always advantageous
in biocatalysis because they can form undesirable side
products, such as acetaldehyde or acetone which are toxic
to the enzyme and can denature the biocatalyst.
Effect of temperature
The reaction temperature is important in catalysis. This
parameter can influence the activity, selectivity and the
stability of the biocatalyst.18,40
In this study four different temperatures, ranging from
35 to 50 °C, were evaluated in the acylation of (RS)-1 with
ethyl acetate in n-hexane applying conventional heating
(CH) or MW irradiation for 3 min. This reaction time was
selected considering that for longer times the conversion
degrees were higher than 50%, and thus unsuitable for
enzymatic resolution (results not presented). Figure 3 shows
the formation of (R)-amide 2 at various temperatures.
When CH was used the (R)-amide 2 was obtained
with conversion degrees of 13-51%, resulting in eep
and E-values of 81-90% and 21-23, respectively, as the
temperature increased from 35 to 45 °C. A decrease in the
conversion degree and the eep and E-values was observed
However, when ethyl acetate was used, the (R)-amide 2
was formed with a conversion degree of 41%, resulting
in eep and E-values of 89% and 32, respectively. Thus,
ethyl acetate was selected to evaluate the molar ratio
between the (RS)-sec-butylamine (1) and the acyl donor,

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Yadav and Borkar41 studied the effect of temperature on
the synthesis of citronellyl acetate catalyzed by CAL-B in
toluene, at 30-60 °C, using CH and MW irradiation. The
results showed that the conversion degrees and reaction
rates were higher when MW irradiation was used compared
to CH. With the use of CH, an increase in the initial rate
from 0.01 to 0.025 mol L-1 min-1 was observed, whereas
under MW irradiation the increase was from 0.015 to
0.045 mol L-1 min-1. In this study the temperature of 50 °C
was selected as optimal.
Thus, considering the above results, the temperature
of 35 °C was selected to evaluate the effect of organic
solvent as well as the immobilization of A. niger lipase in
different supports.
Figure 3. Influence of temperature (°C) on the conversion degrees (%)
in the acylation of (RS)-sec-butylamine (1) with ethyl acetate using free
A. niger lipase. Reaction conditions: (RS)-1 (0.20 mL, 2 mmol), ethyl
acetate (0.20 mL, 2 mmol), n-hexane (10 mL), enzyme (50 mg), 3 min;
() microwave irradiation () conventional heating.
Effect of organic solvent
The organic solvent, in general, influences the selectivity
of an enzyme because it can alter the native conformation
and thus the catalytic activity of the biocatalyst. However,
although there is still no consensus on the main parameters
which influence the enzymatic reaction, the most frequently
considered are the log P (logarithm of the partition
coefficient of the solvent for a standard octanol/water two-
phase system) and the dielectric constant. The hydrophobic
solvents with log P ≥ 4 are considered the most suitable
biocatalysts for reactions. Solvents with log P between 2
and 4 are considered moderate and the polar solvents with
log P < 2 are often inefficient for reactions catalyzed by
enzymes.Although the log P values do not correlate directly
with the efficiency of enzymatic synthesis, this is a valuable
information in terms of the initial selection of solvents.42,43
The solvent of choice also is important for use in
microwave irradiation. With the use of polar solvents,
the energy is transferred from the solvent to the reaction
mixture and thus the results may be similar using MW
irradiation or CH. However, when more apolar solvents
are used, the energy is transferred from the reagents to
the solvent and the results can be improved by using MW
irradiation rather than CH.13,18,44
Thus, to evaluate this effect, solvents with different
polarities, that is, n-heptane (log P 4.00), n-hexane
(log P 3.90), cyclohexane (log P 3.20), toluene (log P 2.50),
chloroform (log P 2.00), tert-butanol (log P 1.45) and
dichloromethane (log P 1.18), were screened in the
resolution of (RS)-1 with ethyl acetate, under CH or MW
irradiation, catalyzed by the free A. niger lipase. The results
are presented in Table 2.
The time of 1 h was selected for the studies applying
CH based on the results obtained using free A. niger lipase
in n-hexane (see Table 1, entry 8). In the case of MW
irradiation the time selected was 1 min, considering that
at 50 °C, these values being 32%, 81% and 13, respectively.
The stability of this lipase was probably lower due to
denaturation of this biocatalyst at higher temperatures. As
previously described, the A. niger lipase presented good
thermostability in the range of 35 to 45 °C.30
Interesting results were obtained with the use of MW
irradiation. The conversion degrees remained almost constant
in the temperature range of 35 to 50 °C, and the (R)-amide 2
was formed with conversion degrees of 45-49%, resulting in
eep and E-values of 80-81% and 18-20, respectively.
Using MW irradiation, the conversion degrees
were higher than those obtained with the use of CH at
temperatures of 35 and 50 °C. At 35 °C the (R)-amide
2 was formed with conversion degrees of 45% under
MW irradiation and 13% under CH, respectively. This
result showed the important influence of MW irradiation,
especially on the degree of conversion into the product.
However, no significant changes in the eep and E-values
were observed.
In order to improve the selectivity under MW irradiation,
the resolution of (RS)-1 was performed for 1 min. When the
reaction was conducted under MW irradiation for 1 min
at 35 °C the (R)-amide 2 was obtained with a conversion
degree of 21%, resulting in eep > 99% and E > 200.
However, when the reaction was carried out for 1 min
using CH, no product was detected (data not presented in
Figure 3). These results also showed the positive influence
of MW irradiation on both conversion and selectivity.
Similar results were obtained by Huang et al.20 in the
enzymatic esterification of n-alcohol homologs and caprylic
acid, and the results were explained considering thermal
and non-thermal effects.

1694
The Influence of Conventional Heating and MW Irradiation on the Resolution of (RS)-sec-Butylamine
J. Braz. Chem. Soc.
Table 2. Effect of organic solvent on the acylation of the (RS)-sec-butylamine (1) with ethyl acetate under conventional heating or MW irradiation
Conventional heating
MW irradiation
entry
solvent
n-heptane
n-hexane
cyclohexane
toluene
log Pa
4.00
3.90
3.20
2.50
2.00
1.45
1.18
c / %
45
eep / %
78
E-value
15
c / %
eep / %
74
E-value
8.7
1
2
3
4
5
6
7
27
21
18
10
84
90
93
41
89
32
> 99
80
> 200
10
52
86
45
63
84
19
83
9
CHCl3
82
70
3.2
1.9
2.6
81
3.7
t-BuOH
CH2Cl2
88
44
70
2
84
62
79
1.9
Reaction conditions: (RS)-sec-butylamine (1) (0.2 mL, 2 mmol), ethyl acetate (0.2 mL, 2 mmol), solvent (10 mL or 7 mL), A. niger lipase (50 mg), 35 ºC,
1 h of conventional heating or 1 min of MW irradiation. aFrom reference 42.
for this reaction time good conversion degrees and eep and
E-values were obtained (see data related to the temperature
effects).
entries 5-7). These values were similar to those obtained
with CH and, as previously discussed, are not appropriate
for enzymatic resolution.
Applying CH and using apolar solvents (n-hexane,
n-heptane and cyclohexane) the (R)-amide 2 was obtained
in moderate to good conversion degrees of 41, 45 and 52%,
resulting in eep and E values of 89, 78 and 86% and 32, 15
and 45, respectively, in 1 h of reaction (Table 2, entries 1-3).
When toluene was used as the organic solvent the amide 2
was obtained with a conversion degree of 63%, resulting
in eep and E-values of 84% and 19, respectively which are
not appropriate for enzymatic resolution (Table 2, entry 4).
When more polar solvents (t-BuOH, CH2Cl2 and CHCl3)
were used, the amide was formed with higher conversion
degrees of 88, 84 and 82%, resulting in eep and E- values of
44, 62 and 70% and 1.9, 2.6 and 3.2, respectively (Table 2,
entries 5-7). These values are considered inadequate for
enzymatic resolution; however these solvents proved to be
good enough for use in the preparation of the racemic amide.
Using MW irradiation, the conversion degrees were also
dependent on the organic solvent. The reaction time was
much lower and in some cases the eep values were higher
than those obtained using CH.
Using apolar solvents (n-hexane, n-heptane and
cyclohexane) the (R)-amide 2 was obtained in moderate
conversion degrees of 21, 27 and 18%, resulting in eep
and E- values of > 99, 74 and 80% and > 200, 8.7 and 10,
respectively, in 1 min of reaction (Table 2, entries 1-3).
When toluene was used as the organic solvent, the amide
was obtained with a conversion degree of 10%, resulting
in eep and E-values of 83% and 9, which are considered
inadequate for enzymatic resolution (Table 2, entry 4).
When more polar solvents (t-BuOH, CH2Cl2 and CHCl3)
were used the amide was formed with higher conversion
degrees of 90, 93 and 84%, resulting in eep and E-values of
70, 79 and 81% and 2, 1.9 and 3.7, respectively (Table 2,
The best result was achieved using n-hexane and applying
MW irradiation for 1 min, where the (R)-amide 2 was
formed with a conversion degree of 21%, resulting in eep
and E-values > 99% and > 200, respectively. On applying
CH, the conversion degree was 30%, with eep and E values
of 85% and 32, respectively, in 30 min of reaction (Table 2,
entry 2). These results showed the positive effect of using
MW irradiation on the enzymatic resolution of (RS)-1.
The data presented herein are consistent with previously
reported results which showed that non-polar organic
solvents do not absorb microwave energy efficiently, and
thus do not warm the reaction medium and may act as
“blockers”, forming barriers to the heat produced by the
interaction of microwave radiation and polar reagents.13,18
Yu et al.18 described the transesterification of (RS)-2-
octanol with vinyl acetate catalyzed by CAL-B, using
solvents with different polarities (n-heptane, n-hexane,
toluene, cyclohexane, acetone, acetonitrile, DMF and
1,4-dioxane) and applying CH and MW irradiation. The
highest activity and enantioselectivity were obtained
using n-heptane under MW irradiation. The enzyme
activity was 203 mmol min-1 mg-1 with an E-value of 352.
With the use of CH the enzyme activity and the E-value
were 55 mmol min-1 mg-1 and 99, respectively. Recently,
the positive effect of ionic liquids associated with MW
irradiation has also been described for this substrate.44
Based on these results, n-hexane was selected to
evaluate the influence of using the lipase from A. niger
immobilized in starch or gelatin films.
Effect of using immobilized A. niger lipase
In this study, the A. niger lipase was immobilized in
yam starch (two types) or gelatin films and then used in

Vol. 23, No. 9, 2012
Pilissão et al.
1695
Table 3. Effect of lipase immobilization on the acylation of (RS)-1 under conventional heating or MW irradiation
Conventional heating
MW irradiation
enzyme/support
c / %
13a
eep / %
90
E
21
c / %
45a
8a
eep / %
81
E
Free A. niger
18
a
A. niger/yam II starch
A. niger/yam II starch
A. niger/yam I starch
A. niger/yam I starch
A. niger/gelatin
-
-
-
> 99
> 99
> 99
> 99
77
> 200
> 200
> 200
> 200
15
2c
8a
> 99
> 99
76
> 200
> 200
10
15b
15a
25b
46a
30c
21a
84
14
Reaction conditions: (RS)-sec-butylamine (1) (0.2 mL, 2 mmol), ethyl acetate (0.2 mL, 2 mmol), n-hexane (10 mL or 7 mL), A. niger lipase/support
(50 mg), 35 ºC, a3 min, b5 min and c30 min.
the acylation of (RS)-1 with ethyl acetate under CH or MW
irradiation. The results are presented in Table 3.
starch and A. niger lipase/yam I starch systems and applying
in longer reaction times.
Firstly, the reactions were carried out for 3 min,
considering that in a shorter reaction time no product was
detected applying CH. The results were also dependent
on the support used. When A. niger lipase was used in the
free form, applying CH or MW irradiation, the (R)-amide 2
was formed with conversion degrees of 13 and 45% and eep
and E-values of 90 and 81% and 21 and 18, respectively.
Under MW irradiation, an increase in the conversion degree
was observed, but both the eep and E-values decreased in
comparison with CH.
In the case of A. niger lipase immobilized in the yam II
starch film, on applying CH no product was formed.
However, under MW irradiation the (R)-amide 2 was
formed with a low conversion degree of 8%, but with
high eep and E-values, these being > 99% and > 200,
respectively.
When the A. niger lipase was immobilized in the
yam I starch film, on applying CH or MW irradiation
the (R)-amide 2 was obtained with conversions degrees
of 8 and 15%, resulting in eep and E-values of > 99%
and > 200, respectively. These results showed that on using
MW irradiation, the conversion degree to (R)-amide 2 was
around two times higher than that obtained using CH.
The best results for the conversion degrees (21 and 46%)
were achieved when the A. niger lipase was immobilized in
gelatin film and either CH or MW irradiation was applied.
However, the eep and E-values were moderate (84 and
77% and 14 and 15, respectively). These data cannot be
considered as appropriate for enzymatic resolution.3
As discussed above, the best results in relation to the
enantioselectivity of the process were obtained using the A.
niger lipase immobilized in yam I starch or yam II starch
films and applying CH or MW irradiation, the positive
effect of protecting the biocatalyst also being observed.
In order to increase the conversion degrees, the acylation
of (RS)-1 was achieved using the A. niger lipase/yam II
When the acylation of (RS)-1 was carried out for 30 min
under CH and using the A. niger lipase immobilized in
yam II or yam I starch films, the (R)-amide 2 was obtained
in conversion degrees of 2 and 30%, respectively. The
eep and E-values were of 81 and 83% and > 200 and 10,
respectively.As observed, the conversion degree increased
and the enantioselectivity of the process was dependent on
the support (data presented on Table 3).
When the reaction was performed under MW irradiation
for 5 min, using the A. niger lipase immobilized in yam II
or yam I starch films, the (R)-amide 2 was obtained in
conversion degrees of 15 and 25%. Interestingly, the eep and
E-values were > 99% and > 200, respectively. These data
show an increase in the conversion degrees in comparison
with those obtained in 3 min of reaction.
The A. niger lipase immobilized in yam II or yam I
starch films was reused in the acylation of (RS)-1 for 5 min
under MW irradiation, a sharp decrease in the E-values
were observed, being about 85%.
In summary, moderate conversion degrees and good
enantioselectivity were obtained using A. niger lipase, free
or immobilized in starch films, and applying conventional
heating or MW irradiation. However, in general, the
conversion degrees were higher using MW irradiation.
Conclusions
In this study, the resolution of (RS)-sec-butylamine (1)
with various acyl donors was investigated using free or
immobilized lipases, particularly A. niger lipase. The
reactions were carried out using conventional heating and
microwave (MW) irradiation, under different experimental
conditions.
When the free A. niger lipase was used applying MW
irradiation the degree of conversion to (R)-amide 2 was
three times higher than that obtained under conventional

1696
The Influence of Conventional Heating and MW Irradiation on the Resolution of (RS)-sec-Butylamine
J. Braz. Chem. Soc.
heating using ethyl acetate as the donor acyl in n-hexane at
35 °C. Using the A. niger lipase/yam I starch and A. niger
lipase/yam II starch systems and applying MW irradiation
the (R)-amide 2 was obtained with conversion degrees lower
than those using the free lipase. However, the selectivity
was much better (eep > 99% and E > 200). Furthermore,
using these systems the conversions, in general, were higher
under MW irradiation (8-25%) in relation to conventional
heating (0-30%).
In conclusion, the results obtained in this study showed
that the use of immobilized lipases in combination with
MW irradiation offers interesting advantages in relation to
biocatalytic reactions, especially in the resolution of (RS)-1.
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Acknowledgments
20. Huang, W.; Xia,Y. M.; Gao, H.; Fang,Y. J.; Wang,Y.; Fang,Y.;
J. Mol. Catal. B: Enzym. 2005, 35, 113.
This work was supported by the Universidade Federal
de Santa Catarina (UFSC-Brazil), Coordenação de
Aperfeiçoamento de Pessoal de Nível Superior (CAPES),
Conselho Nacional de Desenvolvimento Científico e
Tecnológico (CNPq) and INCT-Catalysis, which provided
financial support and scholarships (M. G. N and C. P.).
We also thank Amano Pharmaceutical Co. (Japan),
Novozymes (Brazil) for the donation of lipases, and
Prof. Marcus C. M. Sá (UFSC-Brazil) for the use of the
microwave reactor.
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FAPESP has sponsored the publication of this article.