Convenient enzymatic resolution of (R,S)-2-methylbutyric acid catalyzed by immobilized lipases

Article abstract of DOI:10.1002/chir.22779

The application of several immobilized lipases has been explored in the enantioselective esterification of (R,S)-2-methylbutyric acid, an insect pheromone precursor. With the use of Candida antarctica B, using hexane as solvent, (R)-pentyl 2-methylbutyrat

Full text of DOI:10.1002/chir.22779

Received: 21 June 2017
DOI: 10.1002/chir.22779
Revised: 29 August 2017
Accepted: 1 October 2017
R E G U L A R A R T I C L E
Convenient enzymatic resolution of (R,S)‐2‐methylbutyric
acid catalyzed by immobilized lipases
Mateus Mittersteiner1
| Bruna Luiza Linshalm1 | Ana Paula Furlan Vieira1 |
Patrícia Bulegon Brondani2 | Dilamara Riva Scharf1 | Paulo Cesar de Jesus1
1 Departamento de Química, Universidade
Abstract
Regional de Blumenau, Blumenau, SC,
The application of several immobilized lipases has been explored in the
Brazil
2 Departmento de Ciências Exatas e
Educação, Universidade Federal de Santa
Catarina, Blumenau, SC, Brazil
enantioselective esterification of (R,S)‐2‐methylbutyric acid, an insect phero-
mone precursor. With the use of Candida antarctica B, using hexane as solvent,
(R)‐pentyl 2‐methylbutyrate was prepared in 2 h with c 40%, eep 90%, and
E = 35, while Thermomyces lanuginosus leads to c 18%, eep 91%, and E = 26.
The (S)‐enantiomer was obtained by the use of Candida rugosa or Rhizopus
oryzae (2‐h reaction, c 34% and 35%, eep 75 and 49%, and E = 10 and 4, respec-
tively). Under optimal conditions, the effect of the solvent, the molar ratio, and
the nucleophile were evaluated.
Correspondence
P. C. de Jesus, Departamento de Química,
Universidade Regional de Blumenau,
Antonio da Veiga Street, 140, Blumenau,
SC 89019‐917, Brazil.
Email: pcj@furb.br
KEYWORDS
enantioselective esterification, enzymatic acylation, immobilized enzymes, pheromones, racemic
acids
1 | INTRODUCTION
As an example, consider the (S)‐2‐methylbutyric acid
is found in the sexual pheromones of the pink hibiscus
Enzymes are very efficient catalysts; the rates of enzyme‐
mediated processes are generally faster (by a factor of
approximately 1012) than chemically catalyzed processes.
In addition, enzyme‐catalyzed reactions are environmen-
tally friendly, occurring under mild conditions. Lipases
are considered stable and robust enzymes, even though
they can be sensitive to reaction conditions (such as pres-
sure, temperature, and pH). Given their high specificity,
commercial availability, and versatility to catalyze a wide
range of different reactions (such as esterification,
transesterification, and interesterification), they became
the most explored class of enzymes in organic synthesis.1
Enantiopure alcohols,2-4 amines,5,6 and carboxylic
acids7-9 are key compounds for the synthesis of many
fine‐chemistry derivatives, such as pharmaceuticals, agro-
mealybug, Maconellicoccus hirsutus. It is also a precursor
of (S)‐2‐methyl‐N‐((S)‐2‐methylbutyl)butanamide, the
sexual pheromone of Migdolus fryanus,10,11 a beetle con-
sidered a pest in sugarcane crops. The (R)‐enantiomer
has been successfully applied by Zhang et al12 in the syn-
thesis of liquid crystals, leading to good luminescent and
liquid crystalline properties. Additionally, the esterifica-
tion of carboxylic acids in general leads to ester deriva-
tives that are as well interesting building blocks for the
synthesis of pheromones and flavored compounds used
in the food industry.
In spite of the fact that several racemic carboxylic
acids have been resolved by lipases,13-22 a literature survey
shows only few methodologies for the enantioselective
esterification of 2‐methylbutyric acid. This may be due
to the fact that methyl and ethyl moieties have a small
size difference making it difficult for the efficient recogni-
tion by the lipase.23 In an attempt to resolve the 2‐
methylbutyric acid, Holmberg et al24 prepared (S)‐heptyl
chemicals,
and
pheromones.9
In
particular,
steriochemically pure carboxylic acids are extremely use-
ful building blocks, being easily converted into many of
the other functional groups.
Chirality. 2017;1–6.
wileyonlinelibrary.com/journal/chir
© 2017 Wiley Periodicals, Inc.
1

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MITTERSTEINER ET AL.
2‐methylbutyrate using Candida cylindracea in the free
form as a catalyst. They observed 77% of conversion with
enantioselectivity (E) of 4.6 and the remaining (R)‐acid
with 85% of enantiomeric excess (ee). Lipase originating
300 MHz (1H NMR) and 75 MHz (13C NMR). Data are
reported as follows: chemical shift (δ, ppm), multiplicity
(s = singlet, d = doublet, t = triplet, sex = sextet, dq = dou-
ble quartet, and m = multiplet), and coupling constants
(J) in Hertz and integrated intensity. Gas chromatography
(GC) analysis for the determination of the enantiomeric
excesses of the product (eep) and substrate (ees) was car-
ried out using a GC Agilent 7890B and a Shimadzu 14B
from Chromobacterium viscosum immobilized in
a
microemulsion‐based organogel was also applied in the
preparation of (S)‐ethyl 2‐methylbutyrate. The ester was
obtained with an ee of 31.7% in a reaction time of 21 days,
while the nonreactive (R)‐acid was obtained with 22.2% of
ee.25 Better enantioselectivity values were obtained when
the enzymatic hydrolysis was performed on the (R,S)‐
ethyl 2‐methylbutyrate, obtaining the (R)‐2‐methylbutyric
acid with 95% of enantiomeric excess at 4°C.23 Despite the
high ee values, this protocol requires a previous chemical
preparation of the racemic ester for posterior enzymatic
hydrolysis.
In this context, encouraged by the few publications
regarding methodologies for the enantioselective esterifi-
cation and resolution of this racemic acid, we present
herein an efficient and direct methodology for this
purpose.
(FID
detector).
A
β‐DEX
120
column
(30 m × 0.25 mm × 0.25 μm) (Supelco) was used, and
the method applied was column temperature program of
70°C (3 min) to 200 °C (7°C min−1) and 200°C (15 min).
The injector and detector temperatures were set at
250°C and 300°C, respectively. The flow rate for the car-
rier gas (helium) was 2 mL min−1. The retention times
under these conditions were 9.34 min for (S)‐2‐
methylbutyric acid, 9.41 min for (R)‐2‐methylbutyric acid,
9.58 min for (R)‐pentyl 2‐methylbutyrate, and 9.65 min
for (S)‐pentyl 2‐methylbutyrate. The parameters (eep and
ees) were calculated according the integrated area of the
enantiomers. The absolute configuration was determined
by comparison with data previously reported for these
enzymes.9,26,27
2 | MATERIALS AND METHODS
2.1 | General
2.3 | Immobilization of the lipases
(R,S)‐2‐Methylbutyric acid (98%) was purchased from
Acros Organic. The solvents 1,4‐dioxan (99%), cyclohexane
(99%), heptane (99%), and ethyl acetate (99.5%) were
obtained from Vetec. n‐Hexane (98.5%), acetonitrile
(99%), and chloroform (99.5%) were obtained from
Dinâmica. All reagents and solvents were obtained from
commercial suppliers and used without further
purification. The alcohols 1‐butanol (99.4%), 1‐pentanol
(98%), 1‐hexanol (98%), and 1‐octanol (98%) were obtained
from Vetec. Silica gel (0.063‐0.200 mm; 70‐230 mesh) was
obtained from Macharey‐Nagel. The crude sugarcane
bagasse was obtained from a local commercial source.
Commercially available lipases from Candida antarctica
B (CaLB) immobilized in resin (10 U mg−1), Lipozyme
435 (10 U mg−1), and Lipozyme CaLB‐L (L. CaLB‐L;
5 KLU g−1) were kindly donated by Novozymes and lipases
originating from Rhizopus oryzae (ROL; 150 U g−1), Mucor
javanicus (MJL; 10 U g−1), and Candida rugosa (CRL;
30 U g−1) were donated by Amano Pharmaceuticals Co.
Prior to the immobilization, a pre‐treatment was applied
to the raw bagasse in order to standardize the solid sup-
port. Firstly, the peel was removed and discarded, and
the remaining parts were placed in a flask containing
water for 24 h, changing the water periodically. The fibers
were then cut into pieces of approximately 0.8 cm and
boiled in water for 2 h to remove the saccharose from
the residue. The bagasse was then washed with running
water and left to dry at room temperature. The dry
bagasse was placed in an Erlenmeyer flask (1 g) along
with phosphate buffer (100 mL) (Na2HPO4/KH2PO4
0.07 mol L−1; pH 7.2) and the lipase, that is, CRL, MJL,
or ROL (200 mg). The mixture was shaken (150 rpm) at
35°C (optimal temperature of most lipases herein used)
for 2 h. Lipozyme CaLB‐L and lipase from Thermomyces
lanuginosus were immobilized onto SiO2 according to
Mittersteiner et al.28
2.4 | General procedure for enzymatic
resolution
Lipase from Thermomyces lanuginosus (TLL; 50 U g−1
was obtained from Sigma‐Aldrich.
)
The immobilized lipase was placed in a 125‐mL Erlen-
meyer flask containing an organic solvent (25 mL), (R,
S)‐2‐methylbutyric acid (10 mmol), and an aliphatic alco-
hol (10 mmol). The reaction mixture was then placed in
an orbital shaker (Technal TE‐420) at 150 rpm and 37°C
for pre‐determined times. The formation of the products
2.2 | Analytical methods
NMR was used to calculate the conversion and character-
ize the compounds. Spectra were recorded on a Bruker
Ultrashield spectrometer operating at a frequency of

MITTERSTEINER ET AL.
3
SCHEME 1 Enantioselective
esterification of (R,S)‐1 with aliphatic
alcohols catalyzed by immobilized lipases
was monitored by thin layer chromatography using n‐
hexane and ethyl acetate (15:1 v/v) as eluent. The reac-
tions were interrupted, the enzyme was filtered out, and
0.95 (t, 3H, J = 7.44 Hz); 13C NMR (75 MHz, CDCl3):
δ (ppm) 183.6; 40.9; 26.5; 16.3; 11.4.
1
the reaction medium was analyzed by H NMR and GC‐
3 | RESULTS AND DISCUSSION
FID. Control reactions were carried out in the absence
of the lipases, and no product was detected under the
same experimental conditions. After column chromatog-
raphy (the same eluent as cited above), (R)‐pentyl 2‐
methylbutyrate (yield: 30%) and (S)‐2‐methylbutyric acid
The immobilized lipases from Rhizopus oryzae, Mucor
javanicus, Candida rugosa, Thermomyces lanuginosus,
and Candida antarctica B were applied to the resolution
of (R,S)‐2‐methylbutyric acid. A schematic representation
of the biocatalyzed resolution reaction is presented below
(Scheme 1).
1
(yield: 60%) were isolated and characterized by H and
13C NMR.
25
(R)‐pentyl 2‐methylbutyrate: [α]D = +51 (c 0.1,
CHCl3) ee 90%; IR (KBr) cm−1: 2962, 1734, 1463; 1H
3.1 | Effect of time course in the
NMR (300 MHz, CDCl3):
δ (ppm) 4.06 (t, 2H,
resolution reaction
J = 6.75 Hz); 2.35 (sex, 1H, J = 6.9 Hz); 1.65 (m, 3H);
1.46 (m, 1H); 1.32 (m, 4H); 1.13 (d, 3H, J = 6.96 Hz);
0.90 (t, 6H, J = 6.99 Hz); 13C NMR (75 MHz, CDCl3): δ
(ppm) 176.8; 64.2; 41.1; 28.3; 28.0; 26.7; 22.2; 16.6; 13.9;
11.5.
Initially, in order to obtain the best time conditions
regarding enantiomeric excesses, CaLB was chosen as
the catalyst due to the high enantiomeric excesses it usu-
ally provides. The alcohol 1‐pentanol was chosen to eval-
uate the parameters of this first step, according to Jesus
et al16 where (R,S)‐2‐methylpentanoic acid, a structurally
similar acid, obtained conversion of 28% and ee of 75%
for the corresponding ester. Table 1 presents the data
obtained for the different reaction times.
25
(S)‐2‐methylbutyric acid: [α]D = −10 (c 0.1, CHCl3)
ee 60%; IR (KBr) cm−1: 3032, 2970, 2879, 1707, 1465,
1
1226; H NMR (300 MHz, CDCl3): δ (ppm) 11.0 (s, 1H);
2.42 (sex, 1H, J = 6.81 Hz); 1.72 (dq, 1H, J = 13.62 Hz);
1.50 (dq, 1H, J = 13.71 Hz); 1.18 (d, 3H, J = 6.99 Hz);
The data in Table 1 suggest that a kinetic competition
is occurring between the enantiomers at the active site of
the enzyme. The eep increases for up to 2 h of reaction, in
which case it was possible to obtain a good conversion
(40%) along with high eep (90%), which decays after this
period of time. The longer the reaction time, the higher
the ees value was obtained (Figure 1).
TABLE 1 Effect of time course on the esterification reaction
between (R,S)‐2‐methylbutyric acid and 1‐pentanol catalyzed by
CaLB
(R)‐pentyl 2‐methylbutyrate
Entry
Time, h
c, %
eep, %
ees, %
E
1
0.25
19
39
9
2
2
3
4
5
6
7
8
0.50
1.0
2.0
3.0
4.0
5.0
6.0
36
38
40
60
79
89
94
43
74
90
46
22
11
6
24
45
60
69
81
89
91
3
10
35
5
The results obtained are in agreement with values
reported in the literature24 in which (S)‐heptyl 2‐
3
3
2
Conditions: hexane, 10 mmol of the racemic acid, 10 mmol of the alcohol,
100 mg of CaLB, 37°C, 150 rpm.
aCalculated by using the formula: E = {ln [eep(1 − ees)]/(eep + ees)}/{ln
[eep(1 + ees)]/(eep + ees)}.24
FIGURE 1 Expanded region of the chromatogram of the kinetic
resolution of (R,S)‐2‐methylbutyric acid with 1‐pentanol catalyzed
by CaLB

4
MITTERSTEINER ET AL.
40
35
30
25
20
15
10
5
50
methylbutyrate was prepared with 77% of conversion and
the remaining (R)‐acid was obtained with an enantio-
meric excess of 85%.
40
30
20
10
3.2 | Screening of lipases
Based on the results obtained with CaLB, regarding the
optimal time for the formation of the ester, several lipases
immobilized on different supports were tested for the res-
olution of the racemic acid (Table 2).
0
0
1
2
3
4
As shown in Table 2, all lipases were able to catalyze
the esterification reaction. The best enantiomeric excesses
were observed when using CaLB (entry 4, Table 1) and
TLL (entry 4, Table 2). When CRL and CaLB‐L were the
catalysts, moderated selectivity was observed. ROL and
Lipozyme 435 showed a lower selectivity under the same
conditions, while MJL provided the racemic ester. ROL
and CRL catalyzed the formation of the (S)‐esters, while
TLL, CaLB, CaLB‐L, and Lipozyme 435 formed the (R)‐
esters. In order to confirm the selectivity, (R)‐pentyl 2‐
methylbutyrate catalyzed by CaLB was isolated by
column chromatography (eluent: hexane and AcOEt
Cycle of reaction
FIGURE 2 Reuse of ROL immobilized on sugarcane bagasse
through four cycles of reaction in the preparation of (S)‐pentyl 2‐
methylbutyrate: (■) conversion and (▲) eep
process. Nevertheless, it is important to note that the
enantioselectivity was maintained through all the four
cycles of reaction.
3.3 | Effect of the solvent and molar ratio
90:10) and analyzed by polarimetry, leading to [α]
25
D
The polarity of the solvent, given by log P, considerably
affects the activity and enantioselectivity of enzymes.29-31
For this reason, different solvents with different log P values
were evaluated under the optimal conditions defined in
Table 1. The results can be observed in Figure 3.
Several biocatalytic studies have shown that reactions
using solvents with log P > 3.0 are usually more efficient,
since these non‐polar solvents are able to trap water around
the enzyme, creating a micro‐aqueous layer, maintaining
the conformation of the lipase, therefore preserving its
catalytic activity. Polar solvents, on the other hand, tend
= +51 (0.1, CHCl3). The non‐reactive (S)‐acid of the
corresponding reaction obtained [α]D25 = −10 (0.1, CHCl3).
The reuse of lipases immobilized on SiO2 has been
previously described for our group.28 Here, we evaluated
as well the reuse of ROL immobilized on sugarcane
bagasse. We observed the results for four consecutive
reactions in the preparation of (S)‐pentyl 2‐
methylbutyrate (Figure 2).
As can be seen in Figure 2, the immobilized lipase
could be reused for two cycles without the diminishment
of the conversion rate for the ester formation. After the
second cycle, it was observed a decay of the conversion,
which could indicate that the lipase is suffering inactiva-
tion by the organic solvent, or, due to a desorption
TABLE 2 Effect of different lipases on the preparation of pentyl
2‐methylbutyrate
Entry Enzyme Enantiopreference c, % eep, % ees, %
E
1
2
3
4
5
6
ROLa
MJLa
CRLa
TLLb
(S)
‐
35
32
34
18
09
45
49
‐
26
‐
4
‐
(S)
75
91
81
56
39
20
8
10
26
10
6
(R)
CaLB‐Lb (R)
L. 435 (R)
46
Conditions: immobilized lipases, hexane, 10 mmol of (R,S)‐2‐methylbutyric
acid, 10 mmol of the alcohol, 2 h, 37°C, 150 rpm.
FIGURE 3 Influence of organic solvents on the esterification
reaction for the formation of (R)‐pentyl 2‐methylbutyrate catalyzed
by CaLB. Conditions: 100 mg of immobilized lipase, solvent,
10 mmol of (R,S)‐1, 10 mmol of the alcohol, 2 h, 37°C, 150 rpm
aImmobilized on sugarcane bagasse.
bImmobilized on SiO2.
cThe lipase did not show any selectivity.

MITTERSTEINER ET AL.
5
to alter the amount of water that surrounds the enzyme,
promoting a destabilization of the biocatalyst.29,31,32 The
observed results were in agreement with this information,
and the enzyme activity and selectivity were highly influ-
enced by the choice of the organic solvent.
Having established the best conditions regarding time
(2 h), type of lipase (CaLB), and solvent (hexane), the
molar ratio (alcohol/acid) was studied in the proportions
0.5 (excess of acid) and 2.0 (excess of alcohol).
selectivity only when 1‐pentanol was employed as the
reactant, and moderate to low selectivity was presented
as the alcohol moiety was increased. This suggests that
the lipase is not able to accommodate the substrates with-
out suffering loss of selectivity, thus a conformational
change in the active site might be happening.
4 | CONCLUSION
The excess of acid (ratio 0.5) provided great conversion
but poor selectivity (c 82%, eep 13%, ees 60%, E = 2). A pos-
sible explanation for this is the fact that the media is sat-
urated with the racemic substrate (acyl donor) and the
enzyme is no longer able to selectively differentiate
between the enantiomers before the formation of the
acyl‐enzyme complex. This being, when the nucleophile
attacks the site, there is low selectivity for the product.
On the other hand, when using excess of the alcohol (ratio
2.0), moderate conversion and selectivity were obtained (c
40%, eep 52%, ees 35%, E = 4). We believe that in the excess
of the alcohol, the nucleophilic attack is happening too
fast for full selectivity control, showing that the ratio 1,
as demonstrated in Table 1, is ideal for this substrate.
We have demonstrated a convenient enantioselective
esterification of (R,S)‐2‐methylbutyric acid with several
immobilized lipases. A critical part of the study was the
observation of a kinetic competition occurring in the for-
mation of the enantiopure esters. In this manner, the time
of reaction was proved to be a key variable. The best
results were observed when TLL and CaLB were applied
as catalysts, for 2 h of reaction time. (R)‐pentyl 2‐
methylbutyrate was obtained by TLL with c 18% and eep
of 91% and by CaLB with c 40% and eep of 90%. CRL
and ROL catalyzed the (S)‐ester with conversions of 34%
to 35% and eep of 75% and 49%, respectively.
We expect that this enzymatic protocol will prove to
be useful in the synthesis of enantiopure esters and acid
derivatives, including carboxylic acids where most lipases
showed low selectivity. Further kinetic studies involving
other racemic compounds with small difference of the
substituents should be performed.
3.4 | Effect of the nucleophile
It is well reported in the literature that the chain size of
the alcohol can affect the reaction yield, conversion, and
enantioselectivity in acylation reactions catalyzed by
lipases.24 This being, a homologous series of n‐alcohols
was reacted with (R,S)‐1 to investigate the selectivity of
the lipase towards different alkyl chain sizes. The
obtained results can be observed in Table 3.
The conversion for the corresponding ester decreased
as the chain of the alcohol was increased. This behavior
was already demonstrated by Jesus et al16 in the esterifica-
tion of (R,S)‐2‐methylpentanoic acid with 1‐pentanol,
which had the best conversion for ester. Regarding enan-
tiomeric excesses, it would be expected that in a homolo-
gous series of n‐alcohols, the enantiomeric excess would
be maintained. However, in the preparation of the differ-
ent alkyl 2‐methylbutyrates, the lipase showed good
ACKNOWLEDGMENTS
The authors are grateful to INCT Catalysis Brazil and the
Chemistry Department at the Universidade Regional de
Blumenau (FURB). We are also thankful to Novozymes
Latin America for donating the lipases and Prof. Maria
da Graça Nascimento (UFSC‐Florianópolis—Brazil) for
the polarimetric analysis.
ORCID
Mateus Mittersteiner http://orcid.org/0000-0002-8501-844X
Paulo Cesar de Jesus http://orcid.org/0000-0002-1314-3444
TABLE 3 Effect of the nucleophile moiety in the esterification of
(R,S)‐2‐methylbutyric acid
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How to cite this article: Mittersteiner M,
Linshalm BL, Vieira APF, Brondani PB, Scharf DR,
de Jesus PC. Convenient enzymatic resolution of
(R,S)‐2‐methylbutyric acid catalyzed by
immobilized lipases. Chirality. 2017;1–6. https://
doi.org/10.1002/chir.22779
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