A.C.O. Machado et al. / Journal of Molecular Catalysis B: Enzymatic 69 (2011) 42–46
43
Table 1
◦
Hydrolysis of IPG esters using Novozyme 435 (T = 35 C, 2 h).
O
*
O
lipase
Substrate
R,S)-IPG acetate
Organic solvent
X (%)
ee (P) (%)
ee (S) (%)
O
O
R
O
*
OH
solvent
buffer
(
Hexane
Hexane
Ethyl acetate
Ethyl acetate
50
30
9
0.4
5
30
6
0.4
3
4
IPG
(R,S)-IPG octanoate
O
(
(
R,S)-IPG acetate
R,S)-IPG octanoate
R= CH , IPG acetate
R= C H15, IPG octanoate
3
7
0.4
7
X (conversion,) P and S (refers to product and substrate, respectively).
Fig. 1. Lipase-catalyzed resolution of racemic IPG esters.
Table 2
◦
Hydrolysis of IPG esters using Amano AK (T = 35 C, 2 h).
2
. Experimental
Substrate
Organic solvent
X (%)
ee (P) (%)
ee (S) (%)
2
.1. General
(
(
R,S)-IPG acetate
R,S)-IPG octanoate
Hexane
Hexane
Ethyl acetate
Ethyl acetate
11
50
16
6
23
28
2
4
28
0.4
6
Lipases from Rhizopus orzae (Amano F), Pseudomonas fluorecens
Amano AK, hydrolytic activity of 180U/g), Candida rugosa (Amano
(R,S)-IPG acetate
(R,S)-IPG octanoate
(
99
AY), Aspergillus niger (Amano A) and Pseudomonas cepacia (Amano
PS) were purchased from Sigma Chemical Co. (St. Louis, USA). Can-
dida antarctica lipase B (Novozyme 435) was a gift from NOVO
X (conversion,) P and S (refers to product and substrate, respectively).
(
Bagsvaerd, Denmark). One unit (U) of enzyme activity was defined
IPG, (R)-IPG acetate, (S)-IPG acetate, (R)-IPG octanoate and
(S)-IPG-octanoate were 3.6, 3.8, 4.1, 4.6, 10.8 and 11.0 min, respec-
tively.
Conversions (X) and enantiomeric excesses (ee) (P = hydrolysis
product; S = ester substrate) were determined according to Chen
et al. [15]. Enantioselectivity (E) was determined using conversions
and ee by means of equations by Faber and Hoenig [16].
as the amount of enzyme which was able to catalyze the release
of 1.0 mol of p-nitrophenol per min and was expressed as spe-
cific activity (U/g of protein). The IPG esters were prepared in our
laboratory as described below.
The solvents ethyl acetate, toluene and hexane were purchased
from Vetec Química Fina (Rio de Janeiro, Brazil).
2.2. Esters of (R,S)-IPG
3. Results and discussion
The chemicals used in this study were purchased from Sigma
3
.1. Exploratory experiments of resolution of (R,S)-IPG esters
The acetyl and octanoyl derivatives of racemic IPG were
Chemical Co (St. Louis, USA). (R,S)-IPG-acetate [13] and octanoate
13,14] were synthesized by reaction of IPG with Ac O or octanoyl
[
2
chloride according to literature procedures. The isolated products
were purified by Flash Chromatography (Silica gel neutralized with
screened against two lipases (Novozyme 435 and Amano AK) at
3
◦
5 C, pH = 7 and for 2 h in a biphasic medium (Fig. 1). The concen-
2
% Et N, prior to elution with ethyl acetate–hexane mixtures).
3
tration of the substrates in this step was 4.1 mM and the load of
enzymes, 50 mg. Tables 1 and 2 present the conversions (X) and
enantiomeric excesses (ee) for these substrates obtained in 2 h-
reactions.
The experiments with Novozym 435, a lipase widely employed
for resolutions of racemates, with either hexane or ethyl acetate
as co-solvents, did not afford good results. Despite the observed
high conversions, especially in the reactions in hexane, the selec-
tivities were very low. This result confirms that IPG is a challenging
substrate.
Conversely, Amano AK displayed high selectivity (ee (P) = 99%,
E > 200) in the hydrolysis of the octanoate derivative at a low con-
version (with ethyl acetate), as shown in Table 2. It was found that,
as it had occurred in the reactions of Novozyme 435 with hexane,
Amano AK led to high conversions when IPG octanoate was the
substrate. The activity of the enzyme is dependent on the amount
of water associated with the enzyme, and to a lesser degree on the
water content in the whole system. As long as a minimal amount of
water is associated with the enzyme, its activity in organic media
is retained. It is known that hydrophilic solvents (low Log P val-
2
.3. Hydrolysis of racemic IPG ester derivatives
Hydrolysis of IPG esters was carried out in screw-capped tubes
containing 3 mL of solvent, 3 mL of sodium phosphate buffer at
pH = 7.0 and different enzyme concentrations. The reactions were
initiated by adding 5 l of IPG esters and the tubes were incu-
bated in a thermostatized reactor. The control reaction (Blank)
was carried out at the same condition in the absence of the
enzymes.
2
.4. Analytical methods
Samples (25 l of each phase) were taken at determined
intervals and mixed with 450 l de acetonitrile. The enan-
tiomeric composition and conversion were routinely determined
by gas chromatographic (GC) analysis on a CHROMPACK CP 9000
equipped with a hydrogen flame ionization detector and a chi-
ral capillary column (Hydrodex®--6TBDM, diameter 0.25 mm,
length 25 m, thickness 0.25 ).
ues) may deactivate these enzymes by removing H O molecules
2
The temperature of the injector and detector were maintained
which are essential to their structure [17]. Accordingly, the more
hydrophilic ethyl acetate promoted a lower hydrolysis rate what
might have fostered more effective enantiomer discrimination by
Amano AK in the reaction of IPG octanoate. Activity measurements
did show a steep decrease for the catalyst in the reaction run with
ethyl acetate (vide infra). On the other hand, we did not observe
significant decrease in activity for the lipase which catalyzed the
reaction in hexane (14%-decrease after 48 h), as expected [18]. Pos-
sibly, ethyl acetate might alternatively act as an inhibitor of the
reactions.
◦
◦
at 250 C and 280 C, respectively. The carrier gas was nitrogen
◦
and, after injection, the column temperature was kept at 100 C
◦
◦
for 1 min and then programmed to rise 2 C/min reaching 106 C.
◦
◦
From 106 C, the column temperature was raised to 160 C at a rate
of 40 C/min and then to 165 C at 1 C/min, at which temperature
it was maintained for 15 min.
◦
◦
◦
The absolute configuration of produced IPG was determined
by comparison to samples enantiopure commercially available
samples (Sigma-Aldrich). The retention times for (R)-IPG, (S)-