One-pot lipase-catalyzed enantioselective synthesis of (r)-(?)-n-benzyl-3-(benzylamino)butanamide: The effect of solvent polarity on enantioselectivity

Article abstract of DOI:10.3390/molecules22122189

The use of the solvent engineering has been applied for controlling the resolution of lipase-catalyzed synthesis of β-aminoacids via Michael addition reactions. The strategy consisted of the thermodynamic control of products at equilibrium using the lipase CalB as a catalyst. The enzymatic chemo- and enantioselective synthesis of (R)-(?)-N-benzyl-3-(benzylamino)butanamide is reported, showing the influence of the solvent on the chemoselectivity of the aza-Michael addition and the subsequent kinetic resolution of the Michael adduct; both processes are catalyzed by CalB and both are influenced by the nature of the solvent medium. This approach allowed us to propose a novel one-pot strategy for the enzymatic synthesis of enantiomerically enriched β-aminoesters and β-aminoacids.

Full text of DOI:10.3390/molecules22122189

molecules
Article
One-Pot Lipase-Catalyzed Enantioselective Synthesis
of (R)-(−)-N-Benzyl-3-(benzylamino)butanamide:
The Effect of Solvent Polarity on Enantioselectivity
Marina A. Ortega-Rojas 1 ID , José Domingo Rivera-Ramírez 1, C. Gabriela Ávila-Ortiz 2
,
ID
Eusebio Juaristi 2,3,
*
ID , Fernando González-Muñoz 4, Edmundo Castillo 4,
*
and Jaime Escalante 1,
*
ID
1
The Center for Chemical Research, Autonomous University of Morelos State, Avenida Universidad 1001,
Chamilpa, Cuernavaca 62210, Mexico; marina.ortega@uaem.mx (M.A.O.-R.);
aghomes315@hotmail.com (J.D.R.-R)
Departamento de Química, Centro de Investigación y de Estudios Avanzados, Avenida I.P.N. 2508,
Ciudad de México 07360, Mexico; gabriela@relaq.mx
El Colegio Nacional, Luis Gonzáles Obregón 23, Centro Histórico, Ciudad de México 06020, Mexico
Department of Cell Engineering and Biocatalysis, Institute of Biotechnology, UNAM, Apartado Postal 510-3,
Cuernavaca C.P. 62271, Mexico; gonzalez@ibt.unam.mx
2
3
4
*
Correspondence: juaristi@relaq.mx (E.J.); edmundo@ibt.unam.mx (E.C.); jaime@uaem.mx (J.E.);
Tel.: +52-55-5747-3722 (E.J.); +52-77-7311-4903 (E.C.); +52-77-7329-7997 (J.E.)
Received: 6 November 2017; Accepted: 4 December 2017; Published: 9 December 2017
Abstract: The use of the solvent engineering has been applied for controlling the resolution of
lipase-catalyzed synthesis of
the thermodynamic control of products at equilibrium using the lipase CalB as a catalyst. The enzymatic
chemo- and enantioselective synthesis of (R)-( )-N-benzyl-3-(benzylamino)butanamide is reported,
β-aminoacids via Michael addition reactions. The strategy consisted of
−
showing the influence of the solvent on the chemoselectivity of the aza-Michael addition and
the subsequent kinetic resolution of the Michael adduct; both processes are catalyzed by CalB and
both are influenced by the nature of the solvent medium. This approach allowed us to propose a
novel one-pot strategy for the enzymatic synthesis of enantiomerically enriched β-aminoesters and
β-aminoacids.
Keywords: lipase promiscuity; aza-Michael addition; stereoselectivity; enantioselectivity; chemoselectivity;
solvent engineering
1. Introduction
The synthesis of
have been shown to have a broad spectrum of biological properties [
acids are present in numerous natural products of complex molecules [
the incorporation of -amino acids into -peptides gives them structural characteristics capable of
resisting hydrolytic degradation for long periods of time [ ]. As we know, the particular configuration
of biologically active -amino acids is directly related to their biological activity and therefore to their
β
-amino acids and their derivatives is of great interest because these molecules
]. In addition, -amino
]. On the other hand,
1
–
3
β
1
β
β
4,5
β
therapeutic effect, so that the synthesis of these molecules with a certain absolute configuration
has been an area of great interest. In this regard, many chemical strategies have been explored for
the enantioselective production of
materials in the homologation of natural
synthesis, or the resolution of racemic mixtures of
conventional chemical methods for the resolution of racemic mixtures consists in the use of biocatalysts
β
-amino acids. These methods involve, for example, chiral raw
-amino acids, the use of chiral auxiliaries in asymmetric
-amino acids [ ]. In this context, an alternative to
α
β
1,3
Molecules 2017, 22, 2189; doi:10.3390/molecules22122189
www.mdpi.com/journal/molecules

Molecules 2017, 22, 2189
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in non-aqueous media, a methodology that presents many advantages, including enzymes that show
good to high catalytic activity in organic media [ ]. However, the enzymatic resolution of racemic
mixtures usually involves at least two steps, the synthesis of the racemic -amino acid followed by
enzyme-catalyzed resolution. Among the useful methodologies for the preparation of -amino esters
-unsaturated systems employing enzymes such as
6–8
β
β
is the aza-Michael type addition of amines to α,β
lipases [9–12].
On the other hand, several useful methodologies to control chemoselectivity in Michael addition
have been developed, including the use of mutant enzymes and wild lipases, together with strategies
such as solvent engineering [
type of reaction; however, the use of solvents has not always been intended as a resolutive
process [ 12]. The kinetic resolution of racemic amino esters has usually been accomplished by
enantioselective substitution reactions in acyl-substituted substrates [14], as well as via the resolution
as a substrate for enzymatic N-acylation reactions [15 16]. Within this context, it has been described that
9,11,13]. Some authors have investigated different solvents for this
9–
,
nucleophilic substitution reactions in the acyl group can be achieved by hydrolysis [17], aminolysis [18],
and transesterification [19], affording good yields and high enantioselectivity. In this regard, one of
the most useful lipases is Candida antarctica lipase B (CalB), which is known for its catalytic promiscuity.
In 2009, our research group reported the successful use of solvent engineering strategies to
control the chemoselectivity in the addition of benzylamine (
1
) to methyl crotonate (
) was preferably
-amino ester showed a
= +0.45 (c 1.0, CHCl3), whereas the aminolysis product, crotonamide (3),
2) catalyzed by
CalB [ ]. In that report it was emphasized that the aza-Michael addition product (
9
4
accumulated in a hydrophobic medium such as n-hexane. The desired
β
4
20
specific rotation of [
α
]
D
was preferentially accumulated in polar solvents such as 2-methyl-2-butanol (2M2B), and a small
20
amount of
4
obtained showed a specific rotation of [
α
]
D
= +11 (c 1.0, CHCl3). No other product was
found. At that time, we did not comment on the origin of the observed enantiomeric excess (ee) and
why it was achieved only in 2M2B. In that sense, if two reactions such as a nucleophilic addition and a
substitution reaction were catalyzed by the same enzyme, one could anticipate that enantiopure esters
could be obtained by a one-pot reaction, and that would imply the effect of the polarity of the solvent,
not only for the control of the chemoselectivity but also in the enantioselectivity of the reaction,
that is, in the resolution process. While the groups of Liese and Gröger reported that the origin of
the enantiomeric excess does not result from an enantioselective Michael addition but by an enzymatic
resolution via transamidation, their reactions were carried out in the absence of solvents and in a
chemoenzymatic two-step process [18,20,21].
Therefore, in this work we carry out a one-pot reaction between benzylamine (
1) and methyl
crotonate ( ) catalyzed by CalB to determine the effect of the polarity of the solvent, particularly its
2
effect on the enantioselectivity of the enzymatic process.
2. Results and Discussions
2.1. Profile of Products Obtained in Solvents of Opposite Polarity: n-Hexane and 2M2B
Several experiments were carried out to determine how chemo- and enantioselectivity are
influenced by the polarity of the reaction medium in the addition reaction of benzylamine to
conjugated ester . The experiments were carried out in triplicate in n-hexane and 2M2B (two solvents
1
2
of opposite polarity). Furthermore, a control reaction was carried out in the absence of enzyme.
Gas Chromatography-Mass Spectrometry (GC-MS) analysis of crude reaction products in the presence
of CalB revealed the formation of the aminolysis product (3), the aza-Michael addition (4), and a double
addition (5) (Scheme 1).

Molecules 2017, 22, 2189
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Scheme 1. Lipase-catalyzed addition of benzylamine (1) to methyl crotonate (2) to yield aminolysis
product (3), aza-Michael addition (4), and double addition product (5).
As shown in Figure 1a, the reaction medium determines the differentiated accumulation of
the products. As anticipated, the enzyme is essential for the formation of crotonamide ( ), since in
the absence of enzyme, was not detected (Figure 1b). Interestingly, in the presence of enzyme, was
preferentially accumulated in n-hexane, while in 2M2B the accumulation of was favored. On the other
hand, it was observed that was formed in both media, although the proportion of was slightly higher
in 2M2B than in n-hexane. Finally, in the control reaction (Figure 1b), the aza-Michael adduct ( ) was
observed as the main product in the two media. It is noteworthy that the double-addition product (
3
3
4
3
5
5
4
5
)
accumulated in n-hexane but not in 2M2B, so we can conclude that 2M2B is not a suitable solvent for
the formation of 5 in the absence of enzyme.
3
4
5
3
4
5
Enzymatic
100
90
80
70
60
50
40
30
20
10
0
100
90
80
70
60
50
40
30
20
10
0
Control
n-Hexane
2M2B
n-Henane
2M2B
Solvent
Solvent
(a)
(b)
Figure 1. Influence of the polarity of the solvent in the chemoselective addition of
(a) Lipase-Catalyzed addition; (b) In the absence of enzyme.
1 to 2:
2.2. Enantioselectivity
As discussed above, in addition to the formation of products
was also detected. With n-hexane as the reaction medium , and
yield, respectively. It should be noted that both product and
3
and
4, the double addition product
5
3
4
,
4
5
were obtained in 7, 81, and 12%
5
presented ee values which were
20
determined by Chiral HPLC. Thus,
β
-amino ester
4
exhibited an ee of 1.1% {[
α
]
= +0.3 (c 1.0,
D
20
CHCl3)} and the -aminoamide showed an ee of 3.4% {[
β
5
α
]
D
=
−
1.4 (c 1.0, CHCl3)} (entry 1, Table 1).
, and were 48%, 29%, and 23%,
However, when the medium was 2M2B, the proportions of
3,
4
5
respectively. In contrast to the results obtained in n-hexane, the aza-Michael adduct
4
obtained in 2M2B
20
showed an ee of 96% {[
α
]
= + 15.6 (c 1.0, CHCl3)}. Finally, the product 5 obtained in 2M2B showed
D
20
an ee of 72% {[
α
]
D
=
−
19.2 (c 1.0, CHCl3)} (entry 3, Table 1). These results are consistent with those
obtained in our previous report [9].

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Table 1. Conversion and proportions of
3, 4, and 5 products obtained in the addition of benzylamine 1
to methyl crotonate 2 in n-hexane and 2M2B.
Proportion (%) b
3/4/5
1,4-Addition
4
Double Add
5
Conversion a
Entry
Enzyme
Solvent
[α]D
ee (%) d
[α]D
ee (%) e
c
c
1
2
3
4
5
6
7
8
CalB 435
Control
CalB 435
Control
CalB 435
Control
CalB 435
Control
n-hexane, 100%
n-hexane, 100%
2M2B, 100%
64
67
59
46
67
53
84
74
7/81/12
ND/74/26
48/29/23
ND/100/ND
48/29/23
ND/90/10
25/28/47
ND/80/20
+0.3
0
+15.6
0
+11.6
0
+7.7
0
1.1
0
96
0
79.8
0
75
0
-1.4
0
-19.2
0
-20.8
0
-21
0
3.4
0
72
0
67.8
0
67
0
2M2B, 100%
n-hexane/2M2B, 50:50
n-hexane/2M2B, 50:50
n-hexane/2M2B, 90:10
n-hexane/2M2B, 90:10
Reaction conditions: 3.5 mmol of benzylamine
1, 1.5 eq of methyl crotonate 2, 70 mg of CalB (Novozyme 435),
a
0.1 g of molecular sieves, and 7 mL of solvent. Conversion was calculated in the basis of
1
consumption. Yield
b
d
determined as isolated product. Determined as proportion by CG-MS. c (c 1.0, CHCl3). ee determined by Chiral
HPLC. Chiracel OD-H, hexane/i-PrOH 98:02, 0.8 mL min−1, UV-VIS 210 nm. Retention times: (R)-
4
: 11 min, (S)-
4:
e
13 min. ee determined by Chiral-HPLC. CHIRALPAK® AS-H, hexane/i-PrOH 80:20. 1 mL min−1, UV-VIS 210 nm.
Retention times (S)-5: 20.4 min, (R)-5: 29.4 min.
As shown in Scheme 2, compound
benzylamine on substrate (Path 1) or by the nucleophilic substitution of the acyl group on substrate
(Path 2). To clarify which of the two routes is correct, the following experiments were carried out.
5 could be produced by the aza-Michael addition of
1
3
4
Scheme 2. Possible routes for the formation of product
5 by the aza-Michael addition of 1 upon
the substrate 3 (Path 1), or by the aminolysis process upon the adduct 4 (Path 2).
The first experiment consisted in placing compound
3
(0.5 M in 2M2B) in the presence of CalB
(10 mg mL−1), 0.75 equiv. of benzylamine
1
, and 3
Ǻ
molecular sieves; this mixture was heated to
65 ◦C for 72 h. The enzyme was removed by filtration and washed with dichloromethane (DCM) and
the reaction crude was analyzed by thin layer chromatography (TLC) and 1H-NMR, noting that no
formation of compound 5 took place.
In a second experiment and after synthesizing rac-
placed in 2M2B (0.5 M) together with benzylamine
(0.75 eq), CalB (10 mg mL−1), and molecular sieves
at 65 ◦C for 3 h. The crude product from the reaction was analyzed by TLC and 1H-NMR, observing
the formation of compound in 41%. The latter result confirms the origin of product by nucleophilic
substitution of the acyl group on substrate (Path 2). After the isolation of compound , it showed a
specific rotation of [ 24.5 (c 1.0, CHCl3).
20 = + 7.9 (c 1.0, CHCl3) and for compound
This result is also in agreement with the observations made by Gröger and collaborators [18,20,21].
4 according to the literature [22], rac-4 was
1
5
5
4
4
20
α
]
D
5
of [α
]
D
=
−
The new results clarify the origin of the enantiomeric excess of the compound (S)-(+)-
4, obtained
in 2M2B, by a resolution process with CalB on the compound rac-4 (Scheme 3).

Molecules 2017, 22, 2189
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Scheme 3. Resolution of rac-4 in 2M2B with CalB.
To confirm whether the presence of 2M2B played a significant role in the resolution reaction, it was
decided to repeat the reaction in a 1:1 mixture of n-hexane/2M2B. Under these conditions, the three
products 3, 4, and 5 were again found in yields of 48%, 29%, and 23% respectively, and although
the proportions did not change with respect to those obtained in 2M2B, the ee value of the Michael
adduct and the double addition product decreased to 79.8% and 67.8% respectively (entry 5, Table 1).
Additionally, it is important to mention that in the control reaction both the Michael adduct and
the double addition product were found in a ratio of 90:10, respectively. These results confirm that
only obtained by an enzymatic process in 2M2B, since in the control reaction with 2M2B this was not
observed (entry 4, Table 1). Additionally the low ee of compound obtained in n-hexane is probably
5 is
5
due to the fact that the chemical process is much faster than the enzymatic process.
2.3. Optimization of Enantiomeric Excess
The best values of ee for (S)-(+)-
also favors the formation of aminolysis product
the formation of could be minimized in order to favor the process of resolution of rac-
system was n-hexane/2M2B 90:10. The reaction was monitored by 1H-NMR and proportions were
determined by GC-MS. The products , and were found in yields of 25%, 28%, and 47%, respectively,
showed an ee of 75% {[ , the ee
20 = +7.7 (c 1.0, CHCl3)}. For compound
4
and (R)-(
−
)-
. Thus, it was decided to design a system in which
. This new
5 were obtained in 100% of 2M2B, but this solvent
3
3
4
3,
4
5
and isolated compound
4
α
]
5
D
20
was 67% {[
α
]
=
−
21 (c 1.0, CHCl3)} (entry 7, Table 1). These results were anticipated in terms of
D
the polarity of the solvent, and although the presence of n-hexane inhibited the formation of compound
, the formation of compound was favored. Also, the optical rotation data were slightly smaller with
3
5
respect to the reactions carried out in 50:50 of n-hexane: 2M2B.
3. Materials and Methods
Candida antarctica lipase B (CalB) was obtained as Novozym® 435 (Novozymes Mexico,
Mexico City, Mexico). Methyl crotonate
2 and benzylamine 1 were obtained from Sigma Aldrich
(St. Louis, MI. USA). Thin layer chromatography (TLC) was carried out on pre-coated Merck silica
gel 60 F254 (Merck Mexico, Mexico City, Mexico). Column chromatography was performed on Merck
silica gel 60 (0.040–0.063 mm) (Merck Mexico, Mexico City, Mexico). Analytical grade solvents were
purchased from Tecsiquim (i-PrOH) (Tecsiquim, Toluca, Mexico) or Caledon Laboratory Chemicals
(n-hexane) (Georgetown, ON, Canada).
3.1. Analytical Methods
NMR spectra of products as well as the proportions of each product in the reaction mixture
were recorded on Varian Gemini 200 MHz and Varian Mercury 400 MHz (1H-NMR), and 50 and
100 MHz (13C-NMR) spectrometers, using CDCl3 as a solvent and tretramethylsilane (TMS) as an
internal standard. The proportions of each product in the reaction mixture were determine by Gas
Chromatography-Mass Spectrometry on an Agilent GC system 6890 series coupled to an Agilent
mass selective detector 5973N with an HP-5MS column length of 30 m, internal size of 0.25 mm,
film thickness of 0.25
µ
m, split ration of 5:1, 40 ◦C min−1, 5 ◦C min−1 to 250 ◦C min−1, 10 ◦C min−1
to 280 C min−1. Retention times (min):
= 25.3, = 28, and = 43. Enantiomeric excesses were
measured by an HPLC Dionex Ultimate 3000 model and UV Shodex detector RI-101 model at 210 nm,
using a Chiracel® OD-H column (5
250 mm), hexane/i-PrOH 98/2 (v/v) as eluent at a flow rate of
◦
4
3
5
×

Molecules 2017, 22, 2189
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0.8 mL min−1 at 25 ◦C for
4. Retention times: (R)-
4: 11 min, (S)-
4
: 13 min. A CHIRALPAK® AS-H
column (5
×
250 ◦mm) was used for
5, with hexane/i-PrOH 80/20 (v/v) as eluent at a flow rate of
−1
1 mL min at 25 C. Retention times: (S)-5: 20 min, (R)-5: 29 min. Optical rotations were measured by
means of a Perkin-Elmer 341 polarimeter.
3.2. Chemistry
3.2.1. Enzymatic Reactions
The enzymatic reactions were carried out in sealed glass vials. The reaction consisted in
the preparation of a solution of benzylamine (0.375 g, 3.5 mmol), methyl crotonate (0.525 g, 1.5 eq),
CalB (10 mg mL−1), and 0.01 g of molecular sieves dissolved in 7 mL of a pure dry solvent or a
◦
solvent mixture. The reaction mixture was stirred in a thermostated water bath at 65 C for 72 h.
Enzyme was filtered off and washed with DCM and then methanol. The solvent was evaporated under
reduced pressure. The proportions of 3, 4, and 5 in the reaction mixture were determined by GC-MS.
The products were purified over SiO2 using AcOEt/DCM (98:02 to 60:40) and then AcOEt/MeOH
(90:10 to 50:50), and their structures were confirmed by 1H and 13C-NMR, and compared with available
information in the literature [21–23].
N-Benzylcrotonamide, (
J = 2 Hz, J = 8 Hz, 3H); 4.47 (d, J = 6 Hz, 2H); 5.82 (m, 1H); 5.96 (br s, 1H); 6.86 (m, 1H); 7.21–7.37 (m,
5H). 13C-NMR (100 MHz, CDCl3)
17.9, 43.7, 125.0, 127.6, 128.0, 128.8, 138.5, 140.4, 166.1. Spectral
data 1H and 13C-NMR are consistent with the literature reports [23].
3 δ 1.83 (dd,
): White solid, mp 114–116 ◦C. 1H-NMR (200 MHz, CDCl3)
δ
(S)-(+)-Methyl 3-(benzylamino)butanoate, [(S)-(+)-
4
]: Yellow oil, after isolation. [
α
]
20 = +0.3 (c 1.0,
D
20
CHCl3), for product obtained in n-hexane; [
α
]
= +7.7 (c 1.0, CHCl3), for product obtained in
D
20
n-hexane/2M2B, 9:1; [
α
]
= +11.6 (c 1.0, CHCl3), for product obtained in n-hexane/2M2B, 1:1;
D
20
1
[
α
]
= +15.6 (c 1.0, CHCl3), for product obtained in 2M2B. H-NMR (200 MHz, CDCl3)
δ
1.15
D
(d, J = 8 Hz, 3H); 1.75 (br s, 1H); 2.38 (dd, J = 16 Hz, J = 6 Hz, 1H); 2.51 (dd, J = 15 Hz, J = 6 Hz, 1H);
3.16 (m, 1H); 3.67 (s, 3H); 3.74 (d, J = 14, 1H); 3.84 (d, J = 12 1H); 7.19–7.41 (m, 5H). 13C-NMR (50 MHz,
CDCl3)
consistent with the literature [22].
(R)-( )-N-Benzyl-3-(benzylamino)butanamide, [(R)-(
δ
20.6, 41.6, 49.9, 51.4, 51.7, 127.1, 128.3, 128.6, 140.5, 173.0. Spectral data 1H and 13C-NMR are
20
−
−
)-
5
=
]: Yellow resin, after isolation. [
−
α
]
D
=
−
1.4
20
(c 1.0, CHCl3), for product obtained in n-hexane; [
α
]
21 (c 1.0, CHCl3), for product obtained
D
20
in n-hexane/2M2B, 9:1; [
α
]
=
−
20.8 (c 1.0, CHCl3), for product obtained in n-hexane/2M2B, 1:1;
D
20
1
[
α
]
=
−
19.2 (c 1.0, CHCl3), for product obtained in 2M2B. H-NMR (200 MHz, CDCl3)
δ
1.20
D
(d, J = 8 Hz, 3H); 1.82 (br s, 1H); 2.27 (dd, J = 16 Hz, J = 8 Hz, 1H); 2.46 (dd, J = 16 Hz, J = 4 Hz, 1H);
3.10 (m, 1H); 3.69 (d, J = 12, 1H); 3.80 (d, J = 14 1H); 4.42 (d, J = 6, 2H); 7.08–7.37 (m, 10H); 8.43 (br s,
1H). 13C-NMR (50 MHz, CDCl3)
δ
20.3, 41.8, 43.5, 50.4, 51.0, 127.5, 127.5, 128.1, 128.5, 128.8, 128.9, 138.8,
171.8. Spectral data 1H are consistent with the literature [21].
3.2.2. Synthesis of rac-Methyl 3-(benzylamino)butanoate, rac-
4
was Prepared According to the Literature
, 0.10 g, 1 mmol) in 3 mL of
A solution of benzylamine ( , 0.10 g, 1 mmol) and methyl crotonate (
1
2
methanol were added to a 10-mL glass microwave reaction vessel containing a stir bar. The reaction
vessel was sealed with a cap and then placed into the microwave cavity. The reaction was heated at
T = 150 ◦C and 40 psi for 1 h. After the reaction was completed and the vessel was cooled to below
◦
50 C using a flow of compressed air, the solvent was evaporated in vacuo and the crude material was
purified by column chromatography to give the product as a yellow oil. Yield: 62%. Spectral data 1H
and 13C are consistent with those reported in the literature [22].
4. Conclusions
In this study, the origin of the ee for (S)-(+)-4 obtained in 100% of 2M2B could be clarified as a
resolution process on rac-4. First, the addition of benzylamine to methyl crotonate was catalyzed by

Molecules 2017, 22, 2189
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CalB to obtain rac-
4
, followed by its resolution through an aminolysis reaction to obtain (R)-( )-5.
−
We found that the process in 100% of n-hexane is racemic, and also favors the chemical synthesis of
. In 2M2B, the synthesis of rac- is unfavorable and favors the formation of compound . Finally,
5
4
3
we found a solvent engineering strategy to control the chemoselectivity in the addition of aza-Michael
catalyzed by CalB and at the same time we demonstrated the direct resolution of an intermediate
species through an aminolysis reaction.
Acknowledgments: We would like to thank CONACyT for financial support (Project No. CB2015/256653).
Marina A. Ortega-Rojas gratefully acknowledges CONACyT for a scholarship.
Author Contributions: M.A.O.-R., J.D.R.-R., and J.E. conceived and designed the experiments; E.C. performed
the experiments; C.G.A.-O., and F.G.-M. analyzed the data by Chiral HPLC; J.E., E.J., and E.C. wrote the paper.
Conflicts of Interest: The authors declare no conflict of interest.
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