Effect of the Substituent and Amino Group Position on the Lipase-Catalyzed Resolution of γ-Amino Esters: A Molecular Docking Study Shedding Light on Candida antarctica lipase B Enantioselectivity

Article abstract of DOI:10.1002/ejoc.202100712

The kinetic resolution of N-tert-butoxycarbonyl γ-amino methyl esters bearing different stereocenters at alpha (γ²), beta (γ³), or gamma (γ⁴) positions was carried out by enantioselective hydrolysis with Candida antarctica lipase B (CaLB) in 2-methyl-2-butanol solvent. The results show that the process is significantly less enantioselective for the γ²-amino methyl ester (E=2.5), the γ³-amino methyl ester (E=7.6), and the γ⁴-amino methyl ester (E=8.3) when compared with the kinetic resolution of analogous N-protected β³-amino esters (E>80). Based on these results, molecular docking studies were carried out, through which particular regions in the CaLB catalytic cavity were analyzed. The steric exclusion region composed of Ile189 and Val190 residues, together with the amino bonding region that induces a hydrogen bond with the Asp134 residue, appear to be responsible for the high selectivity in the resolution of carboxylic acid derivatives with beta stereocenters. This interaction is well preserved for β-amino esters as substrates. By contrast, γ-amino esters exhibit greater conformational diversity, so the effectiveness of the interaction is reduced, which apparently is responsible for the loss of enantioselectivity in the resolution process.

Full text of DOI:10.1002/ejoc.202100712

European Journal of Organic Chemistry
Accepted Article
Accepted Article
Title: Effect of the Substituent and Amino Group Position on the
Lipase-Catalyzed Resolution of γ-Amino Esters: A Molecular
Docking Study Shedding Light on CaLB Enantioselectivity
Authors: Marina A. Ortega-Rojas, Edmundo Castillo, Rodrigo Said
Razo-Hernández, Nina Pastor, Eusebio Juaristi, and Jaime
Escalante
This manuscript has been accepted after peer review and appears as an
Accepted Article online prior to editing, proofing, and formal publication
of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: Eur. J. Org. Chem. 10.1002/ejoc.202100712
Link to VoR: https://doi.org/10.1002/ejoc.202100712
01/2020

10.1002/ejoc.202100712
European Journal of Organic Chemistry
FULL PAPER
Effect of the Substituent and Amino Group Position on the
g
Lipase-Catalyzed Resolution of -Amino Esters: A Molecular
Docking Study Shedding Light on Candida antarctica lipase B
Enantioselectivity
Marina A. Ortega-Rojas,[a] Edmundo Castillo,*[b] Rodrigo Said Razo-Hernández,[c] Nina Pastor,[c]
Eusebio Juaristi,[d,e] and Jaime Escalante*[a]
Dedication. Dedicated to the memory of Professor Ferenc Fülöp.
[a]
[b]
[c]
[d]
[e]
Dr. Marina A. Ortega-Rojas; Prof. Dr. Jaime Escalante
Instituto de Investigación en Ciencias Básicas y Aplicadas, Centro de Investigaciones Químicas
Universidad Autónoma del Estado de Morelos
Av. Universidad No. 1001, Col. Chamilpa, C.P. 62210 Cuernavaca, Morelos, México
E-mail: marina.ortega@uaem.mx; jaime@uaem.mx; https://www.ciquaem.mx/escalante-garcia-jaime
Prof. Dr. Edmundo Castillo
Departamento de Ingeniería Celular y Biocatálisis
Instituto de Biotecnología, UNAM
Apartado Postal 510-3, C.P. 62271 Cuernavaca, Morelos, México
E-mail: edmundo@ibt.unam.mx; https://www.ibt.unam.mx/perfil/1436/dr-edmundo-castillo-rosales
Dr. Rodrigo Said Razo-Hernández; Dr. Nina Pastor
Instituto de Investigación en Ciencias Básicas y Aplicadas, Centro de Investigación en Dinámica Celular
Universidad Autónoma del Estado de Morelos
Av. Universidad No. 1001, Col. Chamilpa, C.P. 62210 Cuernavaca, Morelos, México
E-mail: rodrigo.razo@uaem.mx; nina@uaem.mx
Prof. Dr. Eusebio Juaristi
Departamento de Química
Centro de Investigación y de Estudios Avanzados
Av. Instituto Politécnico Nacional No. 2508, 07360 Ciudad de México, México
E-mail: ejuarist@cinvestav.mx
Prof. Dr. Eusebio Juaristi
El Colegio Nacional
Luis González Obregón 23, Centro Histórico, 06020 Ciudad de México, México
Supporting information for this article is available at the end of the document via a link.
Abstract: The kinetic resolution of N-tert-butoxycarbonyl g-amino
methyl esters bearing different stereocenters at alpha (g2), beta (g3),
or gamma (g4) positions was carried out by enantioselective hydrolysis
with Candida antarctica lipase B (CaLB) in 2-methyl-2-butanol solvent.
The results show that the process is significantly less enantioselective
for the g2-amino methyl ester (E = 2.5), the g3-amino methyl ester (E =
7.6), and the g4-amino methyl ester (E = 8.3) when compared with the
kinetic resolution of analogous N-protected b3-amino esters (E > 80).
Based on these results, molecular docking studies were carried out,
through which particular regions in the CaLB catalytic cavity were
analyzed. The steric exclusion region composed of Ile189 and Val190
residues, together with the amino bonding region that induces a
hydrogen bond with the Asp134 residue, appear to be responsible for
the high selectivity in the resolution of carboxylic acid derivatives with
beta stereocenters. This interaction is well preserved for b-amino
esters as substrates. By contrast, g-amino esters exhibit greater
conformational diversity, so the effectiveness of the interaction is
reduced, which apparently is responsible for the loss of
enantioselectivity in the resolution process.
Introduction
The synthesis of pharmacologically active compounds from
suitable substrates has a prominent relevance in the food,
cosmetics, and pharmaceutical industries. In this regard, a high
percentage of pharmacologically active compounds are amines
or their amino acid/amino amide derivatives.[1,2] The specific
connectivity and tridimensional distribution of their different
chemical groups define their functional characteristics. Therefore,
modern drug synthesis considers, as far as possible, the
development of new chiral drugs in the form of a single
enantiomer.[2] In this sense, the importance of preparing
enantiomerically pure amines or their amino acid derivatives is of
critical significance.[1-3]
At present, the two main strategies for obtaining enantiomerically
pure compounds are asymmetric synthesis and the resolution of
racemic mixtures.[4] In both approaches, biocatalysis and/or
biotransformations have been recognized as extremely useful
and convenient synthetic tools for preparing chiral amines and
their derivatives in high enantiopurity owing to their exquisite
1
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10.1002/ejoc.202100712
European Journal of Organic Chemistry
FULL PAPER
selectivity.[5] A particularly successful example is the enzymatic
preparation of enantiomerically pure b- and g-amino acids.[6] The
extraordinary attention paid to the synthesis of enantiomerically
pure b- and g-amino acids arises from their ubiquity in natural
products and their interesting pharmacological and biological
activity, exhibited in antibiotics, numerous chiral drugs, and
valuable building blocks, for example in oligopeptides.[3,7,8] In this
regard, the incorporation of b- and g-amino acids in peptides
frequently induces remarkable stability and interesting structural
and conformational characteristics.[8] On the other hand, the
recent interest in the synthesis of enantiomerically pure g-amino
acids is a consequence of their exceptional pharmacological
applications, for example as g-aminobutyric acid derivatives for
the treatment of various diseases such as neurodegenerative
pathologies, as muscle relaxants,[9] or as anticonvulsants bearing
anxiolytic and analgesic properties.[10]
The enantioselective synthesis of b- and g-amino acids often
involves a multi-step reaction procedure and the use of complex
chiral auxiliaries that are not always commercially available.[3,11]
Recently, several research groups have favored the use of
enzymes such as lipases, for resolving racemic b-amino acids.[6]
By contrast, only in few cases the resolution of g-amino acids or
amino esters has been reported.[12] In the last two decades, the
use of enzymes in organic synthesis has been increasingly
applied to obtain enantiomerically pure compounds, this being
due to their high stereoselectivity, catalytic promiscuity, and broad
substrate specificity among others.13] Lipases are among the
favorite biocatalysts; in particular, CaLB is one of the
commercially available enzymes most used in organic synthesis,
due to its catalytic promiscuity, high selectivity, remarkable
thermostability, and high activity in organic solvents.[14] CaLB has
been successfully employed in the enantioselective synthesis of
pharmaceutical building blocks.[13d,15] Due to its remarkable
catalytic promiscuity, it has been employed in a wide variety of
reactions, such as desymmetrization reactions,[16] 1,4-Michael-
type additions,[17] and particularly in resolution procedures of
racemic chiral substrates such as primary amines,[5b,18] and
secondary alcohols.[18b,19] In this regard, it has been established
that the catalytic cavity of CaLB is formed by two substrate
specific pockets, the acyl pocket and the nucleophile pocket, with
the catalytic active site located at the bottom of the pockets.[14c]
The mechanisms that govern the enantioselectivity of the
nucleophilic resolution reactions of chiral amines and chiral
alcohols catalyzed by means of CaLB lipases have been
adequately described. Molecular dynamics studies of the
observed enantiodiscrimination suggest that the nucleophile
pocket is delimited by a Trp104 residue, which is essential for the
CaLB stereoselectivity.[18b,20] In contrast, in the case of the
resolution of chiral carboxylic acids, very few theoretical studies
have modeled this phenomenon.[21] In general, CaLB has
exhibited low selectivity in the resolution of chiral carboxylic acids
and their ester or amide derivatives, which was ascribed to a
relatively large cavity that results in weaker interactions in the acyl
pocket, when compared with those operative in the nucleophile
specific pocket.[20,22]
(E > 80),[24,25] or in the resolution of β-amino esters through
acylation reactions by using alkyl methoxyacetates as acylating
agents (high ee and moderate conversions).[6h]
These reports have shown that the reaction of b3- and b2,3-amino
methyl esters using CaLB is directly influenced by the nature of
the substituent at the b position of the carboxylic substrate, and is
practically independent of any N-protecting group. On the other
hand, CaLB catalyzed resolution of carboxylic acid derivatives
with g stereocenters have not yet been fully addressed.[22] In this
context we performed the enantioselective hydrolysis reaction in
2-methyl-2-butanol (2M2B)[24] for the resolution of N-tert-
butoxycarbonyl (N-Boc) g-amino methyl esters whit a (g2), b (g3)
and g (g4) stereocenters. Indeed, this tertiary alcohol was selected
as the reaction medium because lipases do not accept it as
substrate. Furthermore, 2M2B increases the simultaneous
solubility of the ester substrates and water in the required
stoichiometric ratio. Moreover, 2M2B facilitates the recovery of
the hydrolyzed products (free acids) that are scarcely soluble in
2M2B. It is worth mentioning that up to now, no molecular docking
models related to lipase-catalyzed resolution reactions of a-, b-
and g-amino acids or amino esters in the acyl pocket have been
reported.
In this work, we report the effect of methyl substituents on CaLB-
catalyzed resolution of N-Boc-protected g-amino methyl esters
bearing a (g2), b (g3), and g (g4) stereocenters. By studying the
enzymatic enantioselective hydrolysis reaction of these b- and g-
amino methyl esters, we define the structural requirements
related to the CaLB -catalyzed resolution of these enantiomeric
substrates in the acyl pocket. In addition, with the help of
computational molecular docking, theoretical elements are
provided for the proper understanding of the structural
characteristics that determine the selectivity of CaLB towards g2-,
g3-, and g4-methyl esters. The information gathered here
constitutes a valuable contribution to the solution of the molecular
puzzle associated with the contrasting experimentally examined
enzymatic resolution of b- and g-amino acids or the corresponding
amino esters catalyzed by CaLB.
Results and Discussion
Chemistry
In order to evaluate the enantioselectivity of CaLB towards the
resolution of chiral g-amino methyl esters, we synthesized
different molecules of g-N-Boc-amino methyl esters bearing
alpha (g2), beta (g3) and gamma (g4) stereocenters. The synthetic
design of the molecules of interest consisted in the incorporation
of a methyl group at different positions (a, b, or g) relative to the
carboxylic group. First, the g-nitro aliphatic methyl esters rac-2a-
c were synthesized by a Michael addition of the corresponding
nitronate ions to commercially available acrylates 1a-c, according
to the previously described method (Scheme 1).[26]
It is noteworthy that in addition to the anticipated products, double
1,4-addition compounds 3a-c were also obtained as already
reported.[26] The direct conversion of g-nitro aliphatic methyl esters
rac-2a-c to N-Boc-amino methyl esters rac-4a-c was achieved via
an efficient one-pot reduction-protection of the nitro group using
H2 and Ni-Ra catalyst in the presence of di-tert-butyl dicarbonate
(Boc2O) as protecting group.
Among the most effective cases described for the resolution of
carboxylic acid derivatives by means of CaLB are those that
involve substrates with a- and/or b-stereocenters.[22] Indeed,
CaLB shows high enantioselectivity in the resolution of N-
protected b3-amino methyl esters via transesterification (E >
98),[23] hydrolysis of N-protected b3-and b2,3-amino methyl esters
2
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10.1002/ejoc.202100712
European Journal of Organic Chemistry
FULL PAPER
Scheme 1. Synthesis of g-N-Boc-amino methyl esters rac-4a-c according to the literature.[26] MW: Microwave conditions
Enzymatic resolution
Finally, in the case of racemate rac-4c, where the g2-substituent
methyl in the a position was evaluated, the expected reaction
proceeded more slowly according to the literature data. The
hydrolysis reactions at 45 ºC achieved only 31 % conversion after
24 h of reaction; hydrolyzed product (R)-6c was obtained with an
ee de 35.6 % and an E of 2.5 (entry 8, Table 1).
For the three racemic g-amino methyl esters rac-4a-c that were
evaluated CaLB showed poor enantiodiscrimination (E value <
10). In order to enhance CaLB enantioselectivity, the hydrolysis
reactions of rac-4a-c were carried out at room temperature.
Unfortunately, the enantioselectivity of these reactions was not
improved (entry 3, 6 and 9; Table 1).
It is worth mentioning that although the values of ee and E
observed for the hydrolyzed product (R)-6a bearing the
stereocenter in g-position are low, CaLB shows enantiopreference
for the stereoisomer that orients the alkyl substituent out of plane
(Scheme 2). In general, these results are in agreement with the
low enantioselectivity shown by CaLB in the resolution of
carboxylic acid derivatives with g-stereocenters.[22]
Similar enantiodiscimination was observed for racemic substrates
rac-4b when the alkyl substituent is located at the b position
(Scheme 2). Although the enantiopreference is in agreement with
observation in the resolution of N-protected and N-benzylated b3-
amino esters,[23-25] the lost in enantioselectivity suggests that, in
addition to the orientation (configuration) of the alkyl group, the
position of the amino group plays an essential role in CaLB
enantiodiscrimination.
The enantioselectivity of CaLB-catalyzed hydrolysis of g-N-Boc-
amino methyl esters rac-4a-c (0.2 M) was evaluated in anhydrous
2M2B as solvent; the same methodology used before for the
hydrolysis of b-amino esters with
b
stereocenters.[24] The
experiments were carried out at 45 °C and at room temperature;
reaction progress was followed by 1H-NMR. Following
chromatographic separation of the crude mixture, the
enantiomeric excesses (ee) for the unreacted amino esters 5a-c
were measured by chiral gas phase chromatography, while the
ee for the hydrolyzed products 6a-c were determined by chiral gas
phase chromatographic analysis of their ester derivatives 7a-c.
The absolute configurations were assigned by comparison of
optical rotation with reported values for the products 6a,[8b] 8a,[27]
6b, and 6c[28] (Scheme 2).
The hydrolysis reactions at 45ºC using g4-rac-4a as substrate
reached conversions of 48 % after 4.5 h, affording the product 6a
with an ee of 64 % and values of E = 8.3 (entry 2, Table 1). Similar
to previous works, these results showed the low selectivity of
CaLB for resolving compounds having g stereocenters.[22]
For the racemate g3-rac-4b bearing the methyl group on the beta
position, conversions of 31 % were achieved after 6 h at 45 ºC;
the hydrolyzed product (R)-6b was obtained with an ee of 70 %
and a low E value of 7.6 (entry 5, Table 1). Unexpectedly,
although CaLB has shown excellent selectivity for derivatives of
b-amino esters with b-stereocenters (E > 80),[23-25] in these
reactions, CaLB showed low selectivity for substrate rac-4b.
Interestingly, while the previously reported resolutions of b-amino
esters carry the amino group at the beta position the rac-4b
substrate of this work carries the amino group at the gamma
position.
Finally, for racemic substrate rac-4c the hydrolyzed product (R)-
6c, presenting the methyl substituent at the a position, the
enantioselectivity is better with respect to their N-benzylated b2-
amino ester analogs,[24] and shows an enantiopreference for the
enantiomer with the methyl substituent inside the plane.
3
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10.1002/ejoc.202100712
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Scheme 2. Enzymatic resolution of g-N-Boc-amino acids rac-4a-c at 45 °C.
Table 1. Resolution of g-amino acids rac-4a-c.
Recovered
Product
5
ee[d]
6
[c]
[c]
Entry[a]
Enzyme
Substrate
T
t (h)
Conversion[b]
[α]D
R/S[e]
[α]D
ee [d],[f]
R/S
E[g]
(°C)
6/7
7
6
1
2
3
4
5
6
7
8
9
Control
CaLB
CaLB
Control
CaLB
CaLB
Control
CaLB
CaLB
45
45
rt
4.5
4.5
20
6
0
0
---
---
R
R
---
S
---
---
64.1 g
40.6
---
---
S
---
8.3
3.6
---
rac-4a
rac-4b
rac-4c
48
54
0
+2
+1.6
0
60.3
47.2
---
+3.5c/-2.3h
+3.1/-1.7
---
S
45
45
rt
---
R
6
31
41
0
+0.16
+0.45
0
30.9
40.6
---
+2.7/-0.96
+2.6/-0.6
---
70
7.6
5.4
---
23
24
24
72
S
57.2
---
R
45
45
rt
---
S
---
R
31
29
+3.1
+3.6
16.2
18.6
-7.6/-9.9
-8.8/-10.5
35.6
44.17
2.5
3.1
S
R
[a]Reaction conditions: substrate rac-4 (0.86 mmol), 2 equivalents of water, 20 mg mL-1 CaLB (Novozym® 435), and 4.32 mL of 2M2B (0.2 M). [b] Calculated from
conv = ees/(ees+eep). [c] (c 1, CHCl3), 20 °C. [d ] Determined by Chiral GC-MS. [e] Absolute configurations were obtained through optical rotation measurements
and literature data comparisons. [f] Determined by ee of methyl esters derived from the corresponding acid. [g] Calculated from E = ln [1-conv(1+eep)]/ln[1-conv(1-
eep)]. [h] (c 0.68, CHCl3), 20 °C.
4
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Scheme 3. Enzymatic resolution of N-protected b3-amino methyl esters (A, previous work [23]), N-benzylated b3-, b3,2- and b2-amino methyl esters (B, previous
works [24,25]), and N-Boc protected g4-, g3-, g2-amino methyl esters (C, this work).
Molecular modeling
step, hydrolysis proceeds via a second tetrahedral intermediate
with water as second substrate (nucleophile) to give the
hydrolyzed product (Figure 1).[22]
Based on the experimental results of the kinetic resolution of N-
protected g-amino methyl esters via enantioselective hydrolysis
by means of CaLB (C, Scheme 3) and the reports described in
the literature for enantioselective transesterification of analogous
N-protected b-amino methyl esters (A, Scheme 3),[23] and
enantioselective hydrolysis of N-benzylated b-amino methyl
esters (B, Scheme 3),[24,25] we carried out a computational
molecular docking study examining the structural, complementary
requirements in the acyl pocket of the enzyme. This study
revealed the non-covalent enzyme-substrate interactions
involved in the recognition and formation of the N-protected g-
amino ester-CaLB complex before the nucleophilic attack takes
place.
The catalytic cavity of CaLB can be visualized as formed by two
pockets, the acyl pocket surrounded by Gln157, Asp134, and
Thr138, and the nucleophilic pocket delimited by Trp104. The two
pockets are separated by several hydrophobic residues with the
catalytic triad (Asp187/His224/Ser105) at the bottom of the cavity
(Figure 1). For a nucleophilic substitution reaction on the
carboxylic group, CaLB acts as chiral catalyst, following an
acylation-deacylation dynamic mechanism. In the resolution of
the b-amino acid ester as first substrate, Ser105 is activated by
the Asp/His residues, thus promoting the nucleophilic attack to the
carbonyl carbon of the substrate. The resulting oxyanion is
stabilized in the oxyanionic cavity (Gln106, Thr40), while His224
donates the proton to the leaving group of the tetrahedral
intermediate. The enzyme generates diastereomeric tetrahedral
intermediates of different energies for each of the enantiomeric
substrates, which results in the resolution of the racemic mixture
(D in Figure 1). Electron movement from the negatively charged
oxygen to the tetrahedral carbon, induces the release of the
leaving group to form the acyl-enzyme intermediate. In the final
The crystal structure of CaLB (PDB: 1LBS) with a phosphonate
inhibitor in the catalytic cavity evidenced the mechanism for
catalysis via transfer of the histidine proton to the leaving group.[20]
It is worth mentioning that in the report describing the 1LBS X-ray
structure, the authors refer to a mixture of conformations, one with
the acyl chain of the substrate in the nucleophilic core of the
pocket and the other with the acyl chain of the substrate in the
acyl pocket. However, the observed electron density showed that
the enzyme was inhibited mainly by the enantiomer whose acyl
segment is oriented towards the nucleophilic pocket (PDB: 1LBS).
Therefore, it does not form a hydrogen bond with His224, which
is essential for a catalytically productive conformation (F in Figure
2).
Validation of the docking protocol
All ligands, including the co-crystallized ligand, were submitted to
conformational and structural optimization before docking (for
details check the supplementary materials).
The co-crystallized ligand in the CaLB (PDB:1LBS) cavity was
well reproduced by its calculated conformation with a value of
RMSD = 1.1 Å (G in Figure 2). This conformation is a non-
productive conformation, as the ethoxy group of the ligand is in
the acyl pocket, therefore it does not form a hydrogen bond with
the histidine, that is necessary for catalysis. For the study of the
active conformation of different ligands, an extra validation
methodology was required. In particular, an alternative catalytic
conformation for the ligand was calculated, in which the ethoxy
group is oriented towards the nucleophile pocket, forming a weak
hydrogen bond with the His224 residue (H in Figure 2).
5
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10.1002/ejoc.202100712
European Journal of Organic Chemistry
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Figure 1. Schematic representation of the mechanism of an enzymatic kinetic resolution by CaLB. D, Diastereomeric tetrahedral intermediates for each of the
enantiomeric substrates, resulting in the required enantiodiscrimination for resolution of the racemic mixture. E, Criteria for evaluating the calculated conformations
in molecular docking: (a) acyl chain of the ligand surrounded by Asp134/Gln157, (b) hydrogen bond interaction between Ser105 and His224 and the methoxy group
at the ligand, (c) the methoxy group of the ligand in the nucleophile pocket delimited by the Trp104 residue.
Figure 2. F, CaLB catalytic cavity (PDB: 1LBS), experimental inhibitor in thick sticks (colored gray), amino acids of the acyl and nucleophilic pockets are displayed.
G, Comparison of the ligand calculated conformation (sticks and spheres colored yellow) with its crystal conformation (sticks colored gray) (RMSD = 1.1 Å). H,
Comparison of the ligand calculated conformation (ball and sticks colored green) for a catalytically productive conformation with a hydrogen bond to His224 with its
crystal conformation (sticks colored gray). All the residues are represented as thin sticks.
The ligand-protein interaction analysis was based on specific
residues, according to the following criteria: a) the ligand’s acyl
segment should be surrounded by Asp134 and Gln157 (acyl
cavity), b) a hydrogen bond interaction between Ser105 and
His224 with the methoxy of the ligand, and c) the methoxy group
of the ligand in the nucleophile pocket is best delimited by Trp104
a stereocenters (Series 3), and the g4-amino ester with
g stereocenters (Figure 3). Figure 3 shows the fast-reacting
enantiomers.
Docking results were divided in two sections. In the first section,
the effect of the size and position of the substituent was analyzed,
and in the second section, the position of the amino group was
(E in Figure 1). Both enantiomers of the substrates were analyzed, studied. Best ligand-protein interaction energies were obtained for
according to the following series: b3- y g3-amino esters with b
stereocenters (Series 1A-C), b2- and g2-amino esters with a
stereocenters (Series 2A and 2B), b2,3-amino esters with b y
the fast-reacting enantiomers; the energy values can be
consulted in the supplementary section.
6
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Figure 3. Fast reacting enantiomers for: (Series 1A-C) b3- and g3-amino esters with b stereocenters, (Series 2A-B) b2- and g2-amino esters with
a
stereocenter, (Series 3) b2,3-amino esters with b and a stereocenters; and (N-Boc protected g4-Amino ester) g4-amino ester with g stereocenter
.
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Effect of size and position of the substituent
Ile189 and Val190, but maintain the steric repulsion with catalytic
Ser105 (J in Figure 4). Such repulsive interaction can be
minimized by optimization of molecular coupling (see
Supplementary Information). At the experimentally, substrates
with substituents at the a position show low conversions, long
reaction times and low enantiodiscrimination (E = 1-2).
The steric repulsion generated by Val190 and Ile189 and the
substituent at b position is also observed with cis-b2,3-amino
methyl esters N-benzylated (cis-4x) with a and b stereocenters (K
in Figure 4). The corresponding trans-isomers (trans-4w) showed
greater conformational diversity orienting the acyl chain outside of
the cavity, minimizing steric repulsions with Val190 and Ile189. At
the experimental level, trans isomers showed loss of selectivity (E
= 11) compared to their cis counterparts (cis-4x) (E > 100). Finally,
the analysis for g4-amino esters with g stereocenters (Series 4,
Figure 3) shows that the substituent at position g does not
generate significant steric repulsion with Va190 and/or Ile189 (L
in Figure 4).
Molecular docking of the Series 1A-C shows the ability of the
ligands to fit into the cavity (shape complementarity) as a
determining factor in the reaction rate. Substituents such as
phenyl exhibit lower experimental reaction rates and substantial
steric congestion in the molecular docking relative to methyl
substituents. In addition, the three-dimensional structure of the
catalytic cavity can restrict the orientation of the substituents at
the stereocenter of the substrate through a steric exclusion effect,
as can be seen for the slow reacting enantiomer of rac-4i.
Interactions with Val190, Ile189, and Ser105 show that the fast-
reacting enantiomer (S)-4i orients the phenyl substituent outside
the cavity, minimizing steric repulsions. In contrast, the slow
reacting (R)-4i enantiomer suffers steric repulsive interactions
with these residues (I in Figure 4). In general, the less active
molecules in Series 1A-1C (Figure 3) present steric hindrance
with these residues, and show greater conformational distortion
(see Supplementary Information).
The calculated conformations for substrates with a stereocenters
in Series 2A-B do not exhibit steric repulsions with the residues of
Figure 4. Steric repulsion effect at CaLB catalytic cavity: I, Substrates with b stereocenters Series 1; fast-reacting enantiomer (S)-4i in sticks (green), slow-reacting
enantiomer (R)-4i in sticks (aqua). J, Substrates with a stereocenters Series 2; fast-reacting enantiomer (S)-4z in sticks (beige), slow-reacting enantiomer (R)-4z in
sticks (brown). K, Substrates with b and a stereocenters Series 3; fast-reacting enantiomer cis (2S,3R)-4x in sticks (Blue), slow-reacting enantiomer (2R,3S)-4x in
sticks (pink). L, Substrates with g stereocenters Series 4; fast-reacting enantiomer (S)-4a in sticks (magenta), slow-reacting enantiomer (R)-4a in sticks (yellow).
Exclusion residues Val190 and Ser105 in ball and sticks representation.
8
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10.1002/ejoc.202100712
European Journal of Organic Chemistry
FULL PAPER
Figure 5. Amino position effect: M, g3- amino ester fast-reacting enantiomer (R)-4b in sticks (dark blue); N, b3-amino ester fast-reacting enantiomer (R)-4f in sticks
(light blue). Hydrogen bonds are displayed as green dashed lines
The docking analysis shows that the substituent at position b
generates the greatest steric hindrance with the residues of
Val190 and Ile189. Nevertheless, the g3-amino ester 4b with a b
stereocenter does not present steric repulsion in the catalytic
cavity with Val 190 or Ile189 (M in Figure 5). These results are in
agreement with experimental observation, that is the loss of
selectivity of the g3-amino ester 4b (E = 7.6) relative to its analog
4f (E = 458).
show a broader conformational diversity, so this interaction is
affected, which may be the reason for the loss of
enantioselectivity for this class of esters.
Experimental Section
General: The lipase Novozym® 435 (Novozymes, Mexico) was used as
the catalyst for all enzymatic reactions. Novozym® 435 is the commercially
available form of the recombinant Lipase B from Candida antarctica,
immobilized by adsorption in a hydrophobic microporous acrylic resin. [14e]
Thin layer chromatography (TLC) was carried out on precoated silica gel
60 F254. Column chromatography was performed on Merck silica gel 60
(0.040-0.063 mm). 1H and 13C NMR spectra were recorded on Varian
Gemini 200 MHz, Varian Mercury 400 MHz or Brucker 500 MHz
equipments. Chemical d values (ppm) are reported relative to internal
tetramethylsilane (TMS) reference (d = 0.0 ppm) for determinations in
CDCl3 or calibrated against the residual impurity of CDCl3, (d = 7.26 ppm)
and CD3OD (d = 3.31 ppm). Gas Chromatography-Mass Spectrometry
(GC-MS) was recorded on an Agilent GC system 6890 series coupled to
an Agilent mass selective detector. Enantiomeric excesses for compounds
5a and 7a were determined by Chiral GC-MS with a SYCLOSIL-B column
length 30 m, internal size 0.25 mm, film thickness 0.25 µm, splitless,
120 °C at 0 min, 2 oC min - 1 to 180 °C by 1 min-1; retention times: (R)-5a
and (R)-7a = 17.62 min; (S)-5a and (S)-7a = 17.77 min; (R)-5b and (R)-7b
= 20.35 min; (S)-5b and (S)-7b = 20.50 min; (R)-5c and (R)-7c = 21.51
min; (S)-5c and (S)-7c = 21.66 min. All N-Boc protected g-amino acids (6a-
c) were converted into the corresponding methyl esters before their GC
analysis. Optical rotations were measured by means of a Perkin-Elmer 341
polarimeter. Mass analyses were carried out by Liquid Chromatography-
Mass Spectrometry (LC-MS) in an Agilent 6545 (LC-MS) spectrometer, by
electrospray ionization and quadrupole time-of-flight (ESI-QTOF).
Microwave Reactions were performed in sealed vessels in a monomode
microwave CEM Discover apparatus.
Amino position effect
Although the steric repulsion effect explains the spatial
requirement of the b3- and b2,3-amino esters with b stereocenters,
it does not justify the loss of selectivity observed in the g3-amino
ester with a b stereocenter (4b). Nevertheless, during the
examination of the calculated conformations we detected that a
stabilizing interaction by hydrogen bonding between Asp134 and
the amino group is possible, and can play an important role during
enantioselective resolutions. This interaction is well operative in
most of the b-amino esters such as 4f (Figure 5N). By contrast,
such interaction is probably lost in the g-amino esters, since they
present greater conformational flexibility (Figure 5M).
Conclusions
In contrast with the results previously reported for the CaLB-
catalyzed enzymatic resolution of N-protected and N-benzylated
b3-amino methyl esters, the resolution of g3-amino methyl esters
exhibited poor enantioselectivity. Based on the experimental and
molecular docking results, two new regions were identified in the
CaLB catalytic cavity: (1) the sterically repulsive region composed
of Val190 and Ile189 residues, which seem to be responsible for
the high selectivity in the resolution of derivatives of carboxylic
acids incorporating b stereocenters. This exclusion effect is not
effective for derivatives of b and g amino esters with a and g
stereocenters, which helps explain the experimentally observed
loss of selectivity. (2) The amino binding region interaction that is
rather efficient with b-amino esters; however, the g-aminoesters
General procedure for the synthesis of methyl 4-nitro alkyl esters via
microwave irradiation (GP1). A solution of a,b-unsaturated ester (1a-c)
and the corresponding nitroalkane (1d or 1e) were added to a glass
microwave reaction vessel containing a stir bar. The vial was placed in an
ice bath before the dropwise addition of the 1,1,3,3-Tetramethylguanidine
(TMG) or 1,8-Diazabicyclo(5.4.0)undec-7-ene (DBU) as base with stirring.
The reaction vessel was sealed and placed into the microwave cavity on
9
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10.1002/ejoc.202100712
European Journal of Organic Chemistry
FULL PAPER
“open vessel” mode, at the desired temperature and power conditions.
Once the reaction was complete, the vessel was allowed to cool to ambient
temperature. The crude mixture was purified by column chromatography.
Raney (0.05 g, 10% mass) and 15.5 mL of MeOH according GP2. The
crude mixture was purified by column chromatography (hexane/EtOAc
95:05) to give the product as a white solid, mp 24-25 °C, Yield: 75 %. Rf=
0.44 (hexane/EtOAc, 70:30). 1H NMR (200 MHz, CDCl3) d= 1.14 (d, J =
6.6 Hz, 3H, CH3CH); 1.44 (s, 9H, [(CH3)C]; 1.64-1.89 (m, 2H,CHCH2); 2.38
(t, J = 7.6 Hz, 2H, CH2CO); 3.68 (s, 4H, CH3CH+OCH3), 4.47 (br s, 1H,
NH). 13C NMR (50 MHz, CDCl3) d= 21.5, 28.5, 31.0, 32.3, 46.4, 51.7, 79.3,
155.5, 174.1. Spectral data 1H are consistent with the literature.[29] LC-
HRMS (ESI-QTOF) calculated for [C11H21NO4]+ requires 231.1471, found
231.1468.
Methyl 4-nitropentanoate [rac-2a] and Dimethyl 4-methyl-4-
nitroheptadionate (3a). Prepared from methyl acrylate (1a, 1.72 g, 20
mmol), nitroethane (1e, 1.87 g, 25 mmol) and (TMG) (1.26 g, 0.55 eq). The
microwave unit was programmed to 60 °C and a power of 50 watts for 5
min. After the reaction was completed, the vessel was cooled to below 50
ºC using a flow of compressed air. The crude mixture was purified by
column chromatography (hexane/EtOAc 90:10), afforded the products 2a
and 3a as colorless oils, with yield of 65 % in 58:42 proportion respectively.
Compound rac-2a: 1H NMR (400 MHz, CDCl3) d= 1.57 (d, J = 6.8 Hz, 3H,
CH3CH); 2.05-2.14 (m, 1H, CHCH2A); 2.23-2.35 (m, 1H, CHCH2B); 2.38-
Methyl 4-(tert-butoxycarbonyl)amino-3-methylbutanoate [rac-4b]:
Prepared from nitro ester rac-2b (0.50 g, 3.9 mmol), Boc2O (0.74 g, 1.1
eq) and Ni-Raney (0.05 g, 10% mass) and 15.5 mL of MeOH according
GP2. The crude mixture was purified by column chromatography
(hexane/EtOAc 90:10) to give the product as a colorless oil Yield: 65 %.
Rf= 0.47 (hexane/EtOAc t, 70:30). 1H NMR (200 MHz, CDCl3) d= 0.96 (d,
J = 6.5 Hz, 3H, CH3CH); 1.44 (s, 9H, [(CH3)3C]); 2.05–2.23 (m, 2H,
CH2ACO+CH); 2.30–2.44 (m, 1H, CH2BCO); 3.05 (pseudo t, J = 5.8 Hz, 2H,
CHCH2); 3.68 (s, 3H, OCH3); 4.74 (br s, 1H, NH). 13C NMR (50 MHz,
CDCl3) d= 17.9, 28.6, 31.4, 39.0, 46.2, 51.7, 79.4, 156.2, 173.5. Spectral
data 1H and 13C NMR are consistent with the literature.[30] LC-HRMS (ESI-
QTOF) calculated for [C11H21NO4]+ requires 231.1471, found 231.147.
2.47 (m, 2H, CH2CO); 3.70 (s, 3H, OCH3); 4.62-4.67 (m, 1H, NO2CH). 13
C
NMR (100 MHz, CDCl3) d= 19.5, 30.0, 30.1, 52.1, 82.6, 172.6. Compound
3a: 1H NMR (400 MHz, CDCl3) d= 1.55 (s, 3H, CH3C); 2.12-2.23 (m, 2H,
CH2AC); 2.27-2.43 (m, 6H, CH2BC+CH2); 3.69 (s, 6H, OCH3). 13C NMR
(100 MHz, CDCl3) d= 21.9, 28.9, 34.3, 52.2, 89.8, 172.6. Spectral data 1H
and 13C-NMR are consistent with the literature.[26]
Methyl 3-methyl-4-nitrobutanoate [rac-2b] and Dimethyl 3,5-dimethyl-
4-nitroheptadionate (3b) Prepared from methyl crotonate (1b, 3.50 g, 35
mmol), nitromethane (1d, 5.34 g, 87.5 mmol,) and DBU (0.26 g, 0.05 eq).
The microwave unit was programmed to 60 °C and a power of 50 watts for
15 min. After the reaction was completed, the vessel was cooled to below
50 °C using a flow of compressed air. The crude mixture was purified by
column chromatography (hexane/EtOAc 98:2 to 90:10), afforded the
products rac-2b and 3b as colorless oils, with yield of 58 % in a 98:2
proportion respectively. Compound rac-2b: 1H NMR (200 MHz, CDCl3) d=
1.10 (d, J = 6.8 Hz, 3H, CH3CH); 2.35 (dd, 2J = 16.3 Hz, 3J = 6.6 Hz, 1H,
CH2ACO) 2.47 (dd, 2J = 16.3 Hz, 3J = 7.1 Hz, 1H, CH2BCO); 2.67-2.90 (m,
1H, CH); 3.69 (s, 3H, OCH3); 4.34 (dd, 2J = 12.1 Hz, 3J = 7 Hz, 1H,
NO2CH2A) 4.48 (dd, 2J = 12.1 Hz, 3J = 6.3 Hz, 1H, NO2CH2B). 13C NMR (50
MHz, CDCl3) d= 17.3. 29.5, 37.7, 51.8, 80.3, 171.8. Spectral data 1H and
13C NMR are consistent with the literature.[26] Compound 3b was obtained
as inseparable mixture of diastereoisomers. See supplementary material.
Methyl 4-(tert-butoxycarbonyl)amino-2-methylbutanoate [rac-4c]:
Prepared from nitro ester rac-2c (0.50 g, 3.1 mmol), Boc2O ( 0.74 g, 1.1
eq), Ni-Raney ( 0.05 g, 10% mass) and 15.5 mL of MeOH according GP2.
The crude mixture was purified by column chromatography
(hexane/EtOAc 90:10) to give the product as a colorless oil Yield: 60 %.
Rf= 0.47 (hexane/EtOAc, 70:30). 1H NMR (500 MHz, CDCl3) d= 1.18 (d, J
= 7.1 Hz, 3H, CH3CH); 1.44 (s, 9H, [(CH3)3C]); 1.59–1.67 (m, 1H,
CH2ACH); 1.84 (m, 1H, CH2BCH); 2.51 (m, 1H, CH3CH); 3.15 (m, 2H,
CH2NH); 3.68 (s, 3H, OCH3); 4.72 (br s, 1H, NH). 13C NMR (50 MHz,
CDCl3) d= 17.2, 28.5, 33.9, 37.2, 38.7, 51.8, 79.3, 156.0, 176.9. Spectral
data 1H and 13C are consistent with the literature.[31] LC-HRMS (ESI-
QTOF) calculated for [C11H21NO4]+ requires 231.1471, found 231.1468.
Enzymatic reactions
Methyl 2-methyl-4-nitrobutanoate [rac-2c] and Dimethyl 2,4-dimethyl-
4-nitroheptadionate (3c) Prepared from methyl methacrylate (1c, 2.00 g,
20 mmol), nitromethane (1d, 1.52 g, 25 mmol) and TMG (1.15 g, 0.5 eq).
The microwave unit was programmed to 60 °C and a power of 50 watts for
20 min. After the reaction was completed, the vessel was cooled to below
50 °C using a flow of compressed air. The crude mixture was purified by
column chromatography (hexane/EtOAc 98:2), afforded the products rac-
2c and 3c as colorless oils, with yield of 66 % in 65:35 proportion
respectively. Compound rac-2c: 1H NMR (400 MHz, CDCl3) d= 1.19 (d, J
= 7.1 Hz, 3H, CH3CH); 2.06-2.14 (m, 1H CH2ACH); 2.23-2.32 (m, 1H,
CH2BCH); 2.50-2.58 (m, 1H, CH3CH); 3.65 (s, 3H, OCH3); 4.35-4.46 (m,
2H, NO2CH2). 13C NMR (100 MHz, CDCl3) d= 17.1, 30.7, 36.6, 52.0, 73.5,
175.3. Compound 3c: 1H NMR (200 MHz, CDCl3) d= 1.19 (d, J = 7.1 Hz,
6H, CH3CH); 1.91-2.19 (m, 4H, CHCH2CH); 2.35- 2.53 (m, 2H, CHCO);
3.69 (s, 6H, OCH3); 4.56-4.69 (m, 1H, NO2CH). 13C NMR (50 MHz, CDCl3)
d= 18.0, 36.4, 37.7, 52.2, 85.6, 175.6. Spectral data 1H and 13C NMR are
consistent with the literature.[26]
General procedure for the enzymatic hydrolysis of N-Boc protected
g
-amino esters (GP3). Enzymatic hydrolysis reactions were carried out in
sealed glass vials. The reactions consisted in the preparation of solution
of the corresponding g-amino esters rac-4a-c (0.20 g, 0.86 mmol), 20 mg
mL-1 of CaLB (Novozym® 435), 2 equivalents of water (31 µL) in 4.32 mL
of anhydrous 2M2B solvent (0.2 M). The reaction mixtures were stirred in
a thermostated water bath at 45 °C or at room temperature. At the end of
reaction the enzyme was filtered off and washed with DCM. The solvent
was evaporated in vacuum. The proportions of 5a-c and 6a-c in the
reaction crude were determined by 1H NMR. The products were purified
by filtration over SO2 using hexane/EtOAc (90:10 to 60:40).
(R)-(+)-Methyl 4-(tert-butoxycarbonyl)amino-pentanoate [(R)-(+)-5a].
Recovered ester: 52%. White solid, mp 24-25 °C, [a]D20 = +2 (c 1, CHCl3);
1
60.3 % ee. H and 13C NMR data are in accordance with those reported
for 4a.[29]
General procedure for the synthesis of N-Boc protected -amino
g
(S)-(+)-Methyl 4-(tert-butoxycarbonyl)amino-3-methylbutanoate [(S)-
(+)-5b]. Recovered ester: 69 %. Colorless oil. [a]D20 = +0.16 (c 1, CHCl3);
30.9 % ee. 1H and 13C NMR data are in accordance with those reported
for 4b.[30]
esters (GP2). The hydrogenation reactions were carried out in a Shaker
Hydrogenation Apparatus. A solution of nitro ester, Boc2O (1.1 eq) and Ni-
Raney (10% mass) in MeOH (5 mL/mmol) was added to a hydrogenation
flask. Reaction flask was placed into the hydrogenation apparatus and
filled with H2 to 60 psi, the reaction was monitored by TLC to observed
disappearance of nitro ester to six hours of reaction time. The catalyst was
filtered off and solvent evaporated under reduced pressure. The resulting
crude product was purified on SiO2 using hexane/EtOAc.
(S)-(+)-Methyl 4-(tert-butoxycarbonyl)amino-2-methylbutanoate [(S)-
20
(+)-5c]. Recovered ester: 69 %. Colorless oil. [a]D = +3.1 (c 1, CHCl3);
16.2 % ee. 1H and 13C NMR data are in accordance with those reported
for 4c [31]
.
Methyl 4-(tert-butoxycarbonyl)amino-pentanoate [rac-4a]: Prepared
from nitro ester rac-2a (0.50 g, 3.1 mmol), Boc2O (0.74 g, 1.1 eq) and Ni-
(S)-(+)-4-(tert-Butoxycarbonyl)amino-pentanoic
acid
[(S)-(+)-6a].
Yield: 48 %. White solid, mp 78-79 °C [lit.[32] (S)-6a: 75-78 °C; [a]D20 = +3.5
10
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10.1002/ejoc.202100712
European Journal of Organic Chemistry
FULL PAPER
(c 1.0, CHCl3); 64.1 % ee {lit.[8b] (S)-6a; [a]D20 = +2.4 (c 4, EtOH)}. 1H NMR
(200 MHz, CDCl3) d= 1.13 (d, J = 6.6 Hz, 3H, CH3CH); 1.42 (s, 9H,
[(CH3)3C]); 1.58-1.88 (m, 2H, CHCH2); 2.38 (t, J = 7.4 Hz, 2H, CH2CO);
3.72 (m, 1H, CHCH3), 4.47 (br s, NH), 9.46 (br s, 1H, OH). 13C NMR (50
MHz, CDCl3) d= 21.5, 28.6, 31.2, 32.3, 46.4, 79.7, 155.9, 178.6. Spectral
data 1H and 13C NMR are consistent with the literature.[32] LC-HRMS (ESI-
QTOF) calculated for [C10H19NO4]+ requires 217.1314, found 217.1326.
178.6, 50.4, 30.7, 29.4, 22.3. Spectral data 1H and 13C NMR are consistent
with the literature.[27]
Computational methods
Ligands preparation
rac-4a-z Structures were constructed in Spartan’18;[33] these molecules
were then submitted to a conformational analysis within the molecular
mechanics level theory employing the MMFF force field. The structures of
all the minimum energy conformers were fully optimized, without symmetry
restrictions, within the density functional theory (DFT) using the B3LYP
hybrid functional and the 6-31*G basis set. All vibrational frequencies were
positive, ensuring that all structures were in a minimum of the potential
energy surface. The ligands for the molecular docking were used with
Mulliken charges.
(R)-(+)-4-(tert-Butoxycarbonyl)amino-3-methylbutanoic acid [(R)-(+)-
6b]. Yield: 31 %. Colorless resin. [a]D20 = +2.7 (c 1, CHCl3); 70 % ee {lit.[28]
(R)-6b; [a]D20 = +4.3 (c 1.05, CHCl3)}, 1H NMR (200 MHz, CD3OD) d= 0.94
(d, J = 6.4 Hz, 3H, CH3CH); 1.44 (s, 9H, [(CH3)3C]); 1.98-2.16 (m, 2H,
CH2ACO + CH); 2.29-2.43 (m, 1H, CH2BCO); 2.96 (d, J = 6.4 Hz, 2H,
CH2NH). 13C NMR (50 MHz, CD3OD) d= 17.8, 28.8, 32.4, 39.8, 46.9, 79.9,
158.6. 176.6. Spectral data 1H and 13C NMR are consistent with the
literature.[28] LC-HRMS (ESI-QTOF) calculated for [C10H19NO4]+ requires
217.1314, found 217.1324.
CaLB structural analysis
All molecular docking calculations were carried out in Molegro Virtual
Docker (MVD).[34] The X-ray structure of CaLB used for molecular docking
was 1LBS PDB code with a phosphonate inhibitor in the catalytic cavity.
1LBS was used as the monomer, all the water molecules as well as the N-
acetyl-d-glucosamine molecule were removed. His224 was diprotonanted
to ensure the formation of the hydrogen bond between His224 and the
oxygen of the ester. The variation of the tertiary structure in 1LBS was
determined using Visual Molecular Dynamics (VMD),[35] by means of a
structural alignment taking as reference the structure of CaLB of higher
resolution (1.55 Å) with PDB code: 1TCA, but without ligand in the cavity.
It was determined that the main chain of the protein in the 1LBS structure
does not undergo important modifications caused by the inductive effect
of the ligand in its cavity (RMSD = 0.33 Å).
(R)-(
-
)-4-(tert-Butoxycarbonyl)amino-2-methylbutanoic acid [(R)-(-)-
20
6c]. Yield: 31 %. Colorless resin. [a]D = -7.6 (c 1, CHCl3); 35.6 % ee
{lit.[28] (R)-[a]D20 = -15.5 (c 1.04, CHCl3)}, 1H NMR (200 MHz, CD3OD) d=
1.16 (d, J = 7 Hz, 3H, CH3CH); 1.43 (s, 9H, [(CH3)3C]); 1.41-1.62 (m, 1H,
CHCH2A); 1.74-1.92 (m, 1H, CHCH2B); 2.36-2.53 (m, 1H, CH); 3.08 (t, J
=7.2 Hz, 2H, CH2NH). 13C NMR (50 MHz, CD3OD) d= 17.5, 28.8, 34.8,
38.1, 39.4, 79.9, 158.5, 180.1. Spectral data 1H and 13C NMR are
consistent with the literature.[28] LC-HRMS (ESI-QTOF) calculated for
[C10H19NO4]+ requires 217.1314, found 217.1323.
General procedure for the esterification of N-Boc protected
g
-amino
acids (GP4). To solution of N-Boc-Protected g-amino acid in
a
MeOH/toluene 3:2 (8 mL mmol-1) was placed in an ice bath then was
added 2 eq of TMS-CHN2 (2 M in hexanes), the reaction mixture was
stirred overnight. After the reaction was completed, the solvent was
evaporated in vacuum and the crude mixture was purified by column
chromatography to give the products as colorless oils.
Molecular Docking
The molecular Docking calculations were performed in Molegro Virtual
Docker.[34] Dockings were done at the phosphonate inhibitor binding site
(cavity volume is 194.05 Å3). The side chains of the amino acid residues
at 6 Å from the ligand were made flexible (34 amino acids). The search
algorithm was MolDock SE (Simplex Evolution) with the following
parameters: a total of 15 runs with a maximum of 2,000 iterations, using a
population of 50 individuals, 2,000 minimization steps for each flexible
residue, and 2,000 global minimization steps per run. The calculated
conformations were evaluated in terms of the enzyme-substrate
interaction energy, through the scoring function used by Molegro®
(MolDoc Score), which is given in kcal/mol and is the sum of the enzyme-
ligand intermolecular energy and the contribution of the internal energy of
the ligand.
(S)-( )-Methyl 4-(tert-butoxycarbonyl)amino-pentanoate [(S)-(-)-7a]:
-
Prepared from 6a (0.06 g, 0.27 mmol), MeOH/toluene 3:2 (2.2 mL) and
TMS-CHN2 2M in hexanes (0.27 mL, 2 eq) according GP4. The crude
mixture was purified by column chromatography (hexane/EtOAc 90:10 to
40:60). Yield: 92 %. [a]D20 = -2.3 (c 0.68, CHCl3); 64.1 % ee. White solid.
1H and 13C NMR data are in accordance with those reported for 4a.[29]
(R)-( )-Methyl 4-(tert-butoxycarbonyl)amino-3-methylbutanoate [(R)-
-
The scoring function to calculate the docking energy value was the
MolDock Score [GRID]. It was set on a 0.2 Å grid and attached the 10 Å
radius search sphere around the cavity. For the energy analysis of the
ligand, internal electrostatic interactions, internal hydrogen bonds and sp2-
sp2 deformations were considered.
The experimental ligand (catalytically non-productive conformation) in the
1LBS, was reproduced by a calculated conformation with a value of RMSD
= 1.1 Å. Additionally, a catalytically productive calculated conformation
was obtained, in which we can observe the ethoxy of the ligand oriented
towards the nucleophile pocket, forming a weak hydrogen bond with the
His224 residue.
(-)-7b]: Prepared from 6b (0.09 g, 0.08 mmol), MeOH/toluene 3:2 (0.680
mL) and TMS-CHN2 2M in hexanes (0.085 mL, 2 eq) according GP4. The
crude mixture was purified by column chromatography (hexane/EtOAc
90:10 to 40:60). Yield: 71 %. Colorless oil. [a]D20 = -0.96 (c 1.04, CHCl3);
70 % ee. 1H and 13C NMR data are in accordance with those reported for
4b.[30]
(R)-( )-Methyl 4-(tert-butoxycarbonyl)amino-2-methylbutanoate [(R)-
-
(-)-7c]: Prepared from 6c (0.03 g, 1.33 mmol), MeOH/toluene 3:2 (1 mL)
and TMS-CHN2 2M in hexanes (0.13 mL, 2 eq) according GP4. The crude
mixture was purified by column chromatography (hexane/EtOAc 90:10 to
40:60). Yield: 77 %. Colorless oil. [a]D20 = -9.9 (c 1.04, CHCl3); 35.6 % ee,
1H and 13C NMR data are in accordance with those reported for 4c.[31]
A docking template was created considering the structural characteristics
of the catalytically productive calculated conformation such as positive and
negative charges, hydrogen bond donors and acceptors, and steric
factors.
(R)-(+)-5-Methyl-2-pyrrolidinona [(R)-(+)-8a]: To a solution of [(R)-(+)-5a
(0.18 g, 0.77 mmol) in DCM was added a solution of TFA/DCM 1:2 (5.4
mL, 1 mL TFA / 100 mg sample); the reaction was monitored by TLC to
observed disappearance of 5a, the reaction mixture was diluted with
hexane to drag the TFA then was evaporated under reducer pressure. The
crude mixture was then refluxed in toluene to obtain 8a as colorless oil.
All the structures of docking of this work were made with Discovery Studio
Visualizer.[36]
Acknowledgements
20
20
Yield: 11 %. [a]D = +4 (c 1, EtOH); {lit.[27] (R): [a]D = +17.2 (c 1.02,
1
EtOH)}; H NMR (200 MHz, CDCl3) d= 1.22 (d, J = 6.3 Hz, 3H, CH3CH);
This research was supported by PAPIIT-UNAM (grant PAPIIT-
IT200220). We are also indebted to CONACYT, Mexico for
1.56-1.73 (m, 1H, CH2ACH); 2.17 -2.4 (m, 3H, CH2BCH+CH2CO); 3.70-
3.86 (m, 1H, CH3CH); 6.27 (br s, 1H, NH)); 13C NMR (50 MHz, CDCl3) d=
11
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10.1002/ejoc.202100712
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[12] a) E. Forró, F. Fülöp, Eur. J. Org. Chem. 2008, 5263–5268. b) F. Felluga,
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financial support via grants CB2015/256653, 220945 and A1-S-
44097, and to fund SEP-CINVESTAV for financial support via
grant 126. M.A.O-R thanks to CONACyT for the graduate
scholarship 266948. M.A.O-R. and R.S.R-H. thanks to Mario A.
Leyva-Peralta from University of Sonora and Zeferino Gómez
Sandoval from University of Colima for allowing access to their
software resources. We thank to Maria G. Medina Pintor for
technical assistance. We are also grateful to the reviewers for
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Keywords: g-Amino methyl esters
•
Biocatalysis
•
Enantiodiscrimination • Kinetic resolution • Molecular Docking
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Entry for the Table of Contents
In contrast to N-protected β3-amino esters, the Candida antarctica lipase B catalyzed kinetic resolution of amino esters holding alpha
(ꢀ2), beta (ꢀ3), and gamma (ꢀ4) stereocenters is strongly compromised by steric factors at the level of the catalytic cavity. Molecular
docking studies helped establish that the steric exclusion region involves the Ile189 and Val190 residues and the amino bonding region
where a hydrogen bond with the Asp134 residue is favored.
14
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