The enantiomeric discrimination of 5-hexyl-2-methyl3,4-dihydro-2h-pyrrole by sulfobutyl ether-β-cyclodextrin: A case study

Article abstract of DOI:10.3390/molecules26092611

1-Pyrrolines are important intermediates of active natural products, such as the 2,5dialkyl-1-pyrroline derivatives found in fire ant venoms. Here, 5-hexyl-2-methyl-3,4-dihydro-2Hpyrrole was synthesized by the enzymatic transamination/cyclization of 2,5-undecadione, and enantiodifferenciation was successfully achieved by capillary electrophoresis with sulfobutyl ether-βcyclodextrin as the chiral selector. The rationale of the enantiomeric discrimination was based on the results of a docking simulation that revealed the higher affinity of (S)-5-hexyl-2-methyl-3,4-dihydro2H-pyrrole for the sulfobutyl ether-β-cyclodextrin.

Full text of DOI:10.3390/molecules26092611

molecules
Article
The Enantiomeric Discrimination of 5-Hexyl-2-methyl-
3,4-dihydro-2H-pyrrole by Sulfobutyl ether-β-cyclodextrin:
A Case Study
Daniel F. Kawano 1,2 , Bruna Z. Costa 1 , Katherine L. Romero-Orejón 1 , Hugo C. Loureiro 1
Dosil P. de Jesus 1 and Anita J. Marsaioli 1,*
,
1
Institute of Chemistry, University of Campinas, Campinas 13083-861, SP, Brazil;
dkawano@unicamp.br (D.F.K.); bzucoloto@gmail.com (B.Z.C.); katherine.romero019@gmail.com (K.L.R.-O.);
hl303999@unicamp.br (H.C.L.); dosil@unicamp.br (D.P.d.J.)
Faculty of Pharmaceutical Sciences, University of Campinas, Campinas 13083-871, SP, Brazil
Correspondence: anita@unicamp.br; Tel.: +55-19-983256715
2
*
Abstract: 1-Pyrrolines are important intermediates of active natural products, such as the 2,5-
dialkyl-1-pyrroline derivatives found in fire ant venoms. Here, 5-hexyl-2-methyl-3,4-dihydro-2H-
pyrrole was synthesized by the enzymatic transamination/cyclization of 2,5-undecadione, and
enantiodifferenciation was successfully achieved by capillary electrophoresis with sulfobutyl ether-β-
cyclodextrin as the chiral selector. The rationale of the enantiomeric discrimination was based on the
results of a docking simulation that revealed the higher affinity of (S)-5-hexyl-2-methyl-3,4-dihydro-
2H-pyrrole for the sulfobutyl ether-β-cyclodextrin.
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Citation: Kawano, D.F. ; Costa, B.Z.;
Romero-Orejón, K.L.; Loureiro, H.C.;
de Jesus, D.P.; Marsaioli, A.J. The
Enantiomeric Discrimination of
5-Hexyl-2-methyl-3,4-dihydro-2H-
pyrrole by Sulfobutyl ether-β-
cyclodextrin: A Case Study. Molecules
2021, 26, 2611. https://doi.org/
10.3390/molecules26092611
Keywords: chiral capillary electrophoresis; pyrroline; enantiodifferenciation; docking; sulfotbutyl
ether-β-cyclodextrin
1. Introduction
2,5-Dialkyl-1-pyrrolines are the synthetic intermediates of several bioactive lipophilic
alkaloids present in ants and frogs [
diketones using enantioselective transaminases (TAs), which are dependent on pyridoxal-
50-phosphate (PLP), leads to enantiomerically enriched pyrrolines
, as shown in Figure 1.
1,2]. Usually, their asymmetric synthesis from 1,4-
1
2
Academic Editor: Roberto Mandrioli
The predominant enantiomer absolute configuration is assumed to follow the transami-
nase preference. However, additional evidence should be obtained during the absolute
configuration evaluation of the enantiomers to prevent future configuration problems.
Received: 9 March 2021
Accepted: 21 April 2021
Published: 29 April 2021
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
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iations.
Copyright:
© 2021 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
Figure 1. Reductive amination reaction of
to obtain enantiopure (S)- and (R)- , respectively. The products were obtained with excellent (>99%)
yields and enantiomeric excess (ee).
1 by S-selective TA (ATA-251) or R-selective TA (ATA-117)
2
2
Molecules 2021, 26, 2611. https://doi.org/10.3390/molecules26092611
https://www.mdpi.com/journal/molecules

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The enantiomeric discrimination of 5-hexyl-2-methyl-3,4-dihydro-2H-pyrrole
2
was
first tested with gas chromatography–mass spectrometry (GC–MS) equipped with a chiral
capillary column of -cyclodextrin, which unfortunately did not efficiently discriminate
the enantiomers [ ]. As a second resource, capillary electrophoresis (CE) analysis was
β
3
exploited for enantiomer resolution.
The CE is an electrodriven separation technique performed in a narrow-bore fused
silica capillary filled with a background electrolyte (BGE). The mechanism of separation in
CE is based on the different velocities of migration of charged species under the influence
of a high-voltage electric field (100 to 700 V/cm) applied to the capillary. The migration
velocity depends on the electrophoretic mobility of the species, which are related to their
charge-to-size ratio. Although enantiomers have the same charge-to-size ratio, CE can
be and is widely used for the separation of chiral molecules. This kind of CE separation
is feasible by adding a chiral selector to the background electrolyte, a compound able to
provide a differential interaction (enantioselectivity) between the enantiomers.
Here, the aim was the enantiomeric discrimination of 5-hexyl-2-methyl-3,4-dihydro-2H-
pyrrole
2, which was synthesized by the transamination/cyclization of 2,5-undecadione 1
applying commercial S-TA (ATA-251) or R-TA (ATA-117) from CODEXIS. It was simply
accomplished by supplying a buffer solution, named the BGE, with a sulfobutyl ether-
cyclodextrin as a chiral selector capable of discriminating the enantiomers of interest.
β-
2. Results and Discussion
2.1. CE Analysis
The better binding between the (S)- or (R)-2 and sulfobutyl ether-
evaluated using the CE technique. Among the available chiral selectors, the cyclodextrin
(CD) derivatives are the most used additives for this purpose [ ]. The separation is based
β-cyclodextrin was
4
upon a complex formation between the enantiomers and the chiral selector, resulting in the
formation of labile diastereoisomers.
Due to the different interaction of the enantiomers with the chiral selector, different
time-dependent mobilities were obtained, so a capillary electrophoretic method for the
enantioseparation of
ether- -cyclodextrin and the analytes. As shown in Figure 2, (R)- and (S)-pyrrolines could
be base-line separated by CE using sulfobutyl ether-β-cyclodextrin as a chiral selector.
2 was achieved, due to a favorable interaction between the sulfobutyl
β
Figure 2.
2H-pyrrole. Separation conditions: bare fused silica capillary with a total length of 60 cm (51.5 cm
effective) and 50
m i.d.; background electrolyte of formate buffer (25 mmol L−1, pH adjusted to
3.8) and sulfobutyl ether- -cyclodextrin sodium salt (3% m/v); hydrodynamic sample injection at
50 mbar for 5 s; separation voltage of −20 kV; UV detection at 210 nm.
(A) Electropherogram of a mixture of (S)-(in excess) and (R)-5-hexyl-2-methyl-3,4-dihydro-
µ
β

Molecules 2021, 26, 2611
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Although the pyrroline isomers were positively charged at the pH (3.8) of the BGE,
these molecules migrated for the anode (positive end) because of their interaction with the
cyclodextrins that are negatively charged. The peak with the highest intensity (peak area)
was identified as the (S)-pyrroline because its concentration in the mixture was higher than
the enantiomer of the (R) configuration (enantiomeric excess). The S-pyrroline showed
a smaller migration time than (R)-pyrroline (Figure 2), indicating that the interaction
between the isomer with (S) configuration and the sulfobutyl ether-β-cyclodextrin was
more favorable. This result is consistent with the predicted model.
2.2. Docking Simulations
Molecular docking simulations were performed to predict and rationalize the inter-
actions (enantiomer recognition) between both enantiomers of and sulfobutyl ether-
cyclodextrin. The binding free energies (∆Gbind) for the docked poses of S-2 and R-2 with
the sulfobutylether- -cyclodextrin were estimated using the Molecular Mechanics General-
ized Born Surface Area (MM/GBSA) method, a highly prevalent method for binding free
energy prediction [ ]. The estimated binding affinities suggest a slight preference of the
sulfobutylether- -cyclodextrin for the S-enantiomer, as the MM/GBSA ∆Gbind predicted
for S-2 was 6.32 kcal/mol (Figure 3A), while the corresponding value observed for R-2
2
β-
β
5
β
−
was −5.84 kcal/mol (Figure 3B).
Although these results suggest that the sulfobutyl ether-β-cyclodextrin would poorly
discriminate the enantiomers, we must highlight that these values should not be considered
as a direct replacement for in vitro results. Since the scoring functions of the docking
programs were developed to be of general use, working across a large set of target proteins,
this does not exclude the possibility that they may perform poorly for a particular drug
target [6] or, in this case, a particular chiral selector. To confirm the feasibility of the
predicted binding modes for the (R)-2 and (S)-2, we also performed a visual inspection of
the interactions between the best docking poses of each enantiomer and the chiral selector.
Analyses of the predicted interactions of the (R)-
2 and (S)-2 with the sulfobutyl ether-
β-cyclodextrin suggest a preferential interaction with the S enantiomer. As depicted in
Figure 2, the only potential interactions performed for both enantiomers are nontraditional
weak hydrogen bonds, where a carbon atom of the pyrroline acts as a hydrogen bond donor.
The C - H ... A hydrogen bonds (where A = O or N) are considerably weaker interactions
than the classical H-bonds, in spite of their recognized importance for the organization of
the biological world [7].
In fact, they are believed to represent one-half of the strength of a traditional H-bond
but are much more frequent in the process of ligand–protein interactions than generally be-
lieved [8]. In spite of both enantiomers displaying the same interaction, the shorter distance
observed for the (S)-pyrroline (2.36 Å versus 2.49 Å) warrants a slightly stronger interaction,
as highlighted by the binding affinities predicted in the molecular docking simulations.
Both enantiomers are predicted to interact with the target with similar binding modes,
with their rings positioned at the entrance of the central cavity, while the alkyl chains enter
the inner channel. Although the sulfobutyl groups do not seem to interact directly with the
pyrrolines, they confer the geometry of a bulky cylinder to this cyclodextrin in contrast to
the traditional β-cyclodextrin, where the side chains tend to form a floor in the structures,
making their geometries resemble one half of a coconut.
Therefore, the ligands are predicted to be more loosely associated with the target,
which reflects the lower predicted
the manufacturer does not clearly state the actual substitution pattern or the configurations
for the sulfobutyl ether- -cyclodextrin [ ], and we assumed for our simulations that all
the hydroxyl groups of -cyclodextrin would be substituted for the sulfobutyl ethers, a
limitation that can seriously compromise the predictivity of this model in particular.
∆Gbind values. However, it is important to notice that
β
9
β

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A
B
Figure 3. Proposed binding modes for the (R)- and (S)-enantiomers of 5-hexyl-2-methyl-3,4-dihydro-
2H-pyrrole with the sulfobutylether- -cyclodextrin, highlighting the main intermolecular inter-
actions and the corresponding binding free energies predicted using the Molecular Mechanics
Generalized Born Surface Area (MM/GBSA) method: ( ) (S)-pyrroline (carbon atoms in yellow;
) (R)-pyrroline (carbon atoms in magenta; weak hydrogen bond of
2
β
A
weak hydrogen bond of 2.36 Å); (
2.49 Å).
B
3. Experimental Section
3.1. General Methods
All commercially available reagents and solvents were purchased from Sigma-Aldrich
or Alfa Aesar and used without further purification. TAs ATA-251 and ATA-117 were
obtained from Codexis. Column chromatography was carried out using silica gel (Sigma-
Aldrich, 230–400 mesh). Thin-layer chromatography (TLC) was performed on Merck
Silica gel 60 F254 on aluminum foil and using a phosphomolybdic acid ethanolic solution
as a spay staining reagent. 1H NMR (400.13 MHz) and 13C NMR (100.63 MHz) were
acquired on a Bruker Avance III 400 (B0 = 9.4 T). Deuterated chloroform (CDCl3) or deuter-
ated methanol (CD3OD) were used as a solvent, and their residual protic solvent was
used as an internal reference. Chemical shifts are reported in δ (ppm), and the coupling
constants (J) are reported in Hertz (Hz). GC analyses were performed on an Agilent
6850 GC (Agilent, Santa Clara, CA, USA) coupled with a flame ionization detector (FID),
equipped with a fused silica capillary column HP-1 (30 m
× 0.32 mm × 0.25 µm, Agilent)
for general purposes or CP-Chirasil-DEX CB (25 m
×
0.25 mm × 0.25 µm, Agilent) for

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enantiomeric discrimination. GC–MS analyses were performed on an Agilent 7890B chro-
matograph coupled with an Agilent 5977B mass spectrometer with an electron ionization
source (EI) operating at 70 eV, and equipped with a fused silica capillary column HP-1ms
(30 m × 0.25 mm × 0.25 µm).
3.2. Synthesis of (R)- and (S)-5-Hexyl-2-methyl-3,4-dihydro-2H-pyrrole 2
Diketone 1 was synthesized based on a procedure previously described in [3]. A so-
lution of 2-octanone (10.6 mmol) in THF (5.0 mL) was added dropwise to a solution of
lithium diisopropylamine (2 M LDA in hexane, 6.35 mL, 12.7 mmol) in THF (40 mL) at
−
78 ◦C. The resulting mixture was stirred for 30 min at
−
78 ◦C. Chloroacetone (1.0 mL,
◦
12.7 mmol) was added dropwise, and the reaction mixture was stirred for 20 min at
−
78 C
◦
and then allowed to warm to 0 C (30 min) and then to r.t. (2–4 h). The reaction was
quenched by adding brine (15 mL) and extracted with dichloromethane (3
combined organic layers were dried over anhydrous MgSO4 and filtered, and the solvent
was removed under reduced pressure. The crude product was purified by silica gel flash
×
15 mL). The
column chromatography (cyclohexane:ethyl acetate gradient 9.5:0.5 to 8:2) to afford dike-
1
tone 2,5-decadione (1) in 74% yield (1.45 g). H NMR (400.13 MHz, CDCl3):
δ 2.71–2.64 (m,
4H), 2.44 (t, J = 7.6, 2H), 2.18 (s, 3H), 1.61–1.53 (quint, J = 7.5, 2H), 1.35–1.20 (m, 6H), 0.88
(t, J = 7.3, 3H). 13C NMR (125 MHz, CDCl3):
δ
209.7 (C), 207.3 (C), 42.8 (CH2), 36.9 (CH2),
36.0 (CH2), 31.4 (CH2), 30.0 (CH3), 28.8 (CH3), 23.5 (CH2), 22.5 (CH2), 13.9 (CH3). EI-MS
(70 eV): 170 (M.+, 184), 127 (9), 114 (81), 99 (100), 71 (96), 55 (10).
The enzymatic reductive amination reactions were carried out in Eppendorf flasks
containing the reaction mixture (450
µL of isopropylamine (1 mol/L) and pyridoxal phos-
phate (PLP, 1 mmol/L) in a triethanolamine buffer (100 mmol/L, pH 7.5 )) and 5 mg
of S-TA (ATA-251) or R-TA (ATA-117). Then, 1,4-diketone
0.3 mmol/L) was added to the reaction mixture. The reactions were maintained at 30
and 200 rpm for 4 h and was finally interrupted by the addition of NaOH (50
and extraction with ethyl ether (3 0.5 mL) afforded (S)- or (R)- in >99% yield and >99%
1
dissolved in DMSO (50
µL,
°C
µL, 5 mol/L),
×
2
2
ee. Subsequently, the organic fraction was analyzed by GC–MS.
3.3. Docking Simulations
The tridimensional structures of the enantiomers were built using the online version
of Corina (https://www.mn-am.com/online_demos/corina_demo) (accessed on 27 March
2021), which ascribes to 3-D structures pre-defined bond lengths and angles depending
on the type of bond, the type of atom, and the hybridization state and defines the most
probable torsional angles according to the nature of the structure [10
geometries of the ligands were then refined via the standard energy minimization in Spartan
14 [12]. The structures of the sulfobutylether- -cyclodextrin were drawn in Discovery
Studio Visualizer 4.1 [13], by modifying the crystallographic model of the -cyclodextrin
,11]. The molecular
β
β
(PDB ID: 1BFN). The geometries of the side chains of the resulting structure were then
refined via energy minimization in Spartan 14 [12], while keeping the geometries of the
D-glucopyranonsyl residues constrained. The lowest-energy conformer was then selected
for the subsequent analyses.
For the molecular docking simulations, the protonation states for both ligands were
assigned using the Epik module available in the Schrödinger suite 10.3. The binding
free energies for the docked poses were then estimated using the Molecular Mechanics
Generalized Born Surface Area (MM/GBSA) method available in the suite [14]. The most
probable binding mode of each structure was then assigned based on the best pose for each
enantiomer, the corresponding intermolecular interactions with the chiral selector being
studied through visual inspection with the aid of the receptor–ligand interactions module
of Discovery Studio Visualizer 4.1 [13].

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3.4. Electrophoresis Procedure
The chiral CE separations were conducted using a 7100 Agilent CE system (Agilent
Technologies, Waldbronn, Germany) equipped with a diode array detector (DAD). A bare
fused silica capillary with a total length of 60 cm (51.5 cm effective) and 50 µm i.d. was used.
The sample solutions were injected into the capillary by pressure (50 mbar for 5 s), and the
separation voltage was
cartridge was kept at 25
composed of a 25 mmol L formate buffer (pH 3.8, adjusted with NaOH solution) and 3%
−
20 kV. During the separations, the temperature of the capillary
C, and the UV detection was conducted at 210 nm. The BGE was
°
−1
(m/v) sulfobutyl ether-β-cyclodextrin sodium salt. The capillary was conditioned daily by
sequential flushes with methanol (10 min), a 0.1 mol L−1 HCl aqueous solution (10 min),
ultra-pure water (2 min), a 1 mol L−1 H3PO4 aqueous solution (10 min), ultra-pure water
(2 min), and finally with the BGE (10 min). The samples were diluted as required with the
BGE before injection into the CE system.
4. Conclusions
In summary, the enantiomeric excess and absolute configuration in picomolar sam-
ples of (R) and (S)-5-hexyl-2-methyl-3,4-dihydro-2H-pyrroles were achieved by capillary
electrophoresis with sulfobutylether-β-cyclodextrin (SB-β-CD) as a chiral selector.
Author Contributions: Methodology: D.F.K., B.Z.C., K.L.R.-O., D.P.d.J., and H.C.L.; writing—
original draft preparation: D.F.K., B.Z.C., K.L.R.-O., D.P.d.J., H.C.L., and A.J.M.; supervision: A.J.M.
All authors have read and agreed to the published version of the manuscript.
Funding: Daniel F. Kawano thanks the Brazilian funding agency Fundação de Amparo à Pesquisa
do Estado de São Paulo (FAPESP) (Grant n
Costa, Katherine L. Romero-Orejón, and Anita J. Marsaioli are grateful to CERSusChem FAPESP
(Grant n 2014/50249-8) and for the Conselho Nacional de Desenvolvimento Científico e Tecnológico
(CNPq) (Grant n 140774/2018-1). Dosil P. de Jesus and Hugo C. Loureiro thank FAPESP (grant n
2013/22127-2 and 2014/50867-3) and CNPq (grant n 311430/2017-1 and 305447/2019-0) and the
º 2018/08585-1) for the financial support. Bruna Z. da
º
º
º
º
National Institute of Science amd Technology in Bioanalysis (INCTBio).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: The data of this study are available in this article.
Conflicts of Interest: The authors have declared no conflict of interest.
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