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Article

Resolution of Racemic Aryloxy-Propan-2-yl Acetates via Lipase-Catalyzed Hydrolysis: Preparation of Enantiomerically Pure/Enantioenriched Mexiletine Intermediates and Analogs

by
Ana Caroline Lustosa de Melo Carvalho
1,
Bruna Rocha de Oliveira
1,
Gledson Vieira Lima
1,
Jonatas Martins Negreiro
1,
Maria Conceição Ferreira Oliveira
1,
Telma Leda Gomes de Lemos
1,
Marcos Reinaldo da Silva
2,
Thiago de Sousa Fonseca
3,
Rayanne Mendes Bezerra
4,
Jose Cleiton Sousa dos Santos
4,
Luciana Rocha Barros Gonçalves
5,
Nathalia Saraiva Rios
6,
Geancarlo Zanatta
7 and
Marcos Carlos de Mattos
1,*
1
Science Center, Laboratory of Biotechnology and Organic Synthesis (LABS), Department of Organic and Inorganic Chemistry, Campus do Pici, Federal University of Ceará, Fortaleza 60455-970, CE, Brazil
2
Institute of Chemistry, Federal University of Rio Grande do Norte, Natal 59072-970, RN, Brazil
3
Crateús Campus—Federal Institute of Ceará, Crateús 63708-260, CE, Brazil
4
Campus das Auroras, Institute of Engineering and Sustainable Development, University of International Integration Lusophone African-Brazilian, Redenção 62790970, CE, Brazil
5
Department of Chemical Engineering, Campus do Pici, Federal University of Ceará, Fortaleza 60455-760, CE, Brazil
6
Department of Chemical Engineering, Campus Central, Federal University of Rio Grande do Norte, Natal 59078-970, RN, Brazil
7
Department of Biophysics, Institute of Biosciences, Federal University of Rio Grande do Sul, Porto Alegre 91501-970, RS, Brazil
*
Author to whom correspondence should be addressed.
Catalysts 2022, 12(12), 1566; https://doi.org/10.3390/catal12121566
Submission received: 11 November 2022 / Revised: 25 November 2022 / Accepted: 28 November 2022 / Published: 2 December 2022
(This article belongs to the Special Issue Applications of Hydrolases in Medicinal Chemistry)
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Abstract

:
The lipase kinetic resolution (KR) of aryloxy-propan-2-yl acetates, via hydrolysis, produced enantiomerically pure/enantioenriched mexiletine intermediates and analogs. Racemic acetates rac-1-(2,6-dimethylphenoxy)propan-2-yl acetate (rac-5a), rac-1-(2,4-dimethylphenoxy)propan-2-yl acetate (rac-5b), rac-1-(o-tolyloxy)propan-2-yl acetate (rac-5c) and rac-1-(naphthalen-1-yloxy)propan-2-yl acetate (rac-5d) were used as substrates. A preliminary screening (24 h, phosphate buffer pH 7.0 with 20% acetonitrile as co-solvent, 30 °C and enzyme:substrate ratio of 2:1, m:m) was carried out with twelve lipases using acetate 5a as substrate. Two enzymes stood out in the KR of 5a, the Amano AK lipase from Pseudomonas fluorescens and lipase from Thermomyces lanuginosus (TLL) immobilized on Immobead 150. Under these conditions, both the (R)-1-(2,6-dimethylphenoxy)propan-2-ol [(R)-4a] and the remaining (S)-1-(2,6-dimethylphenoxy)propan-2-yl acetate [(S)-5a] were obtained with enantiomeric excess (ee) > 99%, 50% conversion and enantiomeric ratio (E) > 200. The KR study was expanded to racemic acetates 5b-d, leading to the corresponding chiral remaining acetates with ≥95% ee, and the alcohols 4b-d with ≥98% ee, and conversion values close to 50%. The best conditions for KRs of rac-5b-d involved the use of lipase from P. fluorescens or TLL immobilized on Immobead 150, 24 or 48 h and 30 °C. These intermediates had their absolute configurations determined using 1H NMR spectroscopy (Mosher’s method), showing that the KRs of these acetates obeyed the Kazlauskas’ rule. Molecular docking studies corroborated the experimental results, indicating a preference for the hydrolysis of (R)-5a-d.

Graphical Abstract

1. Introduction

Hydrolases (E.C. 3) constitute a group of enzymes that catalyze organic reactions in aqueous media, with more than two hundred types of enzymes. These enzymes favor some cellular physiological processes, such as digestion, transport, excretion, regulation and signaling [1]. Moreover, hydrolases play a crucial role in some industrial sectors, accounting for 75% of all enzymes used in the detergent, food, biofuel, pharmaceutical and waste treatment industries [2,3]. The most common subclasses of hydrolases are carbohydrases, proteases, esterases, glycosidases and lipases [4].
Among these subclasses, lipases (EC 3.1.1.3) that naturally catalyze the hydrolysis of triacylglycerol esters, stand out. The lipase market was valued at USD 425.0 million in 2018 and is projected to reach USD 590.2 million by 2023 [5]. This rapid growth in the market for commercial lipases is due to the robustness of these biocatalysts that act under mild conditions of pH and temperature, and in the absence of co-factors [6,7]. In addition to acting in aqueous media, lipases have high stability in organic media, catalyzing esterification, transesterification, interesterification, hydrolysis, aminolysis, ammonolysis, hydrazinolysis and acidolysis reactions, being highly chemo-, regio- and enantioselective [8,9,10,11,12]. Owing to these advantages, lipases have become highly attractive to the pharmaceutical industry [10,11,12], mainly with the development of direct evolution of proteins, allowing adaptation of a recombinant enzyme to act on a specific synthetic substrate, optimizing performance, thermostability and stereospecificity [12,13].
Chiral secondary alcohols are one of the most-used building blocks in drug preparation [14,15,16,17]. One of the approaches to obtain this class of compounds is through the kinetic resolution (KR) of the corresponding racemic acetates, catalyzed by lipases, via hydrolysis reaction, or by the complementary method, the KR of the racemic alcohol, via acetylation reaction. Both approaches have enabled the synthesis of enantiomerically pure active pharmaceutical ingredients (APIs) and their intermediates [9]. An example includes the preparation of the antitumor drug (S)-5-hydroxy-2-(1-hydroxyethyl)naphtho [2,3-b]furan-4,9-dione via a synthetic route starting from commercially available 1,5-dihydroxynaphthalene. The main step consisted in the KR of the secondary alcohol rac-5-hydroxy-2-(1-hydroxyethyl)-naphtho[2,3-b]furan-4,9-dione, via an acetylation reaction, catalyzed by lipases. Both CAL-B and lipase from Pseudomonas cepacia stood out, leading to (S)-alcohol with >99% ee [18]. In another report, rac-indanol was resolved via acetylation reaction in the presence of lipase from Thermomyces lanuginosus, leading to (S)-indanol with >99% ee in just 15 min of reaction. The latter was used to prepare the drug rasagiline mesylate, which is prescribed in the early stages of Parkinson’s disease [19]. The secondary alcohols rac-indanol, rac-1-phenylethanol, rac-1-(3-bromophenyl)-1-ethanol and rac-1-(3-methylphenyl)-1-ethanol were efficiently resolved via an acetylation reaction catalyzed by lipase from Pseudomonas fluorescens immobilized on magnetic nanoparticles. In all cases, both (R)-acetates and the remaining (S)-alcohols were obtained with >99% ee [20]. More recently, rac-1-(3-ethoxy-4-methoxyphenyl)-2-(methylsulfonyl)ethyl acetate was subjected to KR, via a hydrolysis reaction, catalyzed by lipase from Aspergillus niger. Both the remaining (S)-acetate and the (R)-alcohol were obtained with >99% ee. The latter was used in synthesizing apremilast, an effective drug in treating moderate to severe active psoriasis and psoriatic arthritis [21].
Mexiletine [1-(2,6-dimethylphenoxy)-propan-2-amine] (1a, Scheme 1) is a drug that contains a stereogenic center. Pharmacokinetic and pharmacodynamic studies have shown that the enantiomers of mexiletine have different activities, with (R)-mexiletine being the most effective in preventing ventricular arrhythmias, whereas its (S)-enantiomer is more effective in treating allodynia [22]. However, some authors have reported that both enantiomers of mexiletine produce antiallodynia [23]. Catalano and Carocci published an elegant review contemplating synthetic routes to obtain mexiletine in both racemic and chiral forms [22]. Mexiletine enantiomers have been prepared using either chiral starting materials or by the kinetic resolution of racemic mexiletine [22]. In addition, mexiletine analogs have been synthesized using the chiral-pool approach [24,25,26,27] or using ω-transaminases [28,29].
One of the mexiletine intermediates is the chiral secondary alcohol 1-(2,6-dimethylphenoxy)propan-2-ol (4a) or the corresponding acetate (5a) (Scheme 1) [30,31]. In an interesting example, (R)- or (S)-4a were subjected to a Mitsunobu’s Gabriel-type-reaction using a phthalimide, leading to the corresponding (S)- and (R)-phthalimido derivatives, respectively. These latter derivatives, after undergoing hydrazinolysis, resulted in the formation of (S)- and (R)-mexiletine, respectively [30]. In another report, (R)-mexiletine was obtained by a synthetic sequence that included the hydrolytic kinetic resolution of the epoxide 2-[(2,6-dimethylphenoxy)methyl]oxirane, in the presence of the Jacobsen’s catalyst (R,R) salen Co (III)OAc, leading to the corresponding remaining (S)-epoxide. Subsequently, the (S)-epoxide was regioselectivity-opened in the presence of LiAlH4, leading to the alcohol (S)-4a. The last steps consisted of the Mitsunobu reaction of (S)-4a with a phthalimide, followed by hydrazinolysis to generate (R)-mexiletine, [(R)-1a] [31].
Herein, we report the study on the kinetic resolution of rac-1-(2,6-dimethylphenoxy)propan-2-yl acetate (rac-5a), rac-1-(2,4-dimethylphenoxy)propan-2-acetates-yl acetate (rac-5b), rac-1-(o-tolyloxy)propan-2-yl acetate (rac-5c) and rac-1-(naphthalen-1-yloxy)propan-2-yl acetate (rac-5d), catalyzed by lipases, in order to obtain the chiral intermediates of (R)-mexiletine [(R)- or (S)-4a and (R)- or (S)-5a] and its analogs [(R)- or (S)-4b-d and (R)- or (S)-5b-d], Scheme 1. Mosher’s method based on 1H NMR spectroscopy was used to determine the absolute configurations of the new intermediates (the chiral alcohols 4b-d) of the mexiletine analogs, which were obtained from the KRs of the corresponding acetates rac-5b-d. In addition, molecular docking studies were performed to explain the selectivities of lipases that mediated the kinetic resolutions of acetates 5a-d.

2. Results and Discussion

2.1. Preparation of Alcohols rac-4a-d and Their Corresponding Acetates rac-5a-d

The preparation of the secondary alcohols rac-4a-d and their respective acetates rac-5a-d to be used as substrates in lipase-mediated kinetic resolutions (KR) is depicted in Scheme 2.
As the first reaction step, commercial phenols 2a-c and α-naphthol 2d were subjected to reaction with α-chloroketone, leading to ketones 3a-d with yields that varied between 60 and 82%. Next, the ketones 3a-d were reduced in the presence of NaBH4, leading to the alcohols rac-4a-d, with yields ranging from 88 to 98%. Finally, these alcohols were converted into the corresponding acetates rac-5a-d, in yields between 88 and 92%, Scheme 2. The compounds rac-4a, rac-5a, rac-4c and rac-5c were analyzed by chiral GC-FID and the compounds rac-4b, rac-5b, rac-4d and rac-5d were analyzed by chiral HPLC to find the adequate conditions for the separation of their enantiomers.

2.2. Screening of Lipases for the Kinetic Resolution of Acetate rac-5a via Hydrolysis

Initially, lipase screening was performed using acetate rac-5a as substrate, to yield the chiral drug intermediates of mexiletine. Kinetic resolutions (KRs) were performed by evaluating eight commercial lipases and four lipase derivatives from Thermomyces lanuginosus (TLL) immobilized in our laboratory. KRs were carried out via a hydrolytic approach under conditions previously reported by our research group [32,33], such as temperature of 30 °C, 250 rpm and 0.1 M phosphate buffer at pH 7 with 20% of acetonitrile as a co-solvent (buffer/co-solvent 8:2, v:v), in 24 h reaction, Scheme 3 (Path A). The results are summarized in Table 1. As shown in the table, four T. lanuginosus lipase biocatalysts were obtained by immobilization on superparamagnetic nanoparticle (SPMN) by ionic interaction or covalent binding. SPMN was functionalized with 3-amino-propyltriethoxysilane (APTES) or branched-polyethylenimine (BPEI), as well as glutaraldehyde (GA). The biocatalysts are identified by acronyms: SPMN@APTES and SPMN@PEI for ionic interaction, and SPMN@APTES-GA and SPMN@PEI-GA for covalent binding.
Among the lipases evaluated, the most effective in the kinetic resolution of rac-5a were Amano lipase AK from P. fluorescens (entry 1), lipase from T. lanuginosus immobilized in Immobead 150 (entry 2), lipase from C. antarctica B immobilized on acrylic resin (CAL-B) (entry 4) and lipase from T. lanuginosus immobilized on iron nanoparticles (SPMN@PEI-TLL) (entry 10). In the presence of lipase from P. fluorescens, as in the presence of TLL immobilized on Immobead 150, both acetate (S)-5a and alcohol (R)-4a were obtained with >99% ee, 50% conversion and enantiomeric ratio (E) > 200.
The kinetic resolutions followed the empirical rule of Kazlaukas [35], which indicates that in the hydrolysis reactions of racemic acetates from secondary alcohols, catalyzed by lipases, the (R)-acetate undergoes hydrolysis, producing the (R)-alcohol and the remaining (S)-acetate. The absolute configurations were confirmed by the polarimetry method. The specific optical rotation value obtained for (R)-4a was +1.46 (c 8.0, CHCl3) for >99% ee. In reference [25], the reported optical rotation value for (R)-4a is +0.9 (c 5.0, CHCl3) for >99% ee. For acetate (S)-5a, a specific optical rotation value of −10.8 (c 8.0, CHCl3) was obtained for >99% ee. There are no reports of specific optical rotation for (S)-5a.
The KRs of rac-5a, via hydrolysis reaction, were performed in 0.1 M phosphate buffer at pH 7.0 in the presence of 20% acetonitrile as a co-solvent. To investigate the influence of the absence of co-solvent or the presence of other co-solvents, new experiments were carried out in the presence of isopropyl alcohol (IPA), diethyl ether and tetrahydrofuran (THF).

2.3. Influence of the Co-Solvent on the KR of Acetate rac-5a, via Hydrolysis, Catalyzed by Lipases

The reactions were carried out with the lipases that stood out in the previously performed screening (Table 1), such as lipase from P. fluorescens, lipase from T. lanuginosus immobilized on Immobead 150 and immobilized on iron nanoparticles (SPMN@PEI-TLL), along with lipase from C. antarctica B immobilized on acrylic resin. The results are summarized in Table 2.
The KRs of rac-5a were ideal only in the presence of co-solvents, as in the case of the use of P. fluorescens (acetonitrile, Table 2, entry 2), SPMN@PEI-TLL (diethyl ether and THF, Table 2, entries 14 and 15) and CAL-B immobilized on acrylic resin (IPA, Table 2, entry 18). The exception was the TLL immobilized on Immobead 150, which provided an ideal KR for rac-5a in the absence of co-solvent (Table 2, entry 6), and in the presence of acetonitrile, IPA, and diethyl ether (Table 2, entries 7, 8 and 9).
Such results demonstrate that, in some cases, using co-solvent in KRs via hydrolytic process constitutes one of the ways of tuning the enantioselectivity of a lipase. To the best of our knowledge, there is no general rule explaining the co-solvent’s influence on the conversion and enantioselectivity of lipase-mediated hydrolysis reaction in a monophasic aqueous system containing a minor fraction of an organic solvent. It is a fact that co-solvent modifies the microenvironment of the active site of a lipase, altering its catalytic activity [21]. The way to achieve an ideal co-solvent in a KR lipase, catalyzed via hydrolysis reaction, is still an empirical process.
Once the KRs of rac-5a were optimized to produce the chiral mexiletine intermediates (R)-4a and (S)-5a, in enantiomerically pure form, we decided to move forward and explore a complementary methodology, the KR of rac-4a via an acylation approach, Scheme 3 (Path B).

2.4. Kinetic Resolution of rac-4a via Acetylation Reaction, Catalyzed by Lipases

The KRs of rac-4a were performed under reaction conditions previously reported in the literature, using vinyl acetate as acyl donor, 30 °C, rac-4a:lipase ratio of 2:1 (m/m), at 250 rpm [19]. For this approach, the most efficient lipases on KRs of rac-5a were selected, such as the lipase from P. fluorescens, TLL immobilized on Immobead 150 and the lipase from C. antarctica B immobilized on anionic resin. TLL immobilized onto SPMN@PEI was not evaluated as it contains traces of water from the immobilization process, which could influence the acetylation of rac-4a.
Regarding the choice of solvents used, although 1,4-dioxane (log P −0.27) and n-hexane (log P 3.50) are classified in the general solvent selection guides [36] as undesirable, we decided to use them in the KR of rac-4a as representatives of two solvents with a significant difference in polarity. Conversely, THF (log P 0.49) and toluene (log P 2.5) are classified as usable solvents and have intermediate polarities between 1,4-dioxane and n-hexane. Of the three lipases evaluated, only TLL immobilized on Immobead 150 was efficient in the kinetic resolution of rac-4a, via acetylation reaction, Table 3.
Strictly, TLL immobilized on Immobead 150 was less selective in the KR of rac-4a performed in the 1,4-dioxane (Table 3, entry 1) and n-hexane (Table 3, entry 4), both solvents classified as undesirable and with opposite polarities (Table 3, entries 1 and 4). Generally, polar solvents such as 1,4-dioxane can remove part of the hydration layer on the enzyme surface, leading to a decrease in the selectivity of a lipase [37,38], a behavior confirmed by the results presented in Table 3, entry 1. On the other hand, the solvents considered usable by general solvent selection guides [36] and with positive log P values, led to high enantiomeric ratio (E) values, especially toluene, which reached an E value of 139 (Table 3, entry 2). The exception was n-hexane, a nonpolar solvent in which TLL was not selective in the kinetic resolution of rac-4a. The influence of a solvent on the enantioselectivity of a lipase is not restricted only to its polarity. Other factors such as dielectric constant, solvent structure, substrate and product polarities, and enzyme–solvent interphase polarity interfere with the discrimination of one of the enantiomers by the lipase active site. Therefore, predicting the lipase’s ability to mediate a kinetic resolution in a given organic solvent is not a straightforward and obvious process [39].
Since TLL is immobilized on Immobead 150, we evaluated its reuse capacity in several reaction cycles under reaction conditions optimized for KR of rac-4a, via acetylation.

2.5. Reuse Study of TLL Immobilized on Immobead 150 in KR of rac-4a, via Acetylation Reaction

The study on the enzyme reuse in the KR of rac-4a was performed under optimized reaction conditions, such as vinyl acetate as acyl donor, toluene as solvent, 30 °C, 15 min of reaction time, 250 rpm and TLL: rac-4a ratio of 0.5:1. Four reaction cycles were evaluated. The results are shown in Figure 1.
With the analysis of the results presented in Figure 1, it is possible to observe that in the second reaction cycle there is a drastic decrease in the enantiomeric ratio (E), from 139 (first cycle) to only 17 (second cycle), with a conversion of 32%. Conversion values continue to decrease in the third (25%) and fourth cycles (15%), with an E value in these latter two cycles of 25. These results indicate the infeasibility of reusing the enzyme under the reaction conditions optimized for the KR of rac-4a.
The comparison between the KRs to obtain the chiral intermediates mexiletine, shows that the approach via a hydrolytic process (Scheme 3, Path A), which involves the hydrolysis of acetate rac-5a, is more efficient than acetylation of rac-4a (Scheme 3, Path B). In the first case (Path A), both the remaining acetate (S)-5a and the alcohol (R)-4a were obtained enantiomerically pure. In the approach involving the acetylation of rac-4a (Path B), the intermediates were obtained in enantiomerically enriched form. Such results prompted us to extend the KR study of rac-5b-d analogs via a hydrolytic approach (Scheme 3, Path A).

2.6. KR Study of Acetates rac-5b-d via Hydrolysis Reaction in the Presence of Lipases

KR studies of acetates rac-5b-d were performed under the conditions previously established for the KR of rac-5a, such as 0.1 M phosphate buffer at pH 7 with 20% of acetonitrile as a co-solvent (buffer/co-solvent 8:2, v/v) or in the absence of co-solvent, 30 °C, 250 rpm, lipase:substrate (2:1, m/m) in 24 h reaction. The lipases evaluated were those that stood out in the KR of rac-5a, such as TLL immobilized on Immobead 150 and lipase from P. fluorescens. The results are summarized in Table 4.
In general, the KRs of acetates rac-5b-d showed conversion values close to 50% in the presence of both TLL immobilized on Immobead 150 and lipase from P. fluorescens. Depending on the reaction condition employed, the remaining acetates (S)-5b-c or alcohols (R)-4b-d were obtained in enantioenriched or enantiomerically pure forms. In the kinetic resolution of rac-5b, both the alcohol (R)-4b and the remaining acetate (S)-5b were obtained with >99% ee in the presence of TLL immobilized on Immobead 150 and acetonitrile as co-solvent (Table 4, entry 2). The kinetic resolution of rac-5c led to the remaining acetate (S)-5c with >99% ee in the presence of lipase from P. fluorescens and the absence of co-solvent (Table 4, entry 7). For alcohol (R)-4c, the highest enantiomeric excess value (98% ee) was obtained in the presence of TLL immobilized on Immobead 150 and acetonitrile as co-solvent (Table 4, entry 6). The KRs of rac-5d, in 24 h of reaction, led to a maximum conversion of 30% (Table 4, entries 10 and 13), producing the alcohol (R)-4d with >99% ee in the TLL-catalyzed reaction (entry 10) or in the presence of lipase from P. fluorescens (entry 13), in both cases, in the presence of acetonitrile as co-solvent. To increase the conversion values, the reactions were carried out within 48 h, in the presence of acetonitrile as co-solvent. In these cases, conversions reached values close to 50% (entries 11 and 14). Alcohol (R)-4d was obtained with >99% ee in the presence of TLL immobilized on Immobead 150 (entry 11) and lipase from P. fluorescens (entry 14). The best enantiomeric excess value for the remaining acetate (S)-5d (95% ee) was obtained in the presence of lipase from P. fluorescens in acetonitrile as co-solvent (entry 14).
The KRs of acetates rac-5b-d followed Kazlauskas’ rule, leading to novel mexiletine analogs intermediates such as alcohols (R)-4b-d and acetates (S)-5b-d. The absolute configurations of these intermediates were determined by 1H NMR spectroscopy, using the Mosher method.

2.7. Determination of the Absolute Configurations of the New Mexiletine Analogs Intermediates by 1H NMR Spectroscopy, Using the Mosher Method

The absolute configurations of the alcohols 4b-d obtained in the KRs of acetates rac-5b-d, via hydrolysis, were determined by the Mosher method based on double derivatization [40]. As derivatization reagents, (R)-3,3,3-trifluoro-2-methoxy-2-phenylpropanoyl chloride, (R)-MTPA-Cl, and (S)-3,3,3-trifluoro-2-methoxy-2-phenylpropanoyl chloride, (S)-MTPA-Cl, were used. Both (R)-MTPA-Cl and (S)-MTPA-Cl reacted with the chiral alcohols 4b-d, leading to the corresponding ester derivatives, named as (S)- ester derivatives [(S)-ED] and (R)-ester derivatives [(R)-ED], respectively. Then, 1H NMR spectra were obtained for each of the synthesized ester derivatives (Supplementary Materials: Figures S25–S30). The chemical shifts (δ) chosen to be analyzed, i.e., those referring to methylene (L1) and methyl hydrogens (L2), both attached to the stereogenic center of the chiral secondary alcohols. Subsequently, the differences in chemical shifts of L1 (∆δSRL1) between the ester derivatives [(S)-ED] and [(R)-ED] were determined. The same procedure was performed to determine the differences between the chemical shifts of L2 (∆δSRL2) between the [(S)-ED] and [(R)-ED]. The results are summarized in Table 5.
Methylene hydrogens (L1) are more shielded in (S)-ED when compared to (R)-ED, leading to values of ∆δSRL1 < 0 (Table 5, entries 1, 3 and 5). On the other hand, methyl hydrogens (L2) are more shielded in (R)-ED when compared to (S)-ED, leading to values of ∆δSRL2 > 0 (Table 5, entries 2, 4 and 6). These results indicate that in the representative sp conformer of (S)-ED (Figure 2, conformer 1), the anisotropic effect of the phenyl group from the derivatization reagent shields the methylene hydrogens (L1) from the chiral alcohol. Conversely, in the representative sp conformer of (R)-ED (Figure 2, conformer 2), the anisotropic shielding effect of the phenyl group is exerted on the methyl hydrogens (L2). According to the models represented in Figure 2, the results obtained are corroborated by the alcohols 4b-d with (R)-configuration. Therefore, the KRs of acetates rac-5b-d obeyed the Kazlaukas’ rule, following the behavior of the KR of acetate rac-5a.

2.8. Molecular Docking Study

The experimental results revealed the lipases from T. lanuginosus (TLL) immobilized on Immobead 150 and lipase from P. fluorescens as the most active and selective enzymes in the KR of rac-5a-d, showing enantiopreference for the hydrolysis of the (R)-enantiomers. Therefore, molecular docking [41,42] was used as computational modeling to better understand the catalytic process at the atomic level for the enantio-preferred (R)-substrates. Through this in silico approach, the substrates [(R)- and (S)-5a-d] were docked at each enzyme active site, and near attack conformations (NACs) were extracted from visual inspection of the poses obtained from the molecular docking. NAC can be defined as ground-state conformations, in which the substrate and the enzyme present electrostatic interactions with distances and angles very close to those required in the transition state [43,44]. In the context of lipase-mediated hydrolysis of esters, such conformations involve the attack of the catalytic serine (Ser-O) to the electrophilic carbon of the acyl group (Carbonyl-C) in the substrate 44.
The crystallographic structure of TLL (PDB id 1EIN) [45] was employed during simulations, after preparatory steps as described below. In the lack of experimental coordinates for the lipase from P. fluorescens, the technique of molecular modeling by homology was applied, using the primary sequence recovered from UniProt [46] Id P41773. BLAST analysis identified the crystallographic structure PDB Id 2ZVD [47], from Pseudomonas sp. as a suitable template. That structure covers 99% of the lipase from P. fluorescens and shares 79% of identity sequence, showing the lipase in an open conformation. Homology modeling using the SwissModel server [48] generated a model differing only by 0.091 (RMSD value) to the template. Afterward, structural models of TLL and lipase from P. fluorescens were energy-minimized using the GROMACS 2019 package [49], and the stereochemical quality of binding site residues were assessed via the PROCHECK webserver [50].
At total, fifty poses were generated from the docking of (R)- or (S)-5a-d into the binding pocket of each lipase. For consistency, results were analyzed by visual inspection, and those presenting adequate NAC were identified. The NAC cutoff parameters represent the ideal distances and angles for the hydrolysis in the lipase catalytic site and were defined based on literature reports: atom distance between catalytic serine (Ser-O) and the electrophilic carbon of the acyl group (Carbonyl-C) ≤ 4 Å, and attack angles between Ser-O/carbonyl-C/carbonyl-O in the range of 84–110° [43,44,51,52,53]. Interestingly, among selected results, some docking poses exhibited an oxyanion suitable for the stabilization of the substrate in the active site of the lipases, forming a hydrogen bond with the carbonyl oxygen, and contributing to the decrease in the activation energy involved in the formation of the enzyme–substrate complex.
Table 6 shows the best results according to the binding energy of the receptor–ligand complex from molecular docking simulations that were within the NAC criteria. The distance and angle values shown in the table represent the best pose found (lowest binding energy) for each complex. For the (R)-enantiomers (Table 6, entries 1, 2, 5, 6, 9, 10, 13 and 14), a larger number of poses following the NAC criteria were identified when compared with the (S)-enantiomers (Table 6, entries 3, 4, 7, 8, 11, 12, 15 and 16). These suggest that the probability of finding the (R)-esters in a pose close to the nucleophilic attack is higher than for the (S)-esters.
Concerning the molecular docking simulations involving the complexes TLL-(R)-5a-d, there were 8, 6, 2 and 1 poses identified, respectively, which follow the NAC criteria. Compound (S)-5a presented only one pose within the NAC criteria, but with no hydrogen bond with the carbonyl oxygen (Table 6, entry 3); (S)-5b did not present poses attending the NAC criteria (Table 6, entry 7); (S)-5c presented only one, but with no hydrogen bond with the carbonyl oxygen (Table 6, entry 11); and complex (S)-5d (Table 6, entry 15) did not present any pose attending the NAC criteria. Simulations with the complexes P. fluorescens lipase-(R)-5a-d revealed 11, 1, 6 and 1 poses, respectively, which follow the NAC criteria (Table 6, entries 2, 6, 10 and 14). The studies with the (S)-enantiomers and the same lipase yielded five poses for the complex lipase-(S)-5a (Table 6, entry 4) and no pose for lipase-(S)-5b (Table 6, entry 8) attending the NAC criteria. Although five poses that attended the NAC criteria were identified for the complex lipase-(S)-5c (Table 4, entry 12), none of them showed hydrogen bonds with carbonyl oxygen. The complex (S)-5d (Table 6, entry 16) did not present poses attending the NAC criteria. These results corroborate the lipases enantioselectivity, both favoring hydrolyses of the (R)-ester.
The electrostatic interactions involved in complexes TLL-(R)-5a-d are displayed in Figure 3A–D, respectively. In all cases, the amino acid residue Leu147 forms a hydrogen bond with the carbonyl oxygen of the substrate, which is essential for the oxyanion stabilization. Importantly, in the case of TLL, the closest residues around enantiomer (R)-5a-c were Leu93, Phe95, Ser146, Leu147, Pro174, Val203, Leu206 and Pro207, among others, for the (R)-5d enantiomer, we have Ser83, Trp89, His145, Ser146, Leu147 and His258.
Figure 4A–C present the electrostatic interactions involved in complexes P. fluorescens lipase-(R)-5a-c. In these complexes, the amino acid residue Tyr28 is involved in the hydrogen bond with the carbonyl oxygen of the substrates. For lipase from P. fluorescens, the binding site was formed by residues Tyr28, Leu31, Ser206, Trp309, Leu313 and Pro314, among others. For the (R)-5d complex, no hydrogen bonds were found with carbonyl oxygen and the amino acids involved in the electrostatic interactions, which are Thr142, Ser206, Val256, Trp309 and His312 (Figure 4D).

3. Materials and Methods

3.1. Enzymes

(i) Immobilized lipases: Candida antarctica lipase type B immobilized on acrylic resin (CAL-B, Novozym® 435, 7300.0 U/g, Bagsværd, Denmark) and Rhizomucor miehei lipase immobilized on anionic resin (RML, 150.0 U/g) were purchased from Novozymes®. Thermomyces lanuginosus lipase immobilized on Immobead 150 (TLL, 250.0 U/g) and Amano lipase PS from Burkholderia cepacia immobilized on diatomaceous earth (PS-IM, ≥ 30,000 U/g) were acquired from Sigma-Aldrich® (St. Louis, MO, USA) (ii) Crude lipase preparations: Pseudomonas fluorescens lipase (AK, 22,100.0 U/g), Penicillium camemberti lipase (G, 50.0 U/g) and Amano lipase PS from Burkholderia cepacia (PS, ≥ 30,000 U/g) were acquired from Sigma-Aldrich®. Candida rugosa lipase (CRL, 1.4 U/g) was obtained from Sigma®. (iii) Lipase from Thermomyces lanuginosus (Lipozyme® TL 100 L, Novozymes, Bagsværd, Denmark) was immobilized on superparamagnetic iron oxide (Fe3O4) nanoparticles (SPMN).

3.2. Chemical Materials

Chemical reagents were purchased from different commercial sources and used without further purification. Methanol, n-hexane, ethyl acetate, dichloromethane, dimethylformamide (DMF), isopropanol (IPA), acetonitrile and 1,4-dioxane were acquired from Synth® (Diadema, Brazil). Isopropanol (IPA), acetonitrile and n-hexane, HPLC grade, were purchased from TEDIA® (Fairfield, OH, USA). Tetrahydrofuran (THF) was acquired from Sigma-Aldrich® (São Paulo, Brazil). Toluene and diethyl ether were acquired from Vetec® (Odense, Denmark). Solvents used in the reaction of biocatalysis were distilled over an adequate desiccant under nitrogen.

3.3. Analysis

Melting points were determined on a Microquimica hotplate model APF 301 (Marconi, São Paulo, Brazil) and are uncorrected. Analytical TLC analyses were performed on aluminum sheets pre-coated with silica gel 60 F254 (0.2 mm thick) from Merck® (Rahway, NJ, USA). Flash chromatography was performed using silica gel 60 (230–240 mesh). 1H and 13C NMR were obtained using Spectrometer Bruker model Avance DPX-300 (Billerica, MA, USA), operating at 300 MHz frequency for hydrogen and 75 MHz frequency for carbon. The chemical shifts are given in delta (δ) values and the absolute values of coupling constants (J) in Hertz (Hz). Measurement of the optical rotation was done in a Jasco P-2000 polarimeter (Tokyo, Japan). High-performance liquid chromatography (HPLC) analyses were carried out in a Shimadzu chromatograph model LC solution 20A (Kyoto, Japan) using a chiral column Chiralpak® IA from Daicel Company (150 mm × 4.6 mm × 5 μm) for rac-4b and rac-5b with oven set at 35 °C, injection volume: 20 μL. Reaction time related to flow was 0.3 mL/min using hexane/IPA (98/2) as eluent and UV detector at 254 nm. Resolution value (Rs) for rac-4b: 1.5 and Rs for rac-5b: 2.3. Retention times were: (R)-4b 22.2 min; (S)-4b 25.3 min; (R)-5b 7.1 min; (S)-5b 8.4 min. Chiral column Chiralpak® AS-H from Daicel company (150 mm × 4.6 mm × 5 μm) for rac-4d and rac-5d with oven set at 35 °C, injection volume: 20 μL. Rs for rac-4d: 1.6 and Rs for rac-5d: 4.7. Reaction time related to flow was 0.3 mL/min using hexane/IPA (98/2) and UV detector at 254 nm. Retention times were: (R)-4d 33.3 min; (S)-4d 29.3 min; (R)-5d 12.0 min and (S)-5d 13.5 min. Gas chromatography (GC) analysis was carried out with a Shimadzu chromatograph model GC 2010 (Kyoto, Japan) with a flame ionization detector, using a CP-Chirasil-Dex chiral column (25 m × 0.25 mm × 0.25 μm, 0.5 bar N2). Injection volume: 1 μL. Rs for rac-4a: 2.0 and Rs for rac-5a: 3.3. For monitoring the time course of reactions: rac-4a: 100 °C; 0.5 °C/min; 130 °C (hold 15 min); 5 °C/min 140 °C (hold 5 min). Retention times were: (R)-4a 62.8 min; (S)-4a 62.3 min (temperature of separation: 130 °C); rac-5a: 100 °C; 0.5 °C/min; 130 °C (hold 15 min); 5 °C/min 140 °C (hold 5 min). Retention times were: (R)-5a 73.6 min; (S)-5a 71.0 min (temperature of separation: 130 °C). For rac-4c: 120 °C; 1.0 °C/min; 135 °C (hold 5 min); 0.5 °C/min 150 °C (hold 5 min). Rs for rac-4c: 2.7 and Rs for rac-5c: 4.5. Retention times were: (R)-4c 14.8 min (temperature of separation: 134.8 °C); (S)-4c 14.6 min (temperature of separation: 134.6 °C). For rac-5c: 120 °C; 1.0 °C/min; 135 °C (hold 5 min); 0.5 °C/min 150 °C (hold 5 min). Retention times were: (R)-5c 19.0 min; (S)-5c 17.1 min (temperature of separation: 135 °C).

3.4. Procedure for Immobilizing Lipase from Thermomyces lanuginosus

Lipase from Thermomyces lanuginosus (TLL) was immobilized onto superparamagnetic nanoparticles (SPMN) by ionic interaction or covalent binding, according to the protocol previously described [54]. Briefly, both the adsorbed and covalent immobilized biocatalysts were obtained by contacting an enzyme solution and the activated support (3 mg of protein per gram of SPMN) at pH 7 and 25 °C, for 1 h. For the covalent immobilization, 0.01% (v/v) CTAB was added to the solution containing lipase to avoid adsorption.

3.5. General Procedure for the Synthesis of Ketones (3a-d)

A mass of 3.8 mg (0.06 mmol) potassium iodide was dissolved in 11 mmol of α-chloroacetone and allowed to react for 4 h. Then, the reaction mixture was added dropwise into 16.4 mmol of commercial phenols 2a-d and 15 mmol of potassium carbonate in 1.6 mL of dimethylformamide. The reaction was stirred for 24 h at 60 °C. Then, 50 mL of distilled water was added, and the product was extracted with diethyl ether (3 × 40 mL); the organic phases were combined and washed with NaOH (2 M). The organic phase was dried over anhydrous sodium sulfate. After filtration, diethyl ether was evaporated under reduced pressure. The crude product was purified on column chromatography using silica gel as stationary phase and a mixture of hexane/ethyl acetate (80/20%) as mobile phase. With this procedure, the following products were obtained: 1-(2,6-dimethylphenoxy)propan-2-one (3a, 82% yield, colorless oil, Rf = 0.54); 1-(2,4-dimethylphenoxy)propan-2-one (3b, 77% yield, yellow oil, Rf = 0.55) and 1-(naphthalen-1-yloxy)propan-2-one (3d, 75% yield, yellow oil, Rf = 0.60). Hexane/chloroform (20/80%) was used as eluent in the purification of the product 1-(2-methylphenoxy)propan-2-one (2c, 60% yield, brown oil, Rf = 0.50).

3.6. General Procedure for the Synthesis of Racemic Alcohols (rac-4a-d)

Initially, 4.5 mmol of 3a-d was dissolved in 45 mL of methanol. The reaction mixture was then cooled at 0 °C and 0.34 mg (9.0 mmol) of sodium borohydride (NaBH4) was slowly added. The reaction was stirred for 3 h and afterward the methanol was evaporated under reduced pressure. Distilled water (20 mL) was added, and the product was extracted with ethyl acetate (4 × 30 mL); the organic phases were treated with anhydrous sodium sulfate. After filtration, ethyl acetate was evaporated under reduced pressure. Then, the crude product was purified in column chromatography, using silica gel as stationary phase and the mixture hexane/ethyl acetate (80/20%) as mobile phase. With this procedure, the following products were obtained: rac-1-(2,6-dimethylphenoxy)propan-2-ol (rac-4a, 90% yield, yellow oil, Rf = 0.36); rac-1-(2,4-dimethylphenoxy)propan-2-ol (rac-4b, 88% yield, yellow oil, Rf = 0.43) and rac-1-(naphthalen-1-yloxy)propan-2-ol (rac-4d, 92% yield, white solid, mp = 62.8–64.5 °C, Rf = 0.40). Hexane/ethyl acetate (90/10%) was used as an eluent in the purification of the product rac-1-(2-methylphenoxy)propan-2-ol (rac-4c, 98% yield, yellow oil, Rf = 0.40).

3.7. General Procedure for the Synthesis of Racemic Acetates (rac-5a-d)

Initially, 1.7 mmol of rac-4a-d was dissolved in 17 mL of dichloromethane. Then, 0.51 mmol of 4-dimethylaminopyridine (DMAP) was added. Subsequently, 8.8 mmol of acetic anhydride was added. The reaction was processed for 4 h, then the solvent was evaporated, and the product was purified using a silica gel chromatography column, with the mixture hexane/ethyl acetate (80/20%) as mobile phase. With this procedure, the following products were obtained: rac-1-(2,6-dimethylphenoxy)propan-2-yl acetate (rac-5a, 92% yield, yellow oil, Rf = 0.60); rac-1-(2,4-dimethylphenoxy)propan-2-yl acetate (rac-5b, 90% yield, white solid, mp = 40.0–41.7 °C, Rf = 0.65) and rac-1-(naphthalen-1-yloxy)propan-2-yl acetate (rac-5d, 91% yield, white solid, mp = 55.8–56.9 °C, Rf = 0.50). Hexane/ethyl acetate (90/10%) was used as an eluent in the purification of the product rac-1-(o-tolyloxy)propan-2-yl acetate (rac-5c, 88% yield, yellow solid, mp = 88.2–89.0 °C, Rf = 0.50).

3.8. General Procedure for the Synthesis of Mosher Esters

Two-tenths millimole of chiral alcohols 4b-d, 0.1 mmol of dimethylaminopyridine, and 1.2 mmol of triethylamine were added into a 2 mL flask. Then, the flask was sealed, outgassed in vacuum and purged with dry nitrogen gas. Posteriorly, 2.7 mL of dichloromethane, previously dried, and 0.05 mmol of (S)-MTPA-Cl were added. The reaction was processed for 24 h, and then the solvent was evaporated, and the product was purified by column chromatography using chloroform (100%) as eluent, leading to the corresponding (R)-ester derivatives [(R)-ED]. The same procedure was performed with the chiral derivatization reagent (R)-MTPA-Cl, leading to the corresponding (S)-ester derivatives [(S)-ED].

3.9. General Procedure for the Enzymatic Kinetic Resolution of Acetates rac-5a-d, via Hydrolysis, Catalyzed by Lipases

One-tenth millimole of rac-5a-d was dissolved in 0.34 mL of acetonitrile (or in the absence of co-solvent) and 1.36 mL of potassium phosphate buffer (PO43−) (0.1 M, pH 7), followed by the addition of 60 mg of lipase. The reaction was processed at 30 °C and 250 rpm for 24 h. After this period, the products were extracted with ethyl acetate (3 × 1.0 mL) and the organic phases were combined and dried over anhydrous sodium sulfate. After filtration, the solvent was evaporated under reduced pressure and the crude products were purified by flash chromatography on silica gel, using hexane/ethyl acetate (80/20%) as eluent. After this procedure, the (R)-alcohols 4a-d and the remaining (S)-acetates 5a-d were obtained, whose enantiomeric excesses were determined by GC-DIC or HPLC using a chiral column.

3.10. Procedure for the Enzymatic Kinetic Resolution of Alcohol rac-4a, via Acetylation Reaction, Catalyzed by Lipases

Thirty milligrams (0.167 mmol) of rac-4a and 60 mg of lipase was added into a 5 mL flask. Then, the flask was sealed, outgassed in vacuum, and purged with dry nitrogen gas. Subsequently, 1.6 mL of organic solvent, previously dried, and 77 µL (0.835 mmol) of vinyl acetate were added in the flask reaction. The reaction was carried out under stirring at 250 rpm, varying the enzyme:substrate ratio and the reaction time. 1,4-Dioxane, hexane, THF and toluene were evaluated as solvent.

3.11. Procedure for the Reuse Study of TLL Immobilized on Immobead 150 in the Kinetic Resolution of Alcohol rac-4a via Acetylation Reaction

Thirty milligrams (0.167 mmol) of rac-4a and 15 mg of TLL immobilized on Immobead 150 were added into a 5 mL flask. Then, the flask was sealed, outgassed in vacuum, and purged with dry nitrogen gas. Subsequently, 1.6 mL of toluene, previously dried, and 77 µL (0.835 mmol) of vinyl acetate were added to the reaction mixture. The reaction was then stirred for 15 min and, subsequently, the enzyme was filtered off and washed with hexane (3 × 5 mL). The enzyme was thoroughly air-dried to be reused in the following reaction cycles.

3.12. Molecular Docking Study

3.12.1. Computation

The crystallographic coordinates of TLL (PDB id: 1EIN) [45] were retrieved from the Protein Database (PDB) [55]. The lipase was pre-processed with PyMOL (The PyMOL Molecular Graphics System, Version 1.5.0.4 Schrödinger, LLC, New York, NY, USA) to remove water molecules and any small molecule present in the crystallographic structures. The three-dimensional coordinates of lipase from P. fluorescens were modeled by molecular homology using the SwissModel webserver (accessed on 13 October 2022) [48]. The primary sequence (Id P41773) was recovered from UniProt [46]. The template was based on the X-ray structure (2.15 Å resolution) of the crystal structure of the lipase from Pseudomonas sp. in an open conformation (PDB Id: 2ZVD) [47]. Both structures were protonated at pH 7.0, using the PROPKA code in the PDB2PQR webserver [56], which suggested that the ND1 atom of His312 is protonated at indicated pH. The enzymatic mechanism for TLL and P. fluorescens lipase involves a catalytic triad formed by Ser146, Asp201, His258 and Ser206, Asp254, His312, respectively. For histidine residues, the NE2 atom forms a hydrogen bond with the side chain hydroxyl group of the catalytic serine, which can be both a hydrogen bond acceptor and donor. The ND1 atom of this same residue makes a hydrogen bond with OD2 of Aspartate. After the protonation step, each system underwent energy minimization through the GROMACS 2019 [49] package. For that purpose, the enzyme was placed in a cubic water box of volume equal to 437 nm3, followed by the addition of sodium and chloride counter-ions at a concentration of 0.15 mol.L−1 in order to neutralize the system charges. The force field AMBER99SB and the water model TIP3P were employed. The resulting structures of both proteins were employed during docking simulations.
The structures of the (R)-5a-d substrates were drawn in 2D using the program ChemDraw Ultra 12.0 [57], converted into 3D using the program Avogadro version 1.2 (http://avogadro.cc/, (accessed on 13 October 2022)) [58]. Afterward, all structures underwent energy minimization steps using the UFF universal force field. The resulting structures of ligands were employed during docking simulations.

3.12.2. Methodology for Molecular Docking

Molecular docking calculations were performed using Autodock 4.2 [59] and Autodock Tools [60]. The selected binding sites took into consideration the position of the catalytic triad amino acids Serine (Ser146), Aspartate (Asp201) and Histidine (His258) for TLL, and the catalytic triad amino acids Serine (Ser206), Aspartate (Asp254) and Histidine (His312) for P. fluorescens lipase. For TLL, the grid size was defined as 38, 60 and 40, along the x, y and z axes. For P. fluorescens lipase, the grid size was defined with dimensions 40, 40 and 40. For both systems, the grid spacing was set to 0.375 A and the grid map was calculated using the AutoGrid program. For each receptor–ligand complex, 50 poses were generated using the Lamarckian genetic algorithm (GA), with a population of 150 individuals and a maximum number of evaluations of 2,500,000, in addition to a maximum number of generations of 27,000. Results were visually inspected in order to obtain productive poses according to the near attack conformation (NAC) [43,44,51,52,53].

4. Conclusions

In summary, the chiral intermediates of the drug mexiletine such as acetate (S)-5a (>99% ee) and alcohol (R)-4a (>99% ee), as well as the analogs intermediates of this drug, the acetates (S)-5b-d (≥95% ee) and alcohols (R)-4b-d (≥98% ee), were efficiently prepared via lipase-mediated KRs of the corresponding racemic acetates, by a hydrolytic process. Among the evaluated lipases, the lipase from P. fluorescens and the TLL immobilized on Immobead 150 stood out. The complementary process that consisted of KR of alcohol rac-4a, via acetylation reaction, was less efficient when compared to the hydrolytic process. Only TLL immobilized on Immobead 150 was effective in mediating the acetylation of rac-4a, leading to acetate (R)-5a (95% ee) and the remaining alcohol (S)-4a (94% ee) in toluene. Mexiletine analog intermediates such as alcohols (R)-4b-d and acetates (S)-5b-d are new and had their absolute configurations determined by Mosher’s method, confirming that all KRs, via the hydrolysis reaction, followed the empirical rule of Kazlauskas. Molecular docking studies using lipases from P. fluorescens and TLL corroborate the results obtained in the KRs of acetates rac-5a-d, with reaction preference for the enantiomers (R)-5a-d.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/catal12121566/s1, Figure S1: NMR 1H of 3a (300 MHz, CDCl3); Figure S2: NMR 13C of 3a (75 MHz, CDCl3); Figure S3: NMR 1H of 3b (300 MHz, CDCl3); Figure S4: NMR 13C of 3b (75 MHz, CDCl3); Figure S5: NMR 1H of 3c (300 MHz, CDCl3); Figure S6: NMR 13C of 3c (75 MHz, CDCl3); Figure S7: NMR 1H of 3d (300 MHz, CDCl3); Figure S8: NMR 13C of 3d (75 MHz, CDCl3); Figure S9: NMR 1H of rac-4a (300 MHz, CDCl3); Figure S10: NMR 13C of rac-4a (75 MHz, CDCl3); Figure S11: NMR 1H of rac-4b (300 MHz, CDCl3); Figure S12: NMR 13C of rac-4b (75 MHz, CDCl3); Figure S13: NMR 1H of rac-4c (300 MHz, CDCl3); Figure S14: NMR 13C of rac-4c (75 MHz, CDCl3); Figure S15: NMR 1H of rac-4d (300 MHz, CDCl3); Figure S16: NMR 13C of rac-4d (75 MHz, CDCl3); Figure S17: NMR 1H of rac-5a (300 MHz, CDCl3); Figure S18: NMR 13C of rac-5a (75 MHz, CDCl3); Figure S19: NMR 1H of rac-5b (300 MHz, CDCl3); Figure S20: NMR 13C of rac-5b (75 MHz, CDCl3); Figure S21: NMR 1H of rac-5c (300 MHz, CDCl3); Figure S22: NMR 13C of rac-5c (75 MHz, CDCl3); Figure S23: NMR 1H of rac-5d (300 MHz, CDCl3); Figure S24: NMR 13C of rac-5d (75 MHz, CDCl3); Figure S25: NMR 1H of (R)-ED-4b (300 MHz, CDCl3); Figure S26: NMR 1H of (S)-ED-4b (300 MHz, CDCl3); Figure S27: NMR 1H of (R)-ED-4c (300 MHz, CDCl3); Figure S28: NMR 1H of (S)-ED-4c (300 MHz, CDCl3); Figure S29: NMR 1H of (R)-ED-4d (300 MHz, CDCl3); Figure S30: NMR 1H of (S)-ED-4d (300 MHz, CDCl3); Figure S31: Chromatogram obtained by GC from rac-4a; Figure S32: Chromatogram obtained by GC from rac-5a; Figure S33: Chromatogram obtained by GC from (R)-4a and (S)-5a after enzymatic hydrolysis of acetate rac-5a in the presence of TLL immobilized on Immobead 150, using acetonitrile as co-solvent; Figure S34: Chromatogram obtained by HPLC from rac-4b; Figure S35: Chromatogram obtained by HPLC from rac-5b; Figure S36: Chromatogram obtained by HPLC from (S)-5b and (R)-4b after enzymatic hydrolysis of acetate rac-5b in the presence of TLL immobilized on Immobead 150, using acetonitrile as co-solvent; Figure S37: Chromatogram obtained by GC from rac-4c; Figure S38: Chromatogram obtained by GC from rac-5c; Figure S39: Chromatogram obtained by GC from (S)-4c, (R)-4c, (S)-5c and (R)-5c after enzymatic hydrolysis of acetate rac-5c in the presence of TLL immobilized on Immobead 150, using acetonitrile as co-solvent; Figure S40: Chromatogram obtained by GC from (S)-4c, (R)-4c and (S)-5c after enzymatic hydrolysis of acetate rac-5c in the presence of lipase from P. fluorescens in the absence of co-solvent; Figure S41: Chromatogram obtained by HPLC from rac-4d; Figure S42: Chromatogram obtained by HPLC from rac-5d; Figure S43: Chromatogram obtained by HPLC from (R)-5d, (S)-5d and (R)-4d after enzymatic hydrolysis of acetate rac-5d in the presence of TLL immobilized on Immobead 150, using acetonitrile as co-solvent.

Author Contributions

A.C.L.d.M.C.: Conceptualization, Investigation, Data curation, Formal analysis and Writing—original draft. B.R.d.O.: Conceptualization, Investigation, Data curation, Formal analysis and Writing—original draft. G.V.L.: Investigation. J.M.N.: Investigation. M.C.F.O.: Conceptualization, Funding acquisition and Writing—original draft. T.L.G.d.L.: Conceptualization and Writing—original draft. M.R.d.S.: Conceptualization, Investigation, Data curation, Formal analysis and Writing—original draft. T.d.S.F.: Conceptualization, Investigation, Data curation, Formal analysis and Writing—original draft. R.M.B.: Investigation. J.C.S.d.S.: Conceptualization and Writing—original draft. L.R.B.G.: Conceptualization and Writing—original draft. N.S.R.: Investigation and Formal analysis. G.Z.: Conceptualization and Writing—original draft. M.C.d.M.: Conceptualization, Supervision, Resources, Funding acquisition, Project administration, Writing—original draft and Writing—review and editing. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by Coordenação de Aperfeiçoamento de Ensino Superior (CAPES), grant number Finance Code 001-PROEX 23038.000509/2020-82. Nº AUXPE: 1227/2020.

Data Availability Statement

Not applicable.

Acknowledgments

The authors thank the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Coordenação de Aperfeiçoamento de Ensino Superior (CAPES) for fellowships and financial support (Finance Code 001- PROEX 23038.000509/2020-82. Nº AUXPE: 1227/2020). The authors thank the research sponsorships of M. C. de Mattos (Process: 306289/2021-0) and M. C. F. de Oliveira (Process: 310881/2020-0). J. C. dos Santos thanks Fundação Cearense de Apoio ao Desenvolvimento Científico e Tecnológico (FUNCAP) for financial support (Process: BP-0139-00005.01.00/18). N. S. Rios thanks the Programa Nacional de Pós-Doutorado (PNPD/CAPES) for Post-Doctoral fellowship. The authors thank the Northeastern Center for Application and Use of NMR (CENAUREMN) for NMR spectroscopy.

Conflicts of Interest

The authors declare no conflict of interest.

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Catalysts 12 01566 sch001 550
Scheme 1. Retrosynthetic analysis of mexiletine and analogs.
Scheme 1. Retrosynthetic analysis of mexiletine and analogs.
Catalysts 12 01566 sch001
Catalysts 12 01566 sch002 550
Scheme 2. Preparation of alcohols rac-4a-d and their corresponding acetates rac-5a-d. Reagents and conditions: (i) K2CO3, KI, DMF, 60 °C, 24 h (60–82%); (ii) NaBH4, methanol, 0 °C, 3 h (88–98%); (iii) acetic anhydride, DMAP, CH2Cl2, rt, 4 h (88–92%).
Scheme 2. Preparation of alcohols rac-4a-d and their corresponding acetates rac-5a-d. Reagents and conditions: (i) K2CO3, KI, DMF, 60 °C, 24 h (60–82%); (ii) NaBH4, methanol, 0 °C, 3 h (88–98%); (iii) acetic anhydride, DMAP, CH2Cl2, rt, 4 h (88–92%).
Catalysts 12 01566 sch002
Catalysts 12 01566 sch003 550
Scheme 3. Screening of lipases on the KR of acetates rac-5a-d (hydrolytic approach: Path A) and KR of rac-4a (acylation approach: Path B). Conditions Path A: 0.1 M phosphate buffer at pH 7 with 20% of acetonitrile as a co-solvent (buffer/co-solvent 8:2, v/v), 30 °C, 250 rpm, lipase:rac-5a (2:1, m/m) and 24h. Path B: Organic solvent, 30 °C, 250 rpm, lipase and vinyl acetate as acyl donor.
Scheme 3. Screening of lipases on the KR of acetates rac-5a-d (hydrolytic approach: Path A) and KR of rac-4a (acylation approach: Path B). Conditions Path A: 0.1 M phosphate buffer at pH 7 with 20% of acetonitrile as a co-solvent (buffer/co-solvent 8:2, v/v), 30 °C, 250 rpm, lipase:rac-5a (2:1, m/m) and 24h. Path B: Organic solvent, 30 °C, 250 rpm, lipase and vinyl acetate as acyl donor.
Catalysts 12 01566 sch003
Catalysts 12 01566 g001 550
Figure 1. Results from the reuse of TLL immobilized on Immobead 150. Conditions: vinyl acetate as acyl donor, toluene as solvent, 30 °C, 15 min, 250 rpm and TLL: rac-4a ratio of 0.5:1.
Figure 1. Results from the reuse of TLL immobilized on Immobead 150. Conditions: vinyl acetate as acyl donor, toluene as solvent, 30 °C, 15 min, 250 rpm and TLL: rac-4a ratio of 0.5:1.
Catalysts 12 01566 g001
Catalysts 12 01566 g002 550
Figure 2. Mosher models [40] for the assignment of configurations of alcohols 4b-d by 1H NMR using ∆δSR.
Figure 2. Mosher models [40] for the assignment of configurations of alcohols 4b-d by 1H NMR using ∆δSR.
Catalysts 12 01566 g002
Catalysts 12 01566 g003 550
Figure 3. Interactions between TLL and substrate (R)-5a-d that follow the criteria established by the NAC. TLL and substrate (R)-5a (A); TLL and substrate (R)-5b (B); TLL and substrate (R)-5c (C) and TLL and substrate (R)-5d (D). Green dotted lines indicate hydrogen bonds with the oxyanion hole.
Figure 3. Interactions between TLL and substrate (R)-5a-d that follow the criteria established by the NAC. TLL and substrate (R)-5a (A); TLL and substrate (R)-5b (B); TLL and substrate (R)-5c (C) and TLL and substrate (R)-5d (D). Green dotted lines indicate hydrogen bonds with the oxyanion hole.
Catalysts 12 01566 g003
Catalysts 12 01566 g004 550
Figure 4. Interactions between P. fluorescens lipase and substrate (R)-5a-d that follow the criteria established by the NAC. P. fluorescens and substrate (R)-5a (A); P. fluorescens and substrate (R)-5b (B); P. fluorescens and substrate (R)-5c (C) and P. fluorescens and substrate (R)-5d (D). Green dotted lines indicate hydrogen bonds with the oxyanion hole.
Figure 4. Interactions between P. fluorescens lipase and substrate (R)-5a-d that follow the criteria established by the NAC. P. fluorescens and substrate (R)-5a (A); P. fluorescens and substrate (R)-5b (B); P. fluorescens and substrate (R)-5c (C) and P. fluorescens and substrate (R)-5d (D). Green dotted lines indicate hydrogen bonds with the oxyanion hole.
Catalysts 12 01566 g004
Table
Table 1. Screening of lipases in the kinetic resolution of rac-5a via hydrolytic approach.
Table 1. Screening of lipases in the kinetic resolution of rac-5a via hydrolytic approach.
Entry aLipaseees (%) beep (%) bc (%) cE d
1P. fluorescens>99>9950>200
2 eTLL>99>9950>200
3P. camemberti129032
4 fC. antarctica B>999850>200
5C. rugosa2357285
6 fR. miehei95865249
7B. cepacia90865141
8 gB. cepacia>999351135
9SPMN@APTES-TLL18>9915>200
10SPMN@PEI-TLL50>9934>200
11SPMN@APTES-GA5>991713
12SPMN@PEI-GA20>9917>200
a Conditions: 0.1 M phosphate buffer at pH 7 with 20% of acetonitrile as a co-solvent (buffer/co-solvent 8:2, v/v), 30 °C, 250 rpm, lipase:rac-5a (2:1) and 24 h. b ees: Enantiomeric excess of the remaining substrate (S)-5a; eep: enantiomeric excess of product (R)-4a; determined by GC-FID. c Conversion, c = ees/(ees + eep) [34]. d Enantiomeric ratio, E = ln[1 − c(1 + eep)]/ln[1 − c(1 − eep)] [34]. e Immobilized on Immobead 150. f Immobilized on anionic resin. g Immobilized on diatomaceous earth.
Table
Table 2. Influence of the co-solvent on the KR of rac-5a, via hydrolytic approach, catalyzed by lipases.
Table 2. Influence of the co-solvent on the KR of rac-5a, via hydrolytic approach, catalyzed by lipases.
LipaseEntry aCo-solventees (%) beep (%) bc (%) cE d
P. fluorescens1None96>9949>200
2Acetonitrile>99>9950>200
3IPA>999850>200
4Diethyl ether05>9905>200
5THF84>9946>200
TLL immobilized on Immobead 1506None>99>9950>200
7Acetonitrile>99>9950>200
8IPA>99>9950>200
9Diethyl ether>99>9950>200
10THF87>9947>200
TLL immobilized onto SPMN@PEI11None06>9906>200
12Acetonitrile50>9934>200
13IPA12>9911>200
14Diethyl ether>99>9950>200
15THF>99>9950>200
CAL-B immobilized on anionic resin16None55>9936>200
17Acetonitrile>999850>200
18IPA>99>9950>200
19Diethyl ether71>9942>200
20THF88>9947>200
a Conditions: 0.1 M phosphate buffer at pH 7 with 20% of co-solvent (buffer/co-solvent 8:2, v/v), 30 °C, 250 rpm, lipase:rac-5a (2:1) and 24 h. b Determined by GC-FID. c Conversion, c = ees/(ees + eep) [34]. d Enantiomeric ratio, E = ln[1 − c(1 + eep)]/ln[1 − c(1 − eep)] [34].
Table
Table 3. Kinetic resolution of rac-4a via acetylation reaction catalyzed by TLL immobilized on Immobead 150.
Table 3. Kinetic resolution of rac-4a via acetylation reaction catalyzed by TLL immobilized on Immobead 150.
Entry aSolventTime (min)ees (%) beep (%) bc (%) cE d
11,4-dioxane303948454
2 etoluene15949550139
3THF18095925189
4 fn-hexane205745564.5
a Conditions: vinyl acetate as acyl donor, organic solvent, 30 °C, 250 rpm and TLL: rac-4a (2:1). b Determined by GC-FID. c Conversion, c = ees/(ees + eep) [34]. d Enantiomeric ratio, E = ln[1 − c(1 + eep)]/ln[1 − c(1 − eep)] [34]. e TLL:rac-4a (0.5:1). f TLL:rac-4a (1:1).
Table
Table 4. KRs of rac-5b-d via hydrolytic process in the presence of TLL or lipase from P. fluorescens.
Table 4. KRs of rac-5b-d via hydrolytic process in the presence of TLL or lipase from P. fluorescens.
SubstrateLipaseEntry aCo-Solventees (%) beep (%) bc (%) cE d
rac-5bTLL1None3898729
2Acetonitrile>99>9947>200
P. fluorescens3None90>995299
4Acetonitrile989850>200
rac-5cTLL5None949450115
6Acetonitrile929848>200
P. fluorescens7None>99755736
8Acetonitrile92874968
rac-5dTLL9None45243
10Acetonitrile43>9930>200
11 eAcetonitrile84>995459
P. fluorescens12None6912156
13Acetonitrile43>9930>200
14 eAcetonitrile95>9951>200
a Conditions: 0.1 M phosphate buffer at pH 7 with 20% of co-solvent (buffer/co-solvent 8:2, v/v) or in the absence of co-solvent, 30 °C, 250 rpm, lipase:substrate (2:1) and 24 h. b Determined by GC-FID or by chiral HPLC. c Conversion, c = ees/(ees + eep) [34]. d Enantiomeric ratio, E = ln[1 − c(1 + eep)]/ln[1 − c(1 − eep)] [34]. e 48 h of reaction.
Table
Table 5. Values of chemical shifts (δ) of L1 and L2 in esters (S)-ED and (R)-ED and the corresponding values of ∆δSR.
Table 5. Values of chemical shifts (δ) of L1 and L2 in esters (S)-ED and (R)-ED and the corresponding values of ∆δSR.
Starting AlcoholEntry(S)-ED
Catalysts 12 01566 i001		(R)-ED
Catalysts 12 01566 i002		∆δSR
4b1L1 = 3.98L1 = 4.02−0.04
2L2 = 1.49L2 = 1.430.06
4c3L1 = 4.04L1 = 4.08−0.04
4L2 = 1.44L2 = 1.400.04
4d5L1 = 4.20L1 = 4.32−0.12
6L2 = 1.56L2 = 1.480.08
Table
Table 6. Results from the molecular docking simulations of the complex substrate–lipase.
Table 6. Results from the molecular docking simulations of the complex substrate–lipase.
SubstrateEntryLipaseNAC aBinding Energy
(kcal.mol−1) b		Distance (Å) cAngle (°) dHydrogen Bond with the Carbonyl Oxygen
(R)-5a1TLL8−5.043.887.4O:NHLeu147
2P. fluorescens11−4.143.7108.7O:OHTyr28
(S)-5a3TLL1−4.723.4109.8-
4P. fluorescens5−3.953.591.7O:OHTyr28
(R)-5b5TLL6−5.153.690.7O:NHLeu147
6P. fluorescens1−4.183.7103.7O:OHTyr28
(S)-5b7TLL0----
8P. fluorescens0----
(R)-5c9TLL2−4.613.694.6O:NHLeu147
10P. fluorescens6−3.493.799.4O:OHTyr28
(S)-5c11TLL1−4.833.989.4-
12P. fluorescens5−4.133.186.5-
(R)-5d13TLL1−5.023.192.9O:NHLeu147
14P. fluorescens1−3.414.085.7-
(S)-5d15TLL0----
16P. fluorescens0----
a Number of poses following the NAC criteria. b Binding energy from the best pose. c Distance between catalytic serine (Ser-O) and the electrophilic carbon of the acyl group (Carbonyl-C) taken from the best pose. d Angle between Ser-O/carbonyl-C/carbonyl-O taken from the best pose.
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MDPI and ACS Style

Carvalho, A.C.L.d.M.; de Oliveira, B.R.; Lima, G.V.; Negreiro, J.M.; Oliveira, M.C.F.; de Lemos, T.L.G.; da Silva, M.R.; Fonseca, T.d.S.; Bezerra, R.M.; dos Santos, J.C.S.; et al. Resolution of Racemic Aryloxy-Propan-2-yl Acetates via Lipase-Catalyzed Hydrolysis: Preparation of Enantiomerically Pure/Enantioenriched Mexiletine Intermediates and Analogs. Catalysts 2022, 12, 1566. https://doi.org/10.3390/catal12121566

AMA Style

Carvalho ACLdM, de Oliveira BR, Lima GV, Negreiro JM, Oliveira MCF, de Lemos TLG, da Silva MR, Fonseca TdS, Bezerra RM, dos Santos JCS, et al. Resolution of Racemic Aryloxy-Propan-2-yl Acetates via Lipase-Catalyzed Hydrolysis: Preparation of Enantiomerically Pure/Enantioenriched Mexiletine Intermediates and Analogs. Catalysts. 2022; 12(12):1566. https://doi.org/10.3390/catal12121566

Chicago/Turabian Style

Carvalho, Ana Caroline Lustosa de Melo, Bruna Rocha de Oliveira, Gledson Vieira Lima, Jonatas Martins Negreiro, Maria Conceição Ferreira Oliveira, Telma Leda Gomes de Lemos, Marcos Reinaldo da Silva, Thiago de Sousa Fonseca, Rayanne Mendes Bezerra, Jose Cleiton Sousa dos Santos, and et al. 2022. "Resolution of Racemic Aryloxy-Propan-2-yl Acetates via Lipase-Catalyzed Hydrolysis: Preparation of Enantiomerically Pure/Enantioenriched Mexiletine Intermediates and Analogs" Catalysts 12, no. 12: 1566. https://doi.org/10.3390/catal12121566

APA Style

Carvalho, A. C. L. d. M., de Oliveira, B. R., Lima, G. V., Negreiro, J. M., Oliveira, M. C. F., de Lemos, T. L. G., da Silva, M. R., Fonseca, T. d. S., Bezerra, R. M., dos Santos, J. C. S., Gonçalves, L. R. B., Rios, N. S., Zanatta, G., & de Mattos, M. C. (2022). Resolution of Racemic Aryloxy-Propan-2-yl Acetates via Lipase-Catalyzed Hydrolysis: Preparation of Enantiomerically Pure/Enantioenriched Mexiletine Intermediates and Analogs. Catalysts, 12(12), 1566. https://doi.org/10.3390/catal12121566

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