Enantioselective Ester Hydrolysis Research Articles

Enhanced stereodivergent evolution of carboxylesterase for efficient kinetic resolution of near-symmetric esters through machine learning

Carboxylesterases serve as potent biocatalysts in the enantioselective synthesis of chiral carboxylic acids and esters. However, naturally occurring carboxylesterases exhibit limited enantioselectivity, particularly toward ethyl 3-cyclohexene-1-carboxylate (CHCE, S1), due to its nearly symmetric structure. While machine learning effectively expedites directed evolution, the lack of models for predicting the enantioselectivity for carboxylesterases has hindered progress, primarily due to challenges in obtaining high-quality training datasets. In this study, we devise a high-throughput method by coupling alcohol dehydrogenase to determine the apparent enantioselectivity of the carboxylesterase AcEst1 from Acinetobacter sp. JNU9335, generating a high-quality dataset. Leveraging seven features derived from biochemical considerations, we quantitively describe the steric, hydrophobic, hydrophilic, electrostatic, hydrogen bonding, and π-π interaction effects of residues within AcEst1. A robust gradient boosting regression tree model is trained to facilitate stereodivergent evolution, resulting in the enhanced enantioselectivity of AcEst1 toward S1. Through this approach, we successfully obtain two stereocomplementary variants, DR3 and DS6, demonstrating significantly increased and reversed enantioselectivity. Notably, DR3 and DS6 exhibit utility in the enantioselective hydrolysis of various symmetric esters. Comprehensive kinetic parameter analysis, molecular dynamics simulations, and QM/MM calculations offer insights into the kinetic and thermodynamic features underlying the manipulated enantioselectivity of DR3 and DS6.

Simple Plug-In Synthetic Step for the Synthesis of (-)-Camphor from Renewable Starting Materials.

Racemic camphor and isoborneol are readily available as industrial side products, whereas (1R)‐camphor is available from natural sources. Optically pure (1S)‐camphor, however, is much more difficult to obtain. The synthesis of racemic camphor from α‐pinene proceeds via an intermediary racemic isobornyl ester, which is then hydrolyzed and oxidized to give camphor. We reasoned that enantioselective hydrolysis of isobornyl esters would give facile access to optically pure isoborneol and camphor isomers, respectively. While screening of a set of commercial lipases and esterases in the kinetic resolution of racemic monoterpenols did not lead to the identification of any enantioselective enzymes, the cephalosporin Esterase B from Burkholderia gladioli (EstB) and Esterase C (EstC) from Rhodococcus rhodochrous showed outstanding enantioselectivity (E>100) towards the butyryl esters of isoborneol, borneol and fenchol. The enantioselectivity was higher with increasing chain length of the acyl moiety of the substrate. The kinetic resolution of isobornyl butyrate can be easily integrated into the production of camphor from α‐pinene and thus allows the facile synthesis of optically pure monoterpenols from a renewable side‐product.

Overcoming Water Insolubility in Flow: Enantioselective Hydrolysis of Naproxen Ester

Hydrolytic enantioselective cleavage of different racemic non-steroidal anti-inflammatory drugs (NSAIDs) ester derivatives has been studied. An engineered esterase form Bacillus subtilis (BS2m) significantly outperformed homologous enzymes from Halomonas elongata (HeE) and Bacillus coagulants (BCE) in the enantioselective hydrolysis of naproxen esters. Structural analysis of the three active sites highlighted key differences which explained the substrate preference. Immobilization of a chimeric BS2m-T4 lysozyme fusion (BS2mT4L1) was improved by resin screening achieving twice the recovered activity (22.1 ± 5 U/g) with respect to what had been previously reported, and was utilized in a packed bed reactor. Continuous hydrolysis of α-methyl benzene acetic acid butyl ester as a model substrate was easily achieved, albeit at low concentration (1 mM). However, the high degree of insolubility of the naproxen butyl ester resulted in a slurry which could not be efficiently bioconverted, despite the addition of co-solvents and lower substrate concentration (1 mM). Addition of Triton® X-100 to the substrate mix yielded 24% molar conversion and 80% e.e. at a 5 mM scale with 5 min residence time and sufficient retention of catalytic efficiency after 6 h of use.

Enantioselective hydrolysis of amino acid esters by non-chiral copper complexes equipped with bis (β-cyclodextrin)s

Two non-chiral copper(II) complexes equipped with bis(β-cyclodextrin)s (bisCDs) were explored as hydrolase models for the enantioselective hydrolysis of two pairs of alkyl chain-possessing amino acid ester enantiomers. The two bisCD complexes are pyridine-linked with different CD cavity orientations, denoted as CuL1 (L1=2,6-bis(6-mono-amino-β-cyclodextrin-methyl)-pyridine) and CuL2 (L2=2,6-bis(3-mono-amino-β-cyclodextrin-methyl)-pyridine). Kinetic studies indicated that the “back-to-back” bisCD complex CuL1 showed higher catalytic efficiency and more pronounced enantioselectivity for all substrates than the “face-to-face” bisCD complex CuL2. Overall preference of l-isomers was observed for both complexes. In the presence of CuL1, the formation of catalyst-substrate Michaelis complexes during the hydrolysis was demonstrated by saturation kinetic study and Electrospray ionization mass spectrometry (ESI–MS) analysis. Enantiomer selectivity (vmaxL/vmaxD) value for N-Boc-N'-Boc-Lysine 4-nitrophenyl esters (Boc-Lys(Boc)-ONp), the longer alkyl-chain analogs, is twice of that for N-Boc-Alanine 4-nitrophenyl esters (Boc-Ala-ONp). The enantioselective hydrolysis of Boc-Lys(Boc)-ONp promoted by CuL1 was confirmed by chiral high-performance liquid chromatography (HPLC) analysis. The participation of CD cavities during enantioselective hydrolysis was investigated through inhibition assay. The enantioselectivity in hydrolyzing different amino acid esters promoted by CuL1 was compared. The mechanism involved in the cooperation of two adjacent CD cavities of bisCD was proposed.

Enantioselective Hydrolysis of Amino Acid Esters Promoted by Bis(β-cyclodextrin) Copper Complexes

It is challenging to create artificial catalysts that approach enzymes with regard to catalytic efficiency and selectivity. The enantioselective catalysis ranks the privileged characteristic of enzymatic transformations. Here, we report two pyridine-linked bis(β-cyclodextrin) (bisCD) copper(II) complexes that enantioselectively hydrolyse chiral esters. Hydrolytic kinetic resolution of three pairs of amino acid ester enantiomers (S1–S3) at neutral pH indicated that the “back-to-back” bisCD complex CuL1 favoured higher catalytic efficiency and more pronounced enantioselectivity than the “face-to-face” complex CuL2. The best enantioselectivity was observed for N-Boc-phenylalanine 4-nitrophenyl ester (S2) enantiomers promoted by CuL1, which exhibited an enantiomer selectivity of 15.7. We observed preferential hydrolysis of L-S2 by CuL1, even in racemic S2, through chiral high-performance liquid chromatography (HPLC). We demonstrated that the enantioselective hydrolysis was related to the cooperative roles of the intramolecular flanking chiral CD cavities with the coordinated copper ion, according to the results of electrospray ionization mass spectrometry (ESI-MS), inhibition experiments, rotating-frame nuclear Overhauser effect spectroscopy (ROESY), and theoretical calculations. Although the catalytic parameters lag behind the level of enzymatic transformation, this study confirms the cooperative effect of the first and second coordination spheres of artificial catalysts in enantioselectivity and provides hints that may guide future explorations of enzyme mimics.

Enantioselective ester hydrolysis by an achiral catalyst co-embedded with chiral amphiphiles into a vesicle membrane

Co-embedding of an amphiphilic non-chiral hydrolysis catalyst with amphiphilic chiral additives into the membrane of a phospholipid vesicle induces different rates of ester hydrolysis for enantiomeric amino acid esters.

Simultaneous use of in silico design and a correlated mutation network as a tool to efficiently guide enzyme engineering.

In order to improve the efficiency of directed evolution experiments, in silico multiple-substrate clustering was combined with an analysis of the variability of natural enzymes within a protein superfamily. This was applied to a Pseudomonas fluorescens esterase (PFE I) targeting the enantioselective hydrolysis of 3-phenylbutyric acid esters. Data reported in the literature for nine substrates were used for the clustering meta-analysis of the docking conformations in wild-type PFE I, and this highlighted a tryptophan residue (W28) as an interesting target. Exploration of the most frequently, naturally occurring amino acids at this position suggested that the reduced flexibility observed in the case of the W28F variant leads to enhancement of the enantioselectivity. This mutant was subsequently combined with mutations identified in a library based on analysis of a correlated mutation network. By interrogation of <80 variants a mutant with 15-fold improved enantioselectivity was found.

Preparation of New Calix[4]arene-Immobilized Biopolymers for Enhancing Catalytic Properties of Candida rugosa Lipase by Sol–Gel Encapsulation

The article describes preparation of new calixarene biopolymers consisting of the immobilization of convenience calixarene derivative onto cellulose and chitosan biopolymers, and the encapsulation of these calixarene biopolymers with Candida rugosa lipase within a chemical inert sol-gel supported by polycondensation with tetraethoxysilane and octyltriethoxysilane. The catalytic properties of immobilized lipase were evaluated into model reactions employing the hydrolysis of p-nitrophenylpalmitate and the enantioselective hydrolysis of naproxen methyl esters from racemic prodrugs in aqueous buffer solution/isooctane reaction system. The resolution studies using sol-gel support have observed more improvement in the enantioselectivity of naproxen E = 300 with Cel-Calix-E than with encapsulated lipase without calixarene-based materials. Furthermore, the encapsulated lipase (Cel-Calix-E) was still retained about 39 % of their conversion ratios after the fifth reuse in the enantioselective reaction.

ECD spectrometric methods for detecting the enantioselective enzymatic hydrolysis of racemic 3-acetoxy-4-phenyl-β-lactam

(−)-(3R, 4S)-3-Acetoxy-4-phenylazetidin-2-one ((−)-1) was the key intermediate for preparing optical C-13 side-chain moiety in partial synthesis of docetaxel and paclitaxel. It can be successfully prepared via enantioselective hydrolysis of racemic esters ((±)-1) catalyzed by bacterial lipases, but the current reaction evaluation method is tedious and inconvenient. Electronic circular dichroism (ECD) has been widely applied in the stereochemical study of chiral compounds. In this paper, a rapid ECD spectroscopic method has been proposed and established to detect the transformation directly. The absolute configurations of lipase-catalyzed hydrolysis products have also been confirmed by quantum-chemical calculation using time-dependent density functional theory (TDDFT) methodology.

Metadynamics Simulations of Enantioselective Acylation Give Insights into the Catalytic Mechanism of Burkholderia cepacia Lipase

The catalytic mechanism of Burkholderia cepacia lipase (BCL), which catalyzes the enantioselective hydrolysis of racemic esters of primary alcohols, was investigated by modeling the first stage of ...

Following the lipase catalyzed enantioselective hydrolysis of amino acid esters with capillary electrophoresis using contactless conductivity detection

AbstractThe progress of the enzymatic hydrolysis of racemic mixtures of the enantiomers of the methyl esters of serine and threonine was monitored. This was possible in a reaction vessel of 1.5 mL by direct sampling of volumes in the nanoliter‐range directly into an electrophoresis capillary. Contactless conductivity detection was used for quantification as the analytes are not accessible by UV‐detection in capillary electrophoresis. Porcine pancreatic lipase and wheat germ lipase both showed a preference for the L‐enantiomers of both amino acid esters. The selectivity of the porcine lipase between the two L‐esters of the two amino acids was also studied and it was found that the production of L‐threonine had priority over L‐serine. Chirality, 2010. © 2009 Wiley‐Liss, Inc.

Lipase-catalyzed enantioselective hydrolysis of N-protected racemic non-protein amino acid esters

Porcine pancreatic lipase (PPL)-catalyzed enantioselective hydrolysis of N-benzyloxycarbonyl-dl-amino acid esters (Z-dl-AA-ORs) was studied for the optical resolution of a variety of non-protein amino acids. The ester moiety (R) of the substrate affected the rate of hydrolysis significantly. The glyceryl (Gl) and carbamoylmethyl (Cam) esters were found to be highly reactive substrates. The hydrolysis of the Gl esters (Z-dl-AA-OGls) of both aliphatic and aromatic amino acids was examined in acetonitrile containing 70% (v/v) of 0.02 M phosphate buffer (pH 7.0) at 30°C. With all amino acids tested, the corresponding l-enantiomers were hydrolyzed preferentially. PPL favored aromatic amino acids, such as phenylalanine and p-chlorophenylalanine, leading to completion of the hydrolysis within 20 min with excellent enantioselectivities (E>100). The PPL-catalyzed hydrolysis of the corresponding Cam esters (Z-dl-AA-OCams) was also examined under the same reaction conditions. Although the hydrolysis of the Cam esters was rapid, the l-enantioselectivities were rather poor with aromatic amino acids, such as 2-phenylglycine and homophenylalanine.

The First Direct Enzymatic Hydrolysis of Alicyclic β‐Amino Esters: A Route to Enantiopure cis and trans β‐Amino Acids

The first direct enzymatic method is reported for the synthesis of cis and trans beta-amino acid enantiomers through the lipase-catalyzed enantioselective hydrolysis of alicyclic beta-amino esters in organic media. High enantioselectivities (E usually >100) were observed when the Candida antarctica lipase B catalyzed reactions were performed with H2O (0.5 equivalents) in iPr2O at 65 degrees C. The resolved products, obtained in good yields (>or=42%), could be easily separated.

Conjugate Addition of 2- and 4-Pyridylcuprates: An Expeditious Asymmetric Synthesis of Natural (−)-Evoninic Acid

[reaction: see text] The scope and limitations of the conjugate addition of 2- and the first 4-pyridyl Gilman homocuprates to various alpha,beta-unsaturated Michael acceptors are delineated. The conjugate addition of the cuprate of 2-bromo-3-methylpyridine to (E)-methyl crotonate then diastereoselective enolate alkylation and lipase-mediated enantioselective ester hydrolysis have enabled an efficient four-step first asymmetric synthesis of the Celastraceae sesquiterpenoid esterifying ligand (-)-(1'S,2'S)-evoninic acid.

Use of Aspergillus oryzae protease for the resolution of α-hydroxy acids by enantioselective ester hydrolysis

The enzymatic hydrolysis of esters of α-hydroxy acids as non-amino acid substrates for microbial proteases was investigated. The methyl esters of 3-phenyllactic acid and its ring-substituted derivative were hydrolyzed with excellent enantioselectivities (E>110) using Aspergillus oryzae protease to give the (S)-acids. By contrast, only low enantioselectivities (E∼1) were obtained with the esters of mandelic acid. A gram-scale resolution of 3-phenyllactic acid was achieved by adopting this procedure, to afford the (S)-acid with 96% e.e. in 67% yield after recrystallization.

Studies on the enhancement of enantioselectivity in the microbial protease-catalyzed hydrolysis of N-free non-protein amino acid esters

We have recently reported a marked enhancement of enantioselectivity by switching the conventional methyl ester to esters with a longer alkyl chain such as the isobutyl ester in the Aspergillus oryzae protease-catalyzed enantioselective hydrolysis of N-unprotected amino acid esters. The present study reveals that the enantioselectivity enhancement is also attained by changing the pH of the reaction mixture, the optimal pH in terms of enantioselectivity being around 6.2. Kinetic measurements indicate that the most significant factor responsible for the observed enantioselectivity enhancement is the change of the K m value for the l-series.

Newly isolated Streptomyces spp. as enantioselective biocatalysts: hydrolysis of 1,2-O-isopropylidene glycerol racemic esters

To identify microbial strains with esterase activity able to enantioselectively hydrolyse esters of (R,S)-1,2-O-isopropylidene glycerol. The microbial hydrolysis of various racemic esters of 1,2-O-isopropylidene glycerol (IPG) was attempted by screening among Streptomyces spp. previously selected on the basis of their carboxylesterase activity. The best results were observed in the hydrolysis of butyrate ester and two strains appeared promising as they showed opposite enantioselectivity: Streptomyces sp. 90852 gave predominantly (S)-IPG, while strain 90930 mostly gave the R-alcohol. Streptomyces sp. 90930 was identified as Streptomyces violaceusniger, whereas Streptomyces sp. 90852 is a new species belonging to the Streptomyces violaceus taxon. The carboxylesterase belonging to strain 90852 gave a maximum value of enantiomeric ratio (E) of 14-16. This strain was lyophilized and used as dry mycelium for catalysing the synthesis of isopropylidene glycerol butyrate in heptane showing reaction rate and enantioselectivity (E = 6.6) lower than what observed for the hydrolysis. A new esterase with enantioselective activity towards (R,S)-IPG butyrate has been selected. The best enantioselectivity is similar or even better than the highest reported value in the literature with commercial enzymes. The enzyme is produced by a new species belonging to the S. violaceus taxon. New esterases from streptomycetes can be employed for the enantioselective hydrolysis of chiral esters derived from primary alcohols, not efficiently resolved with commercial enzymes.

Synthetic Studies ond-Biotin, Part 8:[1] An Efficient Chemoenzymatic Approach to the Asymmetric Total Synthesis ofd-Biotinvia a Polymer-Supported PLE-Mediated Desymmetrization ofmeso-Symmetic Dicarboxylic Esters

A practical chemoenzymatic method for the asymmetric total synthesis of d-biotin (1) starting from the commercially available cis-1,3-dibenzyl-2-imidazolidone-4,5-dicarboxylic acid (2) has been developed. The key step of the synthesis is the highly enantioselective hydrolysis of meso-dicarboxylic esters by a polymer-supported pig liver esterase and introduction of a formyl group at the C-4 position in 4 via a Grignard reaction. The polymer-supported PLE can be recovered quantitatively from the reaction mixture by simple filtration and reused without significant loss of activity.

Comparison of kinetic resolution between two racemic ibuprofen esters in an enzymic membrane reactor

The enantioselective hydrolysis of racemic esters and simultaneous separation of the corresponding optically pure ( S)-acid as pure isomer is of considerable interest to the pharmaceutical industry as a route to non-steroidal anti-inflammatory drugs. In the present study, an enzymic membrane reactor (EMR) was employed for the optical resolution of racemic ibuprofen ester. EMR consisted of a lipase immobilized polymeric membrane, an organic phase dissolving ester and an aqueous phase to recover the reaction products. The catalytic behaviour of lipase immobilized in a polymeric hollow fibre membrane was investigated using 1-heptyl-ibuprofen ester and 2-ethoxyethyl-ibuprofen ester. The two ester substrates differ in the nature of the alkyl group of the alcohol moiety. The performance of the immobilized enzyme was studied as a function of temperature, pH of phosphate buffer solution and substrate flow rate. The operational conditions that favoured the enzyme selectivity for the ( S)-ibuprofen esters, in order to obtain the corresponding ( S)-ibuprofen acid as an optically pure single enantiomer, were identified. Lipase from Candida rugosa was used in the hydrolysis of racemic ibuprofen ester. ( R)-Ibuprofen ester was found to be less reactive in the reaction. Highest enantioselectivity of enzyme was obtained with phosphate buffer solution of pH 8.0 at temperature of 40 °C and at lower substrate flow rate for both racemic 1-heptyl-ibuprofen and 2-ethoxyethyl-ibuprofen esters. The hydrolysis of racemic 1-heptyl-ibuprofen ester gave 4% ee S , 90% ee P and E value of 1–4, while the hydrolysis of 2-ethoxyethyl-ibuprofen ester resulted in 31% ee S , 85% ee P and E value of 9.5–13 by running the experiment under optimum operating condition in an enzymatic membrane reactor.

Biocatalytic Enantio‐Convergent Preparation of sec‐Alcohols using Sulfatases

AbstractThe deracemization of sec‐alcohols via enantioconvergent processes is of high interest in order to overcome the 50 % yield limitations of kinetic resolution. As enantioconvergent processes are characterized by two reactions with one crossing the plane of symmetry, one of these reactions has to proceed with inversion of configuration. In lipase catalyzed resolutions the enzymatic step proceeds with retention of configuration, and the remaining enantiomer is converted by the Mitsunobu reaction or activated esters under inversion of configuration to achieve complete deracemization. In contrast, alkyl sulfatases, which catalyze the enantioselective hydrolysis of sulfate esters, are one of the rare hydrolytic enzymes which work under inversion of configuration. In this minireview we give an overview of the state of the art in enantioconvergent processes for the deracemization of sec‐alcohols, with the focus on recently developed alkyl‐sulfatases from Rhodococcus spp.