Enantiomeric Substrates Research Articles

Investigating Interaction Dynamics of an Enantioselective Peptide‐Catalyzed Acylation Reaction

Modern nuclear magnetic resonance (NMR) methods like carbon relaxation dispersion in the rotating frame (13C‐R1ρ) and proton chemical exchange saturation transfer (1H‐CEST) are key methods to investigate molecular recognition in biomacromolecules and to detect molecular motions on the µs to s timescale, revealing transient conformational states. Changes in kinetics can be linked to binding, folding, or catalytic events. Here, we investigated whether these methods allow detection of changes in the dynamics of a small, highly selective peptide catalyst during recognition of its enantiomeric substrates. The flexible tetrapeptide Boc‐l‐(π‐Me)‐His‐AGly‐l‐Cha‐l‐Phe‐OMe, used for the monoacetylation of cycloalkane‐diols, is probed at natural abundance using 13C‐R1ρ and 1H‐CEST. Indeed, we detected differences in dynamics of the peptide upon interaction with the diol. Importantly, these differ depending on the enantiomer of the substrate used. These enantiospecific influences of the substrates on the dynamics of the peptide are rationalized using computational techniques. We find that even though one enantiomer reacts faster, as confirmed by reaction monitoring, the other is more tightly bound in DCM (as confirmed by 1H‐saturation transfer difference (STD) measurements). These findings provide insights into the recognition of the substrates and explain the selectivity differences observed between the solvents toluene and DCM.

Bienzymatic Dynamic Kinetic Resolution of Secondary Alcohols by Esterification/Racemization in Water.

Dynamic kinetic resolution (DKR) is a key method used to prepare optically pure compounds in 100% theoretical yield starting from racemic substrates by combining the interconversion of substrate enantiomers with an enantioselective transformation. Various chemoenzymatic DKR approaches have been developed to deracemize secondary alcohols, typically requiring an organic solvent to facilitate enantioselective acylation, primarily catalyzed by lipases, alongside racemization mediated by an achiral, non-enzymatic catalyst. Achieving both steps in an aqueous solution remained elusive. Here, we report a DKR of racemic sec-alcohols in an aqueous solution requiring only two biocatalysts. The first key to success was to achieve fast racemization in a buffer employing a non-stereoselective variant of alcohol dehydrogenase (Lk-ADH-Prince) via a hydrogen-borrowing oxidation-reduction sequence. Engineered variants of the acyltransferase from Mycobacterium smegmatis (MsAcT) enabled enantioselective acyl transfer in water. Besides the appropriate choice of the enzymes, identifying a suitable acyl donor was a second key to the success. The DKR was successfully demonstrated using (R)-selective MsAcT variants for a broad range of racemic (hetero)benzylic alcohols using 2,2,2-trifluoroethyl acetate as the acyl donor, yielding (R)-acetates with up to >99% conv. and high-to-excellent optical purity (83-99.9% ee). The (S)-acetates were accessible using a stereocomplementary (S)-selective MsAcT variant.

Chemodivergent Parallel Kinetic Resolution of Paracyclophanes: Enantiomer Fishing with Different Substrates

AbstractAn unprecedented chemodivergent strategy for parallel kinetic resolution (PKR) is disclosed through which two planar chiral products bearing different structures were simultaneously afforded with opposite stereoselectivities. Two achiral esters are activated by one single chiral N‐heterocyclic carbene (NHC) catalyst to react with the different enantiomers of the racemic imine substrate in a parallel fashion. Two products bearing distinct structures and opposite stereoselectivities are respectively afforded from the same reaction system in good to excellent yields, enantio‐ and diastereoselectivities. Control experiments and kinetic studies are carried out to probe the kinetic and dynamic properties during the reaction progress. The planar chiral pyridine and lactam products show interesting applications in both asymmetric synthesis and pesticide development.

Chemodivergent Parallel Kinetic Resolution of Paracyclophanes: Enantiomer Fishing with Different Substrates.

An unprecedented chemodivergent strategy for parallel kinetic resolution (PKR) is disclosed through which two planar chiral products bearing different structures were simultaneously afforded with opposite stereoselectivities. Two achiral esters are activated by one single chiral N-heterocyclic carbene (NHC) catalyst to react with the different enantiomers of the racemic imine substrate in a parallel fashion. Two products bearing distinct structures and opposite stereoselectivities are respectively afforded from the same reaction system in good to excellent yields, enantio- and diastereoselectivities. Control experiments and kinetic studies are carried out to probe the kinetic and dynamic properties during the reaction progress. The planar chiral pyridine and lactam products show interesting applications in both asymmetric synthesis and pesticide development.

Copper/ruthenium relay catalysis enables 1,6-double chiral inductions with stereodivergence.

The dual catalysis strategy is an efficient and powerful tool to fulfill the stereodivergent synthesis of stereoisomeric products from the same set of starting materials. Great attention has been given to the construction of chiral compounds with two contiguous stereocenters. However, the synthesis of two remote noncontiguous stereocenters is more challenging and is less developed, despite the high demand for synthetic tactics. We herein developed an unprecedented example of the stereodivergent preparation of synthetically useful and biologically important chiral ζ-hydroxy amino ester derivatives containing remote 1,6-noncontiguous stereocenters and a unique β,γ-unsaturation moiety. This cascade dehydrogenation/1,6-Michael addition/hydrogenation protocol between readily-available ketoimine esters and racemic branched dienyl carbinols was rationally realized with bimetallic copper/ruthenium relay catalysis. The key features of the process were atom economy, step economy, and redox-neutrality. All four stereoisomers of chiral ζ-hydroxy amino ester derivatives were easily achieved by the orthogonal permutations of a chiral copper catalyst and chiral ruthenium catalyst. Importantly, a much more challenging stereodivergent synthesis of all eight stereoisomers of chiral peptide products containing three remote stereocenters was accomplished with excellent results through the cooperation of two chiral catalyst pairs and substrate enantiomers.

Rhodium-Catalyzed Asymmetric Synthesis of 1,2-Disubstituted Allylic Fluorides.

Although there are many methods for the asymmetric synthesis of monosubstituted allylic fluorides, construction of enantioenriched 1,2-disubstituted allylic fluorides has not been reported. To address this gap, we report an enantioselective synthesis of 1,2-disubstituted allylic fluorides using chiral diene-ligated rhodium catalyst, Et3 N ⋅ 3HF as a source of fluoride, and Morita Baylis Hillman (MBH) trichloroacetimidates. Kinetic studies show that one enantiomer of racemic MBH substrate reacts faster than the other. Computational studies reveal that both syn and anti π-allyl complexes are formed upon ionization of allylic substrate, and the syn complexes are slightly energetically favorable. This is in contrast to our previous observation for formation of monosubstituted π-allyl intermediates, in which the syn π-allyl conformation is strongly preferred. In addition, the presence of an electron-withdrawing group at C2 position of racemic MBH substrate renders 1,2-disubstituted π-allyl intermediate formation endergonic and reversible. To compare, formation of monosubstituted π-allyl intermediates was exergonic and irreversible. DFT calculations and kinetic studies support a dynamic kinetic asymmetric transformation process wherein the rate of isomerization of the 1,2-disubstituted π-allylrhodium complexes is faster than that of fluoride addition onto the more reactive intermediate. The 1,2-disubstituted allylic fluorides were obtained in good yields, enantioselectivity, and branched selectivity.

Rhodium‐Catalyzed Asymmetric Synthesis of 1,2‐Disubstituted Allylic Fluorides

AbstractAlthough there are many methods for the asymmetric synthesis of monosubstituted allylic fluorides, construction of enantioenriched 1,2‐disubstituted allylic fluorides has not been reported. To address this gap, we report an enantioselective synthesis of 1,2‐disubstituted allylic fluorides using chiral diene‐ligated rhodium catalyst, Et3N ⋅ 3HF as a source of fluoride, and Morita Baylis Hillman (MBH) trichloroacetimidates. Kinetic studies show that one enantiomer of racemic MBH substrate reacts faster than the other. Computational studies reveal that both syn and anti π‐allyl complexes are formed upon ionization of allylic substrate, and the syn complexes are slightly energetically favorable. This is in contrast to our previous observation for formation of monosubstituted π‐allyl intermediates, in which the syn π‐allyl conformation is strongly preferred. In addition, the presence of an electron‐withdrawing group at C2 position of racemic MBH substrate renders 1,2‐disubstituted π‐allyl intermediate formation endergonic and reversible. To compare, formation of monosubstituted π‐allyl intermediates was exergonic and irreversible. DFT calculations and kinetic studies support a dynamic kinetic asymmetric transformation process wherein the rate of isomerization of the 1,2‐disubstituted π‐allylrhodium complexes is faster than that of fluoride addition onto the more reactive intermediate. The 1,2‐disubstituted allylic fluorides were obtained in good yields, enantioselectivity, and branched selectivity.

Anthriscus sylvestris Deoxypodophyllotoxin Synthase Involved inthe Podophyllotoxin Biosynthesis.

Tetrahydrofuran ring formation from dibenzylbutyrolactone lignans is a key step in the biosynthesis of aryltetralin lignans including deoxypodophyllotoxin and podophyllotoxin. Previously, Fe(II)- and 2-oxoglutarate-dependent dioxygenase (2-ODD) from Podophyllum hexandrum (Himalayan mayapple, Berberidaceae) was found to catalyze the cyclization of a dibenzylbutyrolactone lignan, yatein, to give deoxypodophyllotoxin and designated as deoxypodophyllotoxin synthase (DPS). Recently, we reported that the biosynthesis of deoxypodophyllotoxin and podophyllotoxin evolved in a lineage-specific manner in phylogenetically unrelated plant species such as P. hexandrum and Anthriscus sylvestris (cow parsley, Apiaceae). Therefore, a comprehensive understanding of the characteristics of DPSs that catalyze the cyclization of yatein to deoxypodophyllotoxin in various plant species is important. However, for plant species other than P. hexandrum, the isolation of the DPS enzyme gene and the type of the enzyme, e.g.whether it is 2-ODD or another type of enzyme such as cytochrome P-450, have not been reported. In this study, we report the identification and characterization of A. sylvestris DPS (AsDPS). Phylogenetic analysis showed that AsDPS belonged to the 2-ODD superfamily and shared moderate amino acid sequence identity (40.8%) with P. hexandrum deoxypodophyllotoxin synthase (PhDPS). Recombinant protein assay indicated that AsDPS and PhDPS differ in terms of the selectivity of substrate enantiomers. Protein modeling using AlphaFold2 and site-directed mutagenesis indicated that the Tyr305 residue of AsDPS probably contributes to substrate recognition. This study advances our understanding of the podophyllotoxin biosynthetic pathway in A. sylvestris and provides new insight into 2-ODD involved in plant secondary (specialized) metabolism.

The Phenomenon of Self-Induced Diastereomeric Anisochrony and Its Implications in NMR Spectroscopy.

Nuclear magnetic resonance (NMR) spectroscopy is an analytical technique largely applied in the analysis of discrimination processes involving enantiomeric substrates and chiral agents, which can interact with the analyte either via covalent bonding or via formation of diastereomeric solvates. However, enantiodiscrimination has been observed, in some cases, even in the absence of any additional chiral selector. The reasons behind this phenomenon must be found in the capability of some chiral substrates to interact with themselves by forming diastereomeric solvates in solution that can generate nonequivalences in the NMR spectra of enantiomerically enriched mixtures. As a result, differentiation of enantiomers is observed, thus allowing the quantification of the enantiomeric composition of the mixture under investigation. The tendency of certain substrates to self-aggregate and to generate diastereomeric adducts in solution can be defined as Self-Induced Diastereomeric Anisochrony (SIDA), but other acronyms have been used to refer to this phenomenon. In the present work, an overview of SIDA processes investigated via NMR spectroscopy will be provided, with a particular emphasis on the nature of the substrates involved, on the interaction mechanisms at the basis of the phenomenon, and on theoretical treatments proposed in the literature to explain them.

Enantioconvergent 6π Electrocyclization Enabled by Photoredox Racemization.

This study presents a novel photoredox-enabled enantioconvergent catalytic strategy used to construct chiral 2H-1,3-benzoxazines via an unprecedented oxa-6π electrocyclization utilizing racemic α-substituted glycinates as substrates. The approach leverages a cobalt-based chiral Lewis acid catalyst, which promotes the transformation under thermal or photoredox conditions. While the thermal reaction selectively converts only the (S)-configured glycinates into enantioenriched 2H-1,3-benzoxazines (up to 96:4 e.r.), the addition of 0.5 mol % of a commercially available iridium photocatalyst under visible light irradiation transforms the reaction into an enantioconvergent process. Detailed mechanistic and time course studies of optically pure α-deuterated substrates revealed the presence of an enantiospecific kinetic isotope effect, which helped to clarify the role of both the photo- and chiral Lewis acid catalyst in the reaction sequence. In this dual catalytic system, the photocatalyst promotes a dynamic interconversion between the substrate enantiomers─a process not accessible via ground-state chemistry─while the chiral Lewis acid selectively transforms only the (S)-configured substrates. Further mechanistic evidence for the proposed mechanism is provided by linear free energy relationship analysis, which suggests that the stereodetermining step involves a 6π electrocyclization under both thermal and photoredox conditions.

Construction of C-B axial chirality via dynamic kinetic asymmetric cross-coupling mediated by tetracoordinate boron.

Catalytic dynamic kinetic asymmetric transformation (DyKAT) provides a powerful tool to access chiral stereoisomers from racemic substrates. Such transformation has been widely employed on the construction of central chirality, however, the application in axial chirality remains underexplored because its equilibrium of substrate enantiomers is limited to five-membered metalacyclic intermediate. Here we report a tetracoordinate boron-directed dynamic kinetic asymmetric cross-coupling of racemic, configurationally stable 3-bromo-2,1-azaborines with boronic acid derivatives. A series of challenging C-B axially chiral compounds were prepared with generally good to excellent enantioselectivities. Moreover, this transformation can also be extended to prepare atropisomers bearing adjacent C-B and C-C diaxes with excellent diastereo- and enantio-control. The key to the success relies on the rational design of a reversible tetracoordinate boron intermediate, which is supported by theoretical calculations that dramatically reduces the rotational barrier of the original C-B axis and achieves the goal of DyKAT.

Enantioselective Intramolecular Hydroacylation of Alkynes by Rhodium Catalysis through Dynamic Kinetic Resolution. Front Cover Picture: (Adv. Synth. Catal. 20/2023)

The front cover picture illustrates a rhodium-catalyzed enantioselective hydroacylation of racemic alkynals having a substituent at the α-position of the carbonyl group. Mechanistic studies revealed that hydroacylation occurs preferentially from one enantiomer of the racemic substrate via a dynamic kinetic resolution (DKR) process. The picture shows an outline of the DKR process by using an illustration of soccer, in which the player getting the good ball (i. e., enantiomer) successfully scores a goal. Details can be found in the Communication by Yoshihiro Sato and co-workers (Y. Oonishi, K. Takagishi, Y.-M. Liu, Y. Sato, Adv. Synth. Catal. 2023, 365, XXXX–XXXX; DOI: 10.1002/adsc.202300206).

Enantioselective Intramolecular Hydroacylation of Alkynes by Rhodium Catalysis through Dynamic Kinetic Resolution.

The front cover picture illustrates a rhodium-catalyzed enantioselective hydroacylation of racemic alkynals having a substituent at the α-position of the carbonyl group. Mechanistic studies revealed that hydroacylation occurs preferentially from one enantiomer of the racemic substrate via a dynamic kinetic resolution (DKR) process. The picture shows an outline of the DKR process by using an illustration of soccer, in which the player getting the good ball (i. e., enantiomer) successfully scores a goal. Details can be found in the Communication by Yoshihiro Sato and co-workers (Y. Oonishi, K. Takagishi, Y.-M. Liu, Y. Sato, Adv. Synth. Catal. 2023, 365, XXXX–XXXX; DOI: 10.1002/adsc.202300206).

One Molecular Probe with Opposite Enantioselective Fluorescence Enhancement at Two Distinct Emissions.

It is discovered that one enantiomer of a chiral substrate can greatly enhance the fluorescence of one molecular probe at one emitting signal (λ1 = 517 nm), while the opposite enantiomer of the substrate greatly enhances the fluorescence of the same probe at a distinctively different emission (λ2 = 575 nm). This probe is made of a 1,1'-binaphthyl-based chiral dialdehyde that in combination with Zn2+ under slightly acidic conditions shows a chemoselective and enantioselective fluorescent response to histidine. The opposite enantioselective fluorescent responses of the probe at two emissions allow it to be used to determine both the concentration and the enantiomeric composition of the substrate using a single probe. The mechanistic study has revealed two very different reaction pathways when the two enantiomers of the substrate are treated with the probe. These reaction pathways generate two different products, one dimeric and another polymeric, with very different emissions.

Elucidation of the stereocontrol mechanisms of the chemical and biosynthetic intramolecular Diels-Alder cycloaddition for the formation of bioactive decalins.

The intramolecular Diels-Alder reaction (IMDA) is a powerful method for regioselective and stereoselective construction of functionalised decalin skeletons, and the recent discovery of enzymes that catalyse IMDA cycloaddition in biosynthesis has generated considerable interest. This study focused on the role of the absolute configuration of the C-6 carbon of the substrate polyene in the stereocontrol of the IMDA reaction catalysed by Fsa2 and Phm7, which construct different enantiomeric decalin skeletons. Their enantiomeric precursor polyenes were synthesised and subjected to enzymatic or thermal IMDA reactions to isolate various diastereomeric decalines and determine their absolute configuration. Furthermore, density functional theory calculations were performed to elucidate the stereocontrol mechanism underlying the formation of decalin. The results showed that Fsa2 exhibits the same equisetin-type stereoselectivity for enantiomeric substrates regardless of the 6-methyl group configuration of the substrate, while Phm7 shows two types of stereoselectivity depending on the configuration of the 6-methyl group. We also found a unique stereochemistry-activity relationship in antibacterial activity for decalin diastereomers, including new derivatives. This study provides new insights into the stereoselectivity of DAase, which is important in the synthesis of natural product skeletons.

Crystal structure of the α-ketoglutarate-dependent non-heme iron oxygenase CmnC in capreomycin biosynthesis and its engineering to catalyze hydroxylation of the substrate enantiomer

CmnC is an α-ketoglutarate (α-KG)-dependent non-heme iron oxygenase involved in the formation of the l-capreomycidine (l-Cap) moiety in capreomycin (CMN) biosynthesis. CmnC and its homologues, VioC in viomycin (VIO) biosynthesis and OrfP in streptothricin (STT) biosynthesis, catalyze hydroxylation of l-Arg to form β-hydroxy l-Arg (CmnC and VioC) or β,γ-dihydroxy l-Arg (OrfP). In this study, a combination of biochemical characterization and structural determination was performed to understand the substrate binding environment and substrate specificity of CmnC. Interestingly, despite having a high conservation of the substrate binding environment among CmnC, VioC, and OrfP, only OrfP can hydroxylate the substrate enantiomer d-Arg. Superposition of the structures of CmnC, VioC, and OrfP revealed a similar folds and overall structures. The active site residues of CmnC, VioC, and OrfP are almost conserved; however Leu136, Ser138, and Asp249 around the substrate binding pocket in CmnC are replaced by Gln, Gly, and Tyr in OrfP, respectively. These residues may play important roles for the substrate binding. The mutagenesis analysis revealed that the triple mutant CmnCL136Q,S138G,D249Y switches the substrate stereoselectivity from l-Arg to d-Arg with ∼6% relative activity. The crystal structure of CmnCL136Q,S138G,D249Y in complex with d-Arg revealed that the substrate loses partial interactions and adopts a different orientation in the binding site. This study provides insights into the enzyme engineering to α-KG non-heme iron oxygenases for adjustment to the substrate stereoselectivity and development of biocatalysts.

Bis-Thiourea Chiral Sensor for the NMR Enantiodiscrimination of N-Acetyl and N-Trifluoroacetyl Amino Acid Derivatives.

A C2-symmetrical bis-thiourea chiral solvating agent (CSA), TFTDA, for NMR spectroscopy has been obtained by reacting (1R,2R)-1,2-bis(2-hydroxyphenyl)ethylenediamine and 3,5-bis(trifluoromethyl)phenyl isothiocyanate. TFTDA shows remarkable propensity to enantiodiscriminate N-trifluoroacetyl (N-TFA) and N-acetyl (N-Ac) derivatives of amino acids with free carboxyl functions, with the co-presence of 1,4-diazabicyclo[2.2.2]octane (DABCO) as the third achiral additive, which is needed for substrate solubilization. TFTDA shows enhanced enantiodiscriminating efficiency in comparison with the corresponding monomeric counterpart, TFTMA, pointing out cooperativity between its two symmetrical entities. A wide range of amino acid derivatives have been efficiently enantiodiscriminated in CDCl3, with high enantioresolution quotients, which guarantee high quality in applications devoted to the quantification of enantiomers. High enantiodiscriminating efficiency is maintained also in diluted 5 mM conditions or in the presence of sub-stoichiometric amounts of CSA (0.3 equiv). The role of phenolic hydroxyls in the DABCO-mediated interaction mechanism between TFTDA and the two enantiomeric substrates has been pointed out by means of diffusion-ordered spectroscopy (DOSY) and rotating frame Overhauser effect spectroscopy (ROESY) experiments. A conformational model for both the CSA and its diastereomeric solvates formed with the two enantiomers of N-acetyl leucine has also been conceived on the basis of ROE data in order to give a chiral discrimination rationale.

Syn‐Selektive Epoxidierung von chiralen, terminalen Allylalkoholen mit einem Titan‐Salalen‐Katalysator und Wasserstoffperoxid

AbstractIn der asymmetrischen Sharpless‐Epoxidierung von chiralen, sekundären Allylalkoholen wird ein Substratenantiomer bevorzugt zum anti‐Epoxyalkohol umgesetzt. In dieser Arbeit stellen wir die erste hoch syn‐selektive Epoxidierung von terminalen Allylalkoholen vor. Mit einem Titan‐Salalen‐Katalysator und H2O2 als Oxidationsmittel werden enantiomerenreine, terminale Allylalkohole mit bis zu 98 % Ausbeute und >99 : 1 dr (syn) zu Epoxyalkoholen umgesetzt. Diese syn‐Selektivität bleibt auch bei niedrigeren Katalysatorbeladungen (bis zu 1 mol‐%) erhalten. Auch die Modifizierung der Allylalkoholfunktion zu einem Ether beeinflusst die Diastereoselektivität nicht [>99 : 1 dr (syn)]. Wird die Katalysatorkonfiguration invertiert, so wird bevorzugt das anti‐Produkt gebildet, allerdings mit niedrigerem dr (ca. 20 : 1). Das Anwendungspotential wird am Beispiel der Synthese eines THF‐Bausteins mit drei Stereozentren im Grammmaßstab demonstriert, in welcher zwei Ti‐Salalen‐katalysierte Epoxidierungen durchlaufen werden.

Syn‐Selective Epoxidation of Chiral Terminal Allylic Alcohols with a Titanium Salalen Catalyst and Hydrogen Peroxide

In the Sharpless asymmetric epoxidation of chiral secondary allylic alcohols, one substrate enantiomer is predominantly converted to the anti‐epoxy alcohol. We herein report the first highly syn‐selective epoxidation of terminal allylic alcohols using a titanium salalen complex as catalyst, at room temperature, and aqueous hydrogen peroxide as oxidant. With enantiopure terminal allylic alcohols as substrates, the epoxy alcohols were obtained with up to 98 % yield and up to >99 : 1 dr (syn). Catalyst loadings as low as 1 mol % can be applied without eroding the syn‐diastereoselectivity. Modification of the allylic alcohol to an ether does not affect the diastereoselectivity either [>99 : 1 dr (syn)]. Inverting the catalyst configuration leads to the anti‐product, albeit at lower dr (ca. 20 : 1). The synthetic potential is demonstrated by a short, gram‐scale preparation of a tetrahydrofuran building block with three stereocenters, involving two titanium salalen catalyzed epoxidation steps.

Enzymatic Resolution of cis‐Dimethyl‐1‐acetylpiperidine‐2,3‐dicarboxylate for the Preparation of a Moxifloxacin Building Block

AbstractThis work presents the enantioselective resolution of a water‐soluble racemic mixture of cis‐dimethyl 1‐acetylpiperidine‐2,3‐dicarboxylate catalyzed by Candida antarctica lipase B. The separation of the carboxylic acid product in its (2R,3S) configuration from non‐reacted substrate in the (2S,3R) configuration was easily obtained by organic phase extraction. The latter molecule can be used as an enantiomerically pure precursor in the synthesis of (4aS,7aS)‐octahydro‐1H‐pyrrolo[3,4‐b]pyridine, an expensive building block in the production of the antibiotic Moxifloxacin. On the other hand, the hydrolyzed product in the (2R,3S) configuration can be used for the preparation of a neuromediator analogue. The lipase demonstrated enantioselectivity towards the (2R,3S) substrate enantiomer and regioselectivity towards the ester in position 3 of the molecule. Studies of hydrolysis kinetics and the use of the immobilized enzyme were performed to evaluate its industrial application.