Enzymatic dynamic kinetic resolution of α-amino acid esters in miniemulsions

Abstract

The present invention relates to a process for the preparation of chiral alpha-amino acids using a protease enzyme in combination with catalytic amounts of an aldehyde for the racemization of an unwanted isomer in a miniemulsion system.

Description

[0001] ENZYMATIC DYNAMIC KINETIC RESOLUTION OF a-AMINO ACID ESTERS IN MINIEMULSIONS

[0002] FIELD OF INVENTION

[0003] The present invention relates to a process for the production of chiral amino acids using enzymes. The invention particularly relates to the enzymatic dynamic kinetic resolution of amino acid esters. The process of the present invention relates to the production of chiral alpha-amino acids using enzymes in miniemulsions. More particularly, the present invention relates to the effect of surfactants on enzyme activity, substrate loading and process intensification. The invention finds immense application in the biotechnology & pharma sector, especially the pharmaceutical Industries working on chiral APIs.

[0004] BACKGROUND OF THE INVENTION & DESCRIPTION OF PRIOR ART

[0005] Schichl, D. A. et al. (Eur. J. Org. Chem., 2008, 3506-12) discloses the dynamic kinetic resolution of phenylalanine and tyrosine esters in the presence of 3,5-dinitrosalicyaldehyde using alcalase enzyme. However, batch-to-batch variation in conversion rate and e.e were observed due to deactivation of catalysts under the reaction conditions, as well as low substrate solubility (5% w / w) and high enzyme loading (> 20% w / w), are major limiting factors for the practical application of this method.

[0006] The best reaction conditions reported in their studies are a 5% w / w substrate loading in a 1:1 mixture of water and acetonitrile, with an apparent pH of 7.5 at 25°C, resulting in L- phenylalanine with an 80% yield and 96% enantiomeric excess (e.e.) after 30 hours of reaction time. Furthermore, the same research group investigated the reasons for the limited yield of 80% through kinetic analysis and modelling of the reaction system (Organic Process Research & Development 2006, 10, 622-627). Catalyst deactivation was identified as the primary constraint for the low yield. They suggested developing an enzyme with improved stability through directed evolution or screening an alternative enzyme.

[0007] When we followed this method for standard compound preparation, very low conversion and poor e.e. were observed during our studies due to catalyst deactivation, which also hampers product precipitation. In this case, product isolation requires distillation to remove the organic cosolvent in the presence of aldehyde, which may reduce the e.e. of the amino acid product.

[0008] Chen et al. (J. Org. Chem., 1994, 59, 7580-81) teaches a process for the conversion of racemic amino acid to its L-isomer by alcalase-catalyzed resolution of amino acid ester in a mixture of 2-methyl-2-propanol / water (19:1), simultaneously with pyridoxal 5-phosphate catalyzed racemization of the unhydrolyzed antipode. Zimmerman et al., (Org. Proc. Res. & Dev., 2006, 622-27) discloses the reaction course of a dynamic kinetic resolution of amino acid derivatives. Felten et al., (Org. Lett, 2010, 1916-19) disclose the dynamic kinetic resolution of benzyl ester of phenyl glycine in the presence of picolinaldehyde and zinc acetate.

[0009] In summary, despite its potential for achieving a 100% theoretical yield, enzymatic DKR of amino acid esters faces several commercial challenges, mainly enzyme inhibition by aldehydes and organic solvents, low substrate solubility, and operational inefficiencies. Accordingly, the current synthetic routes for optically pure chiral pamino acids remain constrained by low yields, suboptimal enantiomeric purity, environmental concerns, and operational complexities like catalyst deactivation. The present study introduces a novel surfactant-based miniemulsion system to address these challenges, particularly using surfactants or miniemulsion systems to overcome the catalysts deactivation without affecting the enzyme selectivity along with increasing the substrate solubility.

[0010] The present application discloses the kinetic resolution of the alpha-amino ester by using protease (Alcalase®) enzyme and racemization of the unwanted ester by using catalytic amount of an aldehyde in one pot, i.e., dynamic kinetic resolution (DKR) in miniemulsions. The present application also discloses the kinetic resolution of alpha-amino ester by using protease (Alcalase®) enzyme in the presence of miniemulsions for the isolation of both the compounds, chiral amino acids & esters. a) General chemical methods for the kinetic resolution of phenylalanine derivatives

[0011] WO 2019 / 073325 Al discloses the resolution of substituted phenylalanine derivatives using chiral resolving agents such as Cinchonidine alkaloids. While the enantiomeric purity achieved is high, the process suffers from low yields (25-45%) and the generation of a large amount of effluents due to acid / base treatment. Additionally, the use of stoichiometric amounts of chiral resolving agents results in a poor E-factor. b) General enzymatic methods for the kinetic resolution of phenylalanine derivatives Chen S. et al. (Org. Process Res. Dev. 2024, 28, 3, 693-703) disclose the resolution of / V-acetyl- 3-bromo-phenylalanine to produce 3-bromo-L-phenylalanine using acylase enzyme with a 40% yield. The unwanted isomer was isolated and racemized separately using the acetic anhydride / acetic acid (AczO / AcOH) system. A review of the prior art reveals that none of them have reported the usage of miniemulsions for enzymatic dynamic kinetic resolution of organic compounds using protease enzymes which can offer 100% theoretical yield with high enantiomeric purity (>99% e.e).

[0012] Accordingly, keeping in view the drawbacks of the hitherto reported prior art, the inventors of the present invention recognized the critical need to establish an improved process for the preparation of chiral alpha-amino acids using a protease enzyme. This process aims to achieve conversion within 9-16 hours with a conversion rate exceeding 99% while requiring less enzyme loading. The overall theoretical isolated yield in kinetic resolution ranges from 45% to 50%, while in dynamic kinetic resolution, it can reach 85% to 100%.

[0013] OBJECTIVES OF THE INVENTION

[0014] The main objective of the present invention is therefore to provide an enzymatic process for the preparation of chiral alpha-amino acids.

[0015] Another objective of the present invention is to provide a process for the preparation of chiral alpha-amino acids through the dynamic kinetic resolution of ester derivatives using protease enzymes in miniemulsions in the presence of catalytic amounts of aldehyde.

[0016] Yet another objective of the present invention is to provide a process for the preparation of chiral alpha-amino acids through the kinetic resolution of ester derivatives using protease enzymes in miniemulsions.

[0017] Yet another objective of the present invention is to provide a process for the preparation of optionally substituted L-phenylalanine through the dynamic kinetic resolution of ester derivatives using protease enzymes in miniemulsions in the presence of catalytic amounts of aldehyde.

[0018] Yet another objective of the present invention is to provide a process for the preparation of optionally substituted L-phenylalanine through the kinetic resolution of ester derivatives using protease enzymes in miniemulsions.

[0019] Yet another objective of the present invention is to provide a process for the preparation of 3-bromo-L-phenylalanine through the dynamic kinetic resolution of ester derivatives using protease enzymes in miniemulsions in the presence of catalytic amounts of aldehyde.

[0020] Yet another objective of the present invention is to provide a process for the preparation of 3-bromo-L-phenylalanine through the kinetic resolution of ester derivatives using protease enzymes in miniemulsions.

[0021] Yet another objective of the present invention is to provide a process for the preparation of 3-(Methylsulfonyl)-L-phenylalanine through the dynamic kinetic resolution of ester derivatives using protease enzymes in miniemulsions in the presence of catalytic amounts of aldehyde.

[0022] Yet another objective of the present invention is to provide a process for the preparation of 3-(Methylsulfonyl)-L-phenylalanine through the kinetic resolution of ester derivatives using protease enzymes in miniemulsions.

[0023] SUMMARY OF THE INVENTION

[0024] One aspect of the present application relates to a chemoenzymatic process for the preparation of a chiral amino acid derivative of formula (A) from an amino acid ester derivative of formula (B) using a protease enzyme in the presence of catalytic amounts of an aldehyde or the metal complex of aldehyde, and miniemulsions as solvent systems.

[0025] Enzyme aldehyde (cat) iniemulsion' as solvents

[0026] Wherein R represents optionally substituted alkyl, cyclic, arylalkyl, aromatic & heterocyclic compounds. R1represents optionally substituted alkyl, arylalkyl group.

[0027] Yet another aspect of the present application relates to a chemoenzymatic process for the preparation of an amino acid derivative of formula (1) from an amino acid ester derivative of formula (2) using a protease enzyme and in the presence of an aldehyde and miniemulsions as solvent systems.

[0028] Wherein R1represents as described above and X represents H, Halo, Alkyl, Aryl, -CN, -SO2Me, -OH, - CONH2.

[0029] In another embodiment of the present invention, enzymatic dynamic kinetic resolution is conducted in water in the presence of surfactants and aldehyde at a suitable pH. In another embodiment, the present application relates to the kinetic resolution of racemic amino acid ester derivative of formula (B) at a suitable pH using a protease enzyme in miniemulsions as solvent systems. Both isomers of an optically pure amino acid derivative of formula (A) and amino acid ester derivative of formula (C) is isolated.

[0030] R1Protease enzyme 'miniemulsion' as solvents NH2

[0031] B A

[0032] In another embodiment of the present invention, enzymatic kinetic resolution is conducted in water in the presence of surfactants at a suitable pH.

[0033] In still another embodiment of the present invention, the enzyme used is selected from commercially available sources, wild-type microorganisms, isolated, semi-purified and or recombinant enzymes. The enzyme can be used in the form of a crude lysate or in a semipurified form.

[0034] Alternatively, the enzyme may be in the form of whole microbial cells, permeabilized microbial cells, extracts of microbial cells, partially purified enzymes, purified enzymes, and the like. Preferably, the enzyme is used in the form of cell pellet, crude lysate or lyophilisate. Alternatively, the enzyme can be immobilized and used as such, wherein the immobilization techniques are known to a person skilled in the art.

[0035] BRIEF DESCRIPTIONS OF THE ACCOMPANYING DRAWINGS

[0036] Figure 1 represents enzymatic dynamic kinetic resolution of amino acid esters using protease enzyme in miniemulsions.

[0037] Figure 2 represents enzymatic dynamic kinetic resolution of roc-3-bromo phenylalanine using protease enzyme in miniemulsions.

[0038] Figure 3 represents enzymatic dynamic kinetic resolution of roc-3-methylsulfonyl phenylalanine using protease enzyme in miniemulsions.

[0039] DETAIL OF THE BIOLOGICAL RESOURCES USED The enzyme used in the present application is commercially available and supplied by M / s. Novozymes, Denmark in the name of Alcalase®. Currently, it is also marketed as Sustine® by the same company. It is a serine endopeptidase that consists primarily of subtilisin A. This enzyme is produced by submerged fed-batch fermentation using Bacillus licheniformis (Int J Biol Macromol. 2020 Dec 15;165(Pt B):2143-2196). It is an inexpensive enzyme widely used in laundry detergents, pharmaceuticals, and food processing.

[0040] DETAILED DESCRIPTION OF THE INVENTION

[0041] The present application provides a process for the preparation of optically active alpha-amino acids comprising the following steps: a) Prepare a substrate solution with concentrations 100 gL1to 850 gL1dissolved in water containing 1% w / v surfactant. b) Prepare a miniemulsion using ultrasound or high-pressure homogenizers on a mixture or vigorously mixing using mechanical stirring. In addition an inert hydrophobic substance is added as stabilizer, preferably at 0.5 to 2% by weight. c) To the substrate solution of step [b] protease enzyme in a ratio ranging from 10-15% w / w was added at pH of 6 to 8.5 along with catalytic amount of aldehyde and stirred for 10 to 16 h at temperatures ranging from 0 to 40 °C; d) Alternatively to the substrate solution of step [a] protease enzyme in a ratio ranging from 5-15% w / w was added at pH of 6 to 8.5 along with catalytic amount of aldehyde and stirred for 10 to 16 h at temperatures ranging from 0 to 40 °C; e) After completion of the reaction the product stream was separated by precipitation followed by solvent wash.

[0042] The enzymatic process (Biotransformation) of the present application may be performed using any enzyme known in the art that is capable of preferentially cleaving an ester group of a substrate to form an acid. Specifically, the enzymatic process of the present application may be performed using serine endopeptidase enzymes and more specifically, protease enzymes. Such enzymes may be commercially available or prepared by methods known in the art. More specifically, the enzymatic process of the present application may be performed using alcalase enzyme procured from M / s. Novozymes, Denmark.

[0043] Biotransformation may be carried out with whole cells or isolated enzymes. Enzymes are proteins that can be utilized in the transformation of organic compounds. The enzyme or biocatalyst is used in the form of wild-type microorganisms, isolated, semi-purified, purified, immobilized and or recombinant enzymes.

[0044] Alternatively, the enzyme may be in the form of whole broth, cell pellet, crude lysate, permeabilized microbial cells, extracts of microbial cells, partially purified enzymes, purified enzymes, and the like. Preferably, the enzyme is used in the form of cell pellet, crude lysate or lyophilisate. Alternatively, the enzyme can be immobilized and used as such.

[0045] The concentration of substrate for the biotransformation in the present invention may ranges from about 100 g / L to about 850 g / L. The loading of the enzyme may range from about 0.1 wt % to about 100 wt % with respect to the weight of the substrate.

[0046] The quantity of enzyme is selected as a function of the size of the batch, of the reactivity of the substrate, of the desired reaction time, and of the free or immobilized nature of the enzyme. The quantity of enzyme is readily determined by simple preliminary experiments where the reaction is carried out on a pilot scale.

[0047] The continuous stirring is preferred during the reaction. The reaction time depends on the nature of the substrate employed and the amount of enzyme and water / buffer system with surfactants, and is usually only a few hours to 3 days. Progress of the reaction is monitored by using any method known to those skilled in the art of organic synthesis, including thin layer chromatography (TLC) and HPLC.

[0048] Miniemulsion, also known as nanoemulsion, was obtained by shearing a mixture of two immiscible liquid phases (for example, oil and water), one or more surfactants and, possibly, one or more co-surfactants (such as hexadecane or cetyl alcohol) using mechanical stirring, ultrasonication, or high-pressure homogenization (K. Landfester, M. Antonietti, "Miniemulsions the convenient synthesis of organic and inorganic nanoparticles and "single molecule" applications in materials chemistry" in: Colloids and Colloid Assemblies (Ed. : F. Caruso), Wiley VCH Verlag, Weinheim, Germany, 2004, pp. 175-215 and the literature which is cited therein). In general, mix the organic substrate with the chosen surfactants and optionally co-surfactants. Add this mixture to water while stirring or subject it to high-energy methods to disperse the organic phase as small droplets within the aqueous phase. Continue mixing or processing until a stable miniemulsion is formed. The stabilization of miniemulsion formation involves incorporating surfactants or emulsifiers to prevent the coalescence and aggregation of dispersed droplets, thereby maintaining the stability of the emulsion.

[0049] Landfester K. et al. (Macromol. Rapid Commun. 2003, 24, 512-516) discloses the miniemulsion system for the polymerization of lactones as well as for the resolution of amino acid using Pseudomonas cepacia & porcine pancreas enzymes (W02006058595A1 & Angew. Chem. Int. Ed. 2006, 45, 1645-1648).

[0050] In the present invention, a miniemulsion is a mixture containing small, stable droplets, achieved through intense shearing forces such as ultrasound, a steel disperser, or microfluidizer. These droplets are preferably dispersed throughout a second continuous phase for the duration of the reaction. Preferably, hydrophobic droplets are generated in an aqueous medium, where an aqueous medium is a homogeneous phase predominantly composed of water. However, other organic solvents soluble in water, such as alcohols (e.g., methanol, ethanol), ketones (e.g., acetone, MIBK), or DMSO, DMF, NMP, or sulfolane, may be added. The choice of hydrophobic phase liquid depends on whether it forms a two-phase mixture with water under the reaction conditions and remains inert towards enzymatic transformation. The addition of a hydrophobic liquid may be unnecessary if the substrate itself is liquid and can function as the hydrophobic phase. This varies depending on the substrate and must be determined by the person skilled in the art in each individual case. If further hydrophobic liquid is needed, especially when the substrate is solid, the practitioner selects a liquid preferably from ethers, esters, and hydrocarbons. Preferably, MTBE, ethyl acetate, n-hexane, n-heptane, cyclohexane, and toluene are used.

[0051] Emulsion droplets, with a mean diameter of 20 to 1000 nanometers, preferably 30 to 600 nanometers, and most preferably 50 to 500 nanometers, can be formed by treating the mixture with ultrasound, a steel disperser, or a microfluidizer. To prepare miniemulsions, it is advantageous to add a surfactant to the mixture before droplet generation. The surfactant, preferably employed at 1 to 20% by weight, more preferably 1 to 15%, and most preferably 1 to 5%, based on the emulsion quantity, can be any ionic or non-ionic surfactant that does not inactivate the enzymes used. Tween® 80 (nonionic, Polysorbate 80), Ethoxylates of alkyl polyethylene glycol ethers, particularly Lutensol AT* (non-ionic), especially Lutensol AT 50* (Cie / is-EOs to so) and linear block copolymers consisting of polyoxyethylene (EO) and polyoxypropylene (PO) blocks, particularly Pluronics (non-ionic), especially Pluronic® PE 8100 and Pluronic® PE 6800 and linear, saturated fatty alcohol ether sulfate (2EO), sodium salt, particularly Agnique N40I (non-ionic) are preferred. Additionally, the emulsion droplets are stabilized by adding further inert substances, typically hydrophobic. These substances must exhibit lower solubility in water than the hydrophobic phase, with preferred aliphatic hydrocarbons including Ce-Czo-alkanes, particularly hexadecane. These hydrophobic substances are preferably employed at 0.001 to 70% by weight, preferably 0.1 to 3%, and particularly preferably 0.5 to 2% by weight, based on the miniemulsion.

[0052] The biotransformation of the present invention can be conducted in suitable aqueous buffer or in water or in presence of surfactants, miniemulsions at any appropriate pH. Preferably, a pH ranging from about 6 to 8.5, more specifically pH 7.5 is chosen in the present invention. The pH can be adjusted, e.g., by the addition of an inorganic or organic base.

[0053] Suitable buffers include ammonium, alkali or alkaline earth phosphates (e.g., ammonium phosphate, potassium phosphate and sodium phosphate) or ammonium, alkali or alkaline earth acetates (e.g., ammonium acetate and calcium acetate) or Tris buffer or other buffers having a pKa of about 5 to 10. In the present invention, water & phosphate buffer (pH=7.5) is used.

[0054] The chemoenzymatic process of the present application may be carried out in presence of an aldehyde or a metal complex of aldehyde which is capable of racemizing the undesired enantiomer of optically pure amino acid ester of compound of formula (C). Specifically, the aldehyde may include but not limited to 3,5-dinitrosalicylaldehyde, 3,5- dichlorosalicylaldehyde and picolinaldehyde. Alternatively, a mixture of zinc salt such as zinc acetate and aldehyde may be used for racemizing the undesired enantiomer of amino acid ester of compound of formula (C).

[0055] The biotransformation of the present invention may be carried out at a suitable temperature. Specifically, the temperature may vary from 0 °C to 40 °C; more specifically from 20 °C to 35 °C.

[0056] The biotransformation of the present invention may be carried out in suitable equipment, for example, in a reaction vessel made of glass or metal or bioreactors or in a packed bed reactor or in a basket reactor.

[0057] The process of the present application produces amino acid derivative of formula (A) in an enantiomeric purity of more than about 85%. Specifically, the process of the present application produces amino acid derivative of formula (A) in an enantiomeric purity of more than about 90%. More specifically, the process of the present application produces amino acid derivative of formula (A) in an enantiomeric purity of more than about 95%. Most specifically, the process of the present application produces amino acid derivative of formula (A) in an enantiomeric purity of more than about 99%.

[0058] "Halogen" is defined as non-metallic elements found in group VII of the periodic table and is selected from fluorine, bromine, chlorine and iodine.

[0059] The term "alkyl" refers to straight or branched aliphatic hydrocarbon groups having the specified number of carbon atoms, which are attached to the rest of the molecule by a single atom. Preferred alkyl groups include, without limitation, methyl, ethyl, n-propyl, isopropyl, butyl, isobutyl, t-butyl, pentyl, hexyl, heptyl, octyl and the like.

[0060] The term "Cycloalkyl" refers to mono or bicyclic carbocyclic ring functional group including, but not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, bicyclo[2.2.1]heptanyl, bicyclo[3.2.1]octanyl, and bicyclo[5.2.0]nonanyl; wherein the cycloalkyl group may optionally contain 1 or 2 double bonds (i.e., a cycloalkylenyl) including, but not limited to, cyclopentenyl, cyclohexenyl, and cycloheptenyl. The term "cycloalkyl" is intended to include both substituted and unsubstituted cycloalkyl groups.

[0061] The term "aromatic" refers to aryl; arylalkyl; heteroaryl and heteroarylalkyl. The term "substituted" as used herein refers to substitution with any one or any combination of the following substituents: fluorine, chlorine, bromine and iodine; hydroxy; nitro; cyano; oxo (=0); thioxo (=S); azido; nitroso; amino; hydrazino; formyl; alkyl; alkoxy; aryl; haloalkyl groups such as trifluoromethyl, tribromomethyl and trichloromethyl; haloalkoxy groups such as -OCH2CI, -OCHF2 and -OCF3; arylalkoxy groups such as benzyloxy and phenylethoxy; cycloalkyl; -O-cycloalkyl; aryl; alkoxy; heterocyclyl; heteroaryl; alkylamino; -O-CH2-cycloalkyl; -COORa; -C(O)Rb; -C(S)Ra; -C(O)NRaRb; -NRaC(O)NRbRc; -N(Ra)SORb; -N(Ra)SO2Rb; -NRaC(O)ORb; -NRaRb; -NRaC(O)Rb-; NRaC(S)Rb-; -SONRaRb-; -SO2NRaRb; -0Ra; -ORaC(O)ORb-; -OC(O)NRaRb; OC(O)Ra; -OC(O)NRaRb-; -RaNRbRc; -RaORb-; -SRa; -SORaand -SO2Ra; Ra, Rband Rceach independently represents hydrogen atom; substituted or unsubstituted groups selected from alkyl; aryl; arylalkyl; cycloalkyl; heterocyclyl; heteroaryl and heteroarylalkyl; Ra, Rband Rcare also combined to form a 3-7 membered ring having 0-2 hetero atoms.

[0062] The present application can be used for the kinetic resolution of alpha-amino acid derivatives without using aldehyde to isolate both the isomers (optically pure amino acid esters & amino acids) with 50% theoretical yield and > 99% e.e is known to a person skilled in the art.

[0063] An enzymatic process for the preparation of chiral alpha-amino acids compound of formula A using protease enzyme

[0064] Enzyme aldehyde (cat) iniemulsion' as solvents

[0065] Wherein R represents optionally substituted alkyl, cyclic, arylalkyl, aromatic and heterocyclic compounds; R1represents optionally substituted alkyl, arylalkyl group; and the said process comprising the steps:

[0066] (a) Preparing a substrate solution with concentration 100 gL1to 850 gL1in water containing 1% w / v surfactant at suitable pH and catalytic amount of aldehyde in miniemulsions; (b) To the substrate solution of step [a], protease enzyme in a ratio ranging from 5- 15% w / w was added at pH of 6 to 8.5 and stirred for 8 to 24 h at temperatures ranging from 0 to 40 °C;

[0067] (c) Monitoring the product formation at regular intervals by TLC or HPLC analysis;

[0068] (d) Separating the product stream after completion of the reaction by filtration after adjusting the pH at isoelectric point followed by solvent wash.

[0069] The substrate is selected from the group consisting optionally substituted racemic alphaamino acid esters preferably substituted roc-phenylalanine ethyl ester. pH of the reaction is 6-8.5 specifically 7.5.

[0070] Temperature of the reaction is 0 to 40 °C specifically 20-30 ’C.

[0071] Enzyme is endopeptidase, particularly protease.

[0072] Enzyme is isolated from bacteria, particularly Bacillus species.

[0073] Enzyme from wild type microorganisms, whole broth, isolated, semi-purified, purified, immobilized and or recombinant enzymes.

[0074] The enantiomeric purity of compounds of formula A obtained is in the range of 95 to >99%, specifically 99.99 % e.e.

[0075] EXAMPLES

[0076] The following examples are given by way of illustration only and therefore should not be construed to limit the scope of the present invention in any manner.

[0077] The following examples are given by way of illustration and therefore, should not be construed to limit the scope of the invention. For more information on amino acid substrates, structural analogues and applications of chiral alpha-amino acids, please see the review article, Chem. Soc. Rev., 2018, 47, 1516-1561. In this context, the analogues are molecules having substantial biological similarities regardless of structural similarities. In the present invention, the process for the preparation of L-phenylalanine and L-3-bromo phenylalanine was studied as a model case study. The corresponding ester was used as a substrate for the preparation of chiral a-amino acids.

[0078] Example 1: Screening surfactants for the enzymatic DKR of rac-8a

[0079] S. No Solvent system Isolated yield e.e(s)(° / o)

[0080] (%) Before After

[0081] MSA wash

[0082] MSA wash

[0083] 1 Lutensol® AT 50 90 >99 >99

[0084] (BASF, PRD-No. 30043970) Tween 80 (Finar) 66 98 >99 CTAB (Loba Chemie, 0268100500) 65 >99 >99 Triton™ X- 100 (Sigma Aldrich, 47 98 >99 102684862 Agnique® N-40I 49 98 >99

[0085] (BASF, PRD-No. 30783881) Pluronic® PE 6800 58 >99 >99

[0086] (BASF, PRD-No. 30044097) Sokalan® HP 20 45 98 >99

[0087] (BASF, PRD-No. 30516313) Pluronic® PE 8100 60 97 >99

[0088] (BASF, PRD-No. 30044087) '- Dodecyl - '. A'-dimcthyl-3-ammonio- 1 - 55 99 >99 propanesulfonate (Fluka, 40232, Zwitterionic)

[0089] 10 Buffer (0. IM, KPB, pH 7.5) 41 81 ND

[0090] 11 ACN: Water (1: 1) 30-50%* 98 >99

[0091] 12 Water 35 80 ND

[0092] Reaction conditions'. rac-8a (1 g, 3.68 mmol), 0. 1 M potassium phosphate buffer (10 mL, pH 7.5) containing 10% w / w surfactant (100 mg), 3,5-dinitrosalicylaldehyde (19.4 mg, 0.09 mmol), Alcalase enzyme (0.05 mL, 5% w / w) and stirred for 24 h at 27 °C. The reaction progress was monitored, and the e.e. of the isolated product was determined using chiral HPLC. *- Batch-to- batch variation in conversion, yield and 30% w / w enzyme loading was used. MSA wash - DME containing 0.1% v / v MSA. ND - Not done.

[0093] Example 2: Effect of substrate loading.

[0094] The above reactions (example 1) were carried out with substrate concentrations of 5%, 10%, 25%, and 50% w / v, using 5% w / w enzyme loading relative to the substrate. Conversion was monitored at various intervals. The product started to precipitate within 2-4 h in all cases, with the reaction reaching completion within 24 h in the case of 5% and 10% w / v substrate loading.

[0095] In contrast, a conversion of 88% was observed for 25% and 50% w / v substrate loading after 24 h. Increasing the enzyme loading to 10% w / w enabled complete conversion within 24 h even at 25% and 50% w / v substrate concentrations. For comparison, conversion data up to 6 h for all conditions are presented in figure 4. In all cases, the e.e. was >99 %, with isolated yields of 85-90%. Substrate loadings beyond 50% w / v caused agitation issues due to significant product precipitation.

[0096] Figure 4. Effect of substrate loading. Reaction conditions '. racSa (5-50% w / v) in 10 mL buffer containing 10% w / w Lutensol® AT 50 and 1% w / w hexadecane; a). 5% w / w enzyme loading, b). 10% w / w enzyme loading. Example 3: Effect of surfactant concentration.

[0097] The above reactions were carried out by varying the surfactant concentrations (5%, 10%, and 30% w / w) at fixed substrate loading (10% w / v) using 5% w / w enzyme loading relative to the substrate. At 6 hours, with 5%, 10%, and 30% surfactant concentrations, conversions were 43%, 50%, and 56%, respectively. These results indicate that the enzyme retains its activity even at higher surfactant concentrations, with only a marginal improvement observed between 10% and 30% surfactant concentrations. Consequently, further optimization studies were conducted using 50% w / v substrate loading, 10% w / w enzyme loading, and 10% w / w surfactant relative to the substrate.

[0098] Example 4: Dynamic Kinetic resolution of rac-phenyl alanine ester using surfactants

[0099] Ethyl ester of phenylalanine (25 g) was dissolved in water (100 mL) containing 1% w / v lutensol and aldehyde (500 mg). The reaction mixture was treated for 5 min and the pH of the reaction mixture was adjusted to 7.5. After the addition of alcalase enzyme (2.5 mL, 10% w / w), it was stirred for about 16 hours using a magnetic stirrer. After completion of the reaction, the pH of the reaction mixture was then adjusted to 5.5. The enantiomerically pure amino acid was precipitated out and the yellow solid was filtered and then washed with 1,2-dimethoxy ethane (50 mL) to remove the aldehyde and other coloured impurities. Finally, the white solid obtained was dried to provide the L-phenylalanine.

[0100] Isolated yield: 19.2 g, 90%

[0101] Enantiomeric Excess: > 99 % e.e.

[0102] Example 5: Dynamic Kinetic resolution of rac-phenylalanine ester in miniemulsions

[0103] Ethyl ester of phenylalanine (25 g) was dissolved in water (50 mL) containing 1% w / v lutensol and 1% w / v hexadecane and 3,5-dinitrosalicyaldehyde (500 mg). The reaction mixture was treated for 10 min at 200W using an ultrasonic probe. After adjusting the pH of the miniemulsions to 7.5, alcalase enzyme (2.5 mL, 10% w / w) was added and it was stirred for about 16 hours using a magnetic stirrer. After completion of the reaction, the pH of the reaction mixture was then adjusted to 5.5. The enantiomerically pure amino acid was precipitated out and the yellow solid was fdtered and then washed with 1,2-dimethoxy ethane (50 mL) to remove the aldehyde and other coloured impurities, finally, the white solid obtained was dried to provide the L-phenylalanine.

[0104] Isolated yield: 18.5 g, 87%

[0105] Enantiomeric Excess: > 99 % e.e. Example 6: Dynamic kinetic resolution of rac-3-bromo phenylalanine ester in miniemulsions

[0106] Ethyl ester of 3 -bromo phenylalanine (25 g) was dissolved in water (50 mL) containing 1% w / v lutensol and 1% w / v hexadecane and 3,5-dinitrosalicyaldehyde (500 mg). The reaction mixture was treated for 10 min at 200W using an ultrasonic probe. After adjusting the pH of the miniemulsions to 7.5, alcalase enzyme (2.5 mL, 10% w / w) was added and it was stirred for about 16 hours using a magnetic stirrer. After completion of the reaction, the pH of the reaction mixture was then adjusted to 5.5. The enantiomerically pure amino acid was precipitated out and the yellow solid was filtered and then washed with 1,2-dimethoxy ethane (50 mL) to remove the aldehyde and other coloured impurities, finally, the white solid obtained was dried to provide the 3-bromo-L-phenylalanine.

[0107] Isolated yield: 19.7 g, 88%

[0108] Enantiomeric Excess: > 99 % e.e.

[0109] 'H NMR (300 MHz, DMSO-d6+ HC1): 5 8.50 (brs, 2 H), 7.49-7.839 (m, 2 H), 7.29-7.21 (m, 2 H), 4.16-4.04 (m, 1 H), 3.20-3.05 (m, 2 H) ppm;13C NMR (100 MHz, DMSO-d6+ HC1): 8 170.4, 138.2, 132.6, 131.1, 130.5, 129.1, 122.1, 53.4, 35.4 ppm; HRMS (ESI-QTOF) showed an [M + H]+ion at m / z 243.9966 (calculated for CgHnNChBr: 243.9973).

[0110] Example 7: Recovery and re-use of enzyme and surfactant. The recovered supernatant, containing the surfactant, enzyme, and 3,5 -dinitrosalicylaldehyde, was reused for subsequent batches after adjusting the pH to 7.5. Fresh substrate and aldehyde were added to the recovered supernatant, followed by probe sonication for 10 minutes. A small top-up amount of enzyme (2% w / w) was added prior to initiating the next cycle to compensate for any potential loss of enzyme during handling operations such as filtration, sampling, or transfer. The reaction proceeded efficiently, achieving complete conversion with an e. e. of >99%.

[0111] Example 8: DKR of rac-3-(methylsulfonyl)-DL-phenylalanine ethyl ester in miniemulsions.

[0112] The reaction was carried out following the above procedure (Example 6), with a temperature variation (0 °C) and a reaction medium of phosphate buffer and ACN (1:1 v / v). (S')-l b: Isolated yield: 20.26 g, 90.4%, and > 99% e. e. [a]D25= +4.00 (c =1, IN HC1); Mp: 228-230 °C;1H NMR (300 MHz, DMSO-de): 8 = 8.50 (brs, 2 H), 7.90-7.80 (m, 2 H), 7.69-7.59 (m, 2 H), 4.25 (t, J= 6.3 Hz, 1 H), 3.27 (d, J = 6.3 Hz, 2 H), 3.21 (s, 3 H) ppm;13C NMR (100 MHz, CDCh): 8 = 170.5, 141.3, 137.1, 135.2, 130.1, 128.4, 126.2, 53.3, 44.1, 35.6 ppm; HRMS (ESI-QTOF) showed an [M + H]+ion at m / z 244.0634 (calculated for C10H14NO4S: 244.0644).

[0113] Example 9: Comparative data- Dynamic kinetic resolution of rac-3-bromo phenylalanine ester

[0114] Ethyl ester of 3 -bromophenylalanine (1 g) was dissolved in acetonitrile (10 mL) and water (10 mL). To the reaction mixture, 3,5-dinitrosalicylaldehyde (19.4 mg) was added and the pH was adjusted to 7.5. Alcalase enzyme (0.2 mL, 20% w / w) was added, and the mixture was stirred for about 48 hours using a magnetic stirrer. The pH of the reaction was maintained at 7.5 using 2N NaOH solution. The progress of the reaction was monitored by HPLC, and the conversion obtained was 30-50% after 48 hours. After 48 hours of reaction time, the pH of the reaction mixture was adjusted to 5.5. The reaction mixture was then evaporated using a rotary evaporator, reducing the volume to one-fourth. To the reaction mixture, 1,2-dimethoxyethane (20 mL) was added. The enantiomerically pure amino acid precipitated out as a yellow solid, which was filtered and washed with 1,2-dimethoxyethane (10 mL) to remove the aldehyde and other coloured impurities. Finally, the white solid obtained was dried to provide 3-bromo-L- phenylalanine. Experiments were carried out in triplicate.

[0115] The conversion and isolated yield ranged from 30-50% from batch to batch.

[0116] Enantiomeric Excess: 98 % e.e.

[0117] Example 10: Effect of miniemulsion on protease activity of Alcalase

[0118] To further evaluate the impact of sonication on enzyme performance, the protease activity of Alcalase was assessed using casein as the substrate. The tyrosine released was assayed using Folin-Ciocalteu method. It was observed that sonication within the miniemulsion system did not lead to any significant loss of initial enzymatic activity (240 and 242 U / mL, pre- and postsonication, respectively). In contrast, a moderate decrease of approximately 14% in initial activity was observed when sonication was performed in plain buffer (225 and 195 U / g, pre- and post-sonication, respectively). These results indicate that the miniemulsion environment provides a protective effect, preserving enzyme activity during sonication and reinforcing the robustness of the recycling process.

[0119] Example 11: Evaluating the efficiency of the process. A comparative analysis of process efficiency and green chemistry metrics between the present method (enzymatic DKR in miniemulsion) and literature -reported chemical processes (kinetic resolution) provides valuable insights into sustainability and practicality. The enzymatic process demonstrates superior efficiency, achieving higher conversion rates and e.e. exceeding 99% within shorter reaction times compared to traditional methods.

[0120] Green chemistry metrics were calculated for the preparation of chiral phenylalanine derivatives via chemical resolution using Cinchonine alkaloid (WO 2019 / 073325 Al). These metrics were compared with those from the current study via enzymatic DKR in miniemulsions, as summarized below in Figure 5. All calculations were performed using established formulas reported in the literature (Green Chem. 2002, 4, 521-527). Our comparison was intended to serve as a representative example, highlighting the efficiency of the enzymatic DKR process in contrast to the classical chemical resolution method for the preparation of L-phenylalanine derivatives.

[0121] Figure 5. Comparison of green chemistry metrics: Chemical resolution vs enzymatic DKR.

[0122] ADVANTAGES OF THE INVENTION

[0123] 1. The present process serves as a highly efficient and scalable method for the production of chiral alpha-amino acids through enzymatic kinetic or dynamic kinetic resolution by using miniemulsions as solvent system. Increased substrate solubility and enzyme activity were observed when using surfactant or miniemulsion as a solvent system.

[0124] 2. The present invention offers high substrate loading (> 100 - 850 g / L, 10% - 85% w / w) compared to previous reports conducted in aqueous buffers or organic co-solvents. Previous reports have indicated a maximum of 5% w / w substrate loading when using organic co-solvents, primarily due to poor substrate solubility. However, the use of organic co-solvents may result in lower conversion rates due to enzyme inhibition, and complete removal via distillation is necessary for product isolation. This process may lead to racemization and decreased purity.

[0125] 3. The present process is an attractive and economical method for producing chiral alpha-amino acids. It utilizes less enzyme loading (5-10% w / w) and shorter reaction times (9-16 h), achieving 100% substrate conversion to product. This is in contrast to previous reports, which required higher enzyme loading (30% w / w) and longer reaction times (48 h), resulting in only 60-80% conversion due to inhibition of enzyme activity by aldehyde or organic co-solvent systems. 4. The present process is highly stereo-selective, and the isolation and purification of the product are straightforward, achieved through simple filtration and solvent wash, resulting in high yield and purity.

Claims

We Claim1. An enzymatic process for the preparation of chiral alpha-amino acids compound of formula A using protease enzymeEnzyme aldehyde (cat)iniemulsion' as solventsWherein R represents optionally substituted alkyl, cyclic, arylalkyl, aromatic and heterocyclic compounds; R1represents optionally substituted alkyl, arylalkyl group; and the said process comprising the steps:(a) Preparing a substrate solution with concentration 100 gL1to 850 gL1in water containing 1% w / v surfactant at suitable pH and catalytic amount of aldehyde in miniemulsions;(b) To the substrate solution of step [a], protease enzyme in a ratio ranging from 5- 15% w / w was added at pH of 6 to 8.5 and stirred for 8 to 24 h at temperatures ranging from 0 to 40 °C;(c) Monitoring the product formation at regular intervals by TLC or HPLC analysis;(d) Separating the product stream after completion of the reaction by filtration after adjusting the pH at isoelectric point followed by solvent wash.

2. The process wherein, the substrate is selected from the group consisting optionally substituted racemic alpha-amino acid esters preferably substituted roc-phenylalanine ethyl ester.

3. The process, wherein pH of the reaction is 6-8.5 specifically 7.5.

4. The process, wherein temperature of the reaction is 0 to 40 °C specifically 20-30 °C.

5. The process, wherein the enzyme is endopeptidase, particularly protease.

6. The process, wherein the enzyme is isolated from bacteria, particularly Bacillus species.

7. The process, wherein enzyme from wild type microorganisms, whole broth, isolated, semi-purified, purified, immobilized and or recombinant enzymes.

8. The process as claimed in claim 1, wherein the enantiomeric purity of compounds of formula A obtained is in the range of 95 to >99%, specifically 99.99 % e.e.

9. The process, wherein the biotransformation is carried out in suitable bioreactors and the reaction mass was collected from the outlet after completion of the reaction in the range of 4-24 h.

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