Process for preparing amino phenyl compounds

Abstract

The instant invention is directed to an efficient and scalable process for synthesizing Compound 5, (aminomethyl phenylalanine), or a salt thereof. The process utilizes an engineered PAL enzyme to prepare Compound 4 via an enantioselective hydroamination of readily accessible 3-cyanocinnamic acid, which is optimal for industrial scale manufacturing. In the instant invention, Compound 4 then undergoes a hydrogenation step to make Compound 5, or a salt thereof.

Description

PROCESS FOR PREPARING AMINO PHENYL COMPOUNDSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 639,279, filed April 26. 2024, U.S. Provisional Patent Application No. 63 / 754,902, filed February 6, 2025, and U.S. Provisional Patent Application No. 63 / 778,509, filed March 27, 2025, each of which is incorporated by reference in its entirety herein.REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

[0002] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. The XML file, created on September 4, 2024, is named 2593 l-WO-PCT SL. xml and is 60,862 bytes in size.BACKGROUND

[0003] Phenylalanine ammonia lyases (PALs) are enzymes that facilitate the conversion of L- phenylalanine into cinnamic acid through non-oxidative deamination, which plays a crucial role in the synthesis of plant phenylpropanoids. PALs can also perform the reverse reaction, adding ammonia to cinnamic acids to produce L-phenylalanine. This ability makes PALs an efficient method for generating chiral phenylalanine amino acids from economical starting materials, utilizing 100% atom economy and avoiding the need for external co-factors or recycling systems. However, the limited activity’ and low conversion rates of wild-type enzymes, when acting upon non-canonical substrates under conditions relevant to industrial applications, are unattractive and inefficient for large-scale synthesis of non-canonical phenylalanine analogs.

[0004] Existing literature describes the preparation of Compound 44through either enzymatic resolution of the corresponding racemic ester or N-acyl derivative such asor by palladium-catalyzed cross-coupling reactions involving organozinc reagents derived from alanine with ar l bromides (Knittel et al., 1990 Pept. Res. 3: 176-181; Sperl et al.. US Patent Publication US2006 / 0142305; Chien et al., 2018 J. Med. Chem. 61 :7358-7373.) However, these methods suffer from drawbacks such as high step count, low atom economy in the case of racemate resolution, or the requirement for expensive catalysts. Furthermore, approaches utilizing PAL enzy mes have employed whole cell biocatalysts at exceptionally high loadings, rendering them unsuitable for industrial-scale manufacturing (U.S. Patent No. 5,981,239; International Patent Application WO 02 / 16630).

[0005] Engineering of PALs has been demonstrated to improve various biocatalysts’ properties, including activity' and substrate selectivity7.SUMMARY

[0006] The instant invention is directed to an efficient and scalable process for synthesizingCompound 5,(aminomethyl phenylalanine), or a salt thereof. The process utilizes an engineered PAL enzyme from Planctomyces brasiliensis (PbPAL) to prepare Compound 4 via an enantioselective hydroamination of readily accessible 3-cyanocinnamic acid, which is optimal for industrial scale manufacturing. The enzy matic synthesis of Compound 4 utilizes readily available reagents and allows for substantial cost savings as it proceeds in a single, enantioselective step that avoidsracemate resolution. In the instant invention, Compound 4 then undergoes a hydrogenation step to make Compound 5 (aminomethyl phenylalanine), or a salt thereof. The instant invention avoids the challenges of Compound 5, or a salt thereof, being degraded while using a catalyst in the process. The instant invention is directed to a process for preparing Compound 5, or a salt thereof, that includes the advantages of chemoselectivity, chemospecificity, protecting group removal and step count reduction.

[0007] The present invention relates to the synthesis of Compound 5, or a salt thereof, comprising the steps of:

[0008] The processes of the present invention afford advantages over previously know n procedures and include a more efficient route for preparing Compound 5, or a salt thereof, as well as intermediates useful in the preparation of Compound 5. or a salt thereof.

[0009] Other embodiments, aspects and features of the present invention are either further described in or will be apparent from the ensuing description, examples and appended claims.

[0010] The summary of the technology' described above is non-limiting and other features and advantages of the technology will be apparent from the following detailed description, and from the claims.DETAILED DESCRIPTION

[0011] The present disclosure provides enzymatic processes for preparing intermediates used in the manufacturing of Compound 5, or a salt thereof.

[0012] In an embodiment, the process for preparing Compound 55, or a salt thereof, comprises the steps of: a) combining Compound 33 and a PAL enzyme and an ammonium salt to produce Compound 44: and b) combining Compound 4, a Pd catalyst and hydrogen to obtain Compound 5, or a salt thereof.

[0013] In a second embodiment, the instant invention is directed to a process for preparingCompound 3, comprising the steps of: a) combining Compound 11 with Compound 2and an organic solvent to obtain a mixture, wherein the organic solvent is selected from acetonitrile, tetrahydrofuran, propionitrile, dimethyl formamide, and dimethyl acetonitrile; b) adding an amine reagent, wherein the amine reagent is selected from morpholine, pyridine and pyrrolidine; c) heating the mixture to above 65 °C to obtain a first slurry: d) cooling the first slurry to below 30 °C; and e) filtering the first slurry to obtain Compound 3.In a further embodiment of the second embodiment, wherein the mixture of step c is heated to about 75 to about 80°C to obtain a first slurry.

[0014] In a third embodiment, the instant invention is directed to a process for preparing Compound 4, comprising the steps of: a) mixing an ammonium salt and water to create an ammonium buffer solution; b) adding a base to adjust the pH of the ammonium buffer solution to about 8 to about 11 ; c) combining the ammonium buffer solution with a PAL enzyme and Compound 3 to create a resulting solution; d) adding an acid to the resulting solution to adjust the pH to about 6 to about 8 to obtain a second slurry; and e) filtering the second slurry to obtain Compound 4.

[0015] In a further embodiment of the third embodiment, the PAL enzyme is any one of SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, or 20. In a further embodiment, the PAL enzyme is selectedfrom SEQ ID NO: 8. 14. 16. 18 or 20. In a further embodiment, the PAL enzyme is selected from SEQ ID NO: 1 , 18 or 20. In an embodiment, the PAL enzyme is SEQ ID NO: 16. In an embodiment, the PAL enzyme is SEQ ID NO: 18. In an embodiment, the PAL enzy me is SEQ ID NO: 20.

[0016] In a further embodiment of the third embodiment, the process further comprises stirring the resulting solution of step c for about 12 to about 24 hours.

[0017] In a fourth embodiment, the instant invention is directed to a process for preparing Compound 5, or a salt thereof, comprising the steps of: a) combining Compound 4, a polar solvent, a reagent acid and a Pd catalyst to produce a reaction mixture; b) reacting the reaction mixture with hydrogen to obtain a mixture containing Compound 5; c) filtering the mixture containing Compound 5 to obtain a solution containing Compound 5; and e) isolating Compound 5 or a salt thereof.

[0018] In a further embodiment, the process for preparing the mixture containing Compound 5 in step b is performed in a reactor, wherein the reactor is selected from a sealed reactor or a continuous reaction system.

[0019] In an aspect of the embodiments, the process for preparing the mixture containing Compound 5 in step b is performed in a sealed reactor at about 100 to about 600 psi and a temperature of about 25 to about 50 °C. In a further embodiment, the process for preparing the mixture containing Compound 5 in step b is performed in a sealed reactor at about 400 psi and about 25 °C.

[0020] In an aspect of the embodiments, the process for preparing the mixture containing Compound 5 in step b is performed in a flow reactor at about 300 to about 600 psi and a temperature of about 40 to about 85 °C. In an aspect, the process for preparing the mixture containing Compound 5 in step b is performed in a flow reactor at about 400 psi and a temperature of about 50 to about 65 °C. In a further embodiment, the process for preparing the mixture containing Compound 5 in step b is performed in a flow reactor at about 400 psi and about 56 °C.

[0021] In a fifth embodiment, the instant invention is directed to a process for preparing Compound 5Acomprising the steps of: a) combining Compound 4, a polar solvent, a reagent acid and a Pd / carbon catalyst to obtain a reaction mixture, wherein the polar solvent is selected from a mixture of isopropanol and water or a mixture of tetrahydrofuran and water, and the reagent acid is hydrochloric acid; b) reacting the reaction mixture with hydrogen to obtain a mixture containing Compound 5; c) filtering the mixture containing Compound 5 to obtain a solution containing Compound 5; d) distilling the solution containing Compound 5 with isopropanol to obtain a third slurry; and e) filtering the third slurry and isolating Compound 5A.

[0022] In a further embodiment, the process for preparing Compound 5, or Compound 5A, further comprises adding an anti-solvent to the third slurry’ obtained in step d prior to filtering in step e.

[0023] In a further embodiment, the process for preparing the mixture containing Compound 5 in step b is performed in a reactor, wherein the reactor is selected from a sealed reactor or a continuous reaction system. In the embodiments of the instant invention, the processes of the disclosure may be conducted in a single vessel, as a “one-pof?process, or the steps may be conducted sequentially. For clarity, it should be noted that steps and reactions of the instant invention may occur simultaneously, or sequentially, unless otherwise specifically designated. In embodiments, the intermediate products may optionally be isolated.

[0024] In certain aspects of the embodiments, after filtering the third slurry in step e above, the crystalline form of Compound 5, or salt thereof, is isolated.

[0025] In certain aspects of the embodiments, after filtering the third slurry in step e above, the cry stalline form of Compound 5A is isolated.

[0026] In certain aspects, the third slurry can also be referred to as the final slurry'.

[0027] It should also be noted that when a term is used more than once, such as base or acid, the definition at each instance is independent of a prior selection. For example, the same, or a different, alcohol may be chosen for each step of the process independently of a previous selection.Definitions

[0028] Listed below are definitions of various terms used herein. These definitions apply to the terms as they are used throughout this specification and claims, unless otherwise limited in specific instances, either individually or as part of a larger group.

[0029] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Generally, the nomenclature used herein and the laboratory procedures in cell culture, molecular genetics, organic chemistry, and peptide chemistry are those well-known and commonly employed in the art.

[0030] As used herein, the articles “a” and “an” refer to one or to more than one (i. e.. to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element. Furthermore, use of the term “including” as well as other forms, such as “include,” “includes,” and “included,” is not limiting. As used herein, including the appended claims, the singular forms of words such as “a,” “an,” and “the,” include their corresponding plural references unless the context clearly dictates otherwise.

[0031] As used herein, the terms “at least one” item or “one or more” item each include a single item selected from the list as well as mixtures of two or more items selected from the list.

[0032] All ranges disclosed herein are inclusive of the recited endpoint and independently combinable (for example, the range of “from 50 mg to 500 mg” is inclusive of the endpoints, 50 mg and 500 mg, and all the intermediate values). The endpoints of the ranges and any values disclosed herein are not limited to the precise range or value; they are sufficiently imprecise to include values approximating these ranges and / or values. As an example, temperature ranges, percentages, ranges of equivalents, and the like described herein include the upper and lower limits of the range and any value in the continuum there between. “About” when used to modify a numerically defined parameter (e g., the temperature, or the length of time for a reaction, as described herein) means that the parameter may vary by as much as 10% below or above the stated numerical value for that parameter; where appropriate, the stated parameter may be rounded to the nearest whole number. For example, a temperature of about 30°C may vary between 25°C and 35°C. In addition, the term “or,” as used herein, denotes alternatives that may, where appropriate, be combined; that is, the term “or” includes each listed alternative separately.

[0033] As used herein, the term “comprising” may include the embodiments "consisting of’ and “consisting essentially of.” The terms “comprise(s),” “include(s),” “having,” “has,” “may,” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that require the presence of the named ingredients / steps and permit the presence of other ingredients / steps. However, such description should be construed as also describing compositions or processes as “consisting of’ and “consisting essentially of’ the enumerated components, which allows the presence of only the named components or compounds, along with any acceptable carriers or fluids, and excludes other components or compounds.

[0034] For use in medicine, the salts of the compounds described herein may be pharmaceutically acceptable salts. Other salts may, however, be useful in the preparation of the compounds or their pharmaceutically acceptable salts, according to the invention. The instant invention may use salts of primary, secondary and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as arginine, betaine caffeine, choline, N.N'-dibenzylethylenediamine. diethylamine, 2- diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine, ethylenediamine, N- ethylmorpholine. N-ethylpiperidine, glucamine, glucosamine, histidine, hydrabamine, isopropylamine, lysine, methylglucamine, morpholine, piperazine, piperidine, polyamine resins, procaine, purines, theobromine, triethylamine, trimethylamine tripropylamine, tromethamine and the like.

[0035] Salts of the invention may also be prepared using acids such acetic, benzenesulfonic, benzoic, camphorsulfonic, citric, ethanesulfonic, fumaric, gluconic, glutamic, hydrobromic, hydrochloric, isethionic, lactic, maleic, malic, mandelic, methanesulfonic, mucic, nitric, pamoic. pantothenic, phosphoric, succinic, sulfuric, tartaric, p-toluenesulfonic acid and the like. Additional examples of such acids include aryl sulfonic acids, such as but not limited to p- toluenesulfonic acid, 3-methyl-toluenesulfonic acid, 2-methyl-toluenesulfonic acid, benzenesulfonic acid, 2-naphthalene sulfonic acid, 2,6-naphtalene sulfonic acid, as well as hydrochloric acid, hydrobromic acid, sulfuric acid, acetic acid, phenyl acetic acid, trimethylacetic acid, tetrafluoroboric acid, tetraphenylboric acid, maleic acid, fumaric acid, oxalic acid, or camphorsulfonic acid. Specific examples are -toluenesulfonic acid, hydrochloric acid, benzoic acid, oxalic acid and the like. In some embodiments, the acid used may be hydrochloric acid.

[0036] The preparation of the pharmaceutically acceptable salts and other typical pharmaceutically acceptable salts is more fully described by Berg et al., “Pharmaceutical Salts,” J. Pharm. Set., 1977:66: 1-19.

[0037] As used herein, the terms “PAL enzyme’7or “PAL polypeptide” refer to a polypeptide having a capability of catalyzing the non-oxidative deamination of L-phenylalanine to generate a cinnamic acid and / or catalyzing hydroamination of a cinnamic acid to generate a L- phenylalanine. For example, the PAL enzy mes disclosed herein are capable of converting heterocyclic cinnamic acids (e.g., 3-cyanocinnamic acid) into 3-cyano-L-phenylalanine. PAL enzymes or PAL polypetptides as used herein includes non-naturally occurring engineered polypeptides generated by human manipulation. In an embodiment, a PAL enzyme as disclosed herein may have an amino acid sequence selected from any one of SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 23, 24, 25, 26, 27, 28, 29, 30, and 31. In an embodiment, a PAL enzyme as disclosed herein may have an ammo acid sequence selected from any one of SEQ ID NO: 8, 14, 16, 18 and 20. In an embodiment, a PAL enzyme is SEQ ID NO: 8. In an embodiment, a PAL enzyme is SEQ ID NO: 14. In an embodiment, a PAL enzyme is SEQ ID NO: 16. In an embodiment, a PAL enzyme is SEQ ID NO: 18. In an embodiment, a PAL enzyme is SEQ ID NO: 20. A patent application directed to the evolved PAL enzymes, USSN 63 / 639340. was filed contemporaneously on April 26, 2024, and is herein incorporated by reference in its entirety.

[0038] For example, the PbPALRSBB enzyme of SEQ ID NO: 18 was obtained by artificially evolving over multiple generations the gene encoding the wild-type PbPAL enzyme of SEQ ID NO: 2.

[0039] The terms “engineered,” “recombinant,” “variant,” and “non-naturally occurring,” when used with reference to, e.g., a polynucleotide, polypeptide, or cell, refers to a material, or a material corresponding to the natural or native form of the material, or that has been modified in a manner that would not otherwise exist in nature. Non-limiting examples include, among others, recombinant cells expressing genes that are not found within the native (non-recombinant) form of the cell or express native genes that are otherwise expressed at a different level.

[0040] In an embodiment, the Pd catalyst is selected from palladium on carbon catalyst, palladium on silica catalyst, palladium on alumina catalyst, palladium hydroxide on carbon catalyst and the like. In a further embodiment, the Pd catalyst is palladium on carbon (Pd / carbon) catalyst. In a further embodiment, the Pd catalyst is palladium on silica catalyst.

[0041] In an embodiment, the organic solvent is selected from acetonitrile, tetrahydrofuran, , propionitrile, dimethyl formamide, dimethyl acetonitrile and the like. In an embodiment, the organic solvent is acetonitrile.

[0042] As used herein, the term “ammonium salt” refers to a salt of ammonia and an acid. In an embodiment, the ammonium salt is selected from ammonium carbonate, ammonium sulfate, or ammonium carbamate. In an embodiment, the ammonium salt is selected from ammoniumcarbonate or ammonium carbamate. As used herein, a buffer solution refers to a solution which, when added to a liquid mixture, functions to maintain the pH of the liquid mixture at a consistent value.

[0043] In embodiments of the invention, the amine reagent is selected from morpholine, pyridine, pyrrolidine and the like. In a further embodiment, the amine reagent is morpholine.

[0044] As used herein, the term ‘'base” refers to a chemical used to raise pH. In an embodiment, the base is selected from ammonium hydroxide, sodium hydroxide, potassium hydroxide and the like. In an embodiment, the base is ammonium hydroxide.

[0045] As used herein, the term ■acid'’ refers to a chemical used to lower pH. In embodiment, the acid is selected from hydrochloric acid, phosphoric acid, sulfuric acid. In embodiment, the acid is sulfuric acid.

[0046] As used herein, the term “polar solvent” refers to a water-soluble solvent. In an embodiment, the polar solvent is selected from alcohol, water, tetrahydrofuran and mixtures thereof. In an embodiment, the alcohol is selected methanol, ethanol, propanol, isopropanol, and the like. In an embodiment, the polar solvent is mixture of isopropanol and water or a mixture of tetrahydrofuran and water.

[0047] As used herein, the term “reagent acid” refers to a reagent that facilitates a chemical reaction at low pH. In an embodiment, the reagent acid is selected from hydrochloric acid, acetic acid, methane sulfonic acid, trifluoroacetic acid, phosphoric acid or p-toluenesulfonic acid. In an embodiment, the reagent acid is hydrochloric acid.

[0048] As used herein, an antisolvent refers to a water-miscible solvent that reduces the solubility of the solute. In embodiments of this invention, the antisolvent is selected from isopropanol, tetrahydrofuran, acetonitrile or 1.2-dimethoxy ethane, and the like. In an embodiment, the antisolvent is isopropanol.

[0049] In embodiments of the invention, a reactor is used. The reactor may be a sealed reactor or continuous reaction system, such a flow- reactor. Examples of continuous reaction systems include, but are not limited to, packed-bed reactors (PBRs). fixed bed reactors, moving bed reactors, rotating bed reactors, fluidized bed reactors, slurry reactors, batch stirred tank reactors, continuous stirred tank reactors, membrane reactors, tube-in-tube reactors, monolith reactors, microstructured reactors, fluidized bed reactors and the like.

[0050] As used herein, a sealed reactor refers to a reaction vessel in which one can conduct chemical reactions under pressure such as an autoclave reactor. A reactor capable of controlling the reaction pressure includes but is not limited to a reactor equipped with a pressure control valve, or a manual or an automatic backpressure regulator.

[0051] Those skilled in the art will recognize that the reaction pressure is depended on the vessel fill, the volume of the reactor occupied by the reaction mixture, in the reactor.

[0052] As used herein, “polynucleotide’' and “nucleic acid’ refer interchangeably to two or more nucleotides that are covalently linked together. The polynucleotide may be wholly comprised of ribonucleotides (i.e.. RNA), wholly comprised of 2' deoxyribonucleotides (i.e.. DNA), or comprised of mixtures of ribo- and 2' deoxyribonucleotides. While the nucleosides will typically be linked together via standard phosphodiester linkages, the polynucleotides may include one or more non-standard linkages. The polynucleotide may be single-stranded or double-stranded, or the polynucleotide may include both single-stranded regions and doublestranded regions. Moreover, while a polynucleotide will typically be composed of the naturally occurring encoding nucleobases (i.e., adenine, guanine, uracil, thymine, and cytosine), it may include one or more modified and / or synthetic nucleobases, such as, for example, inosine, xanthine, hypoxanthine, etc. In some embodiments, such modified or synthetic nucleobases are nucleobases encoding amino acid sequences.

[0053] The abbreviations used for the genetically encoding nucleosides are conventional and are as follows: adenosine (A); guanosine (G); cytidine (C); thymidine (T); and uridine (U). Unless specifically delineated, the abbreviated nucleosides may be either ribonucleosides or 2'- deoxyribonucleosides. The nucleosides may be specified as being either ribonucleosides or 2'- deoxyribonucleosides on an individual basis or on an aggregate basis. When nucleic acid sequences are presented as a string of one-letter abbreviations, the sequences are presented in the 5' to 3' direction in accordance with common convention, and the phosphates are not indicated.

[0054] As used herein, the terms “protein,” “polypeptide,” and “peptide” are used interchangeably herein to denote a polymer of at least two amino acids covalently linked by an amide bond, regardless of length or post-translational modification (e.g., glycosylation, phosphorylation, lipidation, myristoylation, ubiquitination, and the like). Included within this definition are D- and L-amino acids, and mixtures of D- and L-amino acids, as well as polymers comprising D- and L-amino acids, and mixtures of D- and L-amino acids. Proteins, polypeptides, and peptides may include a tag (e g., an epitope tag), such as a histidine tag.

[0055] As used herein, the terms “amino acid” or “residue” as used in context of the polypeptides disclosed herein refers to the specific monomer at a sequence position. Amino acids are referred to herein by either their commonly known three-letter symbols or by the one-letter symbols recommended by International Union of Pure and Applied Chemistry (IUPAC) - International Union of Biochemistry (IUB) Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single letter codes.

[0056] The abbreviations used for the genetically encoded amino acids are conventional and are as follows: alanine (Ala or A), arginine (Arg or R), asparagine (Asn or N), aspartate (Asp or D), cysteine (Cys or C), glutamate (Glu or E), glutamine (Gin or Q), glycine (Gly or G), histidine (His or H), isoleucine (He or I), leucine (Leu or L), lysine (Lys or K), methionine (Met or M), phenylalanine (Phe or F), proline (Pro or P). serine (Ser or S), threonine (Thr or T), tryptophan (Trp or W), tyrosine (Tyr or Y), and valine (Vai or V).PAL Polypeptides

[0057] This disclosure uses PAL enzymes or PAL polypeptides capable of converting heterocyclic cinnamic acids (e.g., 3-cyanocinnamic acid) into 3-cyano-L-phenylalanine.

[0058] In certain embodiments, a PAL enzyme described herein has an amino acid sequence comprising one or more amino acid differences as compared to a reference amino acid sequence of a wild-type polypeptide that result in an improved enzyme property.

[0059] In some embodiments, a PAL enzyme as described herein is a product of directed evolution from a wild-type sequence (e.g., a wild-type PAL polypeptide as set forth in SEQ ID NO: 2, which includes an N-terminal His tag or a wild-type PAL polypeptide as set forth in SEQ ID NO: 21. which lacks an N-terminal His tag).

[0060] In some embodiments, the PAL enzyme may demonstrate one or more improvements relative the polypeptide of SEQ ID NO: 2 or SEQ ID NO: 21, such as increases in enzyme activity, stereoselectivity (e.g., enantioselectivity), stereospecificity, thermostability, stability under high ammonia concentration, and / or soluble expression (e.g., increased recombinant / soluble expression in E. coll).

[0061] For example, in some embodiments, the PAL enzyme has one or more of the following properties relative to a reference polypeptide: a) increased activity in converting 3- cyanocinnamic acid into 3-cyano-L-phenylalanine, optionally wherein the polypeptide has at least about 1.1-fold, about 5-fold, about 10-fold, or about 100-fold increased activity; b) increased enantioselectivity; c) increased thermostability; and / or d) increased soluble expression. In some embodiments, the polypeptide r about 100-fold increased activity. In some embodiments, the reference polypeptide comprises the amino acid sequence of SEQ ID NO: 2 or 21.

[0062] In some embodiments, such PAL enzymes are capable of converting the substrate to the product with a percent enantiomeric excess of at least about 60% (e.g., 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher). In some embodiments, such polypeptides are capable of converting the substrate to the product with a percent enantiomericexcess of at least 90%. In some embodiments, such polypeptides are capable of converting the substrate to the product with a percent enantiomeric excess of at least 91%. In some embodiments, such polypeptides are capable of converting the substrate to the product with a percent enantiomeric excess of at least 92%. In some embodiments, such polypeptides are capable of converting the substrate to the product with a percent enantiomeric excess of at least 93%. In some embodiments, such polypeptides are capable of converting the substrate to the product with a percent enantiomeric excess of at least 94%. In some embodiments, such polypeptides are capable of converting the substrate to the product with a percent enantiomeric excess of at least 95%. In some embodiments, such polypeptides are capable of converting the substrate to the product with a percent enantiomeric excess of at least 96%. In some embodiments, such polypeptides are capable of converting the substrate to the product with a percent enantiomeric excess of at least 97%. In some embodiments, such polypeptides are capable of converting the substrate to the product with a percent enantiomeric excess of at least 98%. In some embodiments, such polypeptides are also capable of converting the substrate to the product with a percent enantiomeric excess of at least about 99%.

[0063] In some embodiments, the polypeptide (e.g., the PAL polypeptide) is highly enantioselective, wherein the polypeptide can reduce the substrate to the product in greater than about 99%. 99.1%. 99.2%. 99.3%. 99.4%. 99.5%. 99.6%. 99.7%. 99.8%. or 99.9% enantiomeric excess.

[0064] In some embodiments, a PAL enzy me is a polypeptide that comprises an amino acid sequence having at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%. 95%, 96%. 97%. 98%. 99%. or 100%) sequence identity to SEQ ID NO: 2. In other embodiments, an improved PAL enzyme is a polypeptide that comprises an amino acid sequence having at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to SEQ ID NO: 21.

[0065] These differences between these variants and SEQ ID NO:2 or SEQ ID NO: 21 can be amino acid insertions, deletions, substitutions, or any combinations of such changes. In some embodiments, the amino acid sequence differences can comprise non-conservative, conservative, as well as a combination of non-conservative and conservative amino acid substitutions. In some embodiments, the amino acid sequence differences are conservative amino acid substitutions. In other embodiments, the amino acid sequence differences are non-conservative amino acid substitutions.

[0066] In some embodiments, the PAL enzyme further comprises a tag (e.g., an epitope tag). Any suitable tag may be used, e.g., a His tag, a FLAG tag, a fluorescent protein tag (e g., green fluorescent protein (GFP), yellow fluorescent protein (YFP), or red fluorescent protein (RFP)), a hemagglutinin (HA) tag, an ALFA-tag, a V5-tag, a Myc-tag, a SPOT-tag, a T7-tag, or an NE-tag. In some embodiments, the epitope tag is a His tag. In some embodiments, the His tag comprises the amino acid sequence of GSHHHHHHHHSG (SEQ ID NO: 22). In some embodiments, the polypeptide comprises an N-terminal methionine residue, and the epitope tag is inserted immediately following the N-terminal methionine residue, e.g., relative to a reference sequence.

[0067] In another example, provided herein is a PAL enzyme comprising an amino acid sequence having at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to SEQ ID NO: 2, wherein the PAL enzy me comprises at least one substitution at one or more positions selected from 57, 70. 75. 94, 102, 227, 288, 294, 296, 306, 310. 357, 360, 485, 510, and 534, wherein the amino acid positions of the PAL enzyme are numbered with reference to SEQ ID NO: 2

[0068] In some embodiments, the PAL enzy me comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 substitutions at two or more positions selected from 57, 70, 75, 94, 102, 227, 288, 294, 296. 306, 310, 357, 360, 485. 510, and 534, wherein the amino acid positions of the PAL enzyme are numbered with reference to SEQ ID NO: 2.

[0069] In some embodiments, the PAL enzy me comprises: a) a substitution set at positions 57, 75, 94, 102, 227, 288, 360, 485, 510, and 534; b) a substitution at position 102; c) a substitution at position 306; d) a substitution set at positions 94, 102, 288. and 485; e) a substitution set at positions 57. 94. 102, 288. and 485; f) a substitution set at positions 94. 102. 288, and 485; g) a substitution set at positions 57, 75, 94, 102, 288, 360, 485, and 510; h) a substitution set at positions 57, 94, 102, 288, 310, 357, and 485; or i) a substitution set at positions 57, 70, 75, 94, 102, 227. 288, 294, 296, 360, 485, 510, and 534, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 2. In some embodiments, the PAL enzyme comprises a substitution set at positions 57, 75, 94, 102, 227, 288, 360, 485, 510, and 534, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 2. In some embodiments, the PAL enzyme comprises a substitution at position 102, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 2. In some embodiments, the PAL enzyme comprises a substitution at position 306, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 2. In some embodiments, the PAL enzyme comprises a substitution set at positions 94, 102, 288, and 485,wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 2. In some embodiments, the PAL enzyme comprises a substitution set at positions 57, 94, 102, 288, and 485, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 2. In some embodiments, the PAL enzyme comprises a substitution set at positions 94, 102, 288. and 485, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 2. In some embodiments, the PAL enzyme comprises a substitution set at positions 57, 75, 94, 102, 288, 360, 485, and 510, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 2. In some embodiments, the PAL enzyme comprises a substitution set at positions 57, 94, 102, 288, 310, 357, and 485, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 2. In some embodiments, the PAL enzyme comprises a substitution set at positions 57, 70, 75, 94, 102, 227, 288, 294, 296, 360, 485, 510, and 534, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 2.

[0070] In some embodiments, the PAL enzyme comprises one of the following substitutions or substitution sets: a) R57K, I75V, H94M, L102A, S227A, Y288V, M360T, A485R, E510G, and M534V; b) L102V; c) L306N; d) H94M, L102A, Y288V, and A485R; e) R57K, H94M, L102A, Y288P, and A485R; f) H94M, L102A, Y288P, and A485L; g) R57K, I75V, H94M, L102A, Y288V, M360T, A485R, and E510G; h) R57K. H94M. L102A, Y288V, A310P, Y357S. and A485R; or i) R57K, R70D, I75V, H94M, L102A, S227A, Y288V, K294T, N296D, M360T, A485R, E510G, and M534T, wherein the amino acid positions of the PAL enzyme are numbered with reference to SEQ ID NO: 2. In some embodiments, the polypeptide comprises R57K, I75V, H94M, L102A, S227A, Y288V, M360T, A485R, E510G. and M534V substitutions, wherein the amino acid positions of the PAL enzyme are numbered with reference to SEQ ID NO: 2. In some embodiments, the PAL enzyme comprises an LI 02V substitution, wherein the amino acid positions of the PAL enzyme are numbered with reference to SEQ ID NO: 2. In some embodiments, the PAL enzyme comprises an L306N substitution, wherein the amino acid positions of the PAL enzyme are numbered with reference to SEQ ID NO: 2. In some embodiments, the PAL enzyme comprises H94M, L102A, Y288V, and A485R substitutions, wherein the amino acid positions of the PAL enzy me are numbered with reference to SEQ ID NO: 2. In some embodiments, the PAL enzyme comprises R57K, H94M, L102A, Y288P, and A485R substitutions, wherein the amino acid positions of the PAL enzyme are numbered with reference to SEQ ID NO: 2. In some embodiments, the PAL enzyme comprises H94M, L102A, Y288P, and A485L substitutions, wherein the amino acid positions of the PAL enzyme are numbered with reference to SEQ ID NO: 2. In some embodiments, the PAL enzyme comprisesR57K, I75V. H94M. L102A, Y288V, M360T, A485R, and E510G substitutions, wherein the amino acid positions of the PAL enzyme are numbered with reference to SEQ ID NO: 2. In some embodiments, the PAL enzyme comprises R57K, H94M, L102A, Y288V, A310P, Y357S, and A485R substitutions, wherein the amino acid positions of the PAL enzy me are numbered with reference to SEQ ID NO: 2. In some embodiments, the PAL enzyme comprises R57K. R70D, I75V, H94M, L102A, S227A, Y288V, K294T, N296D, M360T, A485R, E510G, and M534T substitutions, wherein the amino acid positions of the PAL enzyme are numbered with reference to SEQ ID NO: 2.

[0071] Provided herein is a PAL enzyme comprising an amino acid sequence comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity to SEQ ID NO: 18. For example, provided herein is a PAL enzy me comprising an amino acid sequence comprising at least 99% sequence identity to SEQ ID NO: 18. For example, provided herein is a PAL enzyme comprising the amino acid sequence of SEQ ID NO: 18. Also provided herein is a PAL enzyme consisting essentially of the amino acid sequence of SEQ ID NO: 18. Also provided herein is a PAL enzyme consisting of the amino acid sequence of SEQ ID NO: 18.

[0072] Provided herein is a PAL enzyme comprising an amino acid sequence comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity to SEQ ID NO: 4. For example, provided herein is a PAL enzyme comprising an amino acid sequence comprising at least 99% sequence identity to SEQ ID NO: 4. In another example, provided herein is a PAL enzyme comprising the amino acid sequence of SEQ ID NO: 4. Also provided herein is a PAL enzyme consisting essentially of the amino acid sequence of SEQ ID NO: 4. Also provided herein is a PAL enzyme consisting of the amino acid sequence of SEQ ID NO: 4.

[0073] Provided herein is a PAL enzyme comprising an amino acid sequence comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity7to SEQ ID NO: 6. For example, provided herein is a PAL enzy me comprising an amino acid sequence comprising at least 99% sequence identity to SEQ ID NO: 6. In another example, provided herein is a PAL enzyme comprising the amino acid sequence of SEQ ID NO: 6. Also provided herein is a PAL enzy me consisting essentially of the amino acid sequence of SEQ ID NO: 6. Also provided herein is a PAL enzy me consisting of the amino acid sequence of SEQ ID NO: 6.

[0074] Provided herein is a PAL enzyme comprising an amino acid sequence comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity7to SEQ ID NO: 8. For example, provided herein is a PAL enzy me comprising an amino acidsequence comprising at least 99% sequence identity to SEQ ID NO: 8. In another example, provided herein is a PAL enzyme comprising the amino acid sequence of SEQ ID NO: 8. Also provided herein is a PAL enzy me consisting essentially of the amino acid sequence of SEQ ID NO: 8. Also provided herein is a PAL enzyme consisting of the amino acid sequence of SEQ ID NO: 8.

[0075] Provided herein is a PAL enzyme comprising an amino acid sequence comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity to SEQ ID NO: 10. For example, provided herein is a PAL enzyme comprising an amino acid sequence comprising at least 99% sequence identity to SEQ ID NO: 10. In another example, provided herein is a PAL enzyme comprising the amino acid sequence of SEQ ID NO: 10. Also provided herein is a PAL enzyme consisting essentially of the amino acid sequence of SEQ ID NO: 10. Also provided herein is a PAL enzy me consisting of the amino acid sequence of SEQ ID NO: 10.

[0076] Provided herein is a PAL enzyme comprising an amino acid sequence comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity to SEQ ID NO: 12. For example, provided herein is a PAL enzy me comprising an amino acid sequence comprising at least 99% sequence identity to SEQ ID NO: 12. In another example, provided herein is a PAL enzyme comprising the amino acid sequence of SEQ ID NO: 12. Also provided herein is a PAL enzyme consisting essentially of the amino acid sequence of SEQ ID NO: 12. Also provided herein is a PAL enzy me consisting of the amino acid sequence of SEQ ID NO: 12.

[0077] Provided herein is a PAL enzyme) comprising an amino acid sequence comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity to SEQ ID NO: 14. For example, provided herein is a PAL enzyme comprising an amino acid sequence comprising at least 99% sequence identity' to SEQ ID NO: 14. In another example, provided herein is a PAL enzyme comprising the amino acid sequence of SEQ ID NO: 14. Also provided herein is a PAL enzyme consisting essentially of the amino acid sequence of SEQ ID NO: 14. Also provided herein is a PAL enzy me consisting of the amino acid sequence of SEQ ID NO: 14.

[0078] Provided herein is a PAL enzyme comprising an amino acid sequence comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity to SEQ ID NO: 16. For example, provided herein is a PAL enzyme comprising an amino acid sequence comprising at least 99% sequence identity' to SEQ ID NO: 16. In another example, provided herein is a PAL enzyme comprising the amino acid sequence of SEQ ID NO: 16. Alsoprovided herein is a PAL enzyme consisting essentially of the amino acid sequence of SEQ ID NO: 16. Also provided herein is a PAL enzy me consisting of the amino acid sequence of SEQ ID NO: 16.

[0079] Provided herein is a PAL enzyme comprising at least 80% (e.g., 80%, 81%, 82%, 83%, 84%. 85%. 86%. 87%. 88%. 89%. 90%. 91%. 92%. 93%. 94%. 95%. 96%. 97%. 98%. 99%. or 100%) sequence identity to the polynucleotide sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, or 33. In some examples, the polynucleotide sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, or 19 is modified to no longer code for an N-terminal His tag having the amino acid sequence of SEQ ID NO: 22.

[0080] Exemplary methods and materials are described herein, although methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure. The materials, methods, and examples are illustrative only and not intended to be limiting. The transaminase enzymes were used as lyophilized cell-free lysate powders. Unless otherwise indicated, solvents and reagents were commercially’ available and were used as received.ABBREVIATIONSMEASUREMENTS: equiv. Equivalent mg Milligram min minutes h hours ml. mL MilliliterL LiterM MolarMHz Megahertz w / w% weight percent mmHg millimeters of Mercury psi pounds per square inchNMR nuclear magnetic resonanceTHF tetrahydrofuranTMS trimethylsilylTsOH p-toluenesulfonic acidAdditional abbreviations may be defined throughout this disclosure.EXAMPLES

[0081] The following examples are meant to be illustrative and should not be construed as further limiting. The contents of the figures and all references, patents, and published patent applications cited throughout this application are expressly incorporated herein by reference.EXAMPLE 1Preparation of CE)-3-Cyanocinnamic acid (Compound 3):

[0082] To a 2-L four-neck round botom flask equipped with a overhead stirrer was added 3- cyanobenzaldehyde (Compound l)(100.0 g, 1.0 equiv.), malonic acid (Compound 2)(159.0 g, 2.0 equiv.) and acetonitrile (500 mL). Morpholine (33.2 g, 0.5 equiv) was added and the mixture heated at 78 °C. After heating for 4.5 h the reaction mixture was cooled to 20-25°C and stirred for 2-3 h. The slurry was filtered and the solids washed with acetonitrile (250 mL). After vacuum drying under nitrogen at ambient temperature. Compound 3 was obtained.NMR (500 MHz, DMSO-de): 5 = 12.57 (s, 1H), 8.22 (d, J = 1.5 Hz, 1H), 8.09 - 7.96 (m, 1H), 7.85 (dt, J = 7.7, 1.2 Hz, 1H), 7.68 - 7.53 (m, 2H), 6.70 (d, J = 16.1 Hz, 1H).13C NMR (126 MHz, DMSO-de): 5 = 167.3, 141.6, 135.6, 133.3. 132.6, 131.8, 130.0, 121.8, 118.4, 112.2.EXAMPLE 2Preparation of L-3-cvanophenylalanine (Compound 4) with seeding

[0083] Ammonium carbonate (216 g), 28-30% aqueous ammonia (264 g) and water (720 g were mixed to create an ammonium carbonate pH 10 buffer solution. Part of this buffer solution(1 L) was added to a 2-Ljacketed reactor vessel. The enzyme having SEQ ID NO: 18 (4.0 g.4 w / w%) was added and the solution heated to 30°C. Next, ( / ’)-3-cyanocinnamic acid (Compound 3) (100.0 g, 1.0 equiv.) was added and the reaction mixture stirred for 21 h. After cooling to 20-25°C, Glanapon 2000 Konz anti-foaming agent (2 mL) was added and the reaction mixture slowly adjusted to pH 8.8 with 50% aqueous sulfuric acid (120 mL). Solid L-3- cy anophenylalanine (compound 4) (2.00 g, 2 w / w%) was added and the reaction mixture stirred for 1.5 h at 20-25 °C. The reaction mixture was further slowly adjusted to pH 6.9 with 50% aqoueous sulfuric acid (180 mL). The resulting slurry was subsequently cooled to 0 °C and stirred overnight. The slurry was filtered and the solids washed with 3: 1 (v / v) water / methanol (400 mL) and methanol (400 mL). After vacuum drying overnight under nitrogen at ambient temperature, Compound 4 was obtained.

[0084] XH NMR (500 MHz, D2O): 5 = 7.76 (d, J = 7.6 Hz, 1H), 7.72 (s, 1H), 7.65 (d, J= 7.9 Hz. 1H), 7.59 (t, J= 7.8 Hz, 1H), 4.07 - 3.98 (m, 1H), 3.34 (dd, J= 14.6. 5.7 Hz, 1H), 3.23 (dd, J = 14.6. 7.6 Hz. 1H).13C NMR (126 MHz. D2O): 5 = 173.4. 136.6, 134.5. 133.0, 131.7. 129.9, 119.5, 111.7, 55.7, 35.9.Preparation of L-3 -cyano phenylalanine (Compound 4) without seeding

[0085] Ammonium carbonate (72 g), 28-30% aqueous ammonia (100 mL) and water (240 g were mixed to create an ammonium carbonate pH 10 buffer solution. Part of the buffer solution (90 mL) were charged to a 500-mL four-neck round bottom flask together with ( / / )-3- cyanocinnamic acid (Compound 3) (10.0 g, 1.0 equiv.) and 28% aqueous ammonia (4 mL). A solution of enzyme having SEQ ID NO: 14 (0.50 g, 5 w / w%) in buffer solution (10 mL) was added and the resulting solution aged at 28-30 °C for 17.5 h. After cooling to 15-25 °C, Glanapon 2000 Konz anti-foaming agent (0.2 mL) was added and the reaction mixture slowly adjusted to pH 7.0 with 50% aqueous sulfuric acid. After stirring for 20 min, the resulting slurry7was cooled to 0 °C for 1.5 h and subsequently filtered. The solids were washed with 3: 1 water / methanol (75 mL) and vacuum dried under nitrogen to afford compound 4.EXAMPLE 3APreparation of L-3-(Aminornethyl)phenylalanine (Compound 5)4 5Batch procedure:

[0086] A 1-L pyrex bottle was charged with L-3-cyanophenylalanine (Compound 4) (34.00 g, 1.0 equiv.). isopropanol (245 mL). water (163 mL) and 12 M aqueous HC1 solution (29.6 mL), and stirred until all solids had dissolved. The obtained solution was transferred into a 1-L autoclave followed by adding 10% Pd / C catalyst (1.7 g). The autoclave was sealed and purged with nitrogen gas three times. Next, it was purged with hydrogen gas three times, the reaction pressure set to 400 psi and the temperature to 25 °C, before aging the reaction overnight. When the reaction was complete, the autoclave was vented and the atmosphere inerted by purging three times with nitrogen gas. Agitation was stopped and the solution discharged into a 1-L Pyrex bottle. The reaction mixture was then filtered with a funnel under vacuum to obtain a solution containing Compound 5.EXAMPLE 3BAlternate process for preparing Compound 5 via Flow procedure:

[0087] A 1-L round bottom flask was charged with L-3 -cyanophenylalanine (Compound 4) (42.00 g, 1 .0 equiv.) with isopropanol (302 mL) and water (201 mL). 12 M Aqueous HC1 solution (36.5 mL) was added, the mixture stirred until all solids had dissolved and the obtained solution transferred to a 500 mL-syringe pump. A 0.5-inch Hastelloy C column reactor (40 micron frit at bottom) was charged with P1241 5% palladium on carbon (3.5 g) and glass beads (1.0 g) addedon top of the catalyst. The flow system was purged with nitrogen gas for 5 min and then hydrogen gas at 237.5 mL / min. The flow reactor temperature was set at 56 °C, the pressure at 400 psi, before flowing with a flush solution for 30 min (302 mL isopropanol, 201 mL water, 18.3 mL 12 M aqueous HC1 solution). The flush stream was turned off and the L-3- cyanophenylalanine reaction stream started at 2.969 mL / min. The reaction stream was collected into 10-mL test tube and Compound 5-containing fractions were combined once all solution had been pumped through the flow reactor.EXAMPLE 4Isolation of Compound 5A:

[0088] The solution containing Compound 5 (as described in Example 3A or 3B) was transferred to a 500-mL jacketed reactor vessel fitted with a distillation apparatus. The reactor vessel was evacuated (100 mmHg) and heated to 65 °C. The reaction mixture was concentrated to approximately 200 mL. Then, the distillation was continued while continuously feeding in isopropanol to maintain a volume of 175-200 mL in the reactor. After feeding a total of 700- 750 mL isopropanol, the distillation was stopped and the solution cooled to 15-20 °C. Solid Compound 5 A (0.65 g) was added and the reaction mixture stirred for 15 min. THF (500 mL) was slowly added and the resulting slurry stirred for 1 h. After cooling to 0 °C and stirring for 2 h, the slurry was filtered and the solids washed with a 15:4: 1 (v / v / v) THF / isopropanol / water (200 mL). After vacuum drying under nitrogen overnight at ambient temperature, Compound 5A was obtained ’H NMR (500 MHz, D2O): 5 = 7.52 (t, J= 7.6 Hz, 1H), 7.45 (d, J= 7.9 Hz, 1H), 7.43 - 7.36 (m. 2H), 4.27 - 4.18 (m. 3H), 3.35 (dd, J= 14.5, 6.0 Hz, 1H), 3.29 (dd, J= 14.5. 7.1 Hz, 1H).13C NMR (126 MHz, D2O): 6 = 171.6, 135.2, 133.5, 130.2, 130.0, 129.8, 128.4, 54.3, 43.0, 35.6.Procedure for seed preparation for compound 5A:

[0089] A 500-mL round-botom flask was charged with (S)-2-((tert-butoxycarbonyl)amino)-3- (3-(((tert-butoxycarbonyl)amino)methyl)phenyl)propanoic acid (10.00 g, 25.4 mmol, 1.0 equiv.) and dichloromethane (100 mL). A 3 M solution of hydrochloric acid in cyclopentyl methyl ether (45 mL, 135 mmol, 5.3 equiv.) was added dropwise over 10 min. The reaction mixture was stirred at ambient temperature for 2 h, and additional 3 M solution of hydrochloric acid in cyclopentyl methyl ether (45 mL, 135 mmol, 5.3 equiv.) was added. The resulting slurry was aged for 5 h at ambient temperature, filtered and the solids were washed with MTBE. After vacuum dry ing under nitrogen overnight at ambient temperature, crude compound 5 was obtained. The obtained crude compound 5 (100 mg) was dissolved in 1: 1 EtOH / water (2 mL). THF was slowly added and the resulting solution aged overnight at 25 °C. The resulting slurry was filtered and the solids vacuum dried under nitrogen overnight at ambient temperature to obtain solid compound 5A.

[0090] It will be appreciated that various of the above-discussed and other features and functions, or alternatives thereof, may be desirably combined into many other different systems or applications. It will also be appreciated that various presently unforeseen or unanticipated alternatives, modifications, variations or improvements therein may be subsequently made by those skilled in the art which are also intended to be encompassed by the following claims.

[0091] The disclosed subject mater is not to be limited in scope by the specific embodiments and examples described herein. Indeed, various modifications of the disclosure in addition to those described will become apparent to those skilled in the art from the foregoing description and accompanying figures. Such modifications are intended to fall w thin the scope of the appended claims.

[0092] All references (e.g.. publications or patents or patent applications) cited herein are incorporated herein by reference in their entirety and for all purposes to the same extent as if each individual reference (e.g., publication or patent or patent application) was specifically and individually indicated to be incorporated by reference in its entirety for all purposes. Other embodiments are within the following claims.

Claims

WHAT IS CLAIMED IS:

1. A process for preparing Compound 5, or a salt thereof, comprising the steps of: a) combining Compound 3and a PAL enzy me and an ammonium salt to produce Compound 4b) combining Compound 4, a Pd catalyst and hydrogen to obtain Compound 5, or a salt thereof.

2. The process of Claim 1, further comprising the step of preparing Compound 4 by: a) mixing ammonium salt and water to create an ammonium buffer solution; b) adding a base to adjust the pH of the ammonium buffer solution to about 8 to about 11; c) combining the ammonium buffer solution with a PAL enzyme and Compound 3 to create a resulting solution;d) adding an acid to the resulting solution to adjust the pH to about 6 to about 8 to obtain a second slurry; and e) filtering the second slurry to obtain Compound 4.

3. The process of Claim 1 or 2. wherein the PAL enzyme is selected from one of SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, or 20.

4. The process of any one of Claim 1-3, wherein the PAL enzyme is SEQ ID NO: 8, 14, 16. 18, or 20.

5. The process of Claim 2, further comprising the steps of preparing Compound 3 by: a) combining Compound 11 with Compound 2and an organic solvent to obtain a mixture, wherein the organic solvent is selected from acetonitrile, tetrahydrofuran, propionitrile, dimethyl formamide, and dimethyl acetonitrile; b) adding an amine reagent, wherein the amine reagent is selected from morpholine, pyridine and pyrrolidine; c) heating the mixture to above 65 °C to obtain a first slurry; d) cooling the first slurry to below 30 °C; and e) filtering the first slurry to obtain Compound 3.

6. The process of Claim 5, wherein the mixture of step c is heated to about 75 to about 80°C to obtain a first slurry.

7. The process of Claim 2, further comprising the step of stirring the resulting solution of step c for about 12 to about 24 hours.

8. The process of Claim 1 for preparing Compound 5, or a salt thereof, comprising the steps of: a) combining Compound 4, a polar solvent, a reagent acid and a Pd catalyst to produce a reaction mixture; b) reacting the reaction mixture with hydrogen to obtain a mixture containing Compound 5; c) filtering the mixture containing Compound 5 to obtain a solution containing Compound 5; and e) isolating Compound 5 or a salt thereof.

9. The process of Claim 8, wherein step b is performed in a reactor, wherein the reactor is selected from a sealed reactor or a continuous reaction system.

10. The process of Claim 9, wherein step b is performed in a sealed reactor at about 400 psi and about 25 °C.

11. The process of Claim 9, wherein step b is performed in a flow reactor at about 400 psi and about 56 °C.

12. A process for preparing Compound 5 A,5A comprising the steps of:a) combining Compound 4a polar solvent, a reagent acid and a Pd / carbon catalyst to obtain a reaction mixture, wherein the polar solvent is selected from a mixture of isopropanol and water or a mixture of tetrahydrofuran and water, and the reagent acid is hydrochloric acid; b) reacting the reaction mixture with hydrogen to obtain a mixture containing Compound 5A; c) filtering the mixture containing Compound 5 to remove the Pd / carbon catalyst and obtain a solution containing Compound 5 A; d) distilling the solution containing Compound 5A with isopropanol to obtain a third slurry; and e) filtering the third slurry to obtain Compound 5A.

13. The process of Claim 12, further comprising the step of adding an anti-solvent to the third slurry obtained in step d prior to filtering in step e.

14. The process of Claim 12, wherein step b is performed in a reactor, wherein the reactor is selected from a sealed reactor or a continuous reaction system.

15. The process of Claim 14, wherein step b is performed in a sealed reactor at about 400 psi and about 25 °C.

16. The process of Claim 14, wherein step b is performed in a flow reactor at about 400 psi and about 56 °C.

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