Compositions, systems, and methods for amino acid synthesis

Abstract

A method of forming a non-canonical amino acid (ncAA) includes utilizing a substrate, an amino acid, an enzyme, actinic radiation, a photocatalyst, and an oxidant to form a non- canonical amino acid, wherein the enzyme includes one or more pyridoxal phosphate (PLP)- dependent enzymes or variants thereof. A method of forming a non-canonical amino acid (ncAA) includes utilizing an organoboron compound, an alpha amino acid, an enzyme, a photocatalyst, and an oxidant to form a sp3-sp3 coupled non-canonical amino acid product, wherein the oxidant includes at least one of a manganese-containing compound, an iron- containing compound, and a copper-containing compound.

Description

COMPOSITIONS, SYSTEMS, AND METHODS FOR AMINO ACID SYNTHESIS CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of and priority to U.S. Provisional Application 63 / 561,913, titled “Compositions, Systems and Methods for Amino Acid Synthesis using Engineered Alpha-Functionalization Pyridoxal Phosphate Dependent Enzymes”, filed March 6, 2024, the contents of which are incorporated by reference herein. STATEMENT OF GOVERNMENT RIGHTS

[0002] This invention was made with government support under GM147387 awarded by the National Institutes of Health. The government has certain rights in the invention. SEQUENCE LISTING

[0003] The instant application contains a Sequence Listing which has been submitted electronically in XML file format and is hereby incorporated by reference in its entirety. Said XML copy, created on January 22, 2025, is named 4059_064PCT1_SL.xml and is 32,712 bytes in size. TECHNICAL FIELD

[0004] The subject matter disclosed herein relates to biocatalytic reactions and enzymes generally, and more particularly to enzyme variants and methods for forming non-canonical amino acids. BACKGROUND

[0005] Capable of realizing chemically inaccessible reactivities and imposing exquisite stereocontrol, biocatalysis has recently emerged as a promising alternative to selective and sustainable chemical synthesis. Drawing inspirations from synthetic chemistry and small- molecule catalysis, biocatalysis researchers successfully repurposed and evolved natural metalloenzymes, and flavin- and nicotinamide-dependent enzymes to catalyze stereoselective unnatural reactions. Collectively, these efforts significantly expanded the catalytic repertoire of enzymes to encompass synthetically valuable reactions not previously known. To furtheradvance the field of biocatalysis, it is thus essential to develop synthetically useful enzymatic activation modes that are both new-to-chemistry and new-to-biology to form novel non- canonical amino acids. These new activation modes have not been previously accessible in conventional enzymology or synthetic chemistry. Nevertheless, despite the structural diversity and broad availability of nucleophilic agents, the oxidativecross- coupling of two distinct nucleophiles has previously been unknown. Therefore, it would be beneficial to provide oxidativecross-coupling of two distinct nucleophiles. SUMMARY

[0006] According to one aspect, a method of forming a non-canonical amino acid (ncAA) includes utilizing a substrate, an amino acid, an enzyme, actinic radiation, a photocatalyst, and an oxidant to form a non-canonical amino acid, wherein the enzyme includes one or more pyridoxal phosphate (PLP)-dependent enzymes or variants thereof.

[0007] According to another aspect, a pyridoxal phosphate (PLP)-dependent enzyme variant includes a variant of a threonine aldolase from Thermotoga maritima, the variant including one or more of the following mutations: E88T, H83F, and W86N.

[0008] According to another aspect, a method of forming a non-canonical amino acid (ncAA) includes utilizing an organoboron compound, an alpha amino acid, an enzyme, a photocatalyst, and an oxidant to form a sp3-sp3coupled non-canonical amino acid product, wherein the oxidant includes at least one of a manganese-containing compound, an iron- containing compound, and a copper-containing compound. BRIEF DESCRIPTION OF DRAWINGS

[0009] This written disclosure describes illustrative embodiments that are non-limiting and non-exhaustive. Reference is made to illustrative embodiments that are depicted in the figures, in which:

[0010] FIG.1 illustrates a method for forming an enzyme variant, according to some embodiments.

[0011] FIG.2 illustrates a method for forming a non-canonical amino acid, according to some embodiments.

[0012] FIG.3 illustrates a method for forming a non-canonical amino acid, according to some embodiments.

[0013] FIG.4A illustrates asymmetric sp3–sp3oxidative cross-coupling via cooperative photobiocatalysis, according to some embodiments.

[0014] FIG.4B illustrates triple photobiocatalytic cycles for sp3–sp3oxidative cross- coupling using an enzyme biocatalyst and a photocatalyst, according to some embodiments.

[0015] FIG.5A illustrates density functional theory (DFT) calculations using a theozyme model, according to some embodiments.

[0016] FIG.5B illustrates density functional theory (DFT) calculations using a theozyme model, according to some embodiments.

[0017] FIG.5C illustrates density functional theory (DFT) calculations using a theozyme model, according to some embodiments.

[0018] FIG.6A illustrates screening of target residues in the α-helix proximal to the PLP covalent intermediate, according to some embodiments.

[0019] FIG.6B illustrates a 96-position photoreactor, according to some embodiments.

[0020] FIG.6C illustrates a high-throughput photobiocatalysis setup, according to some embodiments.

[0021] FIG.6D illustrates asymmetric sp3-sp3oxidative coupling, according to some embodiments.

[0022] FIG.7A illustrates UV-vis spectroscopic analysis (TmPLP^1 pH 8), according to some embodiments.

[0023] FIG.7B illustrates UV-vis spectroscopic analysis (TmPLP^1 pH 9), according to some embodiments.

[0024] FIG.8A illustrates screening of residues, according to some embodiments.

[0025] FIG.8B illustrates diastereomer percentage for enzymes, according to some embodiments.

[0026] FIG.8C illustrates diastereo- and enantioselective sp3–sp3oxidative coupling: synthesis of non-canonical amino acids (ncAAs) with contiguous stereocenters, according to some embodiments.

[0027] FIG.8D illustrates X-ray structures for stereochemistry determination, according to some embodiments.

[0028] FIG.8E illustrates enantioselective synthesis of α-tetrasubstituted amino acids, according to some embodiments.

[0029] FIG.9A illustrates amino acids for Marfey’s analysis, according to some embodiments.

[0030] FIG.9B illustrates Marfey’s analysis with (R)- and (S)-FDAA, according to some embodiments.

[0031] FIG.9C illustrates Marfey’s analysis results, according to some embodiments. DETAILED DESCRIPTION

[0032] As used herein, the term “non-canonical amino acid” (ncAA) refers to amino acids other than canonical amino acids, such as unnatural (non-naturally occurring) amino acids. The term “non-canonical amino acid” may refer to amino acids that have a similar structure to natural amino acids with modified groups or peptide backbones. The term “non- canonical amino acid” may refer to amino acids that have a different chemical structure but function in a similar way to natural amino acids.

[0033] Embodiments of the present disclosure include enzymes, such as enzymes for forming non-canonical amino acids. The enzymes include pyridoxal phosphate (PLP)- dependent enzyme and variants thereof. Generally, the enzymes of the present disclosure include PLP-dependent enzyme variants. The PLP-dependent enzyme variant can include α- functionalization PLP enzyme variants (PLP enzyme variants capable of introduction or removal of bonds and / or functional groups at the α-carbon of amino acids). In one example, the α-functionalization PLP enzyme variant includes threonine aldolase variants. For example, the threonine aldolase variant can be a variant of at least one of Escherichia coli threonine aldolase (EcTA), Aeromonas jandei threonine aldolase (AjTA), and Thermotoga maritima threonine aldolase (TmTA). In one non-limiting example, compared to α- functionalization PLP enzymes from serine hydroxymethyltransferases (SHMTs) and threonine transaldolases (TTAs) that do not provide a desired oxidative coupling product, the threonine aldolase variant of the present disclosure can efficiently provide the desired product.

[0034] The PLP-dependent enzyme variants can be formed by targeting one or more residues in the α-helix proximal to the PLP covalent intermediate. The PLP-dependent enzyme variants can include one or more of the following mutations: E88T, H83F, W86N, W86Q, and W86V. The PLP-dependent enzyme variants can include at least two of the following mutations: E88T, H83F, W86N, W86Q, and W86V. The PLP-dependent enzymevariants can include Thermotoga maritima threonine aldolase variants including one or more of the following mutations: E88T, H83F, W86N, W86Q, and W86V. For example, the PLP- dependent enzyme variant may be selected from TmTA E88T, TmTA E88T W86Q, and TmTA E88T W86V. The PLP-dependent enzyme variant may be selected from TmTA W86N (TmPLPα1), TmTA E88T, TmTA H83F, TmTA E88T H83F (TmPLP^^).

[0035] FIG.1 illustrates a method for forming an enzyme variant, according to some embodiments. Method 100 includes Step 110. Referring to Step 110, one or more mutations are introduced to a PLP enzyme. Introducing one or more mutations can include at least one of (1) Cloning, site-directed mutagenesis and site-saturation mutagenesis; and (2) Expression and purification of threonine aldolase. The PLP enzyme can include PLP-dependent enzymes of the present disclosure. For example, the PLP-dependent enzymes can include at least one of Escherichia coli threonine aldolase (EcTA), Aeromonas jandei threonine aldolase (AjTA), and Thermotoga maritima threonine aldolase (TmTA). The one or more mutations are selected from: E88T, H83F, W86N, W86Q, and W86V. Accordingly, method 100 can be used to form α-functionalization PLP enzyme variants.

[0036] Embodiments of the present disclosure include methods for forming non- canonical amino acids (ncAAs). FIG.2 illustrates a method for forming a non-canonical amino acid, according to some embodiments. Method 200 includes Step 210. Referring to Step 210, a substrate, an amino acid, an enzyme, actinic radiation, a photocatalyst, and an oxidant are utilized to form a non-canonical amino acid. For example, the substrate, amino acid, enzyme, photocatalyst, and oxidant can be mixed, contacted, stirred, and / or placed within close physical proximity. The substrate, amino acid, enzyme, photocatalyst, and oxidant may be added separately, in any order, or simultaneously. One or more non-canonical amino acids can be formed, such as enantiomer products.

[0037] One or more reactants, such as one or more of the substrate, amino acid, enzyme, photocatalyst, and oxidant can be in independent solutions prior to being contacted, such as in degassed DMSO, degassed water, or KPi buffer. For example, the following stock solutions can be prepared (e.g., in a Coy anaerobic chamber): substrate (in degassed DMSO), photocatalyst (in degassed DMSO), amino acid (in degassed KPi buffer), enzyme (in KPi buffer), oxidant (in degassed water). Additionally, a buffer can be added with the substrate, amino acid, enzyme, photocatalyst, and oxidant. For example, a buffer, organoboron reagent stock solution, glycine stock solution, enzyme stock solution, oxidant stock solution, and photocatalyst stock solution can be added to a reactor (e.g., reactor with a stir bar). The pHbuffer may be added to alter / maintain the pH of the solution to about 6.0 to about 8.0. For example, a KPi buffer may be added to the solution.

[0038] The specific volume of the enzyme solution may be dependent on the concentration of the specific enzyme. Contacting may include mixing, stirring, cooling, and / or heating. In one example, the reactants are stirred and heated to a temperature above 40 °C. In another example, the reactants are heated to a temperature ranging from 40 °C to 60 °C. In yet another example, the reactants are stirred at above 500 RPM at about 50 °C. The reactants may be stirred for about 5 hours to about 20 hours. For example, the reactants may be stirred for about 8 hours to about 15 hours. The photocatalyst can be provided with actinic radiation to initiate the reaction.

[0039] The substrate includes one or more organoboron compounds. Organoboron compounds include at least one carbon-boron bond. The organoboron compound can include at least one alkyltrifluoroborate compound. The alkyltrifluoroborate compound can include one or more benzyltrifluoroborates. Examples of benzyltrifluoroborates include Potassium benzyltrifluoroborate and secondary benzyltrifluoroborates. The organoboron compound can include a boronic ester. For example, the organoboron compound can include benzylboronic acid pinacol ester. Examples of organoboron compounds are shown below:benzylboronic acid pinacol Potassiumester (1a’) benzyltrifluoroborate (1a)

[0040] Examples of secondary benzyltrifluoroborates are shown below:Potassium trifluoro(1-(p- Potassium trifluoro(1-(m- Potassium trifluoro(1-(o- tolyl)ethyl)borate (1v) tolyl)ethyl)borate (1w) tolyl)ethyl)borate (1x)Potassium trifluoro(1-(4- fluorophenyl)ethyl)borate (1y)

[0041] The substrate can be provided in a solution at various concentrations. In one example, the substrate is included in a solution, where the concentration of the substrate ranges from about 1 mM to about 1000 mM. In another example, the substrate is included in a solution, where the concentration of the substrate ranges from about 10 mM to about 500 mM. In another example, the substrate is included in a solution, where the concentration of the substrate ranges from about 50 mM to about 200 mM. In another example, the substrate is included in a solution, where the concentration of the substrate ranges from about 75 mM to about 200 mM.

[0042] The amino acid includes one or more amino acids. The amino acid can include one or more alpha amino acids. Alpha amino acids are molecules including an amino group and a carboxylic acid group separated by a carbon. In one example, the alpha amino acid includes glycine or alanine. The amino acid can be provided in a solution at various concentrations. In one example, the amino acid is included in a solution, where the concentration of the amino acid ranges from about 0.1 M to about 3M. In another example, the amino acid is included in a solution, where the concentration of the amino acid ranges from about 0.3 M to about 2 M. In another example, the amino acid is included in a solution, where the concentration of the amino acid ranges from about 0.5 M to about 1.5 M. The amino acid can be provided at concentrations of the present disclosure in degassed KPi buffer. For example, the solution can include the amino acid and degassed 100-500 mM KPi buffer, sufficient for the solution to have a pH value ranging from about 7 to about 9 (e.g., pH value of about 8).

[0043] The enzyme includes one or more enzymes of the present disclosure. For example, the enzyme can include one or more PLP-dependent enzyme variants. The PLP-dependent enzyme variants can include one or more of the following mutations: E88T, H83F, W86N, W86Q, and W86V. The PLP-dependent enzyme variants can include Thermotoga maritima threonine aldolase variants including one or more of the following mutations: E88T, H83F, W86N, W86Q, and W86V. For example, the PLP-dependent enzyme variant may be selected from TmTA E88T, TmTA E88T W86Q, and TmTA E88T W86V. The PLP- dependent enzyme variant may be selected from TmTA W86N (TmPLPα1), TmTA E88T, TmTA H83F, TmTA E88T H83F (TmPLP^^). The enzyme can be provided in a solution at various concentrations, and the volume of the enzyme stock solution can be dependent on the concentration of the specific enzyme utilized.

[0044] The photocatalyst includes one or more substances that initiate and / or accelerate the reaction when exposed to light (e.g., sufficient actinic radiation). For example, the photocatalyst absorbs light energy, becomes excited, and then facilitates chemical transformations of other substances. The photocatalyst may include one or more of [Ru(bpy)3]Cl2, Ru(dtbbpy)3(PF6)2, [Ru(bpz)3](PF6)2, (fac)-Ir(ppy)3, [Ir(ppy)2(dtbbpy)]PF6, [Ir(dF(CF3)ppy(dtbbpy)]PF6, RhB, Eosin Y, rhodamine 6G, 9-mesityl-10-methylacridinium, and 4CzIPN. In one example, performing method 200 without a photocatalyst would lead to no non-canonical amino acid product formation. Structures of examples of photocatalysts are shown below:

[0045] The photocatalyst can be provided in a solution at various concentrations. In one example, the photocatalyst is included in a solution, where the concentration of the photocatalyst ranges from about 0.1 mM to about 40 mM. In another example, the photocatalyst is included in a solution, where the concentration of the photocatalyst ranges from about 0.3 mM to about 10 mM. In another example, the photocatalyst is included in a solution, where the concentration of the photocatalyst ranges from about 1 mM to about 5 mM. The photocatalyst can be provided in degassed DMSO.

[0046] The oxidant accepts an electron from another substance. Generally, the oxidant includes a single-electron oxidant. Single-electron oxidants are chemical species that accept / remove one electron from a substance, at least partially initiating oxidation reactions through a single-electron transfer (SET) mechanism. The oxidant may include one or more of a copper-containing compound, a manganese-containing compound, and an iron-containing compound. For example, the oxidant can include at least one of a manganese-containing salt, an iron-containing salt, and a copper-containing salt. In one example, the oxidant includes one or more of Co(NH3)6Cl3, Cu(OAc)2, (NH4)2Ce(NO3)6, Mn(OAc)3, FeCl3, NaFe(EDTA). In one non-limiting example, the oxidant is Co(NH3)6Cl3. In one example, performing method 200 without an oxidant would decrease the yield of the non-canonical amino acid product, or performing method 200 without an oxidant would not provide a non-canonical amino acid product. In one non-limiting example, the combination of the photocatalyst of thepresent disclosure and using Co(NH3)6Cl3 as an oxidant can promote desirable yields of non- canonical amino acid products.

[0047] The oxidant can be provided in a solution at various concentrations. In one example, the oxidant is included in a solution, where the concentration of the oxidant ranges from about 1 mM to about 500 mM. In another example, the oxidant is included in a solution, where the concentration of the oxidant ranges from about 1 mM to about 200 mM. In another example, the oxidant is included in a solution, where the concentration of the oxidant ranges from about 1 mM to about 150 mM. The oxidant can be provided in degassed water. Method 200 can include utilizing a buffer. For example, the buffer can include potassium phosphate (KPi) buffer. Utilizing a buffer can include initializing the formation of the non-canonical amino acid at a pH value ranging from about 7.5 to about 8.5. Utilizing the buffer can include substantially maintaining a pH value of a reactant solution between 7.5 to 8.5.

[0048] Method 200 can include utilizing pyridoxal phosphate (PLP). Pyridoxal phosphate can be provided in a solution. For example, pyridoxal phosphate can be provided in KPi buffer. In one example, pyridoxal phosphate is included in a solution, where the concentration of the pyridoxal phosphate ranges from about 5 mM to about 100 mM. In another example, pyridoxal phosphate is included in a solution, where the concentration of pyridoxal phosphate ranges from about 10 mM to about 50 mM. In another example, pyridoxal phosphate is included in a solution, where the concentration of pyridoxal phosphate ranges from about 20 mM to about 30 mM. For example, the solution can include pyridoxal phosphate and 100-500 mM KPi buffer, sufficient for the solution to have a pH value ranging from about 7 to about 9 (e.g., pH value of about 7.5). The structure for pyridoxal phosphate is shown below.

[0049] In one example, the substrate, amino acid, enzyme, photocatalyst, oxidant, and PLP can be mixed to form a reaction mixture having the following concentrations: substrate 1-10 mM; amino acid 1-50 mM, enzyme 1-100 ^M, photocatalyst 1-100 ^M, oxidant 1-20 mM, PLP 50-1000 ^M. In another example, the substrate, amino acid, enzyme, photocatalyst, oxidant, and PLP can be mixed to form a reaction mixture having the followingconcentrations: substrate 1-5 mM; amino acid 20-40 mM, enzyme 10-100 ^M, photocatalyst 30-100 ^M, oxidant 2-10 mM, PLP 100-500 ^M. The substrate, amino acid, enzyme, photocatalyst, and oxidant can be selected to form products having the desired enantiomeric ratio (e.r.), the measure of the relative abundance of two enantiomers.

[0050] Actinic radiation is provided to at least the photocatalyst. Actinic radiation is capable of causing a change to one or more substances sufficient to initiate and / or promote the reaction. For example, actinic radiation is provided to the photocatalyst sufficient for the photocatalyst to absorb a photon and to form an excited-state reductant. The excited-state reductant can then be oxidized by the oxidant. The actinic radiation can be provided at a wavelength ranging from about 380 nm to about 550 nm. The actinic radiation can be provided in the visible light wavelength range. In one example, the actinic radiation is provided at a wavelength ranging from about 400 nm to about 500 nm. In another example, the actinic radiation is provided at a wavelength ranging from about 430 nm to about 450 nm. In one example, providing actinic radiation at a wavelength ranging from about 430 nm to about 450 nm can increase the yield of the desirable non-canonical amino acid product. In another example, the actinic radiation is provided at a wavelength of about 440 nm.

[0051] Method 200 can include providing the actinic radiation to the photocatalyst sufficient to form an excited-state reductant. The excited-state reductant can be oxidized by the oxidant to provide a new oxidant. The organoboron substrate can undergo single-electron oxidation with the new oxidant to form a carbon-centered radical and regenerate the photocatalyst. Concurrently, the enzyme can undergo transimination with the amino acid to form an external aldimine. Facile deprotonation can form a quinonoid intermediate. The photocatalytically formed free radical species can travel into the enzyme’s active site and stereoselectively add to the C^^ carbon of the enzymatically formed quinonoid, leading to a nitrogen-centered radical. Single-electron oxidation of the nitrogen-centered radical by the new oxidant can generate a new external aldimine, releasing a sp3–sp3coupled non-canonical amino acid product and regenerating the enzyme.

[0052] The formed non-canonical amino acid includes sp3–sp3coupled non-canonical amino acid products. For example, the formed non-canonical amino acid can include at least one of a α-tri-substituted non-canonical amino acid and a α-tetra-substituted non-canonical amino acid. The non-canonical amino acid may include one or more of L-phenylalanine, L-2- amino-3-(p-tolyl)propanoic acid, L-2-amino-3-(m-tolyl)propanoic acid, L-2-amino-3-(o-tolyl)propanoic acid, L-2-amino-3-(4-methoxyphenyl)propanoic acid, L-2-amino-3-(4- (methylthio)phenyl)propanoic acid, L-tyrosine, L-2-amino-3-(4- (trifluoromethoxy)phenyl)propanoic acid,L-2-amino-3-(4-fluorophenyl)propanoic acid,L-2- amino-3-(4-chlorophenyl)propanoic acid, L-2-amino-3-(3-cyanophenyl)propanoic acid, L-2- amino-3-(3-methoxyphenyl)propanoic acid, L-3-([1,1'-biphenyl]-4-yl)-2-aminopropanoic acid,L-3-(4-(1H-pyrazol-1-yl)phenyl)-2-aminopropanoic acid, andL-2-amino-3-(4-(tert- butyl)phenyl)propanoic acid.

[0053] The non-canonical amino acid may include one or more of L-2-amino-3-(3,4- difluorophenyl)propanoic acid, L-2-amino-3-(3-fluoro-4-methylphenyl)propanoic acid, L-2- aminopent-4-enoic acid,L-2-aminopent-4-enoic acid,L-2-amino-4-phenylbutanoic acid,L-2- amino-3-(naphthalen-2-yl)propanoic acid, (2S, 3S)- 2-amino-3-phenylbutanoic acid, (2S,3S)- 2-amino-3-(o-tolyl)butanoic acid, (2S,3S)-2-amino-3-(m-tolyl)butanoic acid, (2S,3S)-2- amino-3-(o-tolyl)butanoic acid, (2S,3S)-2-amino-3-(4-fluorophenyl)butanoic acid, (S)-2- amino-2-methyl-3-phenylpropanoic acid, (S)-2-amino-3-(4-fluorophenyl)-2-methylpropanoic acid, (S)-2-amino-2-methyl-3-(4-(trifluoromethoxy)phenyl)propanoic acid, (S)-2-amino-3-(3- methoxyphenyl)-2-methylpropanoic acid, and (S)-2-amino-2-benzylbutanoic acid.

[0054] Structures for non-canonical amino acids are shown below:L-phenylalanine (3a) L-2-amino-3-(p-tolyl)propanoic L-2-amino-3-(m- acid (3b) tolyl)propanoic acid (3c)tolyl)propanoic acid methoxyphenyl)propanoic acid (methylthio)phenyl)propano (3d)ic acid (3f)L-tyrosine (3g) L-2-amino-3-(4- L-2-amino-3-(4- (trifluoromethoxy)phenyl)propan fluorophenyl)propanoic oic acid (3h) acid (3i)L-2-amino-3-(4- L-2-amino-3-(3- L-2-amino-3-(3- chlorophenyl)propanoic cyanophenyl)propanoic acid (3k) methoxyphenyl)propanoic acid (3j) acid (3l)L-3-([1,1'-biphenyl]-4- L-3-(4-(1H-pyrazol-1-yl)phenyl)- L-2-amino-3-(4-(tert- yl)-2-aminopropanoic 2-aminopropanoic acid (3n) butyl)phenyl)propanoic acidL-2-amino-3-(3,4-L-2-amino-3-(3-fluoro-4-L-2-aminopent-4-enoic acid difluorophenyl)propanoi methylphenyl)propanoic acid (3q) (3r) c acid (3p)L-2-aminopent-4-enoic L-2-amino-4- L-2-amino-3-(naphthalen-2- acid (3s) phenylbutanoic acid (3t) yl)propanoic acid (3z)

[0055] Additional structures for non-canonical amino acids are shown below.(2S, 3S)- 2-amino-3- (2S,3S)-2-amino-3-(o-tolyl)butanoic (2S,3S)-2-amino-3-(m- phenylbutanoic acid acid (5b) tolyl)butanoic acid (5c)tolyl)butanoic acid (5d) fluorophenyl)butanoic acid (5e) phenylpropanoic acid (8a)(S)-2-amino-3-(4- (S)-2-amino-2-methyl-3-(4- (S)-2-amino-3-(3- fluorophenyl)-2- (trifluoromethoxy)phenyl)propanoic methoxyphenyl)-2- methylpropanoic acid acid (8c) methylpropanoic acid (8d)(S)-2-amino-2- benzylbutanoic acid (8e)

[0056] FIG.3 illustrates a method for forming a non-canonical amino acid, according to some embodiments. Method 300 includes Step 310. Referring to Step 310, an organoboroncompound, an alpha amino acid, an enzyme, a photocatalyst, and an oxidant are utilized to form a sp3-sp3coupled non-canonical amino acid product. The organoboron compound includes organoboron substrates of the present disclosure. The alpha amino acid can be selected from glycine and alanine. The enzyme, photocatalyst, oxidant, and non-canonical amino acid product include enzymes, photocatalysts, oxidants, and non-canonical amino acid products of the present disclosure.

[0057] The organoboron compound, alpha amino acid, enzyme, photocatalyst, and oxidant can be provided as a solution (e.g., individual solutions). The organoboron compound, alpha amino acid, enzyme, photocatalyst, and oxidant can be mixed simultaneously or in various orders. A buffer of the present disclosure can be added to the mixture. Visible light (e.g., visible light actinic radiation) can be provided to the photocatalyst at a wavelength ranging from 380 nm to 550 nm, and at an energy sufficient for the photocatalyst to change to an excited-state. Method 300 can be performed at one or more conditions of method 200, such as pH value, temperature, mixing order, mixing speed, and mixing time. Method 300 can further include utilizing pyridoxal phosphate (PLP).

[0058] Methods 200 and 300 can include triple photobiocatalytic cycles for sp3–sp3oxidative cross-coupling. Method 200 and 300 can form many non-canonical amino acids, including those bearing contiguous stereocenters and α-tetrasubstituted stereocenters. The synergistic use of photocatalysts and enzyme variants enables the development of new radical pyridoxal enzymology, allowing the convergent and stereoselective preparation of valuable non-canonical amino acids, such as without protecting group manipulations. Importantly, non-canonical amino acids bearing contiguous stereocenters or α-tetrasubstituted stereocenters that has previously eluded synthesis can be prepared with excellent enantio- and diastereocontrol. Example 1 – Photobiocatalytic oxidative coupling

[0059] FIG.4A illustrates asymmetric sp3–sp3oxidative cross-coupling via cooperative photobiocatalysis, according to some embodiments. (Ox = oxidant, PC = photocatalyst, En = enzyme catalyst, ET / PT = electron transfer / proton transfer, PCET = proton-coupled electron transfer, ncAA = non-canonical amino acid.) FIG.4B illustrates triple photobiocatalytic cycles for sp3–sp3oxidative cross-coupling using an enzyme and a photocatalyst, according to some embodiments. Under visible light irradiation, (fac)-Ir(ppy)3 (ppy = 2-phenylpyridine, IV) absorbs a photon to furnish a long-lived excited-state reductant (fac)-*Ir(ppy)3 (IV*, E1 / 2(*IrIII / IrIV) = –1.73 V versus saturated calomel electrode (SCE) in CH3CN). (fac)-*Ir(ppy)3 can be rapidly oxidized by [Co(NH3)6]3+(V, E1 / 2 (CoIII / CoII) = –0.18 V versus SCE in H2O), a judiciously selected stoichiometric oxidant, to provide (fac)-Ir(ppy)3+(VI) as a potent oxidant (E1 / 2 (IrIV / IrIII) = 0.77 V versus SCE in CH3CN). Due to the increased ligand lability of the reduced [Co(NH3)6]2+(VI), rapid ligand exchange with H2O results in the formation of [Co(H2O)6]2+(V, E1 / 2 (CoIII / CoII) = 1.68 V versus SCE in H2O), which, in one example, cannot be re-oxidized by (fac)-Ir(ppy)3+(VI).

[0060] Single-electron oxidation of the organoboron substrate (I) with VI can afford a transient carbon-centered radical VIII and regenerate the (fac)-Ir(ppy)3 photocatalyst (I). Concurrent to the radical initiating photoredox cycle I, in the biocatalytic cycle, a PLP- dependent ^−functionalization enzyme IX, such as a threonine aldolase, can first undergo transimination with an abundantly available amino acid substrate II to form an external aldimine X. PLP binding can result in dramatic pKa lowering of the ^-proton, leading to the facile deprotonation to form the quinonoid intermediate (XI). At this stage, the photocatalytically formed free radical species VIII can travel into the enzyme’s active site and stereoselectively add to the C^^ carbon of the enzymatically formed quinonoid XI, leading to a nitrogen-centered radical XII, an elusive species in native PLP enzymology.

[0061] Single-electron oxidation of XII by (fac)-Ir(ppy)3+can generate a new external aldimine XIII, which subsequently releases the sp3–sp3coupled non-canonical amino acid product III and regenerates the PLP biocatalyst IX, thereby completing all the catalytic cycles. This oxidative C(sp3)–C(sp3) cross-coupling can enable the union of two distinct nucleophiles, including an easily available organoboron reagent and an abundant amino acid substrate, via a C(sp3)–H functionalization logic. Furthermore, the enzyme-controlled radical addition and proton transfer steps can occur with excellent stereoselectivity. Accordingly, this ^−pyridoxal radical biocatalysis can allow diverse non-canonical amino acids bearing up to two adjacent stereocenters to be prepared with excellent diastereo- and enantiocontrol. Example 2 – Reaction Enzymes, Photocatalysts, and Oxidants

[0062] Reaction conditions: 1 (1.0 equiv, 3.0 mM), 2a (10 equiv, 30.0 mM), 1 mol% PLP enzyme (30 μM), 10 mol% PLP (300 μM), 2 mol% (fac)-Ir(ppy)3 (60 μM), oxidant (2.0 equiv, 6.0 mM), h^ (440 nm), 200 mM KPi buffer, 50 °C, 12 h. Yields were an average ofthree runs. Enantioselectivities were determined by Marfey’s analysis. Threonine was used in lieu of glycine for ObiH, a threonine transaldolase.

[0063] The enzymes of the present disclosure can facilitate the photobiocatalytic oxidative cross-coupling of benzyltrifluoroborate (1a) and glycine (2a). The enzymes can catalyze C–C bond forming aldol or Mannich reactions at the ^-position of amino acids, as these enzymes can accommodate the incoming carbon-centred radical for productive C–C coupling. Using Ru(bpy)3Cl2 (4a) as the photocatalyst, among the ^-functionalization PLP enzymes evaluated, oxidative coupling activities were discovered in the threonine aldolase (TA) family. An example reaction is shown below.

[0064] While the use of 1 mol% TA from Escherichia coli (EcTA) and Aeromonas jandei (AjTA) furnished the sp3–sp3coupling product 3a in 6% and 1% yield, respectively, the highest initial activity was observed with Thermotoga maritima threonine aldolase (TmTA, 13% yield and 98:2 e.r., e.r. = enantiomeric ratio). For example, the use of ^- functionalization PLP enzymes from other families, including serine hydroxymethyltransferases (SHMTs) and threonine transaldolases (TTAs), did not provide the desired oxidative coupling product, underscoring the importance of enzyme selection. The choice of photocatalysts also influenced the efficiency of this photobiocatalytic coupling, with the highest activity observed with 2 mol% (fac)-Ir(ppy)3 (4d, 49% yield, 97:3 e.r.) and 10 mol% 4CzIPN (4f, 52% yield, 99:1 e.r.). An example reaction is shown below.Table 1. Photocatalyst comparison.

[0065] Moreover, the identity of the oxidant had a significant impact on this oxidative coupling. Among the single-electron oxidants evaluated, Mn(III), Fe(III) and Co(III) salts effectively promoted this sp3–sp3coupling. Co(NH3)6Cl3 was found to be excellent. Both the benzyltrifluoroborate 1a and the benzylboronic acid pinacol ester 1a’ could be efficiently transformed under these conditions, and enhanced yields were observed when 1a’ was used in conjugation with 2 mol% (fac)-Ir(ppy)3 (4d) as the photocatalyst. Table 2. Oxidant comparison.

[0066] FIG.5A illustrates density functional theory (DFT) calculations using a theozyme model, according to some embodiments. For example, the computed energy profile was using a theozyme model at the (U)ωB97X-D / 6-311+G(2d,2p)- SDD(Ir) / SMD(PhCl) / / (U)B3LYP-D3 / 6-31G(d)-SDD(Ir) level of theory. Except those in external aldimine 13, active-site residues are omitted for clarity. Enthalpy values (ΔH) are with respect to 13. FIG.5B illustrates density functional theory (DFT) calculations using a theozyme model, according to some embodiments. Activation enthalpies for radical additions to quinonoid species were computed using theozyme and a cofactor-only model. Enthalpiesare relative to the van der Waals complex (15). FIG.5C illustrates density functional theory (DFT) calculations using a theozyme model, according to some embodiments. This shows optimized structures of regio- and enantioselectivity-determining radical addition transition states. Example 3 – Enzyme Variants and Substrates

[0067] FIG.6A illustrates screening of target residues in the α-helix proximal to the PLP covalent intermediate, according to some embodiments. Furthermore, guided by the crystal structure of TmTA external aldimine (PDB ID: 1LW5), site-saturation mutagenesis (SSM) and screening were performed by targeting residues 83–88 in the ^-helix proximal to the PLP covalent intermediate. In contrast to conventional enzyme engineering, high- throughput technologies to evolve novel photobiocatalytic functions remain underdeveloped. In one single-site saturation library, 88 clones of TmTA variants were chosen and screened. This protein engineering allowed multiple useful enzyme variants to be identified, among which TmTA W86N provided improved yields relative to the wild-type enzyme. TmTA W86N was annotated as TmPLP^1 (PLP ^-radical enzyme 1).Table 3. Enzyme Variants.

[0068] Reaction conditions: 1’ (1.0 equiv, 3.0 mM), 2a (10 equiv, 30.0 mM), 1 mol% TmPLP^1 (30 μM), 10 mol% PLP (300 μM), 2 mol% (fac)-Ir(ppy)3 (60 μM), Co(NH3)6Cl3 (2.0 equiv, 6.0 mM), hν (440 nm), 200 mM KPi buffer, 50 °C, 12 h. Yields were an average of three runs. FIG.6B illustrates a 96-position photoreactor, according to some embodiments. FIG.6C illustrates a high-throughput photobiocatalysis setup, according tosome embodiments. FIG.6D illustrates asymmetric sp3-sp3oxidative coupling, according to some embodiments. Benzylboronates bearing a para-, a meta- and an ortho- substituent were compatible with the engineered PLP biocatalyst, giving rise to the corresponding non- canonical amino acids in excellent yields and excellent enantioselectivities. Electron-donating substituents such as a methoxy, a methylthio and a hydroxy, and electron-withdrawing substituents such as a trifluoromethoxy, a fluorine and a chlorine and a cyano group were also capable under these conditions. Without additional engineering, TmPLP^1 could be applied to convert organoboronates possessing a bulky substituent at the para-position of the phenyl group as well as a naphthyl substrate with good efficiency, further demonstrating the substrate promiscuity of this PLP biocatalyst. For example, benzylboronates bearing a large para-phenyl, pyrazolyl and tert-butyl were found to be suitable substrates.

[0069] Additionally, this dual photobiocatalytic system could also be applied to disubstituted substrates. Further, allylboronates could be transformed to afford the corresponding olefinic non-canonical amino acids with excellent enantiomeric ratio. In particular, prenylboronate was converted with excellent linear to branched ratio (> 95:5 l:b). Unactivated alkylboronates were also accepted by the enzyme when rhodamine B (4e) was used as the photocatalyst, furnishing the non-canonical amino acid product in excellent enantioselectivity albeit with lower yields. Importantly, this photobiocatalytic amino acid synthesis was found to be scalable.1 mmol-scale preparation of 3a was successfully carried out at a 10.0 mM concentration of 1a’ without lowering the enantioselectivity of the biotransformation.

[0070] FIG.7A illustrates UV-vis spectroscopic analysis (TmPLP^1 pH 8), according to some embodiments. FIG.7B illustrates UV-vis spectroscopic analysis (TmPLP^1 pH 9), according to some embodiments. Under the optimized reaction conditions, the use of 1 mol% TmPLP^1, 2 mol% (fac)-Ir(ppy)3 and 200 mol% Co(NH3)6Cl3 provided the desired phenylalanine (3a) in 77% yield and 98.5:1.5 e.r.. Reaction conditions on a 1.0 mmol scale: 1a’ (1.0 equiv, 10 mM), 2a (10 equiv, 100 mM), 1 mol% TmPLP^1 (100 μM), 10 mol% PLP (1.0 mM), 2 mol% (fac)-Ir(ppy)3 (200 μM), Co(NH3)6Cl3 (2.0 equiv, 20.0 mM), hν (440 nm), 200 mM KPi buffer, 50 °C, 12 h. 10 mol% RhB (4e) was used as the photocatalyst in lieu of 2 mol% (fac)-Ir(ppy)3 (4d).

[0071] ^-Methyl non-canonical amino acids encompass an emerging yet largely unchartered chemical space for peptide therapeutics, due to the enhanced proteolytic stabilityand binding affinity of peptides incorporating these structural elements. The efficient catalytic asymmetric synthesis of non-canonical amino acids bearing vicinal ^ and ^- stereocentres without protecting group manipulation represent a nontrivial task facing medicinal chemists. Using racemic 1-phenylethyltrifluoroborate 1u as the radical precursor, it was found that wild-type TmTA was able to catalyse the synthesis of ^-methyl non-canonical amino acids via oxidative C–C coupling, delivering (2S,3S)-5a in 62% yield, 77:23 d.r. (d.r. = diastereomeric ratio) and 99:1 e.r.. Unlike the oxidative coupling of primary alkylboron reagents, for secondary alkyl nucleophiles, the use of alkyltrifluoroborate salts and 4CzIPN afforded superior yields relative to those of alkylboronic acid pinacol esters and (fac)- Ir(ppy)3.

[0072] Reaction conditions: 1 (1.0 equiv, 3 mM), 2a (10 equiv, 30 mM), 1 mol% TmPLP^2 (30 μM), 20 mol% PLP (600 μM), 10 mol% 4CzIPN (300 μM), Co(NH3)6Cl3 (2.0 equiv, 6.0 mM), hν (440 nm), 200 mM KPi buffer, 50 °C, 12 h. To improve the catalytic efficiency and diastereoselectivity of this enantioconvergent oxidative sp3–sp3cross- coupling, directed evolution of TmTA was conducted using iterative rounds of site-saturation mutagenesis (SSM) and screening using in-house built high-throughput photochemistry equipment.Table 4. Enzyme Variants.

[0073] FIG.8A illustrates screening of residues, according to some embodiments. FIG. 8B illustrates diastereomer percentage for enzymes, according to some embodiments. Bytargeting residues 88 and 83 in the key ^-helix, TmTA E88T H83F (TmPLP^2) was formed after two rounds of SSM and screening, affording (2S,3S)-5a in 76% yield and 91:9 d.r.. E88T resulted in enhanced enzyme activity while H83F led to improved diastereocontrol. FIG.8C illustrates diastereo- and enantioselective sp3–sp3oxidative coupling: synthesis of non-canonical amino acids with contiguous stereocenters, according to some embodiments. Using TmPLP^2, 1-phenylethyltrifluoroborate possessing a para-, meta- and ortho-methyl group and a fluorine could be successfully transformed into ^-methylphenylalanine analogs in good yields and excellent diastereo- and enantiocontrol. FIG.8D illustrates X-ray structures for stereochemistry determination, according to some embodiments. The absolute and relative stereochemistry of 5a was determined by single-crystal X-ray diffraction analysis of its amide derivative 7a. Together, this photobiocatalytic oxidative cross-coupling provided a convergent, stereoselective, and protecting-group-free synthesis of valuable ^-methyl non- canonical amino acids in a single operation.

[0074] FIG.8E illustrates the enantioselective synthesis of α-tetrasubstituted amino acids, according to some embodiments. Reaction conditions: 1’ (1.0 equiv, 5 mM), 2 (10 equiv, 50 mM), 1 mol% TmPLP^1 (50 μM), 10 mol% PLP (500 μM), 10 mol% 4CzIPN (500 μM), Co(NH3)6Cl3 (2.0 equiv, 10.0 mM), hν (440 nm), 200 mM KPi buffer, 50 °C, 12 h. Isolated yield on a 1.0 mmol scale. Reaction conditions on a 1.0 mmol scale: 1a’ (1.0 equiv, 10 mM), 2b (10 equiv, 100 mM), 1 mol% TmPLP^1(100 μM), 10 mol% PLP (1.0 mM), 10 mol% 4CzIPN (1.0 mM), Co(NH3)6Cl3 (2.0 equiv, 20.0 mM), hν (440 nm), 200 mM KPi buffer, 50 °C, 12 h.

[0075] TmPLP^1 readily accommodated the transformations of ^-branched amino acid substrates, giving rise to highly enantioenriched ^-tetrasubstituted non-canonical amino acids (8a–8e) that are inaccessible via conventional biocatalysis based on transaminases and ammonia lyases. EitherD- orL-amino acid substrates were accepted by TmPLP^1 under photobiocatalytic conditions, providing the same major enantiomeric non-canonical amino acid product in an enantioconvergent fashion.D-amino acids such asD-alanine underwent more efficient transformations than the L-enantiomers. Finally, a 1 mmol-scale biocatalytic synthesis could be conveniently carried out to afford 8a in 142 mg yield and >99:1 e.r., further demonstrating the synthetic utility of this method. Example 4 – Additional Radical Formation Results

[0076] To gain insights into the mechanism of photobiocatalytic sp3–sp3oxidative cross-coupling, the reaction was carried out in the presence of 3 equivalents of 2,2,6,6- tetramethyl-1-piperidinyloxy (TEMPO). Under the standard conditions, 73% radical trapping product 9a was observed, showing the involvement of benzyl radical in the present process.

[0077] Reaction conditions: 1a’ (1.0 equiv, 3.0 mM), 2a (10 equiv, 30.0 mM), 1 mol% TmPLP^1 (30 μM), 10 mol% PLP (300 μM), 2 mol% (fac)-Ir(ppy)3 (60 μM), Co(NH3)6Cl3 (2.0 equiv, 6.0 mM), TEMPO (3.0 equiv, 9.0 mM), hν (440 nm), 200 mM KPi buffer, 50 °C, 12 h. Reaction conditions: 1a’ (1.0 equiv, 3.0 mM), 2 mol% (fac)-Ir(ppy)3 (60 μM), Co(NH3)6Cl3 (2.0 equiv, 6.0 mM), hν (440 nm), 200 mM KPi buffer, 50 °C, 12 h.

[0078] Additionally, radical generation was studied under these conditions in the absence of the PLP biocatalyst and glycine (2a). In the presence of 2 mol% (fac)-Ir(ppy)3 and 200 mol% Co(NH3)6Cl3, 7% dibenzyl 10a and 23% benzyl alcohol 11a derived from the benzyl radical intermediate were observed, suggesting the facile benzyl radical initiation under these photoredox conditions. In contrast, when the stoichiometric oxidant Co(NH3)6Cl3 was omitted, neither 10a nor 11a was observed, indicating benzyl radical did not form in the absence of Co(NH3)6Cl3. This finding is in accord with the low oxidation potential of excited (fac)-Ir(ppy)3 (E1 / 2 (*IrIII / IrII) = +0.31 V versus saturated calomel electrode (SCE) in CH3CN) relative to those of ate complexes of organoboron reagents. Interestingly, under visible light irradiation, in the absence of 2 mol% (fac)-Ir(ppy)3, the use of 200 mol% Co(NH3)6Cl3 also led to 13% dibenzyl 10a and 15% benzyl alcohol 11a.

[0079] These results showed that Co(NH3)6Cl3 could also trigger radical formation under photochemical conditions. Combined with the finding that omitting (fac)-Ir(ppy)3 in the dual photobiocatalytic process led to no cross-coupling product formation, these results indicated that the single electron oxidation of the PLP-bound amino acid ^-radical XIII likely involves (fac)-Ir(ppy)3 and is not solely promoted by Co(NH3)6Cl3.

[0080] To further elucidate the reaction mechanism of this threonine aldolase-catalysed oxidative coupling, density functional theory (DFT) calculations were performed using atheozyme model prepared from a prior crystal structure of TmTA (PDB ID: 1LW5) including catalytically relevant amino acid residues K199, H83, and other residues within 4.0 Å of the PLP cofactor. To probe the feasibility of deprotonation of the pro-R and pro-S ^^-C–H bonds with engineered TmTA variants, UV-vis spectroscopic analysis was performed. Measurements with both TmPLP^1 and TmPLP^2 showed that the quinonoid species forms rapidly upon the introduction of glycine (495 nm),D-alanine (505 nm) orL-alanine (505 nm). Compared to L-alanine, a more prominent quinonoid signal at 505 nm was observed with D- alanine, suggesting that the pro-S deprotonation occurs more readily than the pro-R deprotonation with K199. Example 5 – Reaction methods

[0081] Biocatalytic reactions were performed in a reaction tube (13 ^ 100 mm, 8 mL) with magnetic stirring (500 rpm) using stir plates. LED-440 nm (max 45 W) was used as the visible light source. Data for1H NMR are reported as follows: chemical shift (δ ppm), multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, p = pentet, sext = sextet, m = multiplet, dd = doublet of doublets, dt = doublet of triplets, ddd = doublet of doublet of doublets, brs = broad singlet), coupling constant (Hz), integration. Sonication on a small scale was performed using an ultrasonic homogenizer equipped with a stepped microtip. Sonication on a large scale was performed using a sonicator equipped with a standard probe (tip diameter = ½’’).

[0082] E. coli cells were grown using Luria-Bertani medium (LB) with 0.05 mg / mL kanamycin (LBkan). Primer sequences for site-directed mutagenesis and site-saturation mutagenesis are provided below. Potassium Phosphate Buffer (abbreviated as KPi buffer) was used as a buffering system for protein purification and storage unless otherwise specified. Analytical reverse phase high performance liquid chromatography-mass spectrometry (HPLC-MS) was carried out using a column (4.6 ^ 50 mm, 5 μm) with water (0.1% formic acid) and acetonitrile (0.1% formic acid) as the mobile phase. Yields were determined by calibration curves using 2-phenylbutyric acid as the internal standard. The enantiomeric ratios (e.r.’s) of non-canonical amino acid products were determined by Marfey’s analysis.

[0083] Stock solutions of products (30 mM in CH3CN / 1 M aq. HCl (v:v = 1:1)) and internal standard (2-phenylbutyric acid) (100 mM in CH3CN / 1 M aq. HCl (v:v = 1:1)) werefreshly prepared. To a 1.5 mL microcentrifuge tube were added 30 μL of internal standard stock solution (3 μmol), 20, 40, 60, 80 and 100 μL of product stock solution was added to each tube, followed by the addition of 950, 930, 910, 890 and 870 μL CH3CN / 1 M aq. HCl to make the total volume to be 1.00 mL. The mixture was vortexed (20 s for 3 times) to ensure good mixing. The mixture was transferred to a 1.5 mL glass vial for HPLC analysis.

[0084] pET-28a(+) was used as the cloning and expression vector for L-threonine aldolase (TA) from Thermotoga maritima (TmTA), including TmTA W86N (TmPLP^1), TmTA E88T H83F (TmPLP^2) and other mutants, as well as other pyridoxal phosphate (PLP)- dependent enzymes described in this study. Genes of PLP-dependent enzymes were codon optimized and purchased as plasmids using pET-28a(+) as the cloning vector. The gene of interest (GOI) was cloned into pET-28a(+) between restriction sites NdeI and HindIII to include a 6X His tag and a thrombin site at the N terminus. Site-directed mutagenesis was carried out using the overlap extension polymerase chain reaction (PCR). Site-saturation mutagenesis was performed using the “22c-trick” method. The PCR products were DpnI digested, gel purified, and ligated using Gibson mix. This Gibson mix was prepared using 5X isothermal (ISO) reaction buffer (25% PEG-8000, 500 mM Tris-HCl pH 7.5, 50 mM MgCl2, 50 mM DTT, 1 mM each of the dNTPs, and 5 mM NAD), T5 exonuclease, Phusion DNA polymerase, and Taq DNA ligase. The ligation mixture was used directly to transform electrocompetent E. coli strain E. cloni BL21(DE3) cells.

[0085] For expression and purification of Thermotoga maritima threonine aldolase (TmTA): single colony from LBkanagar plate was picked using a sterile toothpick and cultured in LBkanmedia (25 mL) at 37 ^C and 230 rpm overnight. This preculture was used to inoculate 1 L of TBkan media (v / v = 2.5%) in a 4 L Erlenmeyer flask, and the expression culture was incubated at 37 ^C and 200 rpm for ca.3 h until the OD600 reached ca.2.0. The culture was cooled on ice for 30 min and induced with 0.5 mM isopropyl-β-D- thiogalatopyranoside (IPTG) (final concentration). Protein expression was conducted at 23 ^C and 180 rpm for 20 h. E. coli cells were harvested by centrifugation at 4 ^C and 6,000 g for 10 min using a superspeed centrifuge. The cell pellet was then flash frozen with liquid nitrogen and stored until further use in a –80 °C freezer or directly used for cell lysis and protein purification.

[0086] For protein purification, frozen cells were suspended in HisTrap buffer A (25 mM KPi, 100 mM NaCl, 20 mM imidazole, pH = 8.0, ca.2–3 mL buffer A was added to 1 gof wet cell pellet), followed by the addition of a PLP stock solution (10 mM in 50 mM KPi buffer, pH = 8.0) to a final PLP concentration of 0.1 mM. The cell suspension was transferred to a 120 mL stainless steel beaker and lysed by sonication using a sonicator equipped with a standard probe (tip diameter = ½’’). Cell lysis conditions: 45% amplitude, 2 secs on, 4 secs off, 18 min in total (sonication time: 6 min). To pellet the cell debris, lysates were centrifuged using a Lynx 6000 superspeed centrifuge (15,000 g, 60 min, 4 °C). Threonine aldolases containing an N-terminal 6^His tag were purified with a column using a protein purification system. Proteins were eluted on a linear gradient from Histrap buffer A (25 mM KPi, 100 mM NaCl, 20 mM imidazole, pH 8.0) to Histrap buffer B (25 mM KPi, 100 mM NaCl, 500 mM imidazole, pH 8.0) over 10 column volumes (CVs). Proteins eluted at an imidazole concentration of ca.200 mM.

[0087] Fractions containing the threonine aldolase variant were combined, followed by the addition of a PLP stock solution (10 mM PLP in 50 mM KPi buffer, pH 8.0) to a final PLP concentration of 0.1 mM. The enzyme solution was concentrated and subjected to three rounds of buffer exchange using the storage buffer (buffer C, 200 mM KPi, pH = 8.0, optimized reaction pH) using an ultracentrifugal filter (10 kDa molecular weight cut-off) to remove excess salt and imidazole. Protein concentration was measured with the Nanodrop A280 method and the bicinchoninic acid (BCA) assay and normalized to ca.30 mg / mL for storage. Typically, 1 L of TBkan expression culture provides ca.50 mg TmTA variant. Concentrated proteins were aliquoted and flash-frozen in liquid N2 and stored at –80 °C until further use.10% glycerol (final concentration) was added to the final storage buffer to enhance long-term protein stability upon storage.

[0088] For Expression of TmTA variants in 96-well plates: single colonies from LBkan agar plates were picked using sterile toothpicks and cultured in deep-well 96-well plates containing LBkan (400 μL / well) at 37 ^C and 250 rpm shaking (12 h – 14 h) in an Eppendorf shaker. TBkan(950 μL / well) in a deep-well 96-well plate was then inoculated with an aliquot (50 μL / well) of these overnight cultures and allowed to shake for 3.0 h at 37 ^C and 250 rpm. The plates were cooled on ice for 20 min and the cultures were induced with 0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG). Expression was conducted at 23 ºC, 220 rpm for 20 h.

[0089] For reaction screening in 96-well plate format: E. coli (E. cloni BL21(DE3)) cells in deep-well 96-well plates were pelleted (3,000 g, 3 min, 4 ^C) using an Eppendorftabletop centrifuge 5910R and resuspended in KPi buffer (400 μL / well) by gentle shaking using a Fisher Scientific microplate shaker (800 rpm, 3–5 min). Then a PLP stock solution (15 μL / well, 10 mM in 50 mM KPi buffer, pH = 8.0) was added. The cell suspension was lysed by sonication using a sonicator equipped with a 24-tip horn. In each round of sonication, cell suspensions in a total of 24 wells in a 96-well plate were lysed. For example, in round 1 of sonication, wells A1, A3, A5, …, A11, C1, C3, C5, …, C11, E1, E3, E5, …, E11, G1, G3, G5, …, and G11 were sonicated. To lyse the cell suspensions in the 96-well plate, four iterations of sonication using this 24-tip horn were required. Cell lysis conditions: 45% amplitude, 2 secs on, 4 secs off, 3 min in total (sonication time: 1 min). To pellet the cell debris, lysates were centrifuged using an Eppendorf 5910R tabletop centrifuge (4000 g, 20 min, 4 °C).

[0090] Using an 12-channel electronic pipette (50–1000 µL), the clarified cell-free lysates (400 ^L per well) were then added to shell vials (400 ^L) in a 96-position reactor from and transferred into a Coy anaerobic chamber. In the Coy chamber, 15 µL of the benzylboron substrate (100 mM in degassed DMSO), 15 µL (fac)-Ir(ppy)3 (2.0 mM in degassed DMSO), 15 µL glycine (1.0 M in degassed 200 mM KPi buffer, pH = 8.0) and 30 µL Co(NH3)6Cl3 (100 mM in degassed water) were added into each well using an Eppendorf Xplorer 12-channel pipette (final concentration of the benzylboron reagent was 3.0 mM). The 96-position reactor was illuminated with a 440 nm 96-position LED array (75 mW) and left on a digital microplate shaker at room temperature and 380 rpm. After 6 h, the 96-well plate was taken out of the anaerobic chamber and the analytical scale reactions were worked up following the appropriate method below.

[0091] For product formation screening and enantioselectivity screening using LC-MS: after 6 h, a solution of 3 mM 2-phenylbutanoic acid (internal standard) in a mixed solvent system (1 M HCl / MeCN = 1:1, 500 μL) was added using an 12-channel, 50–1000 ^L electronic pipette. The reaction mixture was pipetted up and down using the 12-channel pipette several times to ensure complete mixing. This mixture (1000 μL per well) was then transferred to a new deep-well 96-well plate and spun down using a tabletop centrifuge (4500 g, 20 min) to completely separate the solid and liquid layers. The supernatant (200 μL per well) was transferred to 500 μL vial inserts using a 12-channel, 15-300 ^L electronic pipette. The inserts were then placed in 1.5 mL vials and analyzed by reverse-phase LC-MS analysis. A representative LC-MS method is as follows: column (4.6 ^ 50 mm, 5 μm), flow rate = 0.70mL / min, hold at 5% CH3CN (0.1% formic acid) in ddH2O (0.1% formic acid) for 3.0 min, ramp up to 95% CH3CN (0.1% formic acid) in ddH2O (0.1% formic acid) over 1.0 min, hold at 95% CH3CN (0.1% formic acid) in H2O (0.1% formic acid) for 2.0 min.

[0092] TmPLP^(ca.30 mg / mL, 40 μL) was allowed to thaw and kept on top of ice. In a Coy anaerobic chamber, the following stock solutions were prepared: alkylboronic acid pinacol ester (100 mM in degassed DMSO), (fac)-Ir(ppy)3 (2.0 mM in degassed DMSO), glycine (1.0 M in degassed 200 mM KPi buffer, pH = 8.0), PLP (25 mM in 200 mM KPi buffer, pH = 7.5), Co(NH3)6Cl3 (100 mM in degassed water). To a reaction tube (13 ^ 100 mm, 8 mL) containing a stir bar were added the KPi buffer (800 ^L, 200 mM, pH = 9.0), 30 μL organoboron reagent stock solution, 30 μL glycine stock solution, 12 μL PLP stock solution, 60 μL Co(NH3)6Cl3 stock solution, 30 μL photocatalyst stock solution, and ca.40 μL TmPLP^enzyme solution (the volume of the enzyme stock solution is dependent on the concentration of the specific enzyme sample). The total reaction volume was 1.0 mL; the final concentrations of each reaction component were as follows: 3.0 mM organoboron reagent, 30.0 mM glycine, 6.0 mM Co(NH3)6Cl3, 300 ^M PLP, 30 ^M TmPLP^and 60 ^M (fac)-Ir(ppy)3. The reaction tube was then sealed and removed from the Coy anaerobic chamber and submerged in a water bath at 50 °C. The reaction mixture was allowed to stir at 500 rpm and 50 °C and illuminated with two 45 W LED lamps for 12 h.

[0093] Upon completion, the reaction mixture was quenched with 1 mL of CH3CN / 1 M aq. HCl (v:v = 1:1).30 µL of a stock solution of 2-phenylbutyric acid (100 mM in CH3CN / 1 M aq. HCl) was then added as the internal standard. After vigorous mixing, ca.2.0 mL of reaction mixture was transferred into a 2.0 mL Eppendorf tube and centrifuged at 15,000 rpm for 15 min. The supernatant was analyzed by LC-MS. The yield was determined by LC-MS using 2-phenylbutyric acid as the internal standard using a freshly determined calibration curve. The remaining reaction mixture was then used for the determination of enantiomeric ratio by Marfey’s analysis as detailed below. A representative LC-MS analysis method is as follows: column (4.6 ^ 50 mm, 5 μm), flow rate = 0.70 mL / min, hold at 5% CH3CN (0.1% formic acid) in H2O (0.1% formic acid) for 12.0 min, ramp up to 95% CH3CN (0.1% formic acid) in H2O (0.1% formic acid) over 1.0 min, hold at 95% CH3CN (0.1% formic acid) in H2O (0.1% formic acid) for 2.0 min.

[0094] Marfey’s analysis is widely used to determine the stereochemical purity of amino acid products. A modified Marfey’s analysis was applied to determine the enantio- anddiastereomeric ratio of amino acid products. In the protocol, the ratio of Marfey’s reagent to combined amino acid content in the reaction mixture is >10:1 to ensure the complete conversion of biocatalytically formed, enantioenriched amino acids.

[0095] To a 1.5 mL glass vial were added 50 µL of the reaction mixture as described above, 1 M aq. NaHCO3 (100 µL), DMSO (50 µL) and a stock solution of Marfey’s reagent, namely (S)-1-fluoro-2-4-dinitrophenyl-5-L-alanine amide ((S)-FDAA) or (R)-1-fluoro-2-4- dinitrophenyl-5-L-alanine amide ((R)-FDAA) (50 µL, 20 mM in acetone). The vial was then placed in a microplate shaker and shaken at room temperature and 800 rpm for 8–12 h. The reaction mixture was diluted with 1:1 CH3CN / 1 M aq. HCl (800 µL) to afford a clear solution and analyzed by LC-MS.

[0096] A representative LC-MS analysis method is as follows: column (4.6 ^ 50 mm, 5 μm), flow rate = 0.70 mL / min, hold at 5% CH3CN (0.1% formic acid) in H2O (0.1% formic acid) for 1.0 min, ramp up to 60% CH3CN (0.1% formic acid) in H2O (0.1% formic acid) over the course of 14.0 min, then ramp up to 95% CH3CN (0.1% formic acid) in H2O (0.1% formic acid) over the course of 3.0 min, hold at 95% CH3CN (0.1% formic acid) in H2O (0.1% formic acid) for 1.0 min. Example 6 – Determination of enantiomeric ratio (e.r.) of amino acids with one stereocenter.

[0097] FIG.9A illustrates amino acids for Marfey’s analysis, according to some embodiments. FIG.9B illustrates Marfey’s analysis with (R)- and (S)-FDAA, according to some embodiments. First, independently synthesized racemic amino acids using chemical methods were analyzed. Before derivatization, enantiomersL-3 ((S)-3) andD-3 ((R)-3) were inseparable by achiral HPLC. After derivatization with Marfey’s reagent, all the amino acids were consumed as indicated by LC-MS analysis. Enantiomeric (S)-3 and (R)-3 were converted into diastereomeric (S, R)-3’ and (R, R)-3’, and these diastereomers could be easily separated by achiral HPLC. With racemic 3, the peak area ratios, A((S, R)-3’) / A((R, R)-3’), were all found to be 50:50. This indicated that with an excess of Marfey’s reagent, both the L- and the D-amino acid L-3 ((S)-3) and D-3 ((R)-3) were fully converted into the SNAr product (S, R)-3’ and (R, R)-3’, respectively. Furthermore, it showed that diastereomeric (S, R)-3’ and (R, R)-3’ had the same UV responding factor under an appropriate wavelength (e.g., 330 nm). FIG.9C illustrates Marfey’s analysis results, according to some embodiments. Together, these results indicated that the peak area ratio of derivatized diastereomer (S, R)-3’ and (R,R)-3’, namely A((S, R)-3’) / A((R, R)-3’), equals the enantiomeric ratio (e.r.) of 3, namely (S)- 3 : (R)-3.

[0098] Second, to ensure the accuracy of the results, each biocatalytically produced, enantioenriched amino acid product 3 was derivatized with both (R)- and (S)-FDAA and analyzed by LC-MS. Using (R)-FDAA, (S)-3 produces (S, R)-3’ while (R)-3 produces (R, R)- 3’. Similarly, using (S)-FDAA, (S)-3 produces (S, S)-3’ and (R)-3 produces (R, S)-3’. As (S, R)-3’ and (R, S)-3’ are enantiomers, they are indistinguishable by achiral HPLC analysis and have the same retention times. Similarly, (R, R)-3’ and (S, S)-3’ are enantiomers and they are thus indistinguishable by achiral HPLC analysis.

[0099] Using this protocol, for amino acids with a highL-content ((S)-3), when derivatized with (R)-FDAA, the first peak ((S, R)-3’) was found to be the major peak. The e.r. of 3 can be determined by the peak area ratio, namely A((S, R)-3’) / A((R,R)-3’). Using (S)- FDAA for derivatization, the second peak ((S, S)-3’) was found to be the major peak, and the e.r. of 3 can also be determined by the peak area ratio, namely A((S, S)-3’) / A((R, S)-3’). Importantly, in all Marfey’s analyses of enantioenriched amino acids, the measured e.r. of the amino acid product 3 with (R)- and (S)-FDAA were identical. In other words, in all cases, A((S, R)-3’) / A((R, R)-3’) = A((S, S)-3’) / A((R, S)-3’). Together, these results further ensured that no kinetic resolution was involved in Marfey’s analysis. Thus, the e.r. of 3 could be reliably determined. The absolute stereochemistry of model reaction product phenylalanine was determined by comparison with authentic enantiomerically pureL-phenylalanine. The absolute stereochemistry of other non-canonical amino acid products was inferred by analogy to L-phenylalanine. Example 7 - Determination of diastereomeric ratio (d.r.) and enantiomeric ratio (e.r.) of amino acids with two stereocenters.

[0100] The diastereoselective chemical synthesis of racemic amino acids with two contiguous stereocenters ((rac)-5) can require multistep synthesis from starting materials. The diastereomeric ratio (d.r.) of amino acids with two stereocenters can be measured by LC- MS. A representative LC-MS analysis method is as follows: column (4.5 ^ 250 mm, 5 μm), flow rate = 0.70 mL / min, hold at 5% CH3CN (0.1% formic acid) in H2O (0.1% formic acid) for 1.0 min, ramp up to 50% CH3CN (0.1% formic acid) in H2O (0.1% formic acid) over the course of 11.0 min, ramp up to 95% CH3CN (0.1% formic acid) in H2O (0.1% formic acid) over the course of 3.0 min, hold at 95% CH3CN (0.1% formic acid) in H2O (0.1% formicacid) for 1.0 min. The diastereomeric ratio was also measured by1H NMR analysis of the unpurified reaction mixture. LC-MS-determined diastereomeric ratios are in accordance with NMR results. The e.r. of the major diastereomeric non-canonical amino acid products was determined after HPLC separation of the two diastereomers. Example 8 - Determination of enantiomeric ratio (e.r.) of α-tetrasubstituted non-canonical amino acids using Fmoc chloride derivatization and normal phase chiral HPLC analysis.

[0101] Increasing the size of either α-substituent drastically reduced the efficiency of Marfey’s analysis. Fmoc chloride derivatization was used for these α-tetrasubstituted non- canonical amino acid products and resolved the enantiomers of these derivatization products by chiral normal phase HPLC. KPi buffer from the enzymatic reaction system was found to lower the efficiency of the Fmoc chloride derivatization. Accordingly, 10 analytical reactions were combined and purified the product to accurately determine their enantiomeric purity.

[0102] To a one-dram vial containing a stir bar were added the respective α- tetrasubstituted amino acid, 300 mM aq. NaOH (300 µL) and a stock solution of fluorenylmethyloxycarbonyl chloride (Fmoc–Cl, 400 µL, 100 mM in acetonitrile). The reaction mixture was allowed to stir at room temperature for 12 h. The organic solvent was removed in vacuo and the aqueous layer was washed with Et2O (3 mL). The aqueous layer was then acidified with saturated aqueous citric acid solution (2.0 mL, final pH 2–3) and extracted with ethyl acetate (3 × 3 mL). The combined organic layer was dried over Na2SO4, filtered, and evaporated in vacuo with the aid of a rotary evaporator. The residue was subjected to flash column chromatography on silica gel to furnish the desired derivatized compound as a white solid, which was dissolved with 1:1 i-PrOH / hexanes (1.0 mL) and analyzed by chiral HPLC. A representative chiral HPLC analysis method is as follows: column (4.6 mm × 25 cm, 5 micron), 95:5 hexanes: EtOH (0.1% TFA), flow rate = 1.0 mL / min, wavelength = 265 nm, 40 °C.30 min. Example 9 - biocatalytic preparation of non-canonical amino acids.

[0103] TmPLP^^ (TmTA E88T H83F, ca.30 mg / mL, 1 mL / tube) was thawed from the – 80 °C freezer and kept on ice. To a 50 mL round bottom flask, RBF3K 1 (15 μmol), 2a (150 μmol, 10 equiv, 11.3 mg), PLP (3.0 μmol, 20 mol%, 0.74 mg), Co(NH3)6Cl3 (30 μmol, 2 equiv, 8 mg) and 4CzIPN (1.5 μmol, 10 mol%, 1.2 mg) were added in succession. Then, the flask was taken into a Coy anaerobic chamber, where 4.0 mL degassed KPi buffer (0.2 M, pH= 9.0), 800 ^L degassed DMSO (5% final volume) and 200 ^L stock solution of TmPLP^^were added in succession. The total reaction volume was 5.0 mL. The flasks were sealed and removed from the Coy anaerobic chamber, stirred at 500 rpm and 50 °C and illuminated with two 45 W LED lamps (440 nm) for 12 h. The reaction mixture was then quenched with 1 M HCl (20 mL) and MeOH (20 mL) and centrifuged (4 ˚C, 30 min, 4500 rpm). Eight photobiocatalytic reactions were combined for non-canonical amino acid preparation. The combined supernatant was concentrated in vacuo with the aid of a rotary evaporator until the total volume was less than 30 mL. The residue was centrifuged (15 min, 4500 rpm) again to separate the insoluble fraction. The supernatant was further concentrated in vacuo with the aid of a rotary evaporator to close to dryness.

[0104] Approximately 10 mL water was added to dissolve the residue. This resulting mixture was filtered through a 2.2 ^m membrane filter and loaded onto a C18 column (18 g) that had been equilibrated to water with 0.1% HCOOH. The column was washed with 5 column volumes (CV) of water (0.1% HCOOH) to remove salts. Then, the product was eluted with a gradient from 0% to 100% MeOH in water over 12 CV. Product-containing fractions were combined and concentrated in vacuo to remove all the organic solvents. The residual aqueous solution was frozen in liquid nitrogen and lyophilized using a lyophilizer to give the amino acid product as a white solid. If the amino acid product is not UV active, fractions were analyzed by LC-MS and those fractions containing products were pooled and concentrated. Amino acid products were isolated as HCl adducts.

[0105] TmPLP^^(TmTA W86N, ca.30 mg / mL, 1 mL / tube) was thawed from the – 80 °C freezer and kept on ice. In a Coy anaerobic chamber, the following stock solutions were prepared: benzylboronic acid pinacol ester (500 mM in degassed DMSO), (fac)-Ir(ppy)3 (5.0 mM in degassed DMSO), glycine (1.0 M in degassed 200 mM KPi buffer), PLP (50 mM in 200 mM KPi buffer, pH = 7.5), Co(NH3)6Cl3(200 mM in degassed water). To a 250 mL round bottom flask equipped with a stir bar were added the KPi buffer (60 mL, 200 mM, pH = 9.0), 2 mL benzylboronic acid pinacol ester stock solution, 10 mL glycine stock solution, 2 mL PLP stock solution, 10 mL Co(NH3)6Cl3 stock solution, 4 mL (fac)-Ir(ppy)3 stock solution, and ca.12 mL TmPLP^enzyme solution (the volume of the enzyme stock solution is dependent on the specific concentration of the enzyme sample used). The total reaction volume was 100.0 mL; the final concentrations of each reaction component were as follows: 10.0 mM benzylboronic acid pinacol ester, 100.0 mM glycine, 20.0 mM Co(NH3)6Cl3, 1.0mM PLP, 100 ^M TmPLP^and 200 ^M (fac)-Ir(ppy)3. The reaction tube was then sealed and removed from the Coy anaerobic chamber and submerged in a water bath preheated to 50 °C. The reaction mixture was allowed to stir at 500 rpm and 50 °C and illuminated with two LED lamps (440 nm, 45 W) for 12 h. The reaction mixture was then quenched with 1 M HCl (100 mL), diluted with MeOH (100 mL) and then centrifuged (4 ˚C, 30 min, 4,500 rpm). The supernatant was concentrated in vacuo with the aid of a rotary evaporator until the total volume was less than 50 mL. The residue was centrifuged (15 min, 4500 rpm) again to separate the insoluble fraction.

[0106] The supernatant was further concentrated in vacuo with the aid of a rotary evaporator to close to dryness. Approximately 10 mL water was added to dissolve the residue. The resulting mixture was filtered through a 2.2 ^m membrane filter and loaded onto a C18 column (18 g) that had been equilibrated to water with 0.1% HCOOH. The column was washed with 10 column volumes (CV) of water (0.1% HCOOH) to remove salts. Then, the product was eluted with a gradient from 0% to 100% MeOH in water over 12 CV. Product-containing fractions were combined and concentrated in vacuo to remove all the organic solvents. The residual aqueous solution was frozen in liquid nitrogen and lyophilized using a lyophilizer to give the amino acid product as a white solid. If needed, fractions could be analyzed by LC-MS and those containing the desired products were pooled and concentrated. Product 3a was isolated as an HCl adduct as a white powder (123 mg, 61% yield).

[0107] TmPLP^^(TmTA W86N, ca.30 mg / mL, 1 mL / tube) was thawed from the – 80 °C freezer and kept on ice. In a Coy anaerobic chamber, the following stock solutions were prepared: benzylboronic acid pinacol ester (500 mM in degassed DMSO), 4CzIPN (25.0 mM in degassed DMSO),D-alanine (1.0 M in degassed 200 mM KPi buffer), PLP (50 mM in 200 mM KPi buffer, pH = 7.5), Co(NH3)6Cl3(200 mM in degassed water). To a 250 mL round bottom flask equipped with a stir bar were added KPi buffer (60 mL, 200 mM, pH = 9.0), 2 mL benzylboronic acid pinacol ester stock solution, 10 mL D-alanine stock solution, 2 mL PLP stock solution, 10 mL Co(NH3)6Cl3 stock solution, 4 mL 4CzIPN stock solution, and ca.12 mL TmPLP^enzyme solution (the volume of the enzyme stock solution is dependent on the specific concentration of the enzyme sample used). The total reaction volume was 100.0 mL; the final concentrations of each reaction component were as follows: 10.0 mM benzylboronic acid pinacol ester, 100.0 mMD-alanine, 20.0 mM Co(NH3)6Cl3, 1.0 mM PLP,100 ^M TmPLP^and 1.0 mM 4CzIPN. The reaction tube was then sealed and removed from the Coy anaerobic chamber and submerged in a water bath preheated to 50 °C. The reaction mixture was allowed to stir at 500 rpm and 50 °C and illuminated with two LED lamps (440 nm, 45 W) for 12 h.

[0108] The reaction mixture was then quenched with 1 M HCl (100 mL) and MeOH (100 mL) and centrifuged (4 ˚C, 30 min, 4500 rpm). The supernatant was concentrated in vacuo with the aid of a rotary evaporator until the total volume was less than 50 mL. The residue was centrifuged (15 min, 4500 rpm) again to separate the insoluble fraction. The supernatant was further concentrated in vacuo with the aid of a rotary evaporator to close to dryness. Approximately 10 mL water was added to dissolve the residue. The resulting mixture was filtered through a 2.2 ^m membrane filter and loaded onto a C18 column (18 g) that had been equilibrated to water with 0.1% HCOOH. The column was washed with 10 column volumes (CV) of water (0.1% HCOOH) to remove salts. Then, the product was eluted with a gradient from 0% to 100% MeOH in water over 12 CV. Product-containing fractions were combined and concentrated in vacuo to remove all the organic solvents. The residual aqueous solution was frozen in liquid nitrogen and lyophilized using a lyophilizer to give the amino acid product. Product 8a was isolated as an HCl adduct as a white solid (142 mg, 66% yield). Example 10 – Synthesis and Characterization of Substrates

[0109] Compounds 1b−1d, 1f−1i, 1n−1o, 1t and 1z were prepared according to General Procedure A.General Procedure A. Pd(PPh3)4 (0.25 mmol, 5 mol%), bis(pinacolato)diboron (6.0 mmol, 1.2 equiv) and potassium carbonate (15 mmol, 3.0 equiv) were added to an oven-dried three-neck round bottom flask. The flask was evacuated and backfilled with nitrogen (this process was repeated for 3 times). The benzyl bromide substrate S2 (5.0 mmol, 1.0 equiv) was added, followed by the addition of 1,4-dioxane (10 mL). The reaction mixture was heated to 80 °C for 6 h and then cooled to room temperature. The reaction mixture was filtered through a silica plug eluting with EtOAc (30 mL). The organic layer was washed with brine (3 × 30mL), dried over MgSO4, and concentrated in vacuo to provide the crude product, which was purified by silica gel chromatography (hexanes: CH2Cl2 = 3:1) to afford the product 1.

[0110] Compounds 1m were prepared according to General Procedure B.General Procedure B. To an oven-dried 50 mL flask equipped with a stir bar were added 4- formylphenyl acetate (S3, 164 mg, 1.0 mmol, 1.0 equiv) and B2cat2 (710 mg, 3.0 mmol, 3.0 equiv), 6.0 mL DMA (0.33 M), and degassed water (H2O, 200 ^L, 10.0 mmol, 10.0 equiv). The resulting mixture was degassed by three freeze-pump-thaw cycles and allowed to stir at room temperature for 72 h. Pinacol (709 mg, 6.0 mmol, 6.0 equiv) and 3.5 mL Et3N (dried over 4 Å molecular sieves overnight) were added, and the resulting mixture was stirred at ambient temperature for 1 h. The reaction was quenched by the addition of 20 mL brine and extracted with 20 mL EtOAc for 5 times. The combined organic layers were dried over MgSO4 and concentrated in vacuo with the aid of a rotary evaporator. The residue was subjected to flash column chromatography on silica gel to furnish the titled compound 1m as a colorless oil.

[0111] Compounds 1u, 1v, 1w and 1y were prepared following General Procedure C.General Procedure C. CuCl (0.15 mmol, 15 mg), KOtBu (0.3 mmol, 30 mg), and dppbz (0.165 mmol, 73 mg) were added to a 50 mL flask and the flask was evacuated and backfilled with nitrogen. Anhydrous toluene (2 mL) was added and the resulting mixture was stirred for 10 min. Pinacolborane (6.0 mmol, 768 mg, 898 µL) was added, and the reaction mixture was allowed to stir for another 10 min at room temperature. Styrene S4 (5 mmol) was added and the reaction mixture was allowed to stir at 60 °C for 24 h. The mixture was filtered through a celite plug and concentrated in vacuo to provide the crude product S5, which was purified by column chromatography or used directly for the next step without further purification. S5 was dissolved in MeOH (50 mL) and cooled to 0 °C. Saturated aqueous KHF2 (40.5 mmol, 8.1 equiv) was added dropwise over 30 min and the solution was allowed to warm to roomtemperature. The resulting suspension was concentrated in vacuo with the aid of a rotary evaporator. Pinacol and H2O were azeotropically removed by resuspending the mixture in toluene (150 mL) and rotary evaporation at 40 °C. The remaining solid was dried under high vacuum, dissolved in hot acetone (3 × 100 mL) and filtered to remove inorganic salts. The filtrate was concentrated to a minimal volume (5–20 mL) and hexanes (500 mL) was added to provide a white solid. The solid product was isolated by filtration and washed with hexanes (50 mL) and CH2Cl2 (30 mL) to afford the desired benzyltrifluoroborate salt 1.

[0112] Compound 1x was prepared following general procedure D.General Procedure D. A 100 mL round-bottom flask equipped with a stir bar was charged with magnesium turnings (290 mg, 12 mmol). The flask was evacuated and backfilled with nitrogen and this process was repeated for a total of three times. THF was added, followed by a small amount of iodine. The mixture was allowed to stir until the color of I2 disappeared. Pinacolborane (1.2 mL, 12 mmol) was added. Benzyl bromide S6 (10 mmol) in 5 mL of THF was added slowly over 10 min at room temperature such that the Grignard reagent formed gently. The reaction mixture was allowed to stir at room temperature for 3 h, cooled to 0 °C, slowly acidified with 3 M HCl (15 mL), and allowed to stir at room temperature for 30 min. The reaction mixture was then transferred to a separatory funnel and extracted with diethyl ether (3 × 15 mL). The combined organic layers were dried over anhydrous MgSO4, filtered, and dried in vacuo (25 °C, 1 Torr). Residue S7 was used directly for the next step using general procedure C without further purification.

[0113] Characterization data for secondary benzyltrifluoroborates: Potassium trifluoro(1-(p-tolyl)ethyl)borate (1v) 1H NMR (400 MHz, DMSO-d6) δ: 6.92 (m, 2H), 6.85 (m, 2H), 2.18 (s, 3H), 1.51 (m, 1H), 1.00 (d, J = 7.3 Hz, 3H) ppm.13C NMR (101 MHz, DMSO-d6) δ: 149.1, 130.4, 127.4, 127.4, 32.3, 20.6, 17.5. ppm.19F NMR (376 MHz, DMSO-d6) δ: -142.8 ppm.11B NMR (128 MHz, DMSO) δ: 3.9 ppm. IR: 2871, 1511, 1232, 1094, 1016, 910, 626 cm-1. HRMS (ESI) (m / z) for [M+K]+C9H11BF3K2O2 requires 186.0942, observed 186.0948.Potassium trifluoro(1-(m-tolyl)ethyl)borate (1w) 1H NMR (400 MHz, DMSO-d6) δ: 6.93 (m, 1H), 6.87 – 6.79 (m, 2H), 6.69 (m, 1H), 2.20 (s, 3H), 1.64 – 1.42 (m, 1H), 1.02 (d, J = 7.3 Hz, 1H) ppm.13C NMR (101 MHz, DMSO-d6) δ: 152.3, 135.1, 128.3,126.6, 124.7, 122.9, 32.3, 21.3, 17.4. ppm.19F NMR (376 MHz, DMSO-d6) δ: -142.7 ppm.11B NMR (128 MHz, DMSO) δ: 4.0 ppm. IR: 2970, 1738, 1365, 1217, 1003, 916, 713, 528 cm-1. HRMS (ESI) (m / z) for [M+K]+C9H11BF3K2O2 requires 186.0942, observed 186.0937. Potassium trifluoro(1-(o-tolyl)ethyl)borate (1x) 1H NMR (400 MHz, DMSO-d6) δ 7.11 – 7.01 (m, 1H), 6.91 (m, 2H), 6.76 (m, 1H), 2.18 (s, 3H), 1.81 (m, 1H), 1.01 (d, J = 7.2 Hz, 3H) ppm.13C NMR (101 MHz, DMSO-d6) δ: 150.9, 134.7, 128.5, 126.4, 124.7, 121.7,27.6, 20.2, 17.6 ppm.19F NMR (376 MHz, DMSO-d6) δ -142.1 ppm.11B NMR (128 MHz, DMSO) δ 4.0 ppm. IR: 2970, 1738, 1441, 1228, 1217, 528 cm-1. HRMS (ESI) (m / z) for [M+K]+C9H11BF3K2O2 requires 186.0942, observed 186.0941. Potassium trifluoro(1-(4-fluorophenyl)ethyl)borate (1y) 1H NMR (400 MHz, DMSO-d6) δ: δ 7.08 – 6.98 (m, 2H), 6.92 – 6.76 (m, 2H), 1.56 (s, 1H), 1.20 – 0.82 (m, 2H) ppm.13C NMR (101 MHz, DMSO-d6) δ: 158.9 (d, J = 236.2 Hz), 148.2, 128.5 (d, J = 7.2 Hz),113.2 (d, J = 20.0 Hz), 31.2, 17.5. ppm.19F NMR (376 MHz, DMSO- d6) δ: -143.4, -122.7 ppm.11B NMR (128 MHz, DMSO) δ: 3.9 ppm. IR: 2970, 1738, 1506, 1365, 1217, 1022, 836, 523 cm-1. HRMS (ESI) (m / z) for [M+K]+C8H8BF4K2O2 requires 190.0692, observed 190.0696. Example 11 - Synthesis and characterization.

[0114] Compound 3a and 3t were commercially available and were directly used without further purification. Racemic amino acids 3b–3s and 3z were synthesized using General Procedure E. Amino acids 5a–5e, including their racemates, are difficult to synthesize using chemical methods. Thus, preparative biocatalytic synthesis was used to provide these products using the general experimental procedure for non-canonical aminoacid preparation as described above. Compound 8a–8e was were synthesized using general procedure F.General Procedure E. To a solution of diethyl acetamidomalonate S8 (14.4 mol, 2.0 equiv) in DMSO (30 mL) was added Cs2CO3 (18.0 mol, 2.5 equiv) at 0 °C. The reaction solution was stirred at room temperature for 1 h. The alkyl bromide S6 (7.2 mol, 1.0 equiv) was added to the reaction mixture at room temperature and the reaction mixture was stirred at 65 °C for 8 h. Upon the completion of this reaction, the mixture was poured into ice (80 mL) and stirred for 1 h. The precipitate was filtered and dried under vacuum to give the alkylation product S9 as an off-white solid, which was used directly for the next step without further purification. A suspension of this crude product (5.0 mmol, 0.2 M) in 1:1 of aq. HCl:1,4-dioxane was heated to reflux for 12 h. Upon the completion of the reaction as indicated by TLC analysis, the reaction mixture was concentrated in vacuo with the aid of a rotary evaporator and the residue was recrystallized from methanol and diethyl ether. The solid amino acid product was collected by filtration. In many cases, this collected solid was found to be analytically pure. If needed, it can be further purified by C18 silica gel chromatography.

[0115] Compounds 8a–8e was were synthesized using general procedure F.General Procedure F. An oven-dried round-bottom flask equipped with a magnetic stir bar was charged with the corresponding amino acid-derived oxazolone (10 mmol, 1 equiv) and anhydrous THF (10 mL). Triethylamine (2.8 mL, 20 mmol, 2 equiv) was added dropwise into the flask followed by addition of benzyl bromide (20 mmol, 2 equiv) at room temperature. After 12 h, water was added to the reaction mixture. The mixture was extracted by ethyl acetate (10 mL, 3 times), and combined organic phases were dried over anhydrous Na2SO4. The solvent was removed under reduced pressure with the aid of a rotary evaporator, and the residue was purified by silica gel column chromatography (hexane: ethyl acetate = 15:1) togive a colorless or pale-yellow oil. Compound S11 (4.80 mmol) was dissolved in 90:10 trifluoroacetic acid / water (48.0 mL, 0.10 M) and heated to 100 °C for 12 h. After the reaction proceeded to completion as indicated by TLC analysis, the solvent was evaporated under reduced pressure, and the residue was dissolved with water and washed three times with CH2Cl2. The aqueous phase was evaporated under reduced pressure to give the amino acid trifluoroacetic acid salt as a white solid. This solid was added into 5 mL ethanol. Sodium bicarbonate (1.26 g, 3 equiv) was added and the reaction mixture was stirred at room temperature for 1 h. The undissolved solid was filtered off and the collected filtrate was evaporated under reduced pressure. The residue obtained could be further purified by C18 silica gel chromatography. Fractions containing the product was further acidified with 6M HCl to give the corresponding amino acid hydrochloride salt. Example 11 - Characterization data for non-canonical amino acids.

[0116] L-phenylalanine (3a)1H NMR (400 MHz, D2O+NaOH) δ: 6.96 – 6.89 (m, 2H), 6.88 – 6.84 (m, 1H), 6.83 – 6.79 (m, 2H), 3.03 (dd, J = 7.6, 5.4 Hz, 1H), 2.54 (dd, J = 13.5, 5.4 Hz, 1H), 2.34 (dd, J = 13.5, 7.6 Hz, 1H) ppm.13C NMR (101 MHz, D2O+NaOH) δ: 182.1, 138.1, 129.1, 128.4, 126.4, 57.1, 40.7 ppm.3a was prepared according to general procedure using TmTA W86N (1.0 mol%) and derivatized using (R)-FDAA and analyzed by LC-MS. Yield: run 1: 76%; run 2: 77%; run 3: 77%; average yield: 77%. Enantioselectivity: run 1: 98.5:1.5 e.r.; run 2: 98:2 e.r.; run 3: 98.5:1.5 e.r.; average e.r.: 98.5:1.5.

[0117] L-2-amino-3-(p-tolyl)propanoic acid (3b)1H NMR (400 MHz, D2O+NaOH) δ: 6.53 (m, 2H), 6.41 (m, 2H), 2.91 (dd, J = 9.1, 4.4 Hz, 1H), 2.52 (dd, J = 13.5, 4.4 Hz, 1H), 2.07 (dd, J = 13.5, 9.1 Hz, 1H), 1.61 (s, 3H) ppm.13C NMR (101 MHz, D2O+NaOH) δ: 181.5, 135.3, 135.1, 129.0, 128.9, 128.7, 57.2, 40.7 ppm. 3b was prepared according to general procedure using TmTA W86N (1.0 mol%) and derivatized using (R)-FDAA and analyzed by LC-MS. Yield: run 1: 75%; run 2: 70%; run 3: 74%; average yield: 73%. Enantioselectivity: run 1: 98:2 e.r.; run 2: 97:3 e.r.; run 3: 98:2 e.r.; average e.r.: 98:2.

[0118] L-2-amino-3-(m-tolyl)propanoic acid (3c)1H NMR (400 MHz, D2O+NaOH) δ: 6.59 (m, 1H), 6.47 (m, 2H), 6.37 (m, 1H), 2.92 (dd, J = 8.8, 4.6 Hz, 1H), 2.50 (dd, J = 13.5, 4.6 Hz, 1H), 2.10 (dd, J = 13.5, 8.8 Hz, 1H), 1.66 (s, 3H)ppm.13C NMR (101 MHz, D2O+NaOH) δ: 181.7, 138.3, 137.8, 129.7, 128.1, 126.7, 126.0, 57.2, 41.0, 20.4 ppm. IR: 2973, 2896, 1734, 1485, 1226, 1206, 794 cm-1. HRMS (ESI) (m / z) for [M+H]+C10H14NO2 requires 180.1024, observed 180.1020.3c was prepared according to general procedure using TmTA W86N (1.0 mol%) and derivatized using (R)-FDAA and analyzed by LC-MS. Yield: run 1: 66%; run 2: 61%; run 3: 68%; average yield: 65%. Enantioselectivity: run 1: 96:4 e.r.; run 2: 97:3 e.r.; run 3: 97:3 e.r.; average e.r.: 97:3.

[0119] L-2-amino-3-(o-tolyl)propanoic acid (3d)1H NMR (400 MHz, D2O+NaOH) δ: 6.75 – 6.36 (m, 4H), 2.92 (dd, J = 9.0, 5.1 Hz, 1H), 2.56 (dd, J = 13.7, 5.1 Hz, 1H), 2.15 (dd, J = 13.7, 9.0 Hz, 1H), 1.74 (s, 3H) ppm.13C NMR (101 MHz, D2O+NaOH) δ: 182.6, 137.1, 136.8, 130.3, 130.2, 126.8, 125.9, 56.7, 38.4, 18.7 ppm. IR: 2962, 2896, 1733, 1485, 1205, 743 cm-1. HRMS (ESI) (m / z) for [M+H]+C10H14NO2 requires 180.1024, observed 180.1020.3d was prepared according to general procedure using TmTA W86N (1.0 mol%) and derivatized using (R)-FDAA and analyzed by LC-MS. Yield: run 1: 65%; run 2: 69%; run 3: 63%; average yield: 66%. Enantioselectivity: run 1: 96:4 e.r.; run 2: 95:5 e.r.; run 3: 95:5 e.r.; average e.r.: 95:5.

[0120] L-2-amino-3-(4-methoxyphenyl)propanoic acid (3e)1H NMR (400 MHz, D2O+NaOH) δ: 6.65 (d, J = 8.6 Hz, 2H), 6.36 (d, J = 8.6 Hz, 2H), 3.23 (s, 3H), 2.95 (dd, J = 7.9, 5.1 Hz, 1H), 2.46 (dd, J = 13.6, 5.1 Hz, 1H), 2.21 (dd, J = 13.6, 7.9 Hz, 1H) ppm.13C NMR (101 MHz, D2O+NaOH) δ: 182.0, 157.0, 130.6, 130.2, 113.7, 57.2, 55.1, 39.9 ppm.3e was prepared according to general procedure using TmTA W86N (1.0 mol%) and derivatized using (R)-FDAA and analyzed by LC-MS. Yield: run 1: 70%; run 2: 65%; run 3: 68%; average yield: 68%. Enantioselectivity: run 1: 97:3 e.r.; run 2: 97:3 e.r.; run 3: 97:3 e.r.; average e.r.: 97:3.

[0121] L-2-amino-3-(4-(methylthio)phenyl)propanoic acid (3f)1H NMR (400 MHz, D2O+NaOH) δ: 7.19 (m, 2H), 7.11 (m, 2H), 3.37 (dd, J = 5.6 Hz, 7.2 Hz.1H), 2.84 (dd, J = 13.7, 5.6 Hz, 1H), 2.71 (dd, J = 13.7, 7.2 Hz, 1H), 2.39 (s, 3H) ppm.13C NMR (101 MHz, D2O+NaOH) δ: 182.2, 135.4, 135.1, 130.0, 126.6, 57.3, 40.2, 14.8 ppm. IR: 2967, 2878, 1735, 1603, 1485, 1421, 1228, 1195, 819 cm-1. HRMS (ESI) (m / z) for [M+H]+C10H14NO2S requires 212.0745, observed 212.0746.3f was prepared according to general procedure using TmTA W86N (1.0 mol%) and derivatized using (R)-FDAA and analyzed by LC-MS.Yield: run 1: 55%; run 2: 57%; run 3: 58%; average yield: 57%. Enantioselectivity: run 1: 98:2 e.r.; run 2: 98:2 e.r.; run 3: 98:2 e.r.; average e.r.: 98:2.

[0122] L-tyrosine (3g)1H NMR (400 MHz, D2O+NaOH) δ: 7.01 – 6.76 (m, 2H), 6.49 (m, 2H), 3.32 (dd, J = 7.3, 5.2 Hz, 1H), 2.77 (dd, J = 13.7, 5.2 Hz, 1H), 2.59 (dd, J = 13.7, 7.3 Hz, 1H) ppm.13C NMR (101 MHz, D2O+NaOH) δ: 182.9, 164.5, 130.6, 123.7, 118.6, 57.5, 39.8 ppm. L-tyrosine is a known compound.3g was prepared according to general procedure using TmTA W86N (1.0 mol%) and derivatized using (R)-FDAA and analyzed by LC-MS. Yield: run 1: 35%; run 2: 32%; run 3: 31%; average yield: 33%. Enantioselectivity: run 1: 98:2 e.r.; run 2: 98:2 e.r.; run 3: 98:2 e.r.; average e.r.: 98:2.

[0123] L-2-amino-3-(4-(trifluoromethoxy)phenyl)propanoic acid (3h)1H NMR (400 MHz, D2O+NaOH) δ: 7.23 (m, 4H), 3.49 – 3.32 (m, 1H), 2.93 (dd, J = 13.5, 5.6 Hz, 1H), 2.79 (dd, J = 13.6, 7.3 Hz, 1H) ppm.13C NMR (101 MHz, D2O+NaOH) δ: 182.1, 147.5 (q, J = 1.8 Hz), 137.2, 130.7, 121.0, 120.3 (q, J = 255.8 Hz), 57.3, 40.0 ppm.19F NMR (376 MHz, D2O): -57.8 ppm. IR: 2973, 2896, 1735, 1486, 1264, 1206, 1149, 840 cm-1. HRMS (ESI) (m / z) for [M+H]+C10H11F3NO3 requires 250.0691, observed 250.0701.3h was prepared according to general procedure using TmTA W86N (1.0 mol%) and derivatized using (R)-FDAA and analyzed by LC-MS. Yield: run 1: 59%; run 2: 58%; run 3: 62%; average yield: 59%. Enantioselectivity: run 1: 99:1 e.r.; run 2: 98:2 e.r.; run 3: 98:2 e.r.; average e.r.: 98:2.

[0124] L-2-amino-3-(4-fluorophenyl)propanoic acid (3i)1H NMR (400 MHz, D2O+NaOH) δ: 6.83 – 6.68 (m, 2H), 6.58 (m, 2H), 2.98 (dd, J = 7.6, 5.4 Hz, 1H), 2.48 (dd, J = 13.6, 5.4 Hz, 1H), 2.29 (dd, J = 13.6, 7.6 Hz, 1H) ppm.13C NMR (101 MHz, D2O+NaOH) δ: 181.9, 161.0 (d, J = 240.8 Hz), 133.9 (d, J = 2.9 Hz), 130.6 (d, J = 8.0 Hz), 114.8 (d, J = 20.9 Hz), 57.1, 39.9 ppm.19F NMR (376 MHz, D2O+NaOH) δ: -117.7. IR: 2967, 2880, 1737, 1513, 1485, 1223, 1210, 809, 516 cm-1. HRMS (ESI) (m / z) for [M+H]+C9H11FNO2requires 184.0774, observed 184.0766.3i was prepared according to general procedure using TmTA W86N (1.0 mol%) and derivatized using (R)-FDAA and analyzed by LC-MS. Yield: run 1: 71%; run 2: 68%; run 3: 73%; average yield: 71%. Enantioselectivity: run 1: 98:2 e.r.; run 2: 98:2 e.r.; run 3: 99:1 e.r.; average e.r.: 98:2.

[0125] L-2-amino-3-(4-chlorophenyl)propanoic acid (3j)1H NMR (400 MHz, D2O+NaOH) δ: 7.16 (m, 2H), 7.03 (m, 2H), 3.29 (dd, J = 7.3, 5.7 Hz, 1H), 2.77 (dd, J = 13.5, 5.7 Hz, 1H), 2.63 (dd, J = 13.5, 7.3 Hz, 1H) ppm.13C NMR (101 MHz, D2O+NaOH) δ: 182.2, 135.4, 135.1, 130.0, 126.6, 57.3, 40.1ppm.3j was prepared according to general procedure using TmTA W86N (1.0 mol%) and derivatized using (R)- FDAA and analyzed by LC-MS. Yield: run 1: 47%; run 2: 49%; run 3: 41%; average yield: 46%. Enantioselectivity: run 1: 95:5 e.r.; run 2: 97:3 e.r.; run 3: 96:4 e.r.; average e.r.: 96:4.

[0126] L-2-amino-3-(3-cyanophenyl)propanoic acid (3k)1H NMR (400 MHz, D2O+NaOH) δ: 7.36 (m, 2H), 7.30 (s, 1H), 7.23 – 6.80 (m, 2H), 3.12 (d, J = 5.5, 7.5 Hz, 1H), 2.61 (dd, J = 13.5, 5.5 Hz, 1H), 2.44 (dd, J = 13.5, 7.5 Hz, 1H). ppm.13C NMR (101 MHz, D2O+NaOH) δ: 182.1, 175.2, 138.1, 136.1, 131.9, 129.4, 128.3, 126.89, 57.2, 40.7 ppm. IR: 2978, 2902, 2230, 1732, 1488, 1228, 1211, 779, 692 cm-1. HRMS (ESI) (m / z) for [M+H]+C10H11N2O2 requires 191.0820, observed 191.0824.3k was prepared according to general procedure using TmTA W86N (1.0 mol%) and derivatized using (R)- FDAA and analyzed by LC-MS. Yield: run 1: 25%; run 2: 26%; run 3: 29%; average yield: 27%. Enantioselectivity: run 1: 96:4 e.r.; run 2: 96:4 e.r.; run 3: 95:5 e.r.; average e.r.: 96:4.

[0127] L-2-amino-3-(3-methoxyphenyl)propanoic acid (3l)1H NMR (400 MHz, D2O+NaOH) δ: 7.25 (t, J = 7.8 Hz, 1H), 7.00 – 6.64 (m, 3H), 3.76 (s, 3H), 3.56 – 3.34 (m, 1H), 2.92 (dd, J = 13.5, 5.5 Hz, 1H), 2.75 (dd, J = 13.5, 7.5 Hz, 1H) ppm.13C NMR (101 MHz, D2O+NaOH) δ: 182.3, 158.8, 140.1, 129.7, 122.2, 114.8, 112.2, 57.30, 55.3, 40.8 ppm.3l was prepared according to general procedure using TmTA W86N (1.0 mol%) and derivatized using (R)-FDAA and analyzed by LC-MS. Yield: run 1: 82%; run 2: 83%; run 3: 82%; average yield: 82%. Enantioselectivity: run 1: 98:2 e.r.; run 2: 98:2 e.r.; run 3: 98:2 e.r.; average e.r.: 98:2.

[0128] L-3-([1,1'-biphenyl]-4-yl)-2-aminopropanoic acid (3m)1H NMR (400 MHz, D2O+NaOH) δ: 7.09 (m, 4H), 6.92 (m, 4H), 6.85 (m, 1H), 3.25 (dd, J = 9.3, 4.3 Hz, 1H), 2.90 (dd, J = 13.5, 4.3 Hz, 1H), 2.45 (dd, J = 13.5, 9.3 Hz, 1H) ppm.13C NMR (101 MHz, D2O+NaOH) δ: 181.6, 140.1, 138.3, 137.7, 129.7, 128.6, 126.9, 126.7, 126.5, 57.4, 41.0 ppm. IR: 2973, 2875, 1725, 1500, 1230, 1210, 839, 749, 689 cm-1. HRMS (ESI) (m / z) for [M+H]+C15H16NO2 requires 242.1181, observed 242.1186. 3m was prepared according to general procedure using TmTA W86N (1.0 mol%) and derivatized using (R)- FDAA and analyzed by LC-MS.Yield: run 1: 25%; run 2: 29%; run 3: 26%; average yield: 27%. Enantioselectivity: run 1: 99:1 e.r.; run 2: 98:2 e.r.; run 3: 98:2 e.r.; average e.r.: 98:2.

[0129] L-3-(4-(1H-pyrazol-1-yl)phenyl)-2-aminopropanoic acid (3n)1H NMR (400 MHz, D2O+NaOH) δ: 7.84 (d, J = 2.2 Hz, 1H), 7.57 (d, J = 2.2 Hz, 1H), 7.29 (m, 2H), 7.11 (m, 2H), 6.35 (m, 1H), 3.40 – 3.16 (m, 1H), 2.80 (dd, J = 13.5, 5.6 Hz, 1H), 2.65 (dd, J = 13.5, 7.4 Hz, 1H) ppm.13C NMR (101 MHz, D2O+NaOH) δ: 182.1, 141.2, 137.7, 137.0, 130.3, 128.9, 119.4, 107.6, 57.2, 40.2 ppm. IR: 2915, 1736, 1612, 1526, 1398, 758 cm-1. HRMS (ESI) (m / z) for [M+H]+C13H14N3O2 requires 232.1086, observed 232.1089. 3n was prepared according to general procedure using TmTA W86N (1.0 mol%) and derivatized using (R)-FDAA and analyzed by LC-MS. Yield: run 1: 61%; run 2: 58%; run 3: 62%; average yield: 61%. Enantioselectivity: run 1: 97:3 e.r.; run 2: 98:2 e.r.; run 3: 97:3 e.r.; average e.r.: 97:3.

[0130] L-2-amino-3-(4-(tert-butyl)phenyl)propanoic acid (3o)1H NMR (400 MHz, D2O+NaOH) δ: 7.35 – 7.16 (m, 2H), 7.14 – 6.99 (m, 2H), 3.33 (dd, J = 8.7, 4.6 Hz, 1H), 2.93 (dd, J = 13.5, 4.6 Hz, 1H), 2.56 (dd, J = 13.5, 8.7 Hz, 1H), 1.09 (s, 9H) ppm.13C NMR (101 MHz, D2O+NaOH) δ: 181.9, 149.2, 135.6, 129.2, 125.3, 57.4, 40.7, 33.8, 30.8 ppm. IR: 2962, 2874, 1739, 1482, 1227, 1213, 809 cm-1. HRMS (ESI) (m / z) for [M+H]+C13H20NO2 requires 222.1494, observed 222.1495.3o was prepared according to general procedure using TmTA W86N (1.0 mol%) and derivatized using (R)-FDAA and analyzed by LC-MS. Yield: run 1: 53%; run 2: 55%; run 3: 54%; average yield: 54%. Enantioselectivity: run 1: 99:1 e.r.; run 2: 99:1 e.r.; run 3: 99:1 e.r.; average e.r.: 99:1.

[0131] L-2-amino-3-(3,4-difluorophenyl)propanoic acid (3p)1H NMR (400 MHz, D2O+NaOH) δ: 7.21 – 7.03 (m, 2H), 6.94 (m, 1H), 3.40 (m, 1H), 2.86 (dd, J = 13.7, 5.8 Hz, 1H), 2.75 (dd, J = 13.7, 7.2 Hz, 1H) ppm.13C NMR (101 MHz, D2O+NaOH) δ: 182.0, 150.4 (dd, J = 86.1, 12.6 Hz), 148.0 (dd, J = 84.8, 12.7 Hz), 135.3 (dd, J = 6.0, 3.8 Hz), 125.5 (dd, J = 6.5, 3.4 Hz), 117.8 (d, J = 16.9 Hz), 116.9 (d, J = 16.9 Hz), 57.3, 39.9 ppm.19F NMR (376 MHz, D2O+NaOH) δ: -139.5 (d, J = 21.8 Hz), -142.5 (d, J = 21.6 Hz) ppm.3p was prepared according to general procedure using TmTA E88T (1.0 mol%) and derivatized using (R)-FDAA and analyzed by LC-MS. Yield: run 1: 50%; run 2: 43%; run 3: 48%; average yield: 47%. Enantioselectivity: run 1: 96:4 e.r.; run 2: 96:4 e.r.; run 3: 97:3 e.r.; average e.r.: 96:4.

[0132] L-2-amino-3-(3-fluoro-4-methylphenyl)propanoic acid (3q)1H NMR (400 MHz, D2O) δ: 7.27 (m, 1H), 7.10 – 6.84 (m, 2H), 4.31 (dd, J = 7.5, 5.7 Hz, 1H), 3.30 (dd, J = 14.7, 5.7 Hz, 1H), 3.18 (dd, J = 14.7, 7.6 Hz, 1H), 2.24 (s, 3H) ppm.13C NMR (101 MHz, D2O) δ: 171.4, δ 161.1 (d, J = 243.2 Hz), 133.4 (d, J = 7.7 Hz), 132.1 (d, J = 5.7 Hz), 125.0 (d, J = 3.2 Hz), 124.7 (d, J = 17.1 Hz), 115.7 (d, J = 22.7 Hz), 54.0, 35.0, 13.3 (d, J = 3.5 Hz) ppm.19F NMR (376 MHz, D2O) δ: -117.4 ppm. IR: 2849, 1734, 1601, 1484, 1256, 1222, 1190, 853, 821, 760 cm-1. HRMS (ESI) (m / z) for [M+H]+C10H13FNO2 requires 198.0930, observed 198.0921.3q was prepared according to general procedure using TmTA W86N (1.0 mol%) and derivatized using (R)-FDAA and analyzed by LC-MS. Yield: run 1: 69%; run 2: 64%; run 3: 65%; average yield: 66%. Enantioselectivity: run 1: 97:3 e.r.; run 2: 98:2 e.r.; run 3: 97:3 e.r.; average e.r.: 97:3.

[0133] L-2-aminopent-4-enoic acid (3r)1H NMR (400 MHz, D2O+NaOH) δ: 5.71 (m, 1H), 5.45 – 4.99 (m, 2H), 3.23 (m, 1H), 2.28 (m, 2H) ppm.13C NMR (101 MHz, D2O+NaOH) δ: 182.7, 134.5, 117.97, 55.3, 39.1 ppm.3r was prepared according to general procedure using TmTA W86N (1.0 mol%) and derivatized using (R)-FDAA and analyzed by LC-MS. Yield: run 1: 43%; run 2: 41%; run 3: 41%; average yield: 42%. Enantioselectivity: run 1: 88:12 e.r.; run 2: 87:13 e.r.; run 3: 88:12 e.r.; average e.r.: 88:12.

[0134] L-2-aminopent-4-enoic acid (3s)1H NMR (400 MHz, D2O+NaOH) δ: 5.06 (m, 1H), 3.27 – 3.07 (m, 1H), 2.25 (m, 2H), 1.66 (s, 3H), 1.57 (s, 3H) ppm.13C NMR (101 MHz, D2O+NaOH) δ: 183.2, 136.3, 119.3, 55.9, 33.1, 25.0, 17.1 ppm.3s was prepared according to general procedure using TmTA W86N (1.0 mol%) and derivatized using (R)-FDAA and analyzed by LC-MS. Yield: run 1: 43%; run 2: 39%; run 3: 38%; average yield: 40%. Enantioselectivity: run 1: 98:2 e.r.; run 2: 97:3 e.r.; run 3: 98:2 e.r.; average e.r.: 98:2.

[0135] L-2-amino-4-phenylbutanoic acid (3t)1H NMR (400 MHz, D2O+NaOH) δ: 7.32 (m, 2H), 7.28 – 7.18 (m, 3H), 3.20 (m, 1H), 2.59 (t, J = 8.2 Hz, 2H), 2.17 – 1.65 (m, 2H) ppm.13C NMR (101 MHz, D2O+NaOH) δ: 183.3, 142.4, 128.7, 128.5, 126.0, 55.8, 36.9, 31.5 ppm.3t was prepared according to general procedure using TmTA W86N (1.0 mol%) and 10 mol% RhB, derivatized using (R)-FDAA and analyzed by LC-MS. Yield: run 1: 5%; run 2: 4%; run 3: 5%; average yield: 5%. Enantioselectivity: run 1: 98:2 e.r.; run 2: 98:2 e.r.; run 3: 98:2 e.r.; average e.r.: 98:2.

[0136] L-2-amino-3-(naphthalen-2-yl)propanoic acid (3z)1H NMR (400 MHz, D2O+NaOH) δ: 7.84 – 7.71 (m, 3H), 7.61 (s, 1H), 7.51 – 7.37 (m, 2H), 7.31 (m, 1H), 3.62 – 3.40 (m, 1H), 3.08 (dd, J = 13.4, 5.4 Hz, 1H), 2.87 (dd, J = 13.4, 7.8 Hz, 1H) ppm.13C NMR (101 MHz, D2O+NaOH) δ: 182.3, 136.0, 133.1, 131.9, 127.9, 127.8, 127.6, 127.6, 127.5, 126.3, 125.69, 57.3, 41.0. ppm.3t was prepared according to general procedure using TmTA W86N (1.0 mol%), derivatized using (R)-FDAA and analyzed by LC- MS. Yield: run 1: 41%; run 2: 48%; run 3: 49%; average yield: 46%. Enantioselectivity: run 1: 97:3 e.r.; run 2: 96:4 e.r.; run 3: 96.5:3.5 e.r.; average e.r.: 96.5:3.5.

[0137] (2S, 3S)- 2-amino-3-phenylbutanoic acid (5a) Compound 5a was prepared using preparative photobiocatalysis following the General Procedure described above. The reaction mixture was purified by C18 silica using Biotage (10 g Biotage C18 Sfär cartridge, 0–100% MeOH (0.1% acetic acid) in H2O (0.1% acetic acid) over 10 CV) to afford the product as a white solid (16.3 mg, 63% yield).1H NMR (400 MHz, D2O) for major diastereomer (5a) δ: 7.46 (m, 2H), 7.43 – 7.23 (m, 3H), 4.15 (m, 1H), 3.66 – 3.31 (m, 1H), 1.46 (d, J = 7.2 Hz, 3H) ppm.13C NMR (101 MHz, D2O) for major diastereomer (5a) δ: 171.5, 139.2, 129.3, 128.2, 127.8, 58.9, 40.3, 16.9 ppm. The absolute and relative stereochemistry of the major stereoisomer of 5a was determined by derivation and crystallization. (2S, 3S)-5a was prepared according to General Procedure using TmTA E88T H83F (1.0 mol%), derivatized using (R)-FDAA and analyzed by LC-MS. Isolated yield: 63%. Diastereoselectivity determined by NMR analysis: 91:9 d.r. Enantioselectivity of the major diastereomer determined by LC-MS: 99:1 e.r.

[0138] (2S,3S)-2-amino-3-(o-tolyl)butanoic acid (5b) Compound 5b was prepared using preparative photobiocatalysis following the general procedure described above. The reaction mixture was purified by C18 silica using Biotage (10 g Biotage C18 Sfär cartridge, 0–100% MeOH (0.1% acetic acid) in H2O (0.1% acetic acid) over 10 CV) to afford the product as a white solid (15.5 mg, 56% yield).1H NMR (400 MHz, D2O) for major diastereomer (5b) δ: 7.48 – 7.16 (m, 4H), 4.12 (d, J = 7.3 Hz, 1H), 3.40 (m, 1H), 2.34 (s, 3H), 1.44 (d, J = 7.2 Hz, 3H) ppm;13C NMR (101 MHz, D2O) for major diastereomer (5b) δ: 171.6, 138.4, 136.1, 129.8, 127.7, 59.0, 39.9, 20.1, 17.0 ppm.1H NMR (400 MHz, D2O) for minor diastereomer (5b-dia) δ: 7.28 (m, 4H), 3.84 (m,1H), 3.45 (m, 1H), 2.34 (s, 3H), 1.35 (d, J = 7.4 Hz, 3H).13C NMR (101 MHz, D2O) for minor diastereomer (5b-dia) δ: 171.0, 137.7, 137.6, 129.5, 127.760.9, 39.5, 20.0, 13.7. IR: 2972, 2921, 1731, 1515, 1221, 1030, 820 cm-1. HRMS (ESI) (m / z) for [M+H]+C11H16NO2 requires 194.1181, observed 194.1176. (2S, 3S)-5b was prepared according to general procedure using TmTA E88T H83F (1.0 mol%), derivatized using (R)-FDAA and analyzed by LC-MS. Isolated yield: 56%. Diastereoselectivity determined by NMR analysis: 87:13. Enantioselectivity of the major diastereomer determined by LC-MS: 99:1 e.r. Enantioselectivity of the minor diastereomer determined by LC-MS: 88:12 e.r.

[0139] (2S,3S)-2-amino-3-(m-tolyl)butanoic acid (5c) Compound 5c was prepared using preparative photobiocatalysis following the general procedure described above. The reaction mixture was purified by C18 silica using Biotage (10 g Biotage C18 Sfär cartridge, 0–100% MeOH (0.1% acetic acid) in H2O (0.1% acetic acid) over 10 CV) to afford the product as a white solid (16.6 mg, 60% yield).1H NMR (400 MHz, D2O) for major diastereomer (5c) δ: 7.35 (m, 1H), 7.28 – 7.20 (m, 2H), 7.17 (d, J = 7.6 Hz, 1H), 4.13 (d, J = 7.4 Hz, 1H), 3.39 (m, 1H), 2.35 (s, 3H), 1.45 (d, J = 7.4 Hz, 3H) ppm;13C NMR (101 MHz, D2O) for major diastereomer (5c) δ: 171.6, 139.5, 139.3, 129.2, 128.81, 128.3, 124.8, 58.9, 40.3, 20.4, 17.0 ppm.1H NMR (400 MHz, D2O) for minor diastereomer (5c-dia) δ: δ 7.35 (m, 1H), 7.23 (d, J = 7.0 Hz, 2H), 7.18 (d, J = 7.8 Hz, 1H), 4.14 (d, J = 5.5 Hz, 1H), 3.52 (m, 1H), 2.36 (s, 3H), 1.61 – 1.21 (m, 3H) ppm;13C NMR (101 MHz, D2O) for minor diastereomer (5c-dia) δ: 171.8, 139.5, 139.3, 129.0, 128.5, 128.4, 124.7, 59.4, 39.5, 20.4, 14.3 ppm. IR: 3030, 2983, 2915, 1731, 1607, 1492, 1428, 1212, 1033, 790, 703, 512 cm-1. HRMS (ESI) (m / z) for [M+H]+C11H16NO2 requires 194.1181, observed 194.1176. (2S, 3S)-5c was prepared according to general procedure using TmTA E88T H83F (1.0 mol%), derivatized using (R)-FDAA and analyzed by LC-MS. Isolated yield: 60%. Diastereoselectivity determined by NMR analysis: 83:17 d.r. Enantioselectivity of the major diastereomer determined by LC-MS: 97:3 e.r. Enantioselectivity of the minor diastereomer determined by LC-MS: 85:15 e.r.

[0140] (2S,3S)-2-amino-3-(o-tolyl)butanoic acid (5d)Compound 5d was prepared using preparative photobiocatalysis following the general procedure described above. The reaction mixture was purified by C18 silica using Biotage (10 g Biotage C18 Sfär cartridge, 0–100% MeOH (0.1% acetic acid) in H2O (0.1% acetic acid) over 10 CV] to afford the product as a white solid (14.4 mg, 52% yield). Two diastereomers exist (d.r. = 93:7) in this product.1H NMR (400 MHz, D2O) for major diastereomer (5d) δ: 7.46 – 7.17 (m, 4H), 3.98 (d, J = 8.7 Hz, 1H), 3.60 – 3.42 (m, 1H), 2.38 (s, 3H), 1.35 (d, J = 7.0 Hz, 3H) ppm;13C NMR (101 MHz, D2O) for major diastereomer (5d) δ: 173.1, 138.8, 136.9, 131.0, 127.6, 126.9, 126.2, 59.5, 36.1, 18.5, 17.7 ppm. IR: 3020, 2921, 2858, 1732, 1661, 1494, 1330, 792, 628 cm-1. HRMS (ESI) (m / z) for [M+H]+C11H16NO2 requires 194.1181, observed 194.1176. (2S, 3S)-5d was prepared according to general procedure using TmTA E88T H83F (1.0 mol%), derivatized using (R)-FDAA and analyzed by LC-MS. Isolated yield: 52%. Diastereoselectivity determined by NMR analysis: 93:7 d.r. Enantioselectivity of the major diastereomer determined by LC-MS: 97:3 e.r.

[0141] (2S,3S)-2-amino-3-(4-fluorophenyl)butanoic acid (5e) Compound 5e was prepared using preparative photobiocatalysis following the general procedure described above. The reaction mixture was purified by C18 silica using Biotage (10 g Biotage C18 Sfär cartridge, 0–100% MeOH (0.1% acetic acid) in H2O (0.1% acetic acid) over 10 CV) to afford the product as a white solid (19.9 mg, 71% yield). Two diastereomers exist (d.r. = 90:10) in this product.1H NMR (400 MHz, D2O) for major diastereomer (5e) δ: 7.36 (m, 2H), 7.17 (m, 2H), 3.79 (d, J = 7.4 Hz, 1H), 3.33 (m, 1H), 1.41 (d, J = 7.2 Hz, 3H) ppm;13C NMR (101 MHz, D2O) for major diastereomer (5e) δ: 171.5, 162.2 (d, J = 244.0 Hz), 135.0 (d, J = 3.2 Hz), 129.6 (d, J = 8.3 Hz), 115.9 (d, J = 21.6 Hz), 59.0, 39.6, 17.0 ppm.19F NMR (376 MHz, D2O) δ -115.5. IR: 3129, 2981, 2921, 1731, 1513, 1476, 1208, 1060, 838, 762, 700, 508 cm-1. HRMS (ESI) (m / z) for [M+H]+C10H13FNO2requires 198.0930, observed 198.0934. (2S, 3S)-5e was prepared according to general procedure using TmTA E88T H83F (1.0 mol%), derivatized using (R)-FDAA and analyzed by LC-MS. Isolated yield: 71%. Diastereoselectivity determined by NMR analysis: 90:10 d.r. Enantioselectivity of the major diastereomer determined by LC-MS: 98:2 e.r.

[0142] (S)-2-amino-2-methyl-3-phenylpropanoic acid (8a)1H NMR (400 MHz, D2O) δ: 7.53 – 6.80 (m, 5H), 3.10 (d, J = 13.5 Hz, 1H), 2.72 (d, J = 13.5 Hz, 1H), 1.32 (s, 3H) ppm.13C NMR (101 MHz, D2O) δ: 184.2, 137.7, 130.0, 128.4, 126.7, 59.4, 46.4, 25.9 ppm. IR: 2978, 2902, 2230, 1732, 1488, 1228, 1211, 779, 692 cm-1. [α]D25 = - 6.3 (c = 1.0, H2O). L-8a was prepared according to general procedure using TmTA W86N (1.0 mol%) and was derivatized using FmocCl before analyzed by chiral HPLC. HPLC analysis conditions: CHIRALCEL IG column (4.6 mm × 25 cm, 5 micron), 95:5 hexanes: i-PrOH (0.1% TFA), flow rate = 1.0 mL / min, wavelength = 265 nm, 40 °C. tR = 14.13 min. Yield with D-alanine: run 1: 75%; run 2: 78%; run 3: 79%; run 4: 76%; run 5: 72%; average yield: 76%. Enantioselectivity withD-alanine: run 1: >99:1 e.r.; run 2: >99:1 e.r.; run 3: >99:1 e.r.; run 4: >99:1 e.r.; run 5: >99:1 e.r.; average e.r.: >99:1. Yield with DL-alanine: run 1: 69%; run 2: 69%; run 3: 72%; run 4: 71%; run 5: 68%; average yield: 70%. Enantioselectivity with DL-alanine: run 1: >99:1 e.r.; run 2: >99:1 e.r.; run 3: >99:1 e.r.; run 4: >99:1 e.r.; run 5: >99:1 e.r.; average e.r.: >99:1.

[0143] (S)-2-amino-3-(4-fluorophenyl)-2-methylpropanoic acid (8b)1H NMR (400 MHz, D2O) δ: 7.36 – 7.21 (m, 2H), 7.15 (m, 2H), 3.36 (d, J = 14.5 Hz, 1H), 3.09 (d, J = 14.5 Hz, 1H), 1.63 (s, 3H) ppm.13C NMR (101 MHz, D2O) δ: 174.3, 162.3 (d, J = 244.0 Hz), 131.8 (d, J = 8.5 Hz), 129.2 (d, J = 3.2 Hz), 115.7 (d, J = 21.6 Hz), 61.14, 41.54, 21.72 ppm.19F NMR (376 MHz, D2O) δ -115.5. IR: 2928, 1738, 1643, 1602, 1511, 1445, 1184, 1143, 919, 841 cm-1. HRMS (ESI) (m / z) for [M+H]+C10H13FNO2 requires 198.0930, observed 198.0927.L-8b was prepared according to general procedure using TmTA W86N (1.0 mol%) derivatized using Fmoc-Cl and analyzed by chiral HPLC. HPLC analysis conditions: CHIRALCEL IF-3 column (4.6 mm × 25 cm, 5 micron), 95:5 hexanes: EtOH (0.1% TFA), flow rate = 1.0 mL / min, wavelength = 265 nm, 40 °C. tR = 8.08 min. Yield with D-alanine: run 1: 81%; run 2: 83%; run 3: 91%; run 4: 89%; run 5: 85%; average yield: 87%.Enantioselectivity with D-alanine: run 1: >99:1 e.r.; run 2: >99:1 e.r.; run 3: >99:1 e.r.; run 4: >99:1 e.r.; run 5: >99:1 e.r.; average e.r.: >99:1. Yield withDL-alanine: run 1: 77%; run 2: 75%; run 3: 79%; run 4: 81%; run 5: 81%; average yield: 79%. Enantioselectivity with DL-alanine: run 1: >99:1 e.r.; run 2: >99:1 e.r.; run 3: >99:1 e.r.; run 4: >99:1 e.r.; run 5: >99:1 e.r.; average e.r.: >99:1.

[0144] (S)-2-amino-2-methyl-3-(4-(trifluoromethoxy)phenyl)propanoic acid (8c)1H NMR (400 MHz, D2O) δ: 7.35 (m, 4H), 3.38 (d, J = 14.5 Hz, 1H), 3.13 (d, J = 14.5 Hz, 1H), 1.63 (s, 3H) ppm.13C NMR (101 MHz, D2O) δ: 174.2, 148.7 (q, J = 1.8 Hz), 132.4, 131.6, 121.5, 121.3 (q, J = 256.0 Hz), 61.1, 41.6, 21.7 ppm.19F NMR (376 MHz, D2O) δ: - 57.8. IR: 2941, 1746, 1651, 1511, 1440, 1283, 1254, 1188, 1139, 924, 841 cm-1. HRMS (ESI) (m / z) for [M+H]+C11H13F3NO3 requires 264.0848, observed 264.0845.L-8c was prepared according to general procedure using TmTA W86N (1.0 mol%) derivatized using Fmoc-Cl and analyzed by chiral HPLC. HPLC analysis conditions: CHIRALCEL IF-3 column (4.6 mm × 25 cm, 5 micron), 95:5 hexanes: EtOH (0.1% TFA), flow rate = 1.0 mL / min, wavelength = 265 nm, 40 °C. tR = 5.26 min. Yield with D-alanine: run 1: 49%; run 2: 45%; run 3: 50%; run 4: 52%; run 5: 46%; average yield: 48%. Enantioselectivity withD-alanine: run 1: >99:1 e.r.; run 2: >99:1 e.r.; run 3: >99:1 e.r.; run 4: >99:1 e.r.; run 5: >99:1 e.r.; average e.r.: >99:1. Yield with DL-alanine: run 1: 44%; run 2: 42%; run 3: 48%; run 4: 45%; run 5: 42%; average yield: 44%. Enantioselectivity with DL-alanine: run 1: >99:1 e.r.; run 2: >99:1 e.r.; run 3: >99:1 e.r.; run 4: >99:1 e.r.; run 5: >99:1 e.r.; average e.r.: >99:1.

[0145] (S)-2-amino-3-(3-methoxyphenyl)-2-methylpropanoic acid (8d)1H NMR (400 MHz, D2O) δ: 7.37 (m, 1H), 7.00 (m, 1H), 6.93 – 6.79 (m, 2H), 3.83 (s, 3H), 3.36 (d, J = 14.3 Hz, 1H), 3.08 (d, J = 14.3 Hz, 1H), 1.65 (s, 3H) ppm.13C NMR (101 MHz, D2O) δ: 173.9, 159.1, 134.9, 130.3, 122.7, 115.6, 113.6, 60.9, 55.3, 42.3, 21.7 ppm. IR: 2895, 1744, 1666, 1604, 1538, 1535, 1266, 1187, 1142, 1026, 857 cm-1. HRMS (ESI) (m / z) for [M+H]+C11H16NO3 requires 210.1130, observed 210.1127.L-8d was prepared according to general procedure using TmTA W86N (1.0 mol%) derivatized using Fmoc-Cl and analyzed by chiral HPLC. HPLC analysis conditions: CHIRALCEL IF-3 column (4.6 mm × 25 cm, 5 micron), 95:5 hexanes: EtOH (0.1% TFA), flow rate = 1.0 mL / min, wavelength = 265 nm, 40 °C. tR = 7.12 min. Yield withD-alanine: run 1: 80%; run 2: 87%; run 3: 79%; run 4: 82%; run 5: 89%; average yield: 83%. Enantioselectivity with D-alanine: run 1: >99:1 e.r.; run 2: >99:1 e.r.; run 3: >99:1 e.r.; run 4: >99:1 e.r.; run 5: >99:1 e.r.; average e.r.: >99:1. Yield withDL-alanine: run 1: 77%; run 2: 75%; run 3: 73%; run 4: 74%; run 5: 73%; average yield: 74%. Enantioselectivity with DL-alanine: run 1: >99:1 e.r.; run 2: >99:1 e.r.; run 3: >99:1 e.r.; run 4: >99:1 e.r.; run 5: >99:1 e.r.; average e.r.: >99:1.

[0146] (S)-2-amino-2-benzylbutanoic acid (8e)1H NMR (400 MHz, D2O) δ: 7.48 – 7.37 (m, 3H), 7.29 (m, 2H), 3.41 (d, J = 14.5 Hz, 1H), 3.13 (d, J = 14.5 Hz, 1H), 2.15 (dq, J = 15.2, 7.6 Hz, 1H), 2.02 – 1.88 (m, 1H), 1.02 (t, J = 7.6 Hz, 3H) ppm.13C NMR (101 MHz, D2O) δ: 173.2, 133.0, 130.1, 129.1, 128.2, 65.1, 41.0, 28.9, 7.1 ppm. L-8e was prepared according to general procedure using TmTA E88T H83F (1.0 mol%) and derivatized using Fmoc-Cl and analysis using chiral HPLC. HPLC analysis conditions: CHIRALCEL IG column (4.6 mm × 25 cm, 5 micron), 95:5 hexanes: i-PrOH (0.1% TFA), flow rate = 0.7 mL / min, wavelength = 265 nm, 40 °C. tR = 14.94 min. Yield with DL-alanine: run 1: 29%; run 2: 31%; run 3: 32%; run 4: 30%; run 5: 33%; average yield: 31%. Enantioselectivity withDL-alanine: run 1: >99:1 e.r.; run 2: >99:1 e.r.; run 3: >99:1 e.r.; run 4: >99:1 e.r.; run 5: >99:1 e.r.; average e.r.: >99:1. Example 12 – Variations in photocatalysts, oxidants, enzymes, and amino acidsTable 5. Variations in photocatalyst. entry photocatalyst yield of 3a e.r. of 3a 1 Ru(bpy)3Cl2 (2 mol%) 43% 96:4 2 Ru(dtbbpy)3(PF6)2(2 mol%) 24% 99:1 3 [Ru(bpz)3](PF6)2 (2 mol%) 30% 96:4 4 (fac)-Ir(ppy)3 (2 mol%) 49% 97:3 5 [Ir(ppy)2(dtbbpy)]PF6 (2 mol%) 25% 97:3 6 [Ir(dF(CF3)ppy)2(dtbpy)]PF6 (2 mol%) 22% 98.5:1.5 7 rhodamine B (10 mol%) 41% 90:10 8 Eosin Y (10 mol%) 15% 71:29 9 rhodamine 6G (10 mol%) 31% 95:5 10 9-mesityl-10-methylacridinium (10 mol%) 26% 96:4 11 4CzIPN (10 mol%) 52% 99:1 12 (fac)-Ir(ppy) b 3(2 mol%)58% 97:3a1a (3.0 mM), 2a (30 mM), 1.0 mol% TmTA (30 ^M), 2–10 mol% photocatalyst, 200 mol% Co(NH3)6Cl3 (6.0 mM), PLP (0.60 mM), h^ (440 nm), 200 mM KPi buffer, pH = 8.5, DMSO (5% v / v), 50 ^C, 12 h.b1a’ was used in lieu of 1a.Table 6. Variations in oxidant. entry oxidant yield of 3ae.r. of 3a1 no exogenous oxidant 5% 98:2 2 H2O2 0% - 3 NAD+9% 95:5 4 Cu(OAc)2 22% 98:2 5 AgOAc 11% 97:3 6 CAN 12% 96:2 7 FeCl3 12% 98:2 8 NaFe(EDTA)b19% 99:1 9 Mn(OAc)3 27% 99:1 10 Co(NH3)6Cl3 58% 97:3a1a’ (3.0 mM), 2a (30 mM), 1.0 mol% TmTA (30 ^M), 2 mol% (fac)-Ir(ppy)3 (60 ^M), 200 mol% oxidant(6.0 mM),PLP (300 μM), h^ (440 nm), 200 mM KPi buffer, pH 8.5, DMSO (5% v / v), 50 ^C, 12 h.bEDTA = ethylenediaminetetraacetic acid.Table 7. Variations in enzyme. yield e.r. of entry TmTA of 3a (%) 3a 1 wild-type TmTA 58% 97:3 2TmTA W86N (TmPLP^^)77% 98.5:1.53 TmTA E88T 68% 96:4 4 TmTA H83F 5% 78:22 5 TmTA E88T H83F (TmPLP^^)44% 94:6a1a’ (3.0 mM), 2a (30 mM), 1.0 mol% TmTA (30 ^M), 2 mol% (fac)-Ir(ppy)3 (60 ^M), Co(NH3)6Cl3(6.0 mM), PLP (300 μM), h^ (440 nm), 200 mM KPi buffer, pH 8.5, DMSO (5% v / v), 50 ^C, 12 h.Table 8. Additional Variations. variation from the standard yield e.r. of entry conditions of 3a 3a 1 none 77% 98.5:1.5 2 1a instead of 1a’ 67% 98.5:1.5 3 no TmPLP^^ (TmTA W86N)0% -4 no h^ (440 nm)0% -5 No PLP 42% 98.5:1.5 6 no (fac)-Ir(ppy)35% 99:1 7 no Co(NH3)6Cl39% 99:1 8 no Co(NH3)6Cl3and PLP 5% 99:1 9 no (fac)-Ir(ppy)3and PLP <1% - 10390 nm h^ instead of 440 nm62% 97:311525 nm h^ instead of 440 nm26% 99:112FeCl3instead of Co(NH3)6Cl330% 99:1 13Mn(OAc)3instead of Co(NH3)6Cl346% 99:1a1a’ (3.0 mM), 2a (30 mM), Co(NH^3)6Cl3 (6.0 mM), PLP (0.30 mM), 1.0 mol% TmPLP ^, 2 mol% (fac)-Ir(ppy)3, h^ (440 nm), 200 mM KPi buffer, pH 8.5, DMSO (5% v / v), 50 ^C, 12 h.Table 9. Amino Acids.entry Amino acid (2b) yield of 8a e.r. of 8a 1 D-2b 76% >99:1 2 L-2b 55% >99:1 3 (rac)-2b 66% >99:1a1a’ (5.0 mM), 2b (50 mM), Co(NH )^3 6Cl3(10.0 mM), PLP (0.50 mM), 1.0 mol% TmPLP ^, 10 mol% 4CzIPN, h^ (440 nm), 200 mM KPi buffer, pH 8.5, DMSO (5% v / v), 50 ^C, 12 h.Table 10. Amino Acids. entry Amino acid (2b) yield of 14 1 D-2b 0% 2L-2b 0%a13 (3.0 mM), 2b (30 mM), PLP (0.30 mM), 1.0 mol% TmPLP^^, 200 mM KPi buffer, pH 8.5, DMSO (5% v / v), 50 ^C, 12 h. Example 13 – DNA and protein sequences. Table 11. DNA and protein sequences.Example 14 - Primers used for site-saturation mutagenesis for the engineering of TmTA. Table 12. Primers used for site-saturation mutagenesis for the engineering of TmTA.

[0147] In summary, the photobiocatalytic asymmetric sp3–sp3oxidative cross-coupling between organoboron reagents and amino acids was established. The synergistic use of visible-light photocatalysts and engineered enzymes enabled the development of new radical pyridoxal enzymology, allowing the convergent and stereoselective preparation of valuable non-canonical amino acids without protecting group manipulations. Importantly, non- canonical amino acids bearing contiguous stereocenters or ^-tetrasubstituted stereocenters that eluded prior chemical and biosynthesis could be prepared with excellent enantio- and diastereocontrol.Discussion of Possible Embodiments

[0148] A method of forming a non-canonical amino acid (ncAA) includes utilizing a substrate, an amino acid, an enzyme, actinic radiation, a photocatalyst, and an oxidant to form a non-canonical amino acid, wherein the enzyme includes one or more pyridoxal phosphate (PLP)-dependent enzymes or variants thereof.

[0149] The method of the preceding paragraph can optionally include, additionally and / or alternatively any one or more of the following features, configurations and / or additional components.

[0150] The substrate can include an organoboron compound.

[0151] The organoboron compound can include at least one of an alkyltrifluoroborate compound and a boronic ester.

[0152] The amino acid can include an alpha amino acid.

[0153] The alpha amino acid can be selected from glycine and alanine.

[0154] The one or more pyridoxal phosphate (PLP)-dependent enzymes can include a threonine aldolase variant.

[0155] The threonine aldolase variant can include at least one of a W86N, E88T, and H83F mutation.

[0156] The threonine aldolase variant can include two or more of a W86N, E88T, and H83F mutation.

[0157] The photocatalyst can include one or more of [Ru(bpy)3]Cl2, Ru(dtbbpy)3(PF6)2, [Ru(bpz)3](PF6)2, (fac)-Ir(ppy)3, [Ir(ppy)2(dtbbpy)]PF6, [Ir(dF(CF3)ppy(dtbbpy)]PF6, RhB, Eosin Y, rhodamine 6G, 9-mesityl-10-methylacridinium, and 4CzIPN.

[0158] Actinic radiation can be provided to the photocatalyst at a wavelength ranging from about 380 nm to about 550 nm.

[0159] The oxidant can include a single-electron oxidant.

[0160] The oxidant can include at least one of a manganese-containing salt, an iron- containing salt, and a copper-containing salt.

[0161] The oxidant can include Co(NH3)6Cl3.

[0162] The non-canonical amino acid can include a sp3-sp3coupled non-canonical amino acid product.

[0163] The non-canonical amino acid can be selected from L-phenylalanine, L-2- amino-3-(p-tolyl)propanoic acid, L-2-amino-3-(m-tolyl)propanoic acid, L-2-amino-3-(o-tolyl)propanoic acid, L-2-amino-3-(4-methoxyphenyl)propanoic acid, L-2-amino-3-(4- (methylthio)phenyl)propanoic acid, L-tyrosine, L-2-amino-3-(4- (trifluoromethoxy)phenyl)propanoic acid, L-2-amino-3-(4-fluorophenyl)propanoic acid, L-2- amino-3-(4-chlorophenyl)propanoic acid, L-2-amino-3-(3-cyanophenyl)propanoic acid, L-2- amino-3-(3-methoxyphenyl)propanoic acid, L-3-([1,1'-biphenyl]-4-yl)-2-aminopropanoic acid, L-3-(4-(1H-pyrazol-1-yl)phenyl)-2-aminopropanoic acid, L-2-amino-3-(4-(tert- butyl)phenyl)propanoic acid, L-2-amino-3-(3,4-difluorophenyl)propanoic acid, L-2-amino-3- (3-fluoro-4-methylphenyl)propanoic acid, L-2-aminopent-4-enoic acid, L-2-aminopent-4- enoic acid, L-2-amino-4-phenylbutanoic acid, and L-2-amino-3-(naphthalen-2-yl)propanoic acid.

[0164] The non-canonical amino acid can be selected from (2S, 3S)- 2-amino-3- phenylbutanoic acid, (2S,3S)-2-amino-3-(o-tolyl)butanoic acid, (2S,3S)-2-amino-3-(m- tolyl)butanoic acid, (2S,3S)-2-amino-3-(o-tolyl)butanoic acid, (2S,3S)-2-amino-3-(4- fluorophenyl)butanoic acid, (S)-2-amino-2-methyl-3-phenylpropanoic acid, (S)-2-amino-3-(4- fluorophenyl)-2-methylpropanoic acid, (S)-2-amino-2-methyl-3-(4- (trifluoromethoxy)phenyl)propanoic acid, (S)-2-amino-3-(3-methoxyphenyl)-2- methylpropanoic acid, and (S)-2-amino-2-benzylbutanoic acid.

[0165] The method can include generating a quinonoid intermediate.

[0166] The method can include utilizing a buffer and initializing the formation of the non-canonical amino acid at a pH value ranging from about 7.5 to about 8.5.

[0167] A pyridoxal phosphate (PLP)-dependent enzyme variant includes a variant of a threonine aldolase from Thermotoga maritima, the variant including one or more of the following mutations: E88T, H83F, and W86N.

[0168] A pyridoxal phosphate (PLP)-dependent enzyme variant includes a variant of a threonine aldolase from Thermotoga maritima, the variant including two or more of the following mutations: E88T, H83F, and W86N.

[0169] A method of forming a non-canonical amino acid (ncAA) includes utilizing an organoboron compound, an alpha amino acid, an enzyme, a photocatalyst, and an oxidant to form a sp3-sp3coupled non-canonical amino acid product, wherein the oxidant includes at least one of a manganese-containing compound, an iron-containing compound, and a copper- containing compound.

[0170] The method of the preceding paragraph can optionally include, additionally and / or alternatively any one or more of the following features, configurations and / or additional components.

[0171] The enzyme can include a variant of threonine aldolase from Thermotoga maritima, the variant including one or more of the following mutations: E88T, H83F, and W86N.

[0172] The enzyme can include a variant of threonine aldolase from Thermotoga maritima, the variant including two or more of the following mutations: E88T, H83F, and W86N.

[0173] While the disclosure has been described with reference to an exemplary embodiment(s), it will be understood by those skilled in the art that various changes may be made, and equivalents may be substituted for elements thereof without departing from the scope of the embodiment(s). In addition, many modifications may be made to adapt a particular situation or material to the teachings of the embodiment(s) without departing from the essential scope thereof. Therefore, it is intended that the disclosure is not limited to the disclosed embodiment(s), but that the disclosure will include all embodiments falling within the scope of the appended claims. Various examples have been described. These and other examples are within the scope of the following claims.

Claims

CLAIMS:

1. A method of forming a non-canonical amino acid (ncAA), the method comprising: utilizing a substrate, an amino acid, an enzyme, actinic radiation, a photocatalyst, and an oxidant to form a non-canonical amino acid, wherein the enzyme includes one or more pyridoxal phosphate (PLP)-dependent enzymes or variants thereof.

2. The method of claim 1, wherein the substrate includes an organoboron compound.

3. The method of claim 2, wherein the organoboron compound includes at least one of an alkyltrifluoroborate compound and a boronic ester.

4. The method of any one of claims 1-3, wherein the amino acid includes an alpha amino acid.

5. The method of claim 4, wherein the alpha amino acid is selected from glycine and alanine.

6. The method of any one of claims 1-5, wherein the one or more pyridoxal phosphate (PLP)-dependent enzymes includes a threonine aldolase variant.

7. The method of claim 6, wherein the threonine aldolase variant includes at least one of a W86N, E88T, and H83F mutation.

8. The method of any one of claims 1-7, wherein the photocatalyst includes one or more of [Ru(bpy)3]Cl2, Ru(dtbbpy)3(PF6)2, [Ru(bpz)3](PF6)2, (fac)-Ir(ppy)3, [Ir(ppy)2(dtbbpy)]PF6, [Ir(dF(CF3)ppy(dtbbpy)]PF6, RhB, Eosin Y, rhodamine 6G, 9-mesityl-10-methylacridinium, and 4CzIPN.

9. The method of claim 8, wherein actinic radiation is provided to the photocatalyst at a wavelength ranging from about 380 nm to about 550 nm.

10. The method of any one of claims 1-9, wherein the oxidant includes a single-electron oxidant.

11. The method of any one of claims 1-10, wherein the oxidant includes at least one of a manganese-containing salt, an iron-containing salt, and a copper-containing salt.

12. The method of any one of claims 1-10, wherein the oxidant includes Co(NH3)6Cl3.

13. The method of any one of claims 1-12, wherein the non-canonical amino acid includes a sp3-sp3coupled non-canonical amino acid product.

14. The method of any one of claims 1-13, wherein the non-canonical amino acid is selected from L-phenylalanine, L-2-amino-3-(p-tolyl)propanoic acid, L-2-amino-3-(m- tolyl)propanoic acid, L-2-amino-3-(o-tolyl)propanoic acid, L-2-amino-3-(4- methoxyphenyl)propanoic acid, L-2-amino-3-(4-(methylthio)phenyl)propanoic acid, L- tyrosine,L-2-amino-3-(4-(trifluoromethoxy)phenyl)propanoic acid,L-2-amino-3-(4- fluorophenyl)propanoic acid, L-2-amino-3-(4-chlorophenyl)propanoic acid, L-2-amino-3-(3- cyanophenyl)propanoic acid, L-2-amino-3-(3-methoxyphenyl)propanoic acid, L-3-([1,1'- biphenyl]-4-yl)-2-aminopropanoic acid, L-3-(4-(1H-pyrazol-1-yl)phenyl)-2-aminopropanoic acid,L-2-amino-3-(4-(tert-butyl)phenyl)propanoic acid,L-2-amino-3-(3,4- difluorophenyl)propanoic acid, L-2-amino-3-(3-fluoro-4-methylphenyl)propanoic acid, L-2- aminopent-4-enoic acid, L-2-aminopent-4-enoic acid, L-2-amino-4-phenylbutanoic acid, andL-2-amino-3-(naphthalen-2-yl)propanoic acid.

15. The method of any one of claims 1-13, wherein the non-canonical amino acid is selected from (2S, 3S)- 2-amino-3-phenylbutanoic acid, (2S,3S)-2-amino-3-(o-tolyl)butanoic acid, (2S,3S)-2-amino-3-(m-tolyl)butanoic acid, (2S,3S)-2-amino-3-(o-tolyl)butanoic acid, (2S,3S)-2-amino-3-(4-fluorophenyl)butanoic acid, (S)-2-amino-2-methyl-3-phenylpropanoic acid, (S)-2-amino-3-(4-fluorophenyl)-2-methylpropanoic acid, (S)-2-amino-2-methyl-3-(4- (trifluoromethoxy)phenyl)propanoic acid, (S)-2-amino-3-(3-methoxyphenyl)-2- methylpropanoic acid, and (S)-2-amino-2-benzylbutanoic acid.

16. The method of any one of claims 1-15 including generating a quinonoid intermediate.

17. The method of any one of claims 1-16 including utilizing a buffer and initializing the formation of the non-canonical amino acid at a pH value ranging from about 7.5 to about 8.

5.

18. A pyridoxal phosphate (PLP)-dependent enzyme variant, the pyridoxal phosphate (PLP)-dependent enzyme variant comprising: a variant of a threonine aldolase from Thermotoga maritima, the variant including one or more of the following mutations: E88T, H83F, and W86N.

19. A method of forming a non-canonical amino acid (ncAA), the method comprising: utilizing an organoboron compound, an alpha amino acid, an enzyme, a photocatalyst, and an oxidant to form a sp3-sp3coupled non-canonical amino acid product, wherein the oxidant includes at least one of a manganese-containing compound, an iron-containing compound, and a copper-containing compound.

20. The method of claim 19, wherein the enzyme includes a variant of threonine aldolase from Thermotoga maritima, the variant including one or more of the following mutations: E88T, H83F, and W86N.