Compositions and methods for nitrogen migration reaction

Abstract

Embodiments include metalloenzymes and methods for conducting a nitrogen migration reaction catalyzed by a metalloenzyme. Embodiments include metalloenzymes including one or more mutations. A method for forming a non-canonical amino acid includes catalyzing a nitrogen migration reaction to form a non-canonical amino acid using an azanyl moiety-containing substrate and a metalloenzyme catalyst.

Description

2025-367-24059.073PCT1COMPOSITIONS AND METHODS FOR NITROGEN MIGRATION REACTIONCROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of and priority to U. S. Provisional Application 63 / 740,115, titled “COMPOSITIONS AND METHODS FOR NITROGEN MIGRATION REACTION”, filed December 30, 2024, the contents of which are incorporated by reference herein.STATEMENT OF GOVERNMENT RIGHTS

[0002] This invention was made with government support under GM 147387 awarded by the National Institutes of Health, and 2400087 awarded by the National Science Foundation. 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 format and is hereby incorporated by reference in its entirety. Said XML copy, created on December 8, 2025, is named 4059_073PCTl_SL and is 135,532 bytes in size.TECHNICAL FIELD

[0004] The subject matter disclosed herein relates to compositions and methods for performing biocatalytic reactions and more particularly to metalloenzyme catalysts and methods for nitrogen migration reactions.BACKGROUND

[0005] Biocatalysis can be used to form useful compounds for various industries, such as the pharmaceutical and agrochemical industries. Drawing inspiration from synthetic, organic, and organometallic chemistry, biocatalysis researchers have developed a range of unnatural enzymatic activities, including metalloprotein-catalyzed carbene and2025-367-24059.073PCT1nitrene transfer reactions and nicotinamide- and flavin-dependent enzyme-catalyzed reductive radical reactions. To further advance the field of biocatalysis, it would be beneficial to develop enzymes and unnatural enzymatic processes for nitrogen migration reactions to form various products, such as non-canonical amino acids.SUMMARY

[0006] According to one aspect, a method for forming a non-canonical amino acid includes catalyzing a nitrogen migration reaction to form a non-canonical amino acid using an azanyl moiety-containing substrate and a metalloenzyme catalyst.

[0007] According to one aspect, a method for forming a non-canonical amino acid includes catalyzing a nitrogen migration reaction to form a non-canonical amino acid using a nitrogen-containing substrate and a metalloenzyme catalyst derived from 1-aminocyclopropane-1 -carboxylic acid oxidase.

[0008] According to one aspect, a catalyst includes a metalloenzyme catalyst derived from 1 -aminocyclopropane- 1 -carboxylic acid oxidase, wherein the metalloenzyme catalyst includes at least one mutation selected from: a I184A mutation, a K158T mutation, a K172V mutation, a L186V mutation, a G156E mutation, a G156T mutation, a R175P mutation, a V236G mutation, a V236K mutation, a G173R mutation, a G173V mutation, a F250L mutation, a A248T mutation, a S246F mutation, and a A180F mutation.BRIEF DESCRIPTION OF THE DRAWINGS

[0009] FIG. 1 illustrates method 100 for forming a non-canonical amino acid, according to some embodiments.

[0010] FIG. 2A illustrates nonheme Fe enzyme-catalyzed enantioselective amination of secondary C(sp³)–H bonds, according to some embodiments.

[0011] FIG. 2B illustrates enantioconvergent amination of tertiary C(sp3)–H bonds, according to some embodiments.2025-367-24059.073PCT1

[0012] FIG. 2C illustrates enantioconvergent construction of methyl-ethyl stereogenic center, according to some embodiments.

[0013] FIG. 3 illustrates a mechanism of the catalytic cycle of a nonheme Fe enzyme catalyzed nitrogen group migration reaction, according to some embodiments.

[0014] FIG. 4 illustrates high-throughput evaluation of nonheme Fe enzymes across a range of azanyl esters with diverse A’-protecting groups, according to some embodiments.

[0015] FIG. 5 illustrates an active-site of ACCONimi (1 -aminocyclopropane -1- carboxylic acid oxidase), according to some embodiments.

[0016] FIG. 6 illustrates a general mechanism for nitrogen migration using ACCONimi. according to some embodiments.

[0017] FIG. 7 illustrates an active site of ACCONim2, according to some embodiments.

[0018] FIG. 8 illustrates ACCONim2-catalyzed enantioselective synthesis of a-trisubstituted non-canonical amino acids, according to some embodiments.

[0019] FIG. 9 illustrates an active site of ACCONim3, according to some embodiments.

[0020] FIG. 10 illustrates Intramolecular kinetic isotope effects (KIE) studies with enantioenriched (R)-la-di and (S)-1a-d₁ using ACCONim1, according to some embodiments.

[0021] FIG. 11 illustrates Intramolecular kinetic isotope effects (KIE) studies with enantioenriched (R)-la-di and (S)-la-di using ACCONim2, according to some embodiments.

[0022] FIG. 12 illustrates kinetic isotope effects (KIE) and enzymatic enantioinduction effects by quantitative free energy analysis, according to some embodiments.

[0023] FIG. 13 illustrates computed reaction energy profiles of the ACCONimi-catalyzed 1,3-nitrogen migration, according to some embodiments.2025-367-24059.073PCT1

[0024] FIG. 14A illustrates the QM / MM-optimized 1,5-HAT (TS-2) transition state, according to some embodiments.

[0025] FIG. 14B illustrates the QM / MM-optimized 1,5-HAT (TS-2’) transition state, according to some embodiments.

[0026] FIG. 14C illustrates C-N bond forming radical rebound (TS-3) transition state, according to some embodiments.

[0027] F IG. 14D illustrates C-N bond forming radical rebound (TS-3’) transition state, according to some embodiments.DETAILED DESCRIPTION

[0028] Methods and compositions for performing nitrogen migration reactions are described herein. Several embodiments are directed to the use of enzyme variants to perform biocatalytic reactions with enantiocontrol. Particularly, several embodiments are directed to the use of an enzyme variant to perform an enantioselective nitrogen migration reaction. The enzyme variants are tailored to exhibit enhanced activity and stereoselectivity (including enantiocontrol) relative to naturally occurring counterparts or traditional small-molecule catalysts, thereby enabling higher yields and reduced byproduct formation. Several embodiments use non-heme Fe enzymes to serve as the catalyzing enzyme. Further, several embodiments are directed to the ability to perform enantioselective nitrogen migration reactions on substrates featuring azanyl moieties and on substrates having ester moieties. The nitrogen migration reaction can result in the azanyl moieties migrating to a different position on the substrate molecule.

[0029] FIG. 1 illustrates method 100 for forming a non-canonical amino acid, according to some embodiments. Method 100 includes one or more of the following aspects:

[0030] A nitrogen migration reaction is catalyzed 110 to form a non-canonical amino acid using a substrate and a metalloenzyme catalyst. The nitrogen migration reaction can be catalyzed 110 in the presence of at least one of a solvent, a buffer, a reductant, and a metal source. The nitrogen migration reaction can include hydrogen2025-367-24059.073PCT1transfer, such as 1,5-hydrogen atom transfer. The nitrogen migration reaction can include stereoselective radical rebound.

[0031] The substrate is generally a nitrogen-containing substrate. The nitrogencontaining substrate can include one or more nitrogen-containing organic compounds, such as a carboxylic acid derivative. The nitrogen-containing organic compound can include at least one of an ester moiety and an azanyl moiety. The ester moiety follows the general formula: R-COOR', where R may be a hydrogen atom, an alkyl group, or an aryl group, and R' may be an alkyl group or an aryl group. In one example, R’ is not a hydrogen atom. The azanyl moiety includes nitrogen and hydrogen. For example, the azanyl moiety can include an amino group or a substituted amino (-NHR), where R is an organic or inorganic group, such as organic or inorganic groups of the present disclosure.

[0032] In one example, the nitrogen containing organic compound is an azanyl moiety-containing substrate. The azanyl moiety-containing substrate can include an N-alkoxycarbonyl group. In one non-limiting example, the azanyl moiety-containing substrate is an azanyl ester. The azanyl ester can include one or more N-protecting groups, such as te -buty 1 oxycarbonyl (Boc); 2,2,2-trichloroethyloxycarbonyl (Troc); benzyloxycarbonyl (Cbz); and / or methoxycarbonyl (CChMe). In one non-limiting example, the azanyl ester includes at least one of the formulas: R-NHBoc and R-NHTroc, where R can include an alkyl group.

[0033] The azanyl ester can include one or more of: / c / 7- Butyl (2-phenylacetoxy)carbamate; 2,2,2-Trichloroethyl (2-phenylacetoxy)carbamate; tert-Butyl (2-(o-tolyl)acetoxy)carbamate; / - Butyl (2-( -tolyl)acetoxy)carbamate; r -Butyl (2-(j?-tolyl)acetoxy)carbamate; tert-Butyl (2-(4-fhiorophenyl)acetoxy)carbamate; tert- Butyl (2-(4-chlorophenyl)acetoxy)carbamate; / c / v- Butyl (2-(4-bromophenyl)acetoxy)carbamate; tert-Butyl (2-(4-methoxyphenyl)acetoxy)carbamate; fert-Butyl (2-(4-(methylthio)phenyl)acetoxy)carbamate; tert- Butyl (2-(4-(trifluoromethoxy)phenyl)acetoxy)carbamate; / -Butyl (2-(4-(tert-butyl)phenyl)acetoxy)carbamate; tert-Butyl (2-(4-ethynylphenyl)acetoxy)carbamate; Methyl 3-(2-(((terf-butoxycarbonyl)amino)oxy)-2-oxoethyl)benzoate; / - Butyl (2-(4-2025-367-24059.073PCT1hydroxyphenyl)acetoxy)carbamate; rt-Butyl (2-(3-chloro-4-fluorophenyl)acetoxy)carbamate; tert- Butyl (2-(benzo[<f|[l,3]dioxol-5-yl)acetoxy)carbamate; r -Butyl (2-(naphthalen-2-yl)acetoxy)carbamate; / -Butyl (2-(lH-indol-3-yl)acetoxy)carbamate; rt-Butyl (2-(6-methoxypyridin-3-yl)acetoxy)carbamate; te -Butyl (2-(thiophen-2-yl)acetoxy)carbamate; tert-Butyl (2-(thiophen-3-yl)acetoxy)carbamate; t -Butyl (2-(furan-2-yl)acetoxy)carbamate; tert-Butyl (£)-((4-phenylbut-3-enoyl)oxy)carbamate; and z -Butyl (£)-(hex-3-enoyloxy)carbamate.

[0034] The substrate can include a first substrate and a second substrate. The first substrate can include an azanyl moiety, and the second substrate can include an ester moiety. For example, the first substrate and the second substrate may be within a single molecule. For example, the first and second substrate may be in two different molecules.

[0035] The substrate, and optionally one or more additional components, can be mixed with the one or more solvents. In one example, the substrate, and optionally one or more additional components, are mixed with the solvent sufficient to form a solution. In one example, the concentration of substrate in the solution ranges from about 1 mM to about 200 mM. In another example, the concentration of substrate in the solution ranges from about 1 mM to about 40 mM. Method 100 may include utilizing one or more solvents. Examples of solvents include polar aprotic solvents. In one example, the solvent includes at least one of water, acetonitrile, D₂O solvent, and Dimethylsulfoxide (DMSO, e.g., degassed DMSO). In another example, the solvent includes acetonitrile.

[0036] Embodiments of the present disclosure including metalloenzyme catalysts. The metalloenzyme catalyst can be used for biocatalytic reactions, such as nitrogen migration. The metalloenzyme catalyst includes one or more metals (e.g., metal ion(s)). The one or more metals can be bound at / in an active site of the metalloenzyme catalyst. In some embodiments, the metalloenzyme catalyst is a nonheme metal enzyme, such as a nonheme Fe enzyme. In one example, the nonheme Fe enzyme is an enzyme variant derived from (e.g., modified) 1 -aminocyclopropane- 1 -carboxylic acid oxidase. In another2025-367-24059.073PCT1example, the enzyme is an enzyme variant of at least one nonheme Fe enzyme shown in Table 1. The enzyme variant can include at least one mutation.Table 1. Nonheme Fe enzymes.Fe Organism AnnotationenzymeBDDO Pseudomonas biphenyl-2,3-diol 1,2- aeruginosa dioxygenaseTauD Escherichia coli taurine dioxygenaseEvDOi Micromonospora 2OG-dependent oxygenasecarbonaceaEFE Pseudomonas ethylene / succinate-formingsavastanoi enzymeLapB Pseudomonas metapyrocatechasealkylphenolicaHPPD Streptomyces 4-hydroxyphenylpyruvateavermitilis dioxygenaseANS Arabidopsis thaliana leucoanthocyanidindioxygenaseACCO Petunia hybrida 1- aminocycloprop ane-1- carboxylate oxidaseDAOC Streptomyces deacetoxycephalosporin CS clavuligerus synthaseEgtB Mycolicibacterium hercynine oxygenasethermoresistibile

[0037] The enzyme variant can be present as a nonheme Fe enzyme cell-free lysate or a nonheme Fe enzyme whole cell. The enzyme variant can include a mutation at one or more of residue positions 172, 184, and 186. The enzyme variant can include at least one mutation selected from: I184A, K158T, K172V, L186V, G156E, G156T, R175P, V236G,2025-367-24059.073PCT1V236K, G173R, G173V, F250L, A248T, S246F, and A180F. The enzyme variant can include two or more mutations selected from: I184A, K158T, K172V, L186V, G156E, G156T, R175P, V236G, V236K, G173R, G173V, F250L, A248T, S246F, and A180F. The enzyme variant can include one or more mutations selected from: I184A, K158T, K172V. The enzyme variant can include all the following mutations: I184A, K158T, K172V.

[0038] 1 -aminocyclopropane- 1 -carboxylic acid oxidase (ACCO) can possess conformational flexibility, with significant structural changes occurring before and after substrate entry into the active site. In one example, positions 172 and 186 are residues that can create unfavorable interactions with the bulky Boc N-protecting group of the substrate. Mutating K172 and LI 86, such as to valine, can reduce these unfavorable interactions, improving substrate accommodation and contributing synergistically to catalytic performance.

[0039] In one non-limiting example, the enzyme variant includes ACCO I184A K158T K172V L186V G156E R175P V236G G173R (ACCONimi). In another nonlimiting example, the enzyme variant includes ACCO I184A K158T K172V L186V G156E R175P V236K G173V F250L (ACCONim2). In another non-limiting example, the enzyme variant includes ACCO I184A K158T K172V L186V R175P V236K G173V F250L A248T S246F A180F G156T (ACCONims). The enzyme variant can be formed using cloning and expression vectors. Site-saturation mutagenesis can be performed to form a product. The product can be digested, purified, and ligated. The ligation mixture can be used to transform cells. For example, at least one tunnel residue K93X, F91X, T89X, Y162X, S160X, K158X, G156X, and N154X located within the P-sheets and / or at least one active-site residue D169, K172, G173, R175, 1184, L186, A248, and F250 that are in close contact can be selected for site-saturation mutagenesis (SSM).

[0040] Method 100 may include utilizing one or more buffers. Examples of buffers include KPi buffer, Tris-HCl and Na₂HPO₄-NaH₂PO₄ buffer (“NaPi” buffer). Buffers can be utilized to promote precise pH control. In one example, method 100 includes utilizing a buffer having a pH value ranging from about 6 to about 8. In another example, method2025-367-24059.073PCT1100 includes utilizing a buffer having a pH value ranging from about 7 to about 8. In one non-limiting example, utilizing a buffer having a pH value ranging from 7 to 8 increases the yield of desirable product compound(s). In one non-limiting example, performing method 100 at a pH range from 7 to 8 can be important for balancing reactivity and substrate stability. For example, at a pH value below 7, deprotonation of the azanyl substrate is disfavored, which suppresses the reaction. For example, at a pH value above 8, the substrate undergoes hydrolysis more readily. An example concentration range for the buffer compound ranges from about 20 mM to about 250 mM. In one non-limiting example, a 40 mM to 200 mM KPi buffer is utilized.

[0041] Method 100 may include using a reductant. The reductant can act as an electron donor to an electron recipient. In one example, the reductant includes at least one of sodium ascorbate, L-ascorbic acid, and sodium dithionite. In one non-limiting example, the reductant includes sodium ascorbate. A concentration of the reductant utilized can range from about 10 mol% to about 50 mol%. A concentration of the reductant utilized can range from about 10 mol% to about 30 mol%. The reductant can increase the product yield and enhance the e.r. of the product. Method 100 may include using a metal source. The metal source can be an iron source, such as a Fe2+source. In one example, the metal source includes Mohr’s salt (Fe(NH4)2(SO4)2). In one nonlimiting example, Mohr’s salt can provide a stable and well-defined Fe2+source, which facilitates higher and more consistent iron loading into the protein’s active site.

[0042] The one or more solvents (when desirable), one or more buffers, the substrate, and the enzyme (such as a purified enzyme variant) can be contacted and / or mixed to form a reaction mixture (with various orders possible). In one example, the substrate and the one or more solvents are mixed to form a solution prior to adding the one or more buffers and the enzyme. Method 100 can proceed (at least partially) under aerobic or anaerobic conditions. In one non-limiting example, one or more solvents, one or more buffers, the substrate, and the enzyme can be contacted and / or mixed (in any order) in an anaerobic chamber. Any two or more of the components used in method 100 can be mixed in various orders.2025-367-24059.073PCT1

[0043] Any subset or all of the substrate, metalloenzyme catalyst, solvent, buffer, reductant, and metal source can be combined in any order or sequence, including simultaneous or stepwise addition, and can be pre-mixed or contacted in multiple stages The reaction can be conducted in batch, semi-continuous, or continuous formats, and the relative amounts, concentrations, and identities of the substrate, metalloenzyme catalyst, solvent, buffer, reductant, and metal source can be varied independently of one another to achieve formation of the non-canonical amino acid.

[0044] Method 100 can be performed at various temperatures. In one example, method 100 is performed at about 20-40 °C. In some embodiments, the reaction is initiated, conducted, and / or completed at a temperature within a range from about 0° C to about 80° C, such as from about 5° C to about 50° C. from about 10° C to about 40° C, or from about 15° C to about 30° C, including at a temperature of about 20° C. In some embodiments, the reaction is carried out for a period of time sufficient to provide the desired product, such as a non-canonical amino acid, for example for a time ranging from about 0.5 hours to about 48 hours, from about 1 hour to about 24 hours, from about 2 hours to about 12 hours, or from about 3 hours to about 8 hours, including for a period of about 6 hours. The reaction can be started at any of the foregoing temperatures and allowed to proceed for any of the foregoing time periods, and may optionally be performed under conditions in which the temperature is held substantially constant, varied within one or more of the recited ranges, and / or adjusted during the course of the reaction to optimize formation of the non-canonical amino acid.

[0045] In some embodiments, a reaction for forming a desired product, such as a non-canonical amino acid, is carried out by contacting a substrate (e.g., an azanyl moiety -containing substrate) with a metalloenzyme catalyst in the presence of a reductant and a metal source under conditions sufficient to form the desired product. In some embodiments, the reductant is sodium ascorbate, and the metal source is Mohr's salt, and any subset or all of the substrate, metalloenzyme catalyst, sodium ascorbate, and Mohr's salt can be combined in any order or sequence, including simultaneous or stepwise addition, and can be pre-mixed or contacted in multiple stages.2025-367-24059.073PCT1

[0046] Method 100 forms one or more non-canonical amino acids. 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. Method 100 can form at least one of a-trisubstituted amino acid and oc-tetrasubstituted non-canonical amino acid.

[0047] The non-canonical amino acid can be selected from: 2-((tert-Butoxycarbonyl)amino)-2-phenylacetic acid; 2-Phenyl-2-(((2,2,2-trichloroethoxy)carbonyl)amino)acetic acid; 2-((r -Butoxycarbonyl)amino)-2-(o-tolyl)acetic acid; 2-((n?rf-Butoxycarbonyl)amino)-2-(m-tolyl)acetic acid; 2-((tert-Butoxycarbonyl)amino)-2-(p-tolyl)acetic acid; 2-(( / er / -Butoxycarbonyl)amino)-2-(4-fluorophenyl)acetic acid; 2-((tert-Butoxycarbonyl)amino)-2-(4-chlorophenyl)acetic acid; 2-(4-Bromophenyl)-2-(( / -butoxycarbonyl)amino)acetic acid; 2-((n?rt-Butoxycarbonyl)amino)-2-(4-methoxyphenyl)acetic acid; 2-((fert-Butoxycarbonyl)amino)-2-(4-(methylthio)phenyl)acetic acid; 2-((t -Butoxycarbonyl)amino)-2-(4-(trifluoromethoxy)phenyl)acetic acid; and 2-((tert-Butoxycarbonyl)amino)-2-(4-(te / -r-butyl)phenyl)acetic acid.

[0048] The non-canonical amino acid can be selected from: 2-((n?rt-Butoxycarbonyl)amino)-2-(4-ethynylphenyl)acetic acid; 2-((re / -Butoxycarbonyl)amino)-2-(3-(methoxycarbonyl)phenyl)acetic acid; 2-((t -Butoxycarbonyl)amino)-2-(4-hydroxyphenyl)acetic acid; 2-((n?r / -Butoxycarbonyl)amino)-2-(3-chloro-4-fluorophenyl) acetic acid; 2-(Benzo[<f|[l,3]dioxol-5-yl)-2-((rert-butoxycarbonyl)amino)acetic acid; 2-((tert-Butoxycarbonyl)amino)-2-(naphthalen-2-yl)acetic acid; 2-((n?rt-Butoxycarbonyl)amino)-2-(lH-indol-3-yl)acetic acid; 2-((tert-Butoxycarbonyl)amino)-2-(6-methoxypyridin-3-yl)acetic acid; 2- (tert- Butoxycarbonyl)amino)-2-(thiophen-2-yl)acetic acid; 2-((tert-Butoxycarbonyl)amino)-2-(thiophen-3-yl)acetic acid; 2-((tert-Butoxycarbonyl)amino)-2-(furan-2-yl)acetic acid; (E)-2025-367-24059.073PCT12-((fe -Butoxycarbonyl)amino)-4-phenylbut-3-enoic acid; and (E)-2-(( rt-Butoxycarbonyl)amino)hex-3-enoic acid.

[0049] The non-canonical amino acid can be selected from: 2-((tert- Butoxycarbonyl)amino)-2-phenylpropanoic acid; 2-((n?rt-Butoxycarbonyl)amino)-2-( >-tolyl)propanoic acid; 2-(( / -Butoxycarbonyl)amino)-2-(4-methoxyphenyl)propanoic acid; 2-((ter / -Butoxycarbonyl)amino)-2-(4-(trifluoromethyl)phenyl)propanoic acid; 2-(( / erf-Butoxycarbonyl)amino)-2-phenylbutanoic acid; l-(( / ert-Butoxycarbonyl)amino)-2,3-dihydro- 1 / -indene- 1 -carboxylic acid; 2-((tert-Butoxycarbonyl)amino)-2-(thiophen- 3-yl)propanoic acid; Benzyl 2-((7e / 7-butoxycarbonyl)amino)-2-cyclopentylpropanoate; and 2-((terf-Butoxycarbonyl)amino)-2-methylbutanoic acid.

[0050] In some embodiments, the nitrogen migration reaction is represented by the following scheme:nonheme Fe enzymecell-free lysate oO > KPI buffer || I Ji 1 s. J '-L Mohr's saltO R Sodium ascorbatert,6hwherein R can be any number of functional groups. In other embodiments, substrates may exhibit different overall structure. This scheme generally applies to compatible azanyl esters.

[0051] FIG. 2A illustrates nonheme Fe enzyme-catalyzed enantioselective amination of secondary C(sp’)-H bonds, according to some embodiments. FIG, 2B illustrates enantioconvergent amination of tertiary C(sp3)-H bonds, according to some embodiments. FIG, 2C illustrates enantioconvergent construction of methyl-ethyl stereogenic center, according to some embodiments.

[0052] The nonheme Fe enzyme-catalyzed nitrogen migration process is compatible with carboxylic acid derivatives featuring an a-monosubstituted or an a, a- disubstituted a-carbon. Such compatibility enables the enantioselective amination of secondary C(sp3)-H bonds in a prochiral methylene unit (FIG. 2A) and the enantioconvergent amination of tertiary C(sp3)-H bonds of racemic substrates (FIG. 2B) with excellent stereoselectivity. Furthermore, the modification of nonheme Fe nitrogen2025-367-24059.073PCT1migratases can facilitate the asymmetric construction of minimally differentiated methyl¬ ethyl stereogenic center with good enantiocontrol (FIG. 2C). Moreover, evolved nonheme Fe enzymes can exh bit catalytic activities orders of magnitude higher than small-molecule Fe complexes.Example 1

[0053] Expression of ACCOxim variants. E. coli BL21(DE3) cells harboring recombinant plasmid encoding the appropriate ACCO variant were grown overnight at 37 °C and 230 rpm in 4 mL LB media supplemented with 0.05 mg / mL kanamycin (LBkan). Preculture (1.0 mL, 5% v / v) was used to inoculate 20 mL TB media supplemented with 0.05 mg / mL kanamycin (TBkan) in a. 125 mL Erlenmeyer flask. The culture was incubated at 37 °C and 230 rpm for 2 h to reach an ODsoo of ca. 1.5. The culture was then cooled on ice for 20 min and induced with 0.5 mM isopropyl P-D-l- thiogalactopyranoside (IPTG, final concentrations). Protein expression was performed at 20 °C and 200 rpm for 20 h. The cells were then transferred to a conical tube (50 mL) and harvested by centrifugation (3,434 g, 4 min, 4 °C) using a tabletop centrifuge.

[0054] Analytical scale enantioselective biocatalytic nitrogen migration. The suspension of E. coli cells expressing ACCO in KPi buffer (100 mM, pH - 7.4, typically OD600= 5–20, 1.5 mL) was added to a 24- well plate and kept on ice. Cells were then disrupted by sonication using a sonicator equipped with a 24-tip horn (45% amplitude, 2 secs on, 4 secs off, 18 min in total (sonication time: 6 min)). To pellet the cell debris, the resulting lysate was then centrifuged at 21130 g and 4 °C for 30 min using a centrifuge. The clarified cell-free lysate (360 pL per well) was then added to a new 2 mL vial and transferred into a Coy anaerobic chamber. In the anaerobic Coy chamber, the new mixed solution of ferrous ammonium sulfate and sodium ascorbate (20 pL, 20 mM ferrous ammonium sulfate and 40 mM sodium ascorbate in dd H2O), and the azanyl ester substrate (20 pL, 200 mM in MeCN) were added. Final reaction volume was 400 pL; final concentration of the azanyl ester substrate was 10 mM. (Note: reaction performed with E. coli cells resuspended to OD600= 10 indicates that OD600= 10 cells were then disrupted by sonication and 360 µL clarified cell-free lysates was added after centrifuge,2025-367-24059.073PCT1and likewise for other reaction ODeoo descriptions.) The vials were sealed and shaken in a Corning digital microplate shaker at room temperature and 680 rpm for 6 h. The reaction mixture was then analyzed by chiral HPLC.

[0055] Preparative gram-scale enantioselective biocatalytic 1,3-nitrogen migration. E. colt BL21(DE3) cells harboring the recombinant plasmid encoding the specific ACCOxim variants were grown overnight at 37 °C and 230 rpm in 25 mL LB media supplemented with 0.05 mg / mL kanamycin (LBkan). Preculture (10 mL, 4% v / v) was used to inoculate 250 mL TB media supplemented with 0.05 mg / mL kanamycin (TBkan) in a 1 L Erlenmeyer flask. The culture was incubated at 37 °C and 230 rpm for 2 h to reach an OD600of ca. 1.2. The culture was then cooled on ice for 30 min and induced with 0.5 mM isopropyl p-D-l-thiogalactopyranoside (IPTG, final concentrations). Protein expression was performed at 20 °C and 200 rpm for 20 h.

[0056] 500 mL E. coll cells were harvested by centrifugation (4,347 g, 15 min. 4 °C) using a tabletop centrifuge and resuspended in 50 mL KPi buffer (100 mM, pH = 7.4, ODf>oo = 100). Cell suspensions in KPi buffer were kept on ice and lysed by sonication using a sonicator (45% amplitude, 2 secs on, 4 secs off, 18 min in total per cycle (sonication time: 6 min), 2 cycles were applied): samples were carefully submerged in wet ice to avoid overheating during this process. To pellet the cell debris, lysates were centrifuged using a superspeed centrifuge (28,928 g. 30 min, 4 °C). To a 1 L Erlenmeyer flask equipped with a screw cap were added the supernatant. The flask was transferred to a Coy anaerobic chamber, where 200 mL degassed KPi buffer was added to dilute the cell-free lysate suspension. At this point, an aqueous solution of ferrous ammonium sulfate (5 mL, 200 mM stock solution in degassed H2O) and a solution of sodium ascorbate (10.0 mL, 200 mM stock solution in degassed KPi buffer) were added. The resulting mixture was mixed by gentle shaking. 3 minutes later, the azanyl ester substrate (10.0 mmol, 10.0 mL stock solution in MeCN (1.0 M)) was added. The flask was capped, sealed with parafilm, taken out of the Coy anaerobic chamber, and allowed to shake in an shaker at room temperature and 230 rpm for 6 h.2025-367-24059.073PCT1

[0057] Upon the completion of this biotransformation, 100 ml IM HC1 aqueous solution was added to the reaction mixture. The mixture was extracted with 300 mL EtOAc by vigorous mixing. The resulting mixture was transferred to centrifugation buckets and centrifuged (4,347 g, 20 min) using a centrifuge to separate the organic and the aqueous layers. The aqueous layer was extracted with EtOAc for an additional five times. Combined organic layers were dried over MgSO4and an aliquot of the organic layer (400 µL) was used for product enantiomeric ratio determination via chiral HPLC analysis. Combined organic layers were concentrated in vacuo with the aid of a rotary evaporator and purified by column chromatography.Example 2

[0058] FIG. 3 illustrates a mechanism of the catalytic cycle of a nonheme Fe enzyme catalyzed nitrogen group migration reaction, according to some embodiments. The nonheme Fe enzyme (also referred to as “nonheme Fe nitrogen migratase”) (I) first catalyzes the conversion of azanyl ester 1 into an Fe-bound nitrogen centered radical II via α-elimination, This Fe-bound nitrogen radical undergoes an intramolecular 1,5- hydrogen atom transfer (1,5-HAT) to furnish the corresponding carboxylate a-radical. At this stage, intramolecular radical rebound with the Fe-bound nitrogen group (- NHR) allows for enantioselective C-N bond formation (III). Release of the non-canonical amino acid product 2 from III regenerates the ferrous nonheme enzyme, thereby completing the catalytic cycle.

[0059] This leverages the multiple open coordination sites of the nonheme Fe center, enabling simultaneous binding of the reactive nitrogen-centered radical and the substrate carboxylate. This facilitates the intramolecular 1,5-HAT — a transformation that can be difficult to achieve with heme enzymes, which possess only a single available coordination site. As azanyl esters 1 can be prepared from the corresponding carboxylic acids, this enables the synthetically valuable nonheme Fe enzyme-catalyzed stereoselective preparation of non-canonical amino acids with widespread applications as building blocks in peptide therapeutics, bioactive natural products, and functional proteins. The Fe enzyme-catalyzed nitrogen migration process is compatible with2025-367-24059.073PCT1carboxylic acid derivatives featuring either an a-monosubstituted or an a,a-disubstituted a-carbon. This broad applicability of nonheme Fe is demonstrated in FIG. 2A, FIG. 2B, and FIG. 2C.

[0060] FIG. 4 illustrates high-throughput evaluation of nonheme Fe enzymes across a range of azanyl esters with diverse ^-protecting groups, according to some embodiments. In one example, N-alkoxycarbonyl groups, including tert butyloxycarbonyl (Boc), 2,2,2-trichloroethoxycarbonyl (Troc), benzyloxycarbonyl (Cbz) and methoxycarbonyl (CO2Me) exhibited higher activity than N-acyl substituents such as benzoyl (Bz) and pivaloyl (Piv). Among substrates possessing these N-substituents, Boc and Troc were found to display the highest activity across a range of nonheme Fe enzymes evaluated. A plant-derived enzyme, namely 1-aminocyclopropane-l -carboxylic acid oxidase from Petunia hybrida (ACCO, Uniprot ID: Q08506) displayed good initial activity and excellent enantioselectivity in this 1,3-nitrogen migration process.

[0061] FIG. 5 illustrates an active-site of ACCONimt (1 -aminocyclopropane- 1-carboxylic acid oxidase), according to some embodiments. The I184A mutation close to the Fe center can further open up the active site, thus better accommodating the nonnative substrate. As distal residues from the substrate tunnel were found to have impact on the activity of ACCO, arnino acid residues in the 89-93 and 156-160 [3-sheets were targeted at the entrance of the substrate tunnel. Site-saturation mutagenesis (SSM) and screening were employed. In each SSM library, 88 clones were selected, grown in 96- well plates, and analyzed by chiral high performance liquid chromatography (HPLC) for enzyme activity and enantioselectivity. In these P-sheets, a beneficial mutation K158T was identified, affording ca. 1.2-fold improvement in activity ((37 ± 1)% yield, 92:8 e.r., (340 ± 10) TTN).

[0062] Two beneficial mutations KI 72V and LI 86V were completed, leading to notable enhancement in the yield of product 2a (2-((rerr-Butoxycarbonyl)amino)-2-phenylacetic acid). The triple mutant ACCO K158T K172V L186V furnished 2a in (64 ± 2)% yield, 92:8 e.r. and (520 ± 20) TTN. Additional four rounds of SSM and screening by targeting active-site residues led to ACCO I184A K158T K172V L186V G156E2025-367-24059.073PCT1R175P V236G G173R, which is eight mutations away from the wild-type ACCO, affording amino acid product 2a in (71 ± 1)% yield, 99:1 e.r., and (810 ± 10) TTN. All the four newly identified mutations, including G156E, R175P, V236G, and G173R. resulted in a steady improvement of enantioselectivity (92:8 e.r., entry 5 - 99:1 e.r., entry 9). Additionally, the chemoselectivity of the nonheme enzyme also improved throughout directed evolution, as evidenced by improved ratio of amino acid 2a: reduced carboxylic acid 3a. By further lowering the loading of ACCONIMI via decreasing the density of E. coll cell suspension overexpressing ACCONim1(ODsoo = 3, ca. 0.026 mol% enzyme loading), a TTN of (2,260 ± 40) was achieved with a slightly decreased enantioselectivity. This total turnover number was approximately 200-times higher than those of small-molecule Fe catalysts, highlighting the catalytic efficiency of the nonheme Fe enzyme.NHBoc1a 2a3a

[0063] FIG. 6 illustrates a general mechanism for nitrogen migration using ACCONimi. according to some embodiments. Reaction conditions: 10.0 mM azanyl ester 1, ACCONimi cell-free lysate in 100 mM KPi buffer (pH = 7.4, ca. 0.049-0.098 mol%), 1.0 mM Fe(NH4)2(SC>4)2, 2.0 mM sodium ascorbate, MeCN (5% v / v), room temperature, 6 h.

[0064] The non-canonical amino acid can be selected from: 2-((tert-Butoxycarbonyl)amino)-2-phenylacetic acid; 2-Phenyl-2-(((2,2,2-trichloroethoxy)carbonyl)amino)acetic acid; 2-(( / -Butoxycarbonyl)amino)-2-(o-tolyl)acetic acid; 2-((tert-Butoxycarbonyl)amino)-2-(m-tolyl)acetic acid; 2-((tert-Butoxycarbonyl)amino)-2-(p-tolyl)acetic acid; 2-((r -Butoxycarbonyl)amino)-2-(4-fluorophenyl)acetic acid; 2-((tert-Butoxycarbonyl)amino)-2-(4-chlorophenyl)acetic acid; 2-(4-Bromophenyl)-2-((tert-butoxycarbonyl)amino)acetic acid; 2-((tert-Butoxycarbonyl)amino)-2-(4-methoxyphenyl)acetic acid; 2-((tert-Butoxycarbonyl)amino)-2-(4-(methylthio)phenyl)acetic acid; 2-((tert-2025-367-24059.073PCT1Butoxycarbonyl)amino)-2-(4-(trifluoromethoxy)phenyl)acetic acid; and 2-((tert-Butoxycarbonyl)amino)-2-(4-(zerz-butyl)phenyl)acetic acid.

[0065] The non-canonical amino acid can be selected from: 2-((tert-Butoxycarbonyl)amino)-2-(4-ethynylphenyl)acetic acid; 2-((tert-Butoxycarbonyl)amino)-2-(3-(methoxycarbonyl)phenyl)acetic acid; 2-((tert-Butoxycarbonyl)amino)-2-(4-hydroxyphenyl)acetic acid; 2-((tert-Butoxycarbonyl)amino)-2-(3-chloro-4-fluorophenyl)acetic acid; 2-(Benzo[d][1,3]dioxol-5-yl)-2-((tert-butoxycarbonyl)amino)acetic acid; 2-((tert-Butoxycarbonyl)amino)-2-(naphthalen-2-yl)acetic acid; 2-((tert-Butoxycarbonyl)amino)-2-(1H-indol-3-yl)acetic acid; 2-((tert-Butoxycarbonyl)amino)-2-(6-methoxypyridin-3-yl)acetic acid; 2-((tert-Butoxycarbonyl)amino)-2-(thiophen-2-yl)acetic acid; 2-((tert-Butoxycarbonyl)amino)-2-(thiophen-3-yl)acetic acid; 2-((tert-Butoxycarbonyl)amino)-2-(furan-2-yl)acetic acid; (E)-2-((tert-Butoxycarbonyl)amino)-4-phenylbut-3-enoic acid; and (E)-2-((tert-Butoxycarbonyl)amino)hex-3-enoic acid.

[0066] FIG. 7 illustrates an active site of ACCONim2> according to some embodiments. ACCONim2 can be utilized as a nonheme Fe nitrogen migratase to synthesize a-tetrasubstituted amino acids via enantioconvergent C-H functionalization using racemic carboxylic acid derivatives. ACCONim2 was used to synthesize a- tetrasubstituted amino acids via enantioconvergent C-H functionalization using racemic carboxylic acid derivatives. ACCO I184A K158T K 172V L186V G156E R175P, an intermediate variant of ACCOMmi, furnished the corresponding a-tetrasubstituted amino acid 5a with a promising activity and enantioselectivity (67% yield and 88:12 e.r.).V riants possessing mutations at 236 and 173 displayed high activity and / or enantioselectivity compared to the unmutated parent. Thus, these sites were targeted for forming an enantioconvergent C-H functionalizing nitrogen migratase for a- tetrasubstituted amino acid synthesis.2025-367-24059.073PCT1OH5a

[0067] With this ACCO I184A K158T K172V L186V G156E R175P variant as the parent, three rounds of iterative SSM and screening led to the discovery of V236K, G173V and F250L as beneficial mutations, giving rise to a substantially improved enantioconv ergent nitrogen migratase ACCONimi. ACCONim?. catalyzed the formation of a-tetrasubstituted amino acid 5a with (84 ± 2)% yield, 99:1 e.r., and (770 ± 10) TTN in an enantioconvergent fashion. Furthermore, by further lowering the loading of ACCONim2. a TTN of (2210 ± 40) was achieved albeit with a slightly decreased yield and enantioselectivity.

[0068] FIG. 8 illustrates ACCONim1-catalyzed enantioselective synthesis of a-trisubstituted non-canonical amino acids, according to some embodiments. Aryl groups with an electron-donating substituent such as a methyl and a methoxy as well as an electron- withdrawing substituent such as a trifluoromethyl, were found to be compatible with this biocatalytic nitrogen migration. In addition, a- tetrasubstituted amino acid with an ethyl a-substituent, a cyclic moiety at the alpha-position and a thienyl group could also be prepared with excellent enantiocontrol. Furthermore, azanyl esters lacking an a-aryl substituent could also be effectively transformed via the amination of unactivatedC(sp3)-H bond, providing the corresponding fully aliphatic a-tetrasubstituted amino acid with 91:9 er.

[0069] FIG. 9 illustrates an active site of ACCONUI>3, according to some embodiments. Four additional beneficial mutations, including A248T, S246F, A180F and G 156T, were introduced to ACCONim2, resulting in a new nitrogen migratase variant ACCONim2. ACCONim3catalyzed the enantioconvergent C-H functionalization with (55 ± 2)% yield and 88:12 e.r., providing a rare example of enantioconvergent synthesis of a-tetrasubstituted amino acid possessing a methyl-ethyl stereocenter.2025-367-24059.073PCT1

[0070] As part of the mechanistic studies of the nonheme Fe enzyme-catalyzed nitrogen migration process, azanyl esters (Z)-lx and (E)-lx bearing a cis- and trans-olefin, respectively, were synthesized and subjected to this biocatalytic nitrogen migration. Starting from (Z)-lx, (E)-2x bearing a trans-olefin formed exclusively in 27% yield and 97:3 e.r.. The reduction side product carboxylic acid 3x formed with an (E)-configuration.ACCOuimi COOH COOH KPi buffer (pH = 7.4)NHBoc Ph Mohr’s salt (10 mol%), (£)-2x (2)-3x Sodium ascorbate (20 mol%), 24% yield, 97:3 er 31% yield Z! E> 99 / 1 CH3CN (5% v / v), r.t. £7Z>99 / 1 Z7E>99 / 1O ACCONim1COOH Ph^^^COOH KPi buffer (pH =7.4) Ph. NHBocO NHBoc Mohr’s salt (10 mol%), (E)-3x (E)-1x (£)-2xSodium ascorbate (20 mol%), 36% yield, 96:4 er 53% yield SZ> 99 / 1 CH3CN (5% v / v), r.t. E! Z> 99 / 1 EIZ> 99 / 1

[0071] In one example, these results showed the intermediacy of an a radical of carboxylic acid, which can arise from the nonheme Fe enzyme-catalyzed intramolecular 1,5-hydrogen atom transfer (1,5-HAT). Furthermore, the scrambling of olefin geometry indicated that the a-radical is relatively long lived and the C-N bond forming radical rebound is slower than the olefin E / Z isomerization at the a-radical stage.

[0072] Next, the radical clock substrate (rac)-7 was prepared possessing a cyclopropyl group at the benzylic position and applied to the nonheme Fe enzyme-catalyzed process. In addition to the formation of the reduction product 8 (46% yield), dihydropyrone 9 was also observed in 14% yield. It was determined that 9 likely formed via a 1,5-HAT / cyclopropane ring opening / carboxylate rebound process. The discovery of carboxylate rebound product suggested that the newly formed carboxylate derived from the azanyl ester substrate can be bound to Fe center of the nonheme enzyme.2025-367-24059.073PCT1ACCONim2KPI buffer (pH = 7.4) Mohr’s salt (10 mol%), Sodium ascorbate (20 mol%), CH3CN (5% v / v), r.t.914% yieldcarboxylate rebound cyclopropane r O HAT ring-opening Ph His I ^Asp O-Fe— His NHBoc

[0073] FIG. 10 illustrates Intramolecular KIE studies with enantioenricheddi and (S)-1a-d₁ using ACCONim1, according to some embodiments. FIG. 11 illustrates Intramolecular KIE studies with enantioenriched ( / ?)-! a-t?i and ($)- la-di using ACCONim2, according to some embodiments. FIG. 12 illustrates kinetic isotope effects (KIE) and enzymatic enantioinduction effects by quantitative free energy analysis, according to some embodiments.

[0074] First, benzylic deuterated substrate 1 -rfo was prepared and independently determined the initial rates with both la and la-ifc. These independent kinetic measurements with la and la-rfo showed a W: D of (5.5 ± 0.3). These results indicated that the C(5 / ?3)--H cleavage in hydrogen atom transfer is involved in the rate-determining step. Second, enantiomerically pure monodeutero, monoprotic substrates (J?)- la-di and (5)-la- i were prepared by multi step synthesis starting from optically pure mandelic acid.2025-367-24059.073PCT1ACCONim1(0.0125 moi%)KPi buffer (pH = 7.4)Mohr’s salt (10 mol%),Sodium ascorbate (20 mol%),CH3CN (5% v / v), r.t. H NHBocACCONim1(0.0125 mol%)KPi buffer (pH - 7.4)- >Mohr's salt (10 mol%),Sodium ascorbate (20 mol%),CH3CN (5% v / v), r.t.k^kD= 5.5 ± 0.3

[0075] Using (A)- la-rfi and (S)-la- i, ACCONimi and ACCONIHU were evaluated, which were evolved for secondary and tertiary C(sp3)-H amination, respectively. The secondary C(sp3)-H aminating enzyme ACCONimi was found to exhibit a similar kufka value of (5.6 ± 0.3) and (6.4 ± 0.5), respectively, when (R')-la-di and (5)- la-t / j were applied. In contrast, the tertiary C(sp3)–H aminating enzyme ACCONim2 showed a marked difference in ku / ki) values when (A’)- and (S’)-la-dj were used as the substrates. Using (Alla-r / i, a large / cii / fo value of (9.3 ± 0.8) was observed with ACCONim2. By contrast, a much smaller kH / kD of (2.1 ± 0.1) was observed with (S)-1a-d1.ACCONim1 (0.025 mol%) KPi buffer (pH = 7.4) Mohr's salt (10 mol%), Sodium ascorbate (20 mol%), CH3CN (5% v / v), r.t.85% 15% >99:1 e.r. >99:1 e.r. ACCOWim; (0.025 rnoi%> KPS buffer (pH - 7.4) - a*. Cy, Cy, Mohr’s salt (10 mot%), Sodium ascorbate (20 O " NHBoc H 'NHBofiCH3CN (5% v / v), r.t. (R)-1a-d187% 13% >99:1 e.r. >99:1 e.r.

[0076] With these results, quantitative activation free energy analysis was performed for both ACCONimi and ACCONim2. The KIE results with la and la-rC showed that the HAT step in this nitrogen migration reaction is irreversible. The 2a- i / 2a ratio is2025-367-24059.073PCT1thus determined by two energy terms, including DGKIE, which reflects the kinetic isotope effects in HAT, and DGenantioselectivity, which reflects the enzymatic enantiopreference in HAT for non-deuterated substrate (la). When (5)- la-rfi was used as the substrate, both the kinetic isotope effect and the enzymatic enantioselectivity favor the abstraction of the pro-( / )-H, thus resulting in a higher 2a- i / 2a ratio. In contrast, withthe kinetic isotope effect is mitigated by the enzymatic enantiopreference for the abstraction of the pro-(7?)-D, leading to a lower 2a-<7i / 2a ratio.

[0077] Using free energy analysis, the two effects were dissected and the energy terms DGKIEand DGenantioselectivitywere calculated for both ACCONimi and ACCONim2. With nonheme Fe enzyme ACCONimi evolved for secondary C(sp3)-H amination, a close-to-zero DGenantioselectivityof -0.04 kcal / mol was observed with the DGKIEbeing -1.06 kcal / mol. This DGenantioselectivitycorresponds to an enantiomeric ratio (e.r.) of 52:48, indicating a lack of enzymatic enantioselectivity in the HAT step. On the other hand, with ACCONim2 evolved for tertiary CCv / )-H amination, a modest, but non-zero,D G enantioselectivity of -0.43 kcal / mol was determined with a DGKIEof -0.86 kcal / mol.ACCONim2’s DGenantioselectivitycorresponds to an e.r. of 67:33, suggesting a small degree of enantioselectivity in the HAT step. Together, these results demonstrated that directed evolution is a powerful means to manipulate enzymatic stereoselectivity in each individual step in a complex overall catalytic cycle. Furthermore, this data reveals that for the secondary C(sp3)–H aminating nonheme enzyme ACCONim1, the overall enantioselectivity reflects the stereoselective radical rebound step succeeding the HAT step. Finally, the DGKIEterm for ACCONimi and ACCONim2 corresponds to a fc> value of 52:48 and 67:33, respectively. These results are consistent with the kH / kD value obtained from independent initial rate measurements with 1a and 1a-d2.

[0078] This nonheme Fe enzyme was compared with other heme-based enzymes in different C(sp3)–H amination processes. Engineered serine-ligated P450 variant P411Diane2 displayed a DGenantioselectivityof -1.14 kcal / mol for HAT, which corresponds to an e.r. of 87:13. Additionally, histidine-ligated myoglobin variant Mb showed a DGenantioselectivityof -0.28 kcal / mol (62:38 e.r.) for HAT. Collectively, these results2025-367-24059.073PCT1revealed that among all the heme and nonheme Fe enzymes developed for diverse C(sp3)–H amination reactions, the current nonheme enzyme ACCONim1 is the only system where the enzymatic HAT step is almost non-enantioselective while the radical rebound step exhibits excellent enantioselectivity. This finding is consistent with results that ACCONim-catalyzed amination of tertiary C(sp3)–H bonds is fully enantioconvergent with minimal kinetic resolution of (rac)-4.

[0079] F IG. 13 illustrates computed reaction energy profiles of the ACCONimi-catalyzed 1,3-nitrogen migration, according to some embodiments. Hybrid quantum mechanics / molecular mechanics (QM / MM) calculations were performed using the ONIOM method to compute the reaction energy profiles of the enantioselective 1,3-nitrogen migration of azanyl ester la catalyzed by the evolved nonheme enzyme ACCONiml.

[0080] FIG. 14A illustrates the QM / MM-optimized 1,5-HAT (TS-2) transition state, according to some embodiments. FIG. 14B illustrates the QM / MM-optimized 1,5-HAT (TS-2’) transition state, according to some embodiments. FIG. 14C illustrates C-N bond forming radical rebound (TS-3) transition state, according to some embodiments. FIG. 14D illustrates C-N bond forming radical rebound (TS-3’) transition state, according to some embodiments.

[0081] Results demonstrated that after binding of substrate la to the Fe center and N-H deprotonation, intermediate 13 undergoes facile N-0 bond cleavage via TS-1 with a low activation barrier of 4.7 kcal / mol relative to 13, generating a Fe(III)-bound nitrogen radical 14. 14 undergoes 1,5-HAT via TS-2 to form cx-carboxylate radical 15. Subsequent C-N bond forming radical rebound via TS-3 affords the 1,3-nitrogen migration product complex 16. The computed activation barriers for the 1,5-HAT (TS-2) and radical rebound (TS-3) steps are 17.5 and 17.1 kcal / mol relative to 14 and 15, respectively. In one example, the QM / MM-calculated reaction energy profile shows that the C-H cleavage via 1,5-HAT is irreversible and involved in the rate-determining step, which agrees with the primary KIE observed experimentally (kn / ko = 5.5).2025-367-24059.073PCT1

[0082] To investigate the origin of enantioselectivity, the enantio selectivity in both the HAT and radical rebound steps were computed, as well as the rate of conformational change between the two a-carboxylate radical conformers (15 and 15') generated from the competing 1,5-HAT pathways that cleave the enantiotopic C-HRand C-Hsbonds, respectively. The computed activation free energy difference (AAG*) between the competing 1,5-HAT transition states TS-2 and TS-2' is only 0.5 kcal / mol, suggested a low level of enantioselectivity during the C-H cleavage, which would form a mixture of 15 and 15'. Metadynamics simulations using the QM / MM method revealed that the conformational change from 15 to 15' requires a relatively low barrier of 6.4 kcal / mol, which is substantially faster than the C-N radical rebound. Therefore, under these Curtin-Hammett conditions, the enantioselectivity of the overall transformation is determined by the C-N bond forming radical rebound step.

[0083] The disfavored rebound transition state TS-3', which involves the C-N bond formation at the (Sz)-face of the a radical center is destabilized by steric repulsions between the Ph group of the a-carboxylate radical and the F250 residue and the Boc protecting group, whereas in the favored rebound transition state TS-3, these steric repulsions with the Ph group are diminished as the Ph points away from these bulky groups. The lower enantioselectivity between the 1,5-HAT transition states (TS-2 and TS-2') is attributed to their more flexible six-membered ring compared with the fivemembered C-N rebound transition state, which requires minimal energy penalty to distort the Ph group away from F250 and Boc groups in TS-2'. No significant steric interactions with the a-carboxylate radical were found in either TS-2 or TS-2', leading to nearly identical barriers for these competing transition states. In this nonheme Fe enzyme-catalyzed reaction, the prochiral a-carboxylate radical intermediate undergoes a rapid stereoablative conformational change. The high level of enantioselectivity is determined by the C-N bond formation radical rebound.Primer Sequences2025-367-24059.073PCT1Table 2. Primers used for the engineering of ACCO I184A K158T K172V L186V G156E R175P V236G G173R (ACCONimi).DNA Primer name Primer sequencetemplateACCO K158X_fwd_TGG (SEQ ID NO: 1)I184A K158X_fwd_NDT (SEQ ID NO: 2)K158X_fwd_VHG (SEQ ID NO: 3)K158X_rev (SEQ ID NO: 4)ACCO K172X_fwd_TGG (SEQ ID NO: 5)I184A K172X_fwd_NDT (SEQ ID NO: 6)K158T K172X_fwd_VHG (SEQ ID NO: 7)K172X_rev (SEQ ID NO: 8)ACCO L174X_fwd_TGG (SEQ ID NO: 9)I184A L174X_fwd_NDT (SEQ ID NO: 10) K158T L174X_fwd_VHG (SEQ ID NO: 11)L174X_rev (SEQ ID NO: 12)ACCO E87X_fwd_TGG (SEQ ID NO: 13)I184A E87X_fwd_NDT (SEQ ID NO: 14)K158T E87X_fwd_VHG (SEQ ID NO: 15)E87X_rev (SEQ ID NO: 16)ACCO F91X_fwd_TGG (SEQ ID NO: 17)I184A F91X_fwd_NDT (SEQ ID NO: 18)K158T F91X_fwd_VHG (SEQ ID NO: 19)F91X_rev (SEQ ID NO: 20)ACCO L186X_fwd_TGG (SEQ ID NO: 21)I184A L186X_fwd_NDT (SEQ ID NO: 22)K158T L186X_fwd_VHG (SEQ ID NO: 23)K172V L186X_rev (SEQ ID NO: 24)2025-367-24059.073PCT1ACCO K93X_fwd_TGG (SEQ ID NO: 25) I184A K93X_fwd_NDT (SEQ ID NO: 26) K158T K93X_fwd_VHG (SEQ ID NO: 27) K172V K93X_rev (SEQ ID NO: 28) ACCO N154X_fwd_TGG (SEQ ID NO: 29) I184A N154X_fwd_NDT (SEQ ID NO: 30) K158T N154X_fwd_VHG (SEQ ID NO: 31)K172V N154X_rev (SEQ ID NO: 32) L186VACCO G156X_fwd_TGG (SEQ ID NO: 33)I184A G156X_fwd_NDT (SEQ ID NO: 34) K158T G156X_fwd_VHG (SEQ ID NO: 35) K172V GI56X_rev (SEQ ID NO: 36) L186VACCO S160X_fwd_TGG (SEQ ID NO: 37) I184A S160X_fwd_NDT (SEQ ID NO: 38) K158T S160X_fwd_VHG (SEQ ID NO: 39) K172V S160X_rev (SEQ ID NO: 40) L186VACCO Y162X_fwd_TGG (SEQ ID NO: 41) I184A Y162X_fwd_NDT (SEQ ID NO: 42) K158T Y162X_fwd_VHG K172V Y162X_rev (SEQ ID NO: 44) L186VACCO R175X_fwd_TGG (SEQ ID NO: 45)I184A R175X_fwd_NDT (SEQ ID NO: 46) K158T R175X_fwd_VHG (SEQ ID NO: 47) K172V RI 75X_rev (SEQ ID NO: 48) L186V2025-367-24059.073PCT1G156EACCO G173X_fwd_TGG (SEQ ID NO: 49)I184A G173X_fwd_NDT (SEQ ID NO: 50)K158T G173X_fwd_VHG (SEQ ID NO: 51)K172V G173X_rev (SEQ ID NO: 52)L186VG156EACCO D169X_fwd_TGG (SEQ ID NO: 53) I184A D169X_fwd_NDT (SEQ ID NO: 54) K158T D169X_fwd_VHG (SEQ ID NO: 55) K172V D169X_rev (SEQ ID NO 56) L186VG156EACCO V236X_fwd_TGG (SEQ ID NO: 57)I184A V236X_fwd_NDT (SEQ ID NO: 58) K158T V236X_fwd_VHG (SEQ ID NO 59) K172V V236X_rev (SEQ ID NO 60) L186VG156ER175PACCO G173X_fwd_TGG (SEQ ID NO 61) I184A G173X_fwd_NDT (SEQ ID NO 62) K158T G173X_fwd_VHG (SEQ ID NO: 63) K172V G173X_rev (SEQ ID NO 52) L186VG156ER175PV236G2025-367-24059.073PCT1Table 3. Primers used for engineering of ACCO I184A K158T K172V L186V G156E R175P V236K G173V F250L (ACCONimi).DNA template primer name primer sequenceACCO I184A V236X_fwd_TGG (SEQ ID NO: 57)K158T K172V V236X_fwd_NDT (SEQ ID NO: 58)L186V G156E V236X_fwd_VHG (SEQ ID NO: 59)R175P V236X_rev (SEQ ID NO: 60)ACCO I184A G173X_fwd_TGG (SEQ ID NO: 61)K158T K172V G I 73X_fwd_NDT (SEQ ID NO: 62)L186V G156E ^GlTSjTfw^ R175P G173X_rev (SEQ ID NO: 52)ACCO I184A F250X_fwd_TGG (SEQ ID NO: 64)K158T K172V F250X_fwd_NDT (SEQ ID NO: 65)L186V G156E F250X_fwd_VHG (SEQ ID NO: 66)R175P V236K F250X_rev (SEQ ID NO: 67)G173VTable 4. Primers used for engineering of ACCO I184A K158T K172V L186V R175P V236K G173V F250L A248T S246F A180F G156T (ACCONims).DNA template primer name primer sequenceACCO I184A N252X_fwd_TGG (SEQ ID NO: 68)K158T K172V N252x fwd_NDT (SEQ ID NO: 69)L186V G156E N252X_fwd_VHG (SEQ ID NO: 70)R175P V236K N252X_rev (SEQ ID NO: 67)G173V F250LACCO I184A A248X_fwd_TGG (SEQ ID NO: 71)K158T K172V A248X_fwd_NDT (SEQ ID NO: 72)L186V G156E A248X_fwd_VHG (SEQ ID NO: 73)R175P V236K A248X_rev (SEQ ID NO: 74)2025-367-24059.073PCT1G173V F250LACCO I184A S246X_fwd_TGG (SEQ ID NO: 75)K158T K172V S246X_fwd_NDT (SEQ ID NO: 76)L186V G156E S246X_fwd_VHG (SEQ ID NO: 77)R175P V236K S246X_rev (SEQ ID NO: 78)G173V F250LA248TACCO I184A A180X_fwd_TGG (SEQ ID NO 79)K158T K172V A180X_fwd_NDT (SEQ ID NO 80)L186V G156E A180X_fwd_VHG (SEQ ID NO 81)R175P V236K A180X_rev (SEQ ID NO 82)G173V F250LA248T S246FACCO I184A G156X_fwd_TGG (SEQ ID NO: 83)K158T K172V G156X_fwd_NDT (SEQ ID NO: 84) L186V G156E G156X_fwd_VHG (SEQ ID NO: 85)R175P V236K G156X_rev (SEQ ID NO: 86) G173V F250LA248T S246FA180FDNA and Protein Sequences

[0084] DNA and amino acids sequence of WT ACCO

[0085] DNA sequence: (SEQ ID NO: 87)

[0086] Protein sequence:(SEQ ID NO: 88)

[0087] DNA and protein sequence of ACCO I184A:

[0088] DNA sequence: (SEQ ID NO: 89)

[0089] Protein sequence: (SEQ ID NO: 90)

[0090] DNA and protein sequence of ACCO I184A K158T:2025-367-24059.073PCT1

[0091] DNA sequence:(SEQ ID NO: 91)

[0092] Protein sequence: (SEQ ID NO: 92)

[0093] DNA and protein sequence of ACCO I184A K158T K172V:

[0094] DNA sequence:(SEQ ID NO: 93)

[0095] Protein sequence: (SEQ ID NO: 94)

[0096] DNA and protein sequence of ACCO I184A K158T K172V L186V:

[0097] DNA sequence: (SEQ ID NO: 95)

[0098] Protein sequence:(SEQ ID NO: 96)

[0099] DNA and protein sequence of ACCO I184A K158T K172V L186V G156E:

[0100] DNA sequence:(SEQ ID NO: 97)

[0101] Protein sequence:(SEQ ID NO: 98)

[0102] DNA and protein sequence of ACCO I184A K158T K172V L186V G156E R175P:

[0103] DNA sequence: (SEQ ID NO: 99)

[0104] Protein sequence: (SEQ ID NO: 100)

[0105] DNA and protein sequence of ACCO I184A K158T K172V L186V G156E R175P V236G:

[0106] DNA sequence: (SEQ ID NO: 101)

[0107] Protein sequence: (SEQ ID NO: 102)

[0108] DNA and protein sequence of ACCO I184A K158T K172V L186V G156E R175P V236G G173R (ACCONim1):

[0109] DNA sequence: (SEQ ID NO: 103)

[0110] Protein sequence: (SEQ ID NO: 104)

[0111] DNA and protein sequence of ACCO I184A K158T K172V L186V G156E R175P V236K:

[0112] DNA sequence:(SEQ ID NO: 105)

[0113] Protein sequence: (SEQ ID NO: 106)2025-367-24059.073PCT1

[0114] DNA and protein sequence of ACCO I184A K158T K172V L186V G156E R175P V236K G173V:

[0115] DNA sequence:(SEQ ID NO: 107)

[0116] Protein sequence: (SEQ ID NO: 108)

[0117] DNA and protein sequence of ACCO I184A K158T K172V L186V G156E R175P V236K G173V F250L (ACCONim2):

[0118] DNA sequence: (SEQ ID NO: 109)

[0119] Protein sequence:(SEQ ID NO: 110)

[0120] DNA and protein sequence of ACCO I184A K158T K172V L186V G156E R175P V236K G173V F250L A248T:

[0121] DNA sequence:(SEQ ID NO: 111)

[0122] Protein sequence:(SEQ ID NO: 112)

[0123] DNA and protein sequence of ACCO I184A K158T K172V L186V G156E R175P V236K G173V F250L A248T S246F:

[0124] DNA sequence: (SEQ ID NO: 113)

[0125] Protein sequence:(SEQ ID NO: 114)

[0126] DNA and protein sequence of ACCO I184A K158T K172V L186V G156E R175P V236K G173V F250L A248T S246F A180F:

[0127] DNA sequence:(SEQ ID NO: 115)

[0128] Protein sequence:(SEQ ID NO: 116)

[0129] DNA and protein sequence of ACCO I184A K158T K172V L186V R175P V236K G173V F250L A248T S246F A180F G156T (ACCONim3):

[0130] DNA sequence: (SEQ ID NO: 117)

[0131] Protein sequence: (SEQ ID NO: 118)Discussion of Possible Embodiments

[0132] Clause 1. A method for forming a non-canonical amino acid, including: catalyzing a nitrogen migration reaction to form a non-canonical amino acid using an azanyl moiety-containing substrate and a metalloenzyme catalyst.2025-367-24059.073PCT1

[0133] The clause of the preceding paragraph can optionally include, additionally and / or alternatively any one or more of the following features, configurations and / or additional components.

[0134] Clause 2. The method of clause 1, wherein the azanyl moiety-containing substrate includes an azanyl ester.

[0135] Clause 3. The method of clause 1, wherein the azanyl moiety-containing substrate includes an N-alkoxycarbonyl group.

[0136] Clause 4. The method of any one of clauses 1-3, wherein the metalloenzyme catalyst is a nonheme Fe enzyme.

[0137] Clause 5. The method of clause 4, wherein the nonheme Fe enzyme is an enzyme variant derived from 1 -aminocyclopropane- 1 -carboxylic acid oxidase.

[0138] Clause 6. The method of clause 5, wherein the enzyme variant includes a 1184 A mutation.

[0139] Clause 7. The method of clause 5, wherein the enzyme variant includes at least one of a K158T mutation, a K172V mutation, and a L186V mutation.

[0140] Clause 8. The method of clause 5, wherein the enzyme variant includes at least one of a V236K mutation, a G173V mutation, and an F250L mutation.

[0141] Clause 9. The method of any one of clauses 1-3, wherein the non-canonical amino acid includes an a-tetrasubstituted amino acid.

[0142] Clause 10. The method of clause 1, including utilizing a reductant.

[0143] Clause 11. The method of clause 10, wherein the reductant includes at least one of sodium ascorbate, L-ascorbic acid, and sodium dithionite.

[0144] Clause 12. The method of clause 1, including utilizing an iron source.

[0145] Clause 13. The method of clause 12, wherein the iron source includes a Fe2+source.

[0146] Clause 14. The method of clause 1, including catalyzing the nitrogen migration reaction at a pH value ranging from 7 to 8.

[0147] Clause 15. A method for forming a non-canonical amino acid, including: catalyzing a nitrogen migration reaction to form a non-canonical amino acid using a2025-367-24059.073PCT1nitrogen-containing substrate and a metalloenzyme catalyst derived from 1-aminocyclopropane-1 -carboxylic acid oxidase.

[0148] The clause of the preceding paragraph can optionally include, additionally and / or alternatively any one or more of the following features, configurations and / or additional components.

[0149] Clause 16. The method of clause 15, wherein the metalloenzyme catalyst includes an I184A mutation.

[0150] Clause 17. The method of clause 15, wherein the metalloenzyme catalyst includes a mutation at one or more of residue positions 172, 184, and 186.

[0151] Clause 18. The method of clause 15, wherein the metalloenzyme catalyst includes an I184A mutation and at least one of a K172V mutation and a LI 86V mutation.

[0152] Clause 19. The method of any one of clauses 15-18, wherein the non-canonical amino acid includes an a-tetrasubstituted amino acid.

[0153] Clause 20. A catalyst, including: a metalloenzyme catalyst derived from 1-aminocyclopropane- 1 -carboxylic acid oxidase, wherein the metalloenzyme catalyst includes at least one mutation selected from: a I184A mutation, a K.158T mutation, a K172V mutation, a L186V mutation, a G156E mutation, a G156T mutation, a R175P mutation, a V236G mutation, a V236K mutation, a G173R mutation, a G173V mutation, a F250L mutation, a A248T mutation, a S246F mutation, and a A180F mutation.

[0154] The clause of the preceding paragraph can optionally include, additionally and / or alternatively any one or more of the following features, configurations and / or additional components.

[0155] Clause 21. The catalyst of clause 20, wherein the metalloenzyme catalyst includes the I184A mutation.

[0156] Clause 22. The catalyst of clause 21, wherein the metalloenzyme catalyst includes at least one of the K158T mutation, the K172V mutation, and the L186V mutation.2025-367-24059.073PCT1

[0157] Clause 23. The catalyst of clause 20, wherein the metalloenzyme catalyst includes at least one of the K158T mutation, the K172V mutation, and the L186V mutation.

[0158] Clause 24. The catalyst of clause 20, wherein the metalloenzyme catalyst includes the I184A mutation and the K158T mutation.

[0159] Clause 25. The catalyst of clause 20, wherein the metalloenzyme catalyst includes the I184A mutation, the K158T mutation, the K172V mutation, and the L186V mutation.

[0160] While the invention 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 invention. In addition, many modifications may be made to adapt a particular situation or material to the teachings of the invention without departing from the essential scope thereof. Therefore, it is intended that the invention not be limited to the particular embodiment(s) disclosed, but that the invention will include all embodiments falling within the scope of the appended claims.

Claims

1. 2025-367-24059.073PCT1CLAIMS:

1. A method for forming a non-canonical amino acid, comprising:catalyzing a nitrogen migration reaction to form a non-canonical amino acid using an azanyl moiety-containing substrate and a metalloenzyme catalyst.

2. The method of claim 1, wherein the azanyl moiety-containing substrate includes an azanyl ester.

3. The method of claim 1, wherein the azanyl moiety-containing substrate includes an N-alkoxycarbonyl group.

4. The method of any one of claims 1-3, wherein the metalloenzyme catalyst is a nonheme Fe enzyme.

5. The method of claim 4, wherein the nonheme Fe enzyme is an enzyme variant derived from 1 -aminocyclopropane- 1 -carboxylic acid oxidase.

6. The method of claim 5, wherein the enzyme variant includes a I184A mutation.

7. The method of claim 5, wherein the enzyme variant includes at least one of a K158T mutation, a K172V mutation, and a L186V mutation.

8. The method of claim 5, wherein the enzyme variant includes at least one of a V236K mutation, a G173V mutation, and an F250L mutation.

9. The method of any one of claims 1-3, wherein the non-canonical amino acid includes an a-tetrasubstituted amino acid.

10. The method of claim 1, including utilizing a reductant.2025-367-24059.073PCT111. The method of claim 10, wherein the reductant includes at least one of sodium ascorbate, L-ascorbic acid, and sodium dithionite.

12. The method of claim 1, including utilizing an iron source.

13. The method of claim 12, wherein the iron source includes a Fe2+source.

14. The method of claim 1, including catalyzing the nitrogen migration reaction at a pH value ranging from 7 to 8.

15. A method for forming a non-canonical amino acid, comprising:catalyzing a nitrogen migration reaction to form a non-canonical amino acid using a nitrogen-containing substrate and a metalloenzyme catalyst derived from 1 -aminocyclopropane- 1 -carboxylic acid oxidase.

16. The method of claim 15, wherein the metalloenzyme catalyst includes an 1184A mutation.

17. The method of claim 15, wherein the metalloenzyme catalyst includes a mutation at one or more of residue positions 172, 184, and 186.

18. The method of claim 15, wherein the metalloenzyme catalyst includes an I184A mutation and at least one of a K172V mutation and a LI 86V mutation.

19. The method of any one of claims 15-18, wherein the non-canonical amino acid includes an a-tetrasubstituted amino acid.

20. A catalyst, comprising:2025-367-24059.073PCT1a metalloenzyme catalyst derived from 1 -aminocyclopropane- 1 -carboxylic acid oxidase, wherein the metalloenzyme catalyst includes at least one mutation selected from: a I184A mutation, a K158T mutation, a K172V mutation, a L186V mutation, a G156E mutation, a G156T mutation, a R175P mutation, a V236G mutation, a V236K mutation, a G173R mutation, a G173V mutation, a F250L mutation, a A248T mutation, a S246F mutation, and a A180F mutation.

21. The catalyst of claim 20, wherein the metalloenzyme catalyst includes the I184A mutation.

22. The catalyst of claim 21, wherein the metalloenzyme catalyst includes at least one of the K158T mutation, the K172V mutation, and the L186V mutation.

23. The catalyst of claim 20, wherein the metalloenzyme catalyst includes at least one of the K158T mutation, the K172V mutation, and the L186V mutation.

24. The catalyst of claim 20, wherein the metalloenzyme catalyst includes the I184A mutation and the K158T mutation.

25. The catalyst of claim 20, wherein the metalloenzyme catalyst includes the I184A mutation, the K158T mutation, the K172V mutation, and the L186V mutation.

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