Compositions, systems, and methods for stereoselective decarboxylative radical cyclization

Abstract

A method for radical cyclization includes converting a substrate to a product compound by catalyzing the formation of a C-C bond using an enzyme, wherein the enzyme includes a mutated variant of a fatty acid photodecarboxylase (FAP). Radical photocyclases (RAP) for catalyzing stereocontrolled cyclization are included in the present disclosure.

Description

COMPOSITIONS, SYSTEMS, AND METHODS FOR STEREOSELECTIVE DECARBOXYLATIVE RADICAL CYCLIZATION CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of and priority to U.S. Provisional Application 63 / 542,560, titled “Compositions, systems, and methods for stereoselective decarboxylative radical cyclization”, filed October 5, 2023, 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 September 25, 2024, is named 4059_062PCT1_SL.xml and is 215,322 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 radical C-C bond formation. 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 and nitrene transfer reactions. Due to the challenge of imposing stereocontrol over free radical intermediates, achieving catalytic asymmetric radical transformations has long remained a daunting task facing synthetic organic chemists. To further advance the field of biocatalysis, it would be beneficial to developenzymes for controlling the stereochemistry of free radical-mediated reactions. Further, it would be beneficial to develop enzymes for catalyzing radical C-C bond formation. SUMMARY

[0006] According to one aspect, a method for radical cyclization includes: converting a substrate to a product compound by catalyzing the formation of a C-C bond using an enzyme, wherein the enzyme includes a mutated variant of a fatty acid photodecarboxylase (FAP).

[0007] According to another aspect, a radical photocyclase (RAP) for catalyzing stereocontrolled cyclization includes: a mutated variant of a fatty acid photodecarboxylase (FAP) including at least one mutation selected from Y466A, Y427A, Y428A, G462A, and Y431A.

[0008] According to another aspect, a method for converting fatty acid photodecarboxylases (FAP) to radical photocyclases includes: incorporating a tyrosine-to-alanine mutation to a fatty acid photodecarboxylase (FAP) sufficient to form a radical photocyclase.

[0009] According to another aspect, a mutated enzyme variant includes: a mutated variant of a fatty acid photodecarboxylase (FAP) including a Y466A and G462A mutation. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] FIG.1 illustrates a method for radical cyclization, according to some embodiments.

[0011] FIG.2 illustrates a method for converting fatty acid photodecarboxylases (FAP) to radical photocyclases, according to some embodiments.

[0012] FIG.3A illustrates a catalytic cycle of a radical photocyclase (RAP), according to some embodiments.

[0013] FIG.3B illustrates enzyme controlled enantioconvergent radical cyclization, according to some embodiments.

[0014] FIG.3C illustrates enzyme-controlled diastereo- and enantioconvergent radical cyclization, according to some embodiments.

[0015] FIG.4A illustrates phylogenetic analysis of putative fatty acid decarboxylases (FAPs), according to some embodiments.

[0016] FIG.4B illustrates CvFAP variants as chemo- and enantioselective radical photocyclases, according to some embodiments.

[0017] FIG.5 illustrates an example reaction of RAP-catalyzed asymmetric decarboxylative radical cyclization, according to some embodiments.

[0018] FIG.6 illustrates orthogonal photoenzyme variants CvRAP3-6 for stereodivergent asymmetric radical cyclization of 1p with CvRAP3-6, according to some embodiments.

[0019] FIG.7 illustrates enantiodivergent radical cyclization of 1a with CvRAP1 and CvRAP6, according to some embodiments.

[0020] FIG.8A illustrates representative conformations of alkyl radical intermediate 4a, according to some embodiments.

[0021] FIG.8B illustrates distribution of C3–C7 distance of 4a in the enzyme active sites from classical MD simulations, according to some embodiments.

[0022] FIG.8C illustrates potential of mean force (PMF) of 4a in the enzyme active sites from GaMD simulations, according to some embodiments.

[0023] FIG.8D illustrates an overlay of representative conformations of 4a in wt CvFAP, according to some embodiments.

[0024] FIG.8E illustrates an overlay of representative conformations of 4a in CvRAP1, according to some embodiments. DETAILED DESCRIPTION

[0025] Embodiments of the present disclosure provide novel enzymes and radical mechanisms for C-C bond formation in organic compounds. The enzymes of the present disclosure include mutated enzymes for enhanced selectivity and efficiency in these processes. These enzymes can be used for controlling the stereochemistry of free radical-mediated reactions, such as for catalyzing radical C-C bond formation. Accordingly, enzymes of the present disclosure include radical photocyclases. Products of radical C-C bond formation can include esters, amides, and / or ketones, such as α,B-unsaturated esters, α,B-unsaturated amides, and / or α,B-unsaturated ketones.

[0026] Enzymes can be used to promote radical cyclization (e.g., ring formation), where these enzymes can act as photoenzymes. Accordingly, these enzymes can be radical photocyclases (RAP) for catalyzing stereocontrolled cyclization. Enzymes of the present disclosure generally include a fatty acid photodecarboxylases (FAP), or mutated variants thereof. In one example, fatty acid photodecarboxylases of the present disclosure can be derived from analgae species. Examples of fatty acid photodecarboxylases include CvFAP, CrFAP, CsuFAP, GpFAP, and RsFAP. CvFAP, CrFAP, CsuFAP, GpFAP, and RsFAP can be from sources Chlorella variabilis, Chlamydomonas reinhardtii, Coccomyxa subellipsoidea, Gonium pectorale, and Raphidocelis subcapitata, respectively.

[0027] The enzyme can include a mutated variant of a fatty acid photodecarboxylase, such as fatty acid photodecarboxylases of the present disclosure. The enzyme can include high-energy intermediates, since these enzymes can be flavin adenine dinucleotide (FAD)-dependent photoenzymes. In one example, the mutated variant includes a G462A mutation. For example, the G462A mutation can enhance cyclization chemoselectivity. In another example, the mutated variant includes a Y466A mutation. For example, the introduction of the Y466A mutation can improve total activity and chemoselectivity. For example, the tyrosine residue at 466 (Y466) can play an important role in shaping the substrate tunnel, and replacing this tyrosine with an alanine can broaden the active site of the fatty acid photodecarboxylase (FAP), promoting the facilitation of radical cyclization. In yet another example, the mutated variant includes a S464A mutation. For example, the S464A mutation can increase the efficiency of the mutated variant for C-C bond formation reactions. G462A, S464A and Y466A mutations reside in the α-helix proximal to the substrate. This α-helix can promote modulating the photoenzyme activity.

[0028] The mutated variant can include two or more of, or all, G462A, S464A and Y466A mutations. In one example, the mutated variant includes at least one of Y466A, G462A, S574G, S574A, S464A, Y431A, Y434F, G431W, I130F, T465S, A466F, S465M, T484S, T131A, A131V, T131S, V463A, and V463N. In one example, the mutated variant can include two or more of Y466A, G462A, S574G, S574A, S464A, Y431A, Y434F, G431W, I130F, T465S, A466F, S465M, T484S, T131A, A131V, T131S, V463A, and V463N. In another example, the mutated variant includes residues C432 and R451, without mutations at these C432 and R451 residues. Residues C432 and R451 can promote the production of product compounds of the present disclosure. In one example, compared to a fatty acid photodecarboxylases without any mutation that are unable to facilitate radical cyclization, the mutated variants of the present disclosure promote desired yields and enantiopreference of product compounds during radical cyclization.

[0029] In one example, the mutated variant includes CvFAP Y466A G462A S574G S464A T131A (referred to herein as CvRAP1). In another example, the mutated variant includes CvFAP Y466A G462A S574A S464A T131A (referred to herein as CvRAP2). In another example, themutated variant includes CvRAP2G431W I130F A131V (referred to herein as CvRAP3). In another example, the mutated variant includes CvFAP Y466A G462A S574A S464A T465S T131S (referred to herein as CvRAP4). In another example, the mutated variant includes CvRAP4 A466F S465M V463A (referred to herein as CvRAP5). In another example, the mutated variant includes CvFAP Y466A G462A S574A S464A I130F T484S V463N (referred to herein as CvRAP6).

[0030] FIG.1 illustrates method 100 for radical cyclization, according to some embodiments. Method 100 includes at least Step 110.

[0031] Referring to Step 110, a substrate is converted to a product compound by catalyzing the formation of a C-C bond using an enzyme. For example, the enzyme can include an enzyme of the present disclosure, such as a mutated variant of a fatty acid photodecarboxylase (e.g., FAD-dependent FAPs). In one example, the enzyme can include one or more of cell-free lysates, whole cell, and purified enzymes. In one non-limiting example, performing the reaction without the enzyme would lead to no product formation. Further, in another non-limiting example, performing the reaction without the enzyme and with a free coenzyme FAD does not promote this radical cyclization. Supplementing the enzyme of the present disclosure with free coenzyme FAD can provide desirable product compounds. The substrate generally includes an organic compound. The substrate can include a carboxylic acid. In one example, the carboxylic acid substrate is an α-β-unsaturated ester carboxylic acid.

[0032] In one example, the substrate is selected from (E)-8-ethoxy-7-methyl-8-oxooct-6- enoic acid; (Z)-8-ethoxy-7-methyl-8-oxooct-6-enoic acid; (E)-8-methoxy-7-methyl-8-oxooct-6- enoic acid; (E)-7-methyl-8-oxo-8-propoxyoct-6-enoic acid; (E)-8-(2-bromoethoxy)-7-methyl-8- oxooct-6-enoic acid; (E)-6-(2-oxodihydrofuran-3(2H)-ylidene)hexanoic acid; (E)-8-ethoxy-7- fluoro-8-oxooct-6-enoic acid; (Z)-7-chloro-8-ethoxy-8-oxooct-6-enoic acid; (Z)-7-bromo-8- ethoxy-8-oxooct-6-enoic acid; (E)-7-(ethoxycarbonyl)non-6-enoic acid; (E)-7-methyl-8- (methylamino)-8-oxooct-6-enoic acid; (E)-7-methyl-8-oxodec-6-enoic acid; (E)-9-ethoxy-8- methyl-9-oxonon-7-enoic acid; (E)-9-ethoxy-4,4-difluoro-8-methyl-9-oxonon-7-enoic acid; (E)- 9-ethoxy-4,4,8-trimethyl-9-oxonon-7-enoic acid; (E)-3-((5-ethoxy-4-methyl-5-oxopent-3-en-1- yl)oxy)propanoic acid; (E)-2-((5-ethoxy-4-methyl-5-oxopent-3-en-1-yl)oxy)acetic acid; and (Z)- 8-ethoxy-6,7-dimethyl-8-oxooct-6-enoic acid.

[0033] Any two or more of the components used in method 100 can be mixed in various orders. Method 100 can be performed at various temperatures. One example, method 100 is performed at about 20–40 ℃. 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, D2O solvent, and Dimethylsulfoxide (DMSO, e.g., degassed DMSO). In another example, the solvent includes degassed DMSO. 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 50 mM to about 200 mM. In another example, the concentration of substrate in the solution is about 100 mM.

[0034] Method 100 may include utilizing one or more buffers. Examples of buffers include 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 9. In another example, method 100 includes utilizing a buffer having a pH value ranging from about 7 to about 9. In yet another example, method 100 includes utilizing a buffer having a pH value ranging from about 8 to about 9. For example, degassed Tris-HCl buffer can be utilized having a pH value of about 8.5. In one non-limiting example, utilizing a buffer having a pH value of greater than about 7 increases the yield of desirable product compound(s). An example concentration range for the buffer compound ranges from about 20 mM to about 70 mM.

[0035] The one or more solvents, one or more buffers, the substrate, and the enzyme (such as a purified enzyme) 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 an anaerobic chamber. Anaerobic conditions can increase the yield of desirable product compound(s). Example concentrations of components are as follows: 2-20 mM substrate, 2-20% (v / v) solvent, and 0.1–5.0 mol% enzyme. Example concentrations of components are as follows: about 10 mM substrate, about 10% (v / v) solvent, and about 0.1–2 mol% enzyme.

[0036] Method 100 generally includes providing light during at least a portion of the reaction process. This light can be provided to the enzyme after a reaction mixture has been formed. For example, method 100 can include providing actinic radiation to one more components used for, or produced from, the reaction. Actinic radiation can be sufficient for the enzyme (or at least of the enzyme) to form an excited state. Accordingly, actinic radiation can exhibit sufficient energy to induce the reaction using the enzyme, where actinic radiation can include visible light.

[0037] In one example, visible light irradiation is provided to produce desired product compounds. In one example, visible light having a wavelength ranging from about 400 nm to about 600 nm is provided. In another example, visible light having a wavelength ranging from about 400 nm to about 500 nm is provided. In another example, visible light having a wavelength of 440 nm is utilized. In one non-limiting example, providing visible light having a wavelength greater than 430 nm and less than 500 nm (such as 440 nm) improves product compound yields. Exposing the enzyme to sufficient visible light can be sufficient to form an excited-stated flavoquinone cofactor. In one non-limiting example, light is provided to the enzyme under aerobic conditions.

[0038] To quench the reaction, one or more liquids can be added. For example, the reaction can be quenched using an acid, such as hydrochloric acid. Additionally, or alternatively, one or more solvents can be added, such as mesitylene in ethyl acetate. Shaking, mixing, and / or centrifuging can be used to separate organic and aqueous layers. Organic solvents can be allowed to evaporate, and a dissolving agent, such as hexanes, can be used to dissolve the product compound. If the product compound is an amide product, an organic nitrile, such as acetonitrile can be added to the formed reaction product mixture.

[0039] The product compound generally includes an organic compound. In one example, the product compound is selected from esters, amides, and ketones. In another example, the product compound is selected from an α,B-unsaturated ester, α,B-unsaturated amide, and α,B- unsaturated ketone.

[0040] In one example, the product compound is selected from Ethyl (S)-2- cyclopentylpropanoate (also referred to herein as “(2a)”); Ethyl (E)-2-methylhept-2-enoate (3a); Methyl (S)-2-cyclopentylpropanoate (2b); Propyl (S)-2-cyclopentylpropanoate (2c); 2- bromoethyl (S)-2-cyclopentylpropanoate (2d); (S)-3-cyclopentyldihydrofuran-2(3H)-one (2e);Ethyl (S)-2-cyclopentyl-2-fluoroacetate (2f); Ethyl (S)-2-chloro-2-cyclopentylacetate (2g); Ethyl (S)-2-bromo-2-cyclopentylacetate (2h); Ethyl (S)-2-cyclopentylbutanoate (2i); (S)-2-cyclopentyl- N-methylpropanamide (2j); (S)-2-cyclopentylpentan-3-one (2k); Ethyl (S)-2- cyclohexylpropanoate (2l); Ethyl (S)-2-(4,4-difluorocyclohexyl)propanoate (2m); Ethyl (S)-2- (4,4-dimethylcyclohexyl)propanoate (2n); and Ethyl (S)-2-(tetrahydro-2H-pyran-4-yl)propanoate (2o).

[0041] In another example, the product compound is selected from Ethyl 2- (tetrahydrofuran-3-yl)propanoate, Ethyl (S)-2-((S)-tetrahydrofuran-3-yl)propanoate, Ethyl (R)-2- ((S)-tetrahydrofuran-3-yl)propanoate, Ethyl (S)-2-((R)-tetrahydrofuran-3-yl)propanoate, and Ethyl (R)-2-((R)-tetrahydrofuran-3-yl) propanoate.

[0042] Chemical structures for example product compounds are shown below:2024-843-2 4059.062PCT1

[0043] Additional chemical structures for example product compounds are shown below:and ethyl (R)-2-((S)-tetrahydrofuran-3-yl)propanoate and ethyl (S)-2-((S)-tetrahydrofuran-3-yl)propanoate (2p) (2p)and ethyl (S)-2-((R)-tetrahydrofuran-3-yl)propanoate (2p) ethyl (S)-2-((S)-tetrahydrofuran-3-yl)propanoate (2p)- - and ethyl (R)-2-((S)-tetrahydrofuran-

[0044] As discussed, method 100 can be used for radical cyclization. Light can be provided sufficient for photoexcitation of the enzyme. The enzyme can include high-energy intermediatessince these enzymes can be flavin adenine dinucleotide (FAD)-dependent photoenzymes. Starting the cycle, visible light irradiation can produce a photoexcited strongly oxidizing flavoquinone FADq*. Single electron transfer (SET) from the carboxylate substrate to FADq* followed by rapid decarboxylation can generates an alkyl radical and a flavosemiquinone FAD•−(FADsq). At this stage, this nucleophilic alkyl radical species can undergo addition to a pendent ^, ^-unsaturated carbonyl to furnish a new carbon-centered radical. Subsequent proton-coupled electron transfer (PCET) involving the flavosemiquinone FAD•−and conserved amino acid residue(s) can furnish the product compound in a stereocontrolled manner and regenerate the FAD cofactor, completing the cycle. For example, mutated variants of the present disclosure can promote the interception of the photoenzymatically formed carbon-centered radical with a pendant C=C double bond moiety to outcompete the PCET process, preventing or reducing undesired protodecarboxylation products.

[0045] Importantly, method 100 can utilize mutated enzyme variants for enhanced selectivity and efficiency in these processes. These enzymes can be used for controlling the stereochemistry of free radical-mediated reactions, such as for catalyzing radical C-C bond formation. By using mutated enzyme variants of the present disclosure, these enzymes exhibit improved activity, chemoselectivity, and enantioselectivity for the formation of product compounds. Products of radical C-C bond formation can include esters, amides, and / or ketones, such as α,B-unsaturated esters, α,B-unsaturated amides, and / or α,B-unsaturated ketones.

[0046] FIG.2 illustrates a method for converting fatty acid photodecarboxylases (FAP) to radical photocyclases, according to some embodiments. Mutated variants of the present disclosure can be formed by Method 200, where Method 200 includes at least Step 210.

[0047] Referring to Step 210, a tyrosine-to-alanine mutation is incorporated to a fatty acid photodecarboxylase (FAP) sufficient to form a radical photocyclase. The fatty acid photodecarboxylase can include residues C432 and R451, and these residues can promote the production of product compounds of the present disclosure. These residues can be unmodified and / or present without any mutations. The formed radical photocyclase can include mutated variants of the present disclosure.

[0048] For example, the tyrosine residue at 466 (Y466) can play an important role in shaping the substrate tunnel, and replacing this tyrosine with an alanine can broaden the active site of the fatty acid photodecarboxylase (FAP), promoting the facilitation of radical cyclization.Accordingly, method 200 can include generating the mutated variant with Y466A. For example, the introduction of the Y466A mutation into CvFAP can promote a three-fold improvement in total activity. Additional examples include Y427A, Y431A, and / or Y428A (such as CrFAP Y427A, CsuFAP Y431A and GpFAP Y428A). Embodiments of the present disclosure include methods for using these mutated variants to produce various product compounds of the present disclosure.

[0049] Mutated variants of the present disclosure can be used to catalyze C-C bond formation to form various product compounds. In contrast to flavophotoenzymology with ‘ene’ reductases that operates through a strongly reducing excited-state flavohydroquinone, the mutated variants from fatty acid photodecarboxylases capitalize on the strongly oxidizing power of the excited-state flavoquinone to facilitate radical biotransformations. Collectively, mutated enzyme variants of fatty acid photodecarboxylases address longstanding challenges in free radical chemistry and asymmetric catalysis. Example 1 – Radical Cyclization

[0050] FIG.3A illustrates a catalytic cycle of a radical photocyclase (RAP), according to some embodiments. FIG.3B illustrates enzyme controlled enantioconvergent radical cyclization, according to some embodiments. FIG.3C illustrates enzyme-controlled diastereo- and enantioconvergent radical cyclization, according to some embodiments. In these processes, carboxylic acids are used as the radical precursor, allowing overall redox-neutral radical cyclization reactions, thereby eliminating the need for stoichiometric reductants. At the commencement of the catalytic cycle, visible light irradiation produces a photoexcited strongly oxidizing flavoquinone FADq*. Single electron transfer (SET) from the carboxylate substrate to FADq* followed by rapid decarboxylation generates an alkyl radical and a flavosemiquinone FAD•−(FADsq). At this stage, this nucleophilic alkyl radical species undergoes addition to a pendent ^, ^-unsaturated carbonyl to furnish a new carbon-centered radical. Subsequent proton- coupled electron transfer (PCET) involving the flavosemiquinone FAD•−and conserved amino acid residue(s) furnishes the product compound in a stereocontrolled manner and regenerates the FAD cofactor, thus completing the catalytic cycle.

[0051] Conventionally, this biocatalytic cycle would have several challenges to overcome. First, the interception of the photoenzymatically formed carbon-centered radical with a pendant C=C double bond moiety should preferably outcompete the native PCET process leading toundesired protodecarboxylation products. Second, the development of stereoselective radical photocyclases should preferably exhibit both the C–C bond forming radical addition step and the PCET step with excellent stereocontrol. Such reaction engineering is a nontrivial task, largely due to the limited stability of natural FAPs posing significant challenges. Accordingly, a panel of fully enantio- and diastereodivergent radical C–C bond forming enzymes were developed.

[0052] E. coli cells were grown using Luria-Bertani medium (LB) or Terrific Broth (TB) with 0.05 mg / mL kanamycin (LBkanor TBkan). pET-28a(+) was used as the cloning and expression vector for enzymes. T5 exonuclease, Phusion DNA polymerase, and T4 DNA ligase were purchased. The gene fragment containing 6×His tag, TrxA tag, TEV protease cleavage site, and the codon-optimized truncated FAP was assembled and inserted into the vector pET-28a(+). E. coli BL21 (DE3) was subsequently transformed with this recombinant plasmid using the heat shock method. Plasmid containing the FAP gene was extracted using a plasmid prep kit. The PCR product was digested with DpnI, gel purified and ligated using mix prepared from 5×isothermal 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 T4 DNA ligase. The ligation mixture was used to transform electrocompetent E. coli BL21(DE3) cell.

[0053] Colonies from LBkanagar plate were picked using sterile toothpicks and cultured in 96-well deep-well plates containing LBkan (400 μL / well) at 37 ^C and 250 rpm shaking overnight. In 24-well plates, TBkan media (4 mL / well) was then inoculated with an aliquot of overnight culture (200 μL / well) and allowed to shake at 37 ^C and 230 rpm for 2.5 h. The plates were cooled on ice for 20 min and the cultures were then induced with 0.1 mM isopropyl β-D-1- thiogalactopyranoside (IPTG). Expression was conducted at 18 ºC and 200 rpm for 18 h.

[0054] E. coli cells in 24-well plates were harvested using a centrifuge (3,000 x g, 10 min, 4 ^C) and resuspended in Tris-HCl buffer (50 mM, pH 8.5, 1 mL / well) by pipetting up and down for several times using an electronic pipette. Cells were then disrupted by sonication using a sonicator equipped with a 24-tip horn (1 sec on, 2 sec off, 40% amplitude for 2 min. This sequence was repeated for a total of three cycles). To pellet the cell debris, the resulting lysate was then centrifuged at 3,000 x g and 4 ℃ for 20 min using a centrifuge.

[0055] One-dram vials (1 dram = 4 mL) charged with a magnetic stir bar (7 × 2 mm) were put into each well of a 24-well plate.450 µL of cell lysate supernatant was transferred to eachvial using an electronic pipette. The 24-well plate with 24 one-dram vials was then transferred into an anaerobic chamber. In the anaerobic chamber, 50 µL of the substrate stock solution (100 mM in DMSO) was added into each vial. Final reaction volume was 500 μL, and final concentrations were 10 mM substrate and 10% (v / v) DMSO. The vials were sealed, taken out of the chamber, and illuminated with an LED lamp (440 nm). The reactions were allowed to stir at room temperature with a fan (e.g., about 27–30 ℃). After 5–12 h, the vials were collected into 24-well plate.

[0056] After 5–12 h, the reaction mixtures were quenched with 50 μL of HCl (2 M). An extraction mixture containing 1.0 mM mesitylene (internal standard) in EtOAc (750 μL) was added using an electronic pipette. The vials were then tightly sealed, shaken vigorously for 60 times, and centrifuged (3,000 x g, 5 min) to separate (e.g., completely separate) the organic and aqueous layers. The organic layers (350 μL / vial) were transferred to 500 μL vial inserts and analyzed by chiral GC or GC-MS.

[0057] A single colony was used to inoculate LBkan(25 mL), and grown at 37 ℃ and 230 rpm shaking for 12−14 h.10 mL of the culture was used to inoculate TBkan (1 L) in a 4 L flask. The culture was shaken at 37 ^C and 230 rpm for 3 h to reach an OD600of approximately 1.0. After cooling on ice for 20 min, the culture was induced with 0.1 mM IPTG. Protein expression was performed at 18 ℃ and 200 rpm for 18 h. The cell culture was then centrifuged (5,000 x g, 15 min, 4 °C) using a superspeed centrifuge and the cell pellet was stored in a –80 ℃ freezer or used.

[0058] E. coli cell pellet was resuspended in NaPi buffer (50 mM NaPi buffer, pH 7.5, 300 mM NaCl, 20 mM imidazole) and lysed by sonication. To pellet the cell debris, the resulting lysate was centrifuged at 12,000 x g and 4 ℃ for 20 min using a superspeed centrifuge. The supernatant was filtered through a 0.45 ^m PES syringe filter and loaded onto a column (5 mL, in a protein purification system. Proteins were eluted with an increasing gradient of imidazole from 20 to 500 mM in NaPi buffer (50 mM NaPi buffer, pH 7.5, 300 mM NaCl) at a flow rate of 5.0 mL / min. Fractions containing the desired protein were pooled and subjected to three exchanges of NaPi buffer (50 mM, pH 8.0) using an ultracentrifugal filter (30 kDa molecular weight cut-off) to remove excess salt and imidazole. Concentrated proteins were aliquoted, flash- frozen in liquid nitrogen and stored at –80 °C with 20% glycerol as the cryoprotectant until further use.

[0059] FIG.4A illustrates phylogenetic analysis of putative fatty acid decarboxylases (FAPs), according to some embodiments. Putative FAPs from the glucose-methanol-choline (GMC) oxidoreductase superfamily possessing functionally important conserved cysteine and arginine residues corresponding to C432 and R451 of CvFAP were initially selected. The mined putative FAPs showed an identity ranging from 45% to 73% relative to CvFAP, thus constituting a structurally diverse initial screening for the development of unnatural decarboxylative photoenzymatic reactions. The phylogenetic tree of a subset of the selected FAP homologs is delineated in FIG.4A, and the substrate tunnel highlighting Y466 is shown.

[0060] In one example, CvFAP is sourced from Chlorella variabilis NC64A with GenBank ID A0A248QE08.1. In another example, CrFAP is sourced from Chlamydomonas reinhardtii with GenBank ID A8JHB7. In another example, CsuFAP is sourced from Coccomyxa subellipsoidea with GenBank ID XP_005642926.1. In another example, GpFAP is sourced from Gonium pectorale with GenBank ID KXZ47408.1. In another example, RsFAP is sourced from Raphidocelis subcapitata with GenBank ID GBF88787.1.

[0061] In one example, it is desirable to effectively suppress the undesired premature PCET termination of the carbon-centred radical formed from photoenzymatic decarboxylation with photoexcited FADq*. However, (wt) wild-type FAPs are naturally evolved to accommodate hydrophobic long-chain fatty acids in an extended linear conformation. In the crystal structure of wild-type (wt) CvFAP in complex with the fatty acid substrate, the conformationally flexible fatty acid substrate is forced to adopt a slightly kinked but largely extended binding mode in CvFAP’s hydrophobic tunnel. In this binding conformation, the carboxylate radical precursor and the ^, ^-unsaturated radical acceptor are distal to each other, disfavoring radical cyclization.

[0062] The tyrosine residue at 466 (Y466) was found to play an important role in shaping the substrate tunnel. Replacing this tyrosine with an alanine significantly broadens the active site of CvFAP, thereby facilitating radical cyclization. Accordingly, site-directed mutagenesis was performed to generate CvFAP Y466A as well as the analogous tyrosine-to-alanine mutants of the FAP homologs and tested their activities toward photobiocatalytic radical cyclization of 1a. Initial photoenzyme activity evaluation was performed using the cell-free lysates of FAP variants.

[0063] To prepare the substrate stock solution (100 mM in DMSO), the substrate was added to a 4 mL vial. One or more of the following general procedures can be used for synthesisof carboxylic acid substrates: TBS protection, Swern oxidation, Wittig reaction, Deprotection, PDC oxidation, Fisher esterification, Amidation, and Ester Hydrolysis. The vial was then transferred into an anaerobic chamber where degassed DMSO was added. Purified enzyme used for the reaction was kept on ice in the dark before it was transferred into the anaerobic chamber. Vials (4 mL) charged with a stir bar (7 × 2 mm) were transferred into the anaerobic chamber. In the anaerobic chamber, degassed Tris·HCl buffer (50 mM, pH = 8.5), a stock solution of the substrate (100 mM in DMSO, 50 μL), and the FAP variant were added into the vial in succession. Final reaction volume was 500 μL, and final concentrations were 10 mM substrate, 10% (v / v) DMSO, and 0.1–2.0 mol% FAP. The vials were tightly sealed, taken out of the anaerobic chamber, and illuminated with a LED lamp for 20 h. The reaction mixture was then analyzed by chiral GC and / or HPLC.

[0064] For ester and ketone products, the reaction mixtures were quenched with 50 μL of HCl (2 M). A solution of 1 mM mesitylene (internal standard) in EtOAc (750 μL) was added. The vials were tightly sealed with screw caps, shaken vigorously for 60 times and centrifuged (3,000 x g, 5 min) to separate the organic and aqueous layers. The organic layers (350 μL / vial) were transferred to 500 μL inserts, which were then placed in 2 mL vials and analyzed by chiral GC. To determine the enantioselectivity of the product (2m), the organic solvents were allowed to evaporate in a fume hood. Hexanes were added to dissolve the product and the samples were analyzed by chiral HPLC using the OJ-H column. For the amide product 2j, 100 μL MeCN was added to 100 μL of reaction mixture. The mixture was shaken vigorously and centrifuged at 12,000 x g for 5 min. The supernatant was filtered through a PTFE membrane filter (0.22 μm) into a 500 μL vial insert, which was then analyzed by reverse-phase HPLC.

[0065] Table 1 shows the results of various FAPs and mutated variants thereof. It was found that CvFAP was capable of promoting the radical cyclization with low levels of initial activity and modest enantioselectivity (0.5% yield and 42:58 enantiomeric ratio (e.r.) of 2a, (entry 1). Furthermore, the introduction of the Y466A mutation into CvFAP furnished a three- fold improvement in total activity (1.5% yield) and chemoselectivity (entry 6). CvFAP Y466A also displayed reversed enantiopreference (67:33 e.r. in favor of (S)-2a), allowing the opposite enantiomer to form upon radical cyclization. Table 1. Results of FAPs and mutated variants. FAP / variant Yield of 2a e.r. of 2a Yield of 3a2024-843-2 4059.062PCT1 1 CvFAP 100% 0.5% 42:58 3.3% 2 CrFAP 57% 0% - 0.2% -found to be an activating mechanism to convert FAPs into radical photocyclases (entries 6–10). For example, while wild-type Chlamydomonas reinhardtii FAP (CrFAP), Coccomyxa subellipsoidea FAP (CsuFAP), and Gonium pectorale FAP (GpFAP) were unable to facilitate the radical cyclization to provide 2a, CrFAP Y427A, CsuFAP Y431A and GpFAP Y428A furnished 2a in 0.1% yield, 39:61 e.r. (Table 1 - entry 7), 0.1% yield, 69:31 e.r. (Table 1 - entry 8), and 0.1% yield, 34:66 e.r. (Table 1 - entry 9), respectively. Among these single mutants, CrFAP Y427A and GpFAP Y428A displayed opposite enantiopreference compared to others (Table 1 - entry 9). The highest enantioselectivity was observed with (RsFAP) Y431A, which afforded 2a in 0.8% yield and 94:6 e.r., further showcasing the utility of mutated enzyme variants. Together, among these FAP analogs, the tyrosine-to-alanine mutation led to at least 3- fold enhancement in total activity, underscoring its activating effect.

[0067] Finally, control experiments using free FAD (1.0 mol%) in lieu of the FAP provided no cyclization product (Table 2). In one example, performing this biocatalytic reaction in the absence of light also afforded no product formation, confirming the photoenzymatic nature of this transformation (Table 2). In another example, visible light irradiation at 440 nm provided higher yields of 2a than other wavelengths (Table 3). Table 2. Radical Cyclization of 1a.1 standard conditions 91 96:4 5 2no h ^0 - 03 no CvRAP10 - 0 4 FAD (1 mol%) instead of CvRAP1 0 - 02024-843-2 4059.062PCT1 5 CvRAP1 (0.25 mol%) 52 96:4 3 6 CvRAP1(0.25 mol%) 90 96:4 33 456 nm 84 96:4 4 4 525 nm 83 96:4 4

[0068] As can be seen from Table 3, visible light irradiation at 440 nm provided desirable results for the present photobiotransformation. As can be seen from Table 4, this photobiocatalytic radical cyclization is more efficient under basic conditions. Reduced yields were observed when the reaction was performed under acidic conditions. Preferred pH of this process was determined to be 8.5. Table 5 showed that performing this photobiocatalytic radical cyclization under air led to reduced yields. As can be seen from Table 2, performing the reaction in the absence of light or photoenzyme led to no product formation, and free coenzyme FAD did not promote this radical cyclization. Running this biotransformation using 0.25 mol% evolved radical photocyclase CvRAP1 led to a reduced yield of 2a. Supplementing additional FAD could recover the yield of 2a, providing a total turnover number (TTN) of 360 under these conditions. Table 4. Effect of pH on photobiocatalytic radical cyclization of 1a.Table 5. Effect of air on photobiocatalytic radical cyclization of 1a.2024-843-2 4059.062PCT1

[0069] FIG.4B illustrates directed evolution of CvFAP as chemo- and enantioselective radical photocyclases, according to some embodiments. Conventionally, FAPs have limited stability precluding convenient processing in a high-throughput manner. Indeed, upon the completion of FAP expression, canonical high-throughput cell lysis protocol using lysozyme or detergents led to minimal enzyme activity, suggesting FAP denaturation under these cell lysis conditions. The use of intact E. coli cells harboring FAP also furnished reduced yields and enantioselectivities (Table 6). To circumvent these issues, a mild and high-throughput cell lysis protocol was optimized using a 24-tip horn sonicator. Additionally, 96 photoenzymatic reactions were performed in parallel at ambient temperature (e.g., about 20 °C). Together, the newly developed protocol allowed high-throughput experimentation to be carried out in a 96-well plate format, thereby permitting directed evolution of FAPs to be successfully performed. Table 6. Comparison of photobiocatalytic reactions with whole cells, cell-free lysates, and purified enzymes.2 wt CvFAP whole cells 0.2 49:51 1.6 11:89 3 wt CvFAP purified enzyme 0.4 46:54 1.7 19:81 4 CvFAP Y466A lysate 1.5 67:33 4.6 25:75 5 CvFAP Y466A whole cells 0.9 61:39 6.8 12:88 6 CvFAP Y466A purified enzyme 1.0 67:33 2.7 27:73

[0070] The enzyme concentration in cell-free lysates, whole cell, and purified enzyme was normalized to be about 3 μM. As shown in Table 6, compared to cell-free lysates, the use of intact E. coli cells led to a lower yield. Furthermore, in screening reactions, the enantioselectivity with whole E. coli cells was found to be much lower than that with the cell-free lysate. Toaddress this problem, a new workflow was developed to enable the directed evolution of FAPs using cell-free lysates.

[0071] With these advances, iterative rounds of site-saturation mutagenesis (SSM) and screening were performed to form new-to-nature radical photocyclases. Using CvFAP Y466A as the template, directed evolution allowed beneficial mutations including G462A, S574G / S574A, S464A, and T131A to be identified, leading to highly efficient radical photocyclases CvFAPAAGAAand CvFAPAAAAAto be developed (Table 7). In this disclosure, CvFAPAAGAAand CvFAPAAAAAare designated as CvRAP1 and CvRAP2, respectively (RAP = radical photocyclase). Table 7. Directed evolution of CvFAP Y466A G462A S574G S464A T131A (CvFAPAAGAA, CvRAP1) and CvFAP Y466A G462A S574A S464A T131A (CvFAPAAAAA, CvRAP2).CvFAP Y466A A171X, I398X, enantioselectivity G462A G462X, Q486X and activity CvFAP Y466A G462A H572X, S573X, enantioselectivity S574G S574X, N575X and activity S574A CvFAP Y466A G462A S574G V463X, S464X, enantioselectivity S464A CvFAP Y466A G462A S574A T465X, F469X CvFAP Y466A G462A S574G S429X, T430X, enantioselectivity T131A S464A G431X, T131X CvFAP Y466A G462A S574A S464A CvFAP Y466A G462A S574G - - S464A T131A (CvRAP1) CvFAP Y466A G462A S574A S464A T131A (CvRAP2)2024-843-2 4059.062PCT1

[0072] Formed CvRAP1afforded radical cyclization product 2a in 91% yield, 96:4 e.r. and 95:5 chemoselectivity. CvRAP2 provided 2a in 92% yield, 94:6 e.r. and 96:4 chemoselectivity. Among these beneficial mutations, G462A played an important role in enhancing the cyclization chemoselectivity (Table 8, 2a:3a = 98:2, entry 2). Thus, the newly engineered CvFAP Y466A G462A effectively supressed the undesired native protodecarboxylation reactivity. Beneficial mutations G462A, S464A and Y466A all reside in the 461–472 ^-helix proximal to the substrate. This ^-helix was found to play an important role in modulating the photoenzyme activity. A131 is located in the 129–134 ^-helix of the active site, while G / A574 resides in a flexible loop close to both the substrate and the FAD cofactor. From a mechanistic perspective, the successful formation of radical photocyclases demonstrated that the proton-coupled electron transfer (PCET) step in the catalytic cycle could be rendered highly enantioselective. Table 8. Chemo- and enantioselective radical photocyclases. entry FAP mutated variant Yield of 2a e.r. of 2a Yield (E / Z) of 3a 1 CvFAP Y466A 13% 67:33 15% (96:4) 2 CvFAP Y466A G462A 48% 87:13 1% (74:26)were as 9). To identify important active-site residues of RsFAP, multiple sequence alignment results of RsFAP, CvFAP and other FAP homologs were analyzed. The final variant RsRAP carried three beneficial mutations relative to RsFAP, including Y431A, Y434F, and Q429D, all of which resides in the ^-helix that aligns with the 461–472 ^-helix of CvFAP. In one example, RsRAP (RsFAP Y431A Y434F Q429D) catalyzed radical cyclization of 1a in 48% yield and 96:4 e.r. with lower chemoselectivity (2a:3a = 51:49) relative to that of CvRAP. Table 9. Directed evolution of RsRAP: photoenzyme activity and selectivity summary.RsFAP variant sites targeted by selection criteria beneficial SSM mutation RsFAP Y363X, Q429X enantioselectivity Y434F Y431A K433X, Y434X and activity RsFAP L93X, Q429X, enantioselectivity and Q429D Y431A Y434F S430X, Y421X activity RsFAP - - Y431A Y434F Q429D RsFAP variant e.r. of 2a yield of 2a (%) yield of 3a (%) RsFAP Y431A 94:6 6% 19% RsFAP Y431A 95:5 45% 47%decarboxylative radical cyclization, according to some embodiments. In addition to ethyl ester 2a, methyl (2b), n-propyl (2c), and 2-bromoethyl (2d) esters represented excellent substrates for the photobiocatalytic radical cyclization, leading to cyclized products with good to excellent levels of enantioselectivity. Lactone 2e could also be converted with good yield and enantiocontrol. Importantly, ^-halogen substituted ^ ^ ^-unsaturated esters were also excellent radical acceptors, giving rise to ^-fluoro (2f), ^-chloro (2g) and ^-bromoesters (2h) in a highly enantiomerically enriched form. Thus, these evolved radical photocyclases afford a new method to prepare enantioenriched ^-haloesters in a catalytic asymmetric manner.

[0075] Increasing the size of the ^-substituent to an ethyl (2i) did not lower the enzymatic enantiocontrol over the radical cyclization process. For example, in addition to ^ ^ ^-unsaturated esters, ^ ^ ^-unsaturated amide 2j could also be transformed into the cyclized product, albeit with lower conversion and enantioselectivity. In another example, ^ ^ ^-Unsaturated ketones (2k) underwent smooth photoenzymatic transformation, but the cyclization product formed with no or low levels of enantiocontrol. Furthermore, in addition to five-membered cyclization products (2a–2k), six-membered products could be readily prepared without additional photoenzyme engineering (2l–2o). Diverse substituents of the cyclohexyl moiety, including difluoromethyl (2m), gem-dimethyl (2n), and an oxygen (2o), were compatible with this 6-exo-trig photoenzymatic cyclization. Taken together, these results demonstrated the utility of the mutated2024-843-2 4059.062PCT1 variant in controlling the ^-stereochemistry of products through the PCET step.2a, 2b, 2e, and 2k were all formed with yields above 85% yields.

[0076] FIG.6 illustrates directed evolution of orthogonal photoenzyme variants CvRAP3-6 for stereodivergent asymmetric radical cyclization of 1p with CvRAP3-6, according to some embodiments. The engineered radical photocyclases were challenged to set two contiguous stereogenic centers at the ^ and ^ positions in a stereodivergent fashion (FIG.6). The highly stereoselective formation of stereochemical dyads can require the photocyclase to exert exquisite control over both the C–C bond forming radical cyclization step and the subsequent PCET step. 1p was selected as the substrate to provide product 2p with adjacent ^- and ^-stereocenters. Starting from CvRAP2 (CvFAPAAAAA), three additional rounds of site-saturation mutagenesis and screening led to CvRAP2G431W I130F A131V (CvRAP3) (Table 10) with further improved diastereo- and enantioselectivity towards the formation of (2S,3R)-2p (83% yield, 90:10 d.r., >99:1 e.r.). (Table 11) Table 10. Summary of directed evolution of CvRAP2 G431W I130F A131V (CvRAP3).V453X, G455X diastereoselectivity CvRAP2I130X, F134X, enantioselectivity I130F G431W A171X, M456X diastereoselectivity CvRAP2 A131X, A171X, enantioselectivity A131V G431W I130F V463X, T484X diastereoselectivity activity CvRAP2- - - G431W I130F A131V Table 11. Directed evolution of CvRAP3: photoenzyme activity and selectivity summary.2024-843-2 4059.062PCT1 CvRAP268:32 92:8 79:21 78 CvRAP2G431W 69:31 97:3 77:23 39CvFAPAAAA(CvFAP Y466A G462A S574A S464A) and the final variant CvFAPAAAAAdisplayed similar diastereo- and enantioselectivities, surpassing those with CvRAP1 (CvFAPAAGAA). It was found that by inverting the ^-stereochemistry, CvFAPAAAAT465S resulted in the reversal of diastereoselectivity along with a significant improvement in enantioselectivity for (2S,3S)-2p (72% yield, 33:67 d.r., 98:2 e.r.). Further protein engineering led to CvFAPAAAAT465S T131S (CvRAP4), giving rise to (2S,3S)-2p in 86% yield, 20:80 d.r. and >99:1 e.r. (Tables 12 and 13) Table 12. Summary of directed evolution of (CvRAP4).2024-843-2 4059.062PCT1

[0078] Moreover, both the ^- and the ^-stereochemistry could be reversed, and all the four possible stereoisomers of 2p could be accessed in an enzyme-controlled fashion. Specifically, starting from CvRAP4, CvRAP4 A466F S465M V463A (CvRAP5) was advanced, providing (2R,3S)-2p with inverted ^-stereochemistry in 55% yield, 59:41 d.r. and 90:10 e.r. (FIG.6, Tables 14 and 15). Table 14. Summary of directed evolution of CvRAP4 A466F S465M V463A (CvRAP5).L458X, A466X, diastereoselectivity CvRAP4 T172X, P460X, enantioselectivity S465M A466F S465X, R468X, diastereoselectivity CvRAP4 S131X, S429X, enantioselectivity A466F S465M T430X, V463X diastereoselectivity V463A activity CvRAP4- - - A466F S465M V463A Table 15. Directed evolution of CvRAP5: photoenzyme activity and selectivity summary.CvRAP4A466F 30:70 69:31 97:3 43 CvRAP4 A466F S465M 51:49 86:14 81:19 26 CvRAP4 A466F S465M 59:41 90:10 66:34 55 V463A (CvRAP5)

[0079] FIG.7 illustrates enantiodivergent radical cyclization of 1a with CvRAP1 and CvRAP6, according to some embodiments. CvRAP6allowed the conversion of 1a into 2a in 86%2024-843-2 4059.062PCT1 yield and 25:75 e.r. with inverted enantioselectivity (FIG.7, Tables 16 and 17). The fourth stereoisomer (2R,3R)-2p could also be synthesized with another evolved radical photocyclase CvFAPAAAAI130F T484S V463N (CvRAP6) in 89% yield, 78:22 d.r. and 96:4 e.r. The biocatalyst-controlled, fully diastereo- and enantiodivergent access of all four possible stereoisomers shows the exceptional tunability of natural FAP photoenzymes. Together, CvRAP1 and CvRAP6 constituted an orthogonal set of biocatalysts for the enantiodivergent synthesis of cyclization products with a single stereo centre. Table 16. Summary of directed evolution of (CvRAP6).L386X, I398X diastereoselectivity CvFAPAAAAA399X, W479X enantioselectivity T484S I130F T484X, Q486X diastereoselectivity activity CvFAPAAAAT131X, D402X enantioselectivity V463N I130F T484S V463X, Y466X, diastereoselectivity activity CvFAPAAAA- -summary.CvFAPAAAAI130F 59:41 91:9 83:17 75 CvFAPAAAAI130F T484S 32:68 76:24 91:9 81 CvFAPAAAAI130F T484S V463N 22:78 53:47 96:4 89 (CvRAP6) Table 18. Evaluation of CvRAP1-6 and RsRAP for radical cyclization of 1q: photoenzyme activity and selectivity summary.2024-843-2 4059.062PCT1CvRAP457 74:26 CvRAP551 97:3 CvRAP6 56 86:14 RsRAP 53 82:18

[0080] It was found that E- and Z-olefins were transformed with almost identical yields and enantioselectivities favoring the same major enantiomeric products, permitting radical acceptors as a E / Z mixture to be transformed in a stereoconvergent fashion. Example reactions are shown below:photobiotransformation, the photoactivity of (E)- and (Z)-1a was evaluated using free FAD under blue light (440 nm) irradiation. It was found that (E)-1a maintained its geometric integrity in the presence of free FAD and did not undergo radical cyclization. In contrast, (Z)-1a underwent (Z / E)-scrambling under these conditions, and the photochemical equilibration was found to largely favor (E)-1a. Starting from (Z)-1a, no radical cyclization product 2a was observed. Example reactions are shown below:ratio of recovered (E)-1a and (Z)-1a was measured at varying conversions. Similar to the findings with free FAD, when (E)-1a was applied, no erosion of olefin configuration was observed. With (Z)-1a, stereochemical scrambling of 1a was observed, and a notable amount of (E)-1a was present in recovered 1a. For example, taken together, these results show that under photochemical conditions, the engineered photoenzyme CvRAP1and the free FAD cofactor can catalyze the olefin isomerisation of (Z)-1a, but not (E)-1a. Furthermore, the initial rate of this photobiocatalytic decarboxylative radical cyclization was compared with (E)-1a and (Z)-1a. Although (Z)-1a was found to isomerize to (E)-1a under visible light irradiation as evidenced by a lower Z / E ratio of recovered 1a (Z / E = 72:28), the conversion of (Z)-1a into the cyclized product 2a was about twice as fast as that with (E)-1a, as revealed by the higher initial rate of the 1a → 2a conversion with (Z)-1a ((Z)-1a: 6.4 μmol·L-1· min-1; (E)-1a: 3.4 μmol·L-1· min-1). Together, these results showed that although (Z)- 1a underwent isomerization to (E)-1a, it could also be directedly converted into 2a by CvRAP1 at a faster rate. The same e.r. of 2a starting from (E)-1a and (Z)-1a indicated that the rotation along the C–C ^ bond within the active site of is faster relative to the enantiodetermining PCET step. Example reactions are shown below:chemoselectivity of CvRAP1favoring C–C bond forming radical cyclization, classical molecular dynamics (MD) simulations were conducted to explore the conformational space of alkyl radical 4a formed after the FAD- promoted decarboxylation of 1a. FIG.8A illustrates representative conformations of alkyl radical intermediate 4a, according to some embodiments.

[0084] FIG.8B illustrates distribution of C3–C7 distance of 4a in the enzyme active sites from classical MD simulations, according to some embodiments. The MD simulations reveal that in the active site of wt CvFAP, the alkyl radical intermediate 4a prefers an anti-anti conformation with a long distance between the radical center (C7) and the β carbon (C3) (d = 4.8–5.3 Å. By contrast, because of its expanded active-site volume compared to wt CvFAP, in the active site of CvRAP1, alkyl radical 4a is conformationally more flexible. The most populated gauche−-anti conformation has a shorter C3–C7 distance of ca.4.1–4.7 Å.

[0085] FIG.8C illustrates potential of mean force (PMF) of 4a in the enzyme active sites from GaMD simulations, according to some embodiments. The difference between conformational profiles of 4a in the active sites of wt CvFAP and CvRAP1is corroborated by Gaussian accelerated molecular dynamics (GaMD) enhanced sampling simulations, indicating a preference for the anti-anti conformation in wt CvFAP and the gauche−-anti conformation in CvRAP1. Both conventional MD and GaMD simulations indicate the near-attack conformations2024-843-2 4059.062PCT1 [d(C3–C7) ≤ 3.1 Å] are rarely populated in wt CvFAP (< 0.3 % population and > 5.5 kcal / mol less stable than the most stable conformer), but are present in relatively large populations in CvRAP1 (2.1 % and 1.5 kcal / mol).

[0086] FIG.8D illustrates an overlay of representative conformations of 4a in wt CvFAP, according to some embodiments. FIG.8E illustrates an overlay of representative conformations of 4a in CvRAP1, according to some embodiments. Representative snapshots from MD simulations indicate that in wt CvFAP, Y466 and Q486 residues block both π-faces of the C=C double bond of 4a. Therefore, the Y466A mutation increases the size of the binding pocket, allowing the radical intermediates to adopt the bent near-attack conformation, and removing the steric shield preventing radical cyclization in the wt CvFAP enzyme.

[0087] In conclusion, natural fatty acid photodecarboxylases (FAPs) were repurposed as unnatural radical photocyclases (RAPs) to catalyze stereocontrolled cyclization via an open-shell mechanism. The advancement of a high-throughput experimentation platform allowed for directed evolution of FAPs to be carried out, allowing a collection of highly chemo- and stereoselective RAPs to be developed. Notably, a set of diastereo- and enantiodivergent RAP variants were formed, permitting all the four possible stereoisomers of stereochemical dyads to be prepared. In contrast to flavophotoenzymology with ‘ene’ reductases that operates through a strongly reducing excited-state flavohydroquinone, the RAPs repurposed from FAPs capitalize on the strongly oxidizing power of the excited-state flavoquinone to facilitate radical biotransformations. Collectively, mutated variants of fatty acid photodecarboxylases address longstanding challenges in free radical chemistry and asymmetric catalysis. Example 2 – Primer Sequences Table 19. Primer sequences. template primer name SEQ ID NO:2024-843-2 4059.062PCT1 CvFAP Y466A Q486X_NDT_fwd 13 Q486X_VHG_fwd 14 4 XT f 12024-843-2 4059.062PCT1 S464A T131X_TGG_fwd 63 T131X_rev 64template primer name SEQ ID NO: RsFAP Y363X_NDT_fwd 65Table 21. Primer sequences. template primer name SEQ ID NO:2024-843-2 4059.062PCT1 G431X_rev 60 CvRAP2 V453X_NDT_fwd 93 V453XVHGfwd 94Table 22. Primer sequences. templa primer name SEQ ID NO:2024-843-2 4059.062PCT1 CvFA S464X_NDT_fwd 37 PAAAAS464X_VHG_fwd 38 44XT fTable 23. Primer sequences. template primer name SEQ ID NO: CvRAP N170XNDTfwd 1342024-843-2 4059.062PCT1 T172X_TGG_fwd 148 T172X_rev 149 CRAP P460XNDTfwd 150Table 24. Primer sequences. template primer name SEQ ID NO: CFAPAAA128XNDTfwd 1692024-843-2 4059.062PCT1 CvFAPAAA399X_NDT_fwd 177 AAA399X_VHG_fwd 178 I130F A399X TGG fwd 179Example 3 –DNA and amino acid sequences Table 25. DNA and Amino Acid Sequences. Description Sequence Type SEQ ID NO:amino acid 201 RsFAP (GBF88787.1) DNA 202Discussion of Possible Embodiments

[0088] The following are non-exclusive descriptions of possible embodiments of the present invention.

[0089] According to one aspect, a method for radical cyclization includes converting a substrate to a product compound by catalyzing the formation of a C-C bond using an enzyme, wherein the enzyme includes a mutated variant of a fatty acid photodecarboxylase (FAP).

[0090] The method of the preceding paragraph can optionally include, additionally and / or alternatively, any one or more of the following features / steps, configurations, and / or additional components.

[0091] The substrate can include a carboxylic acid.

[0092] The product compound can be selected from an α,B-unsaturated ester, α,B- unsaturated amide, and α,B-unsaturated ketone.

[0093] The product compound can be selected from Ethyl (S)-2-cyclopentylpropanoate, Methyl (S)-2-cyclopentylpropanoate, Propyl (S)-2-cyclopentylpropanoate, 2-bromoethyl (S)-2- cyclopentylpropanoate, (S)-3-cyclopentyldihydrofuran-2(3H)-one, Ethyl (S)-2-cyclopentyl-2- fluoroacetate, Ethyl (S)-2-chloro-2-cyclopentylacetate, Ethyl (S)-2-bromo-2-cyclopentylacetate, Ethyl (S)-2-cyclopentylbutanoate, (S)-2-cyclopentyl-N-methylpropanamide, (S)-2- cyclopentylpentan-3-one, Ethyl (S)-2-cyclohexylpropanoate, Ethyl (S)-2-(4,4- difluorocyclohexyl)propanoate, Ethyl (S)-2-(4,4-dimethylcyclohexyl)propanoate, and Ethyl (S)- 2-(tetrahydro-2H-pyran-4-yl)propanoate.

[0094] The product compound can be selected from Ethyl 2-(tetrahydrofuran-3- yl)propanoate, Ethyl (S)-2-((S)-tetrahydrofuran-3-yl)propanoate, Ethyl (R)-2-((S)- tetrahydrofuran-3-yl)propanoate, Ethyl (S)-2-((R)-tetrahydrofuran-3-yl)propanoate, and Ethyl (R)-2-((R)-tetrahydrofuran-3-yl) propanoate.

[0095] The mutated variant of the fatty acid photodecarboxylase (FAP) can include a G462A mutation.

[0096] The mutated variant of the fatty acid photodecarboxylase (FAP) can include a Y466A mutation.

[0097] The mutated variant of the fatty acid photodecarboxylase (FAP) can include at least one of Y431A, Y434F, and Q429D mutations.

[0098] The mutated variant of the fatty acid photodecarboxylase (FAP) can be a variant of wild-type Chlorella variabilis FAP, Chlamydomonas reinhardtii FAP, Coccomyxa subellipsoidea FAP, Gonium pectorale FAP, or Raphidocelis subcapitata FAP.

[0099] The method can include exposing the enzyme to actinic radiation sufficient to form an excited-stated flavoquinone cofactor.

[0100] The method can include generating an alkyl radical and a flavosemiquinone.

[0101] The method can include proton-coupled electron transfer.

[0102] According to another aspect, a radical photocyclase (RAP) for catalyzing stereocontrolled cyclization includes a mutated variant of a fatty acid photodecarboxylase (FAP) including at least one mutation selected from Y466A, Y427A, Y428A, G462A, and Y431A.

[0103] The RAP of the preceding paragraph can optionally include, additionally and / or alternatively, any one or more of the following features / steps, configurations, and / or additional components.

[0104] The RAP can further include one or more of a S574A, S574G, S464A, and T131A mutation.

[0105] The RAP can further include one or more of a G431W, I130F, A131V, T465S, T131S, A466F, S465M, V463A, T484S, and V463N mutation.

[0106] The RAP can further include one or more of a Y434F and a Q429D mutation.

[0107] The mutated variant of the fatty acid photodecarboxylase (FAP) can be a variant of wild-type Chlorella variabilis FAP, Chlamydomonas reinhardtii FAP, Coccomyxa subellipsoidea FAP, Gonium pectorale FAP, or Raphidocelis subcapitata FAP.

[0108] According to another aspect, a method for converting fatty acid photodecarboxylases (FAP) to radical photocyclases includes incorporating a tyrosine-to-alanine mutation to a fatty acid photodecarboxylase (FAP) sufficient to form a radical photocyclase.

[0109] The method of the preceding paragraph can optionally include, additionally and / or alternatively, any one or more of the following features / steps, configurations, and / or additional components.

[0110] The mutation can include a Y466A mutation.

[0111] The fatty acid photodecarboxylase (FAP) can be selected from wild-type Chlorella variabilis FAP, Chlamydomonas reinhardtii FAP, Coccomyxa subellipsoidea FAP, Gonium pectorale FAP, and Raphidocelis subcapitata FAP.

[0112] According to another aspect, a mutated enzyme variant includes a mutated variant of a fatty acid photodecarboxylase (FAP) including a Y466A and G462A mutation.

[0113] The mutated enzyme variant of the preceding paragraph can optionally include, additionally and / or alternatively, any one or more of the following features / steps, configurations, and / or additional components.

[0114] The mutated enzyme variant can include a S464A mutation.

[0115] The mutated enzyme variant can include a S574G mutation.

[0116] The mutated enzyme variant can include a S574A mutation.

[0117] The mutated enzyme variant can include a T131A mutation.

[0118] The mutated enzyme variant can include at least one of G431W, I130F, and A131V mutations.

[0119] The mutated enzyme variant can include at least one of T465S and T131S mutations.

[0120] The mutated enzyme variant can include at least one of A466F, S465M, and V463A mutations.

[0121] The mutated enzyme variant can include at least one of I130F, T484S, and V463N mutations.

[0122] The mutated enzyme variant can be mutated from a Chlorella variabilis fatty acid photodecarboxylase.

[0123] 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

CLAIMS:

1. A method for radical cyclization, the method comprising: converting a substrate to a product compound by catalyzing the formation of a C-C bond using an enzyme, wherein the enzyme includes a mutated variant of a fatty acid photodecarboxylase (FAP).

2. The method of claim 1, wherein the substrate includes a carboxylic acid.

3. The method of claim 1, wherein the product compound is selected from an α,B- unsaturated ester, α,B-unsaturated amide, and α,B-unsaturated ketone.

4. The method of claim 1, wherein the product compound is selected from Ethyl (S)-2- cyclopentylpropanoate, Methyl (S)-2-cyclopentylpropanoate, Propyl (S)-2- cyclopentylpropanoate, 2-bromoethyl (S)-2-cyclopentylpropanoate, (S)-3- cyclopentyldihydrofuran-2(3H)-one, Ethyl (S)-2-cyclopentyl-2-fluoroacetate, Ethyl (S)-2-chloro- 2-cyclopentylacetate, Ethyl (S)-2-bromo-2-cyclopentylacetate, Ethyl (S)-2-cyclopentylbutanoate, (S)-2-cyclopentyl-N-methylpropanamide, (S)-2-cyclopentylpentan-3-one, Ethyl (S)-2- cyclohexylpropanoate, Ethyl (S)-2-(4,4-difluorocyclohexyl)propanoate, Ethyl (S)-2-(4,4- dimethylcyclohexyl)propanoate, and Ethyl (S)-2-(tetrahydro-2H-pyran-4-yl)propanoate.

5. The method of claim 1, wherein the product compound is selected from Ethyl 2- (tetrahydrofuran-3-yl)propanoate, Ethyl (S)-2-((S)-tetrahydrofuran-3-yl)propanoate, Ethyl (R)-2- ((S)-tetrahydrofuran-3-yl)propanoate, Ethyl (S)-2-((R)-tetrahydrofuran-3-yl)propanoate, and Ethyl (R)-2-((R)-tetrahydrofuran-3-yl) propanoate.

6. The method of claim 1, wherein the mutated variant of the fatty acid photodecarboxylase (FAP) includes a G462A mutation.

7. The method of claim 1, wherein the mutated variant of the fatty acid photodecarboxylase (FAP) includes a Y466A mutation.

8. The method of claim 1, wherein the mutated variant of the fatty acid photodecarboxylase (FAP) includes at least one of Y431A, Y434F, and Q429D mutations.

9. The method of claim 1, wherein the mutated variant of the fatty acid photodecarboxylase (FAP) is a variant of wild-type Chlorella variabilis FAP, Chlamydomonas reinhardtii FAP, Coccomyxa subellipsoidea FAP, Gonium pectorale FAP, or Raphidocelis subcapitata FAP.

10. The method of claim 1, including exposing the enzyme to actinic radiation sufficient to form an excited-stated flavoquinone cofactor.

11. The method of claim 10, including generating an alkyl radical and a flavosemiquinone.

12. The method of claim 11, including proton-coupled electron transfer.

13. A radical photocyclase (RAP) for catalyzing stereocontrolled cyclization, the radical photocyclase comprising: a mutated variant of a fatty acid photodecarboxylase (FAP) including at least one mutation selected from Y466A, Y427A, Y428A, G462A, and Y431A.

14. The radical photocyclase of claim 13 further including one or more of a S574A, S574G, S464A, and T131A mutation.

15. The radical photocyclase of claim 13 further including one or more of a G431W, I130F, A131V, T465S, T131S, A466F, S465M, V463A, T484S, and V463N mutation.

16. The radical photocyclase of claim 13 further including one or more of a Y434F and a Q429D mutation.

17. The radical photocyclase of claim 13, wherein the mutated variant of the fatty acid photodecarboxylase (FAP) is a variant of wild-type Chlorella variabilis FAP, Chlamydomonasreinhardtii FAP, Coccomyxa subellipsoidea FAP, Gonium pectorale FAP, or Raphidocelis subcapitata FAP.

18. A method for converting fatty acid photodecarboxylases (FAP) to radical photocyclases, the method comprising: incorporating a tyrosine-to-alanine mutation to a fatty acid photodecarboxylase (FAP) sufficient to form a radical photocyclase.

19. The method of claim 18, wherein the mutation includes a Y466A mutation.

20. The method of claim 18, wherein the fatty acid photodecarboxylase (FAP) is selected from wild-type Chlorella variabilis FAP, Chlamydomonas reinhardtii FAP, Coccomyxa subellipsoidea FAP, Gonium pectorale FAP, and Raphidocelis subcapitata FAP.

21. A mutated enzyme variant, the enzyme variant comprising: a mutated variant of a fatty acid photodecarboxylase (FAP) including a Y466A and G462A mutation.

22. The mutated enzyme variant of claim 21, further including a S464A mutation.

23. The mutated enzyme variant of claim 21, further including a S574G mutation.

24. The mutated enzyme variant of claim 21, further including a S574A mutation.

25. The mutated enzyme variant of claim 21, further including a T131A mutation.

26. The mutated enzyme variant of claim 21, further including at least one of G431W, I130F, and A131V mutations.

27. The mutated enzyme variant of claim 21, further including at least one of T465S and T131S mutations.

28. The mutated enzyme variant of claim 21, further including at least one of A466F, S465M, and V463A mutations.

29. The mutated enzyme variant of claim 21, further including at least one I130F, T484S, and V463N mutations.

30. The mutated enzyme variant of claim 21, wherein the mutated variant is mutated from a Chlorella variabilis fatty acid photodecarboxylase.

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