Artificial light enzyme, its preparation and application in photo-biocatalytic chiral synthesis

Abstract

This invention belongs to the field of photobiological chiral catalytic synthesis, and relates to an artificial photoenzyme, its preparation, and its application in photobiological chiral catalytic synthesis. This artificial photoenzyme is constructed by inserting an organic photosensitizer into a natural protein backbone using site-directed protein chemical modification technology. It can catalyze a [2+2] asymmetric photocycloaddition reaction of a 2-functionalized alkenylindole derivative under visible light to generate a chiral cyclobutane-fused tetracyclic indole spirocyclic compound. The artificial photoenzyme provided by this invention, constructed using site-directed protein chemical modification, can achieve highly efficient asymmetric photocatalytic reactions through energy transfer pathways, and features green environmental protection, mild conditions, and high reactivity.

Description

Technical Field

[0001] This invention belongs to the field of photobiological chiral catalytic synthesis, and more specifically, relates to an artificial photoenzyme, its preparation, and its application in photobiological chiral synthesis. Background Technology

[0002] Photocatalytic synthesis, as a highly efficient and green synthetic method, has developed rapidly in recent years and has been widely used in various types of organic reactions, including oxidation, reduction, radical atom transfer, and cycloaddition, making it an important branch of current organic synthetic chemistry. For example, the [2+2] cycloaddition of olefins is a key photocatalytic asymmetric synthetic reaction that has been studied extensively. This reaction is the most efficient and direct route for preparing cyclobutane molecules, and cyclobutane is an important intermediate in the synthesis of many chiral drugs. However, due to the high reactivity of the intermediates during the reaction, it is often difficult to control the stereoselectivity of small-molecule chiral catalysts. The three-dimensional chiral environment of protein macromolecules holds promise for providing an effective means to solve this problem. Therefore, asymmetric photobiocatalysis is attracting increasing attention from researchers.

[0003] Asymmetric photobiocatalysis, a cutting-edge field at the intersection of organic chemistry and chemical biology, combines the advantages of photochemical reactions and biological enzymes, making it an ideal driving force for organic reactions. However, naturally occurring photosensitive enzymes are scarce, exhibit strong catalytic specificity, and are insufficient to meet the needs of non-natural organic reactions. Therefore, developing artificial photosensitive enzymes with non-natural active sites is of significant research importance for expanding the types of photobiocatalytic reactions and their applications. Recently, Professor Green and our research group reported on the construction of artificial photosensitive enzymes by introducing the non-natural amino acid (BpA) of benzophenone, which has excellent photocatalytic performance, into the natural protein backbone using gene codon expansion technology (APGreen et al., Nature, 2022, 611, 709-714; Y. Wu et al., Nature, 2022, 611, 715-720). This demonstrated that the artificial photoenzyme encoded by this gene can be further optimized for its cavity microenvironment using directed evolution technology, thereby providing hydrogen bonds and multiple weak bond interactions with the substrate, enabling the catalytic synthesis of non-natural chiral compounds. Although this study demonstrates the great potential of artificial photosensitizers in broadening the types of photocatalytic reactions, asymmetric photocatalysis is still in its early stages. The main reason is that the types of photocofactors available in nature are limited. Although synthetic chemistry has developed a variety of chemical photosensitizers, codon expansion technology is only suitable for introducing specific photosensitizing groups into the protein backbone. It is difficult to use it to efficiently screen a large number of different photosensitizing groups to meet the catalytic requirements of different substrates. In particular, the photosensitizers that can be introduced by codon expansion technology are still limited to ultraviolet light as the reaction driving force, making it difficult to develop and utilize the abundant visible light resources. Summary of the Invention

[0004] To address the shortcomings or improvement needs of existing technologies, the main objective of this invention is to provide an application of artificial photoenzyme CMP in photobiocatalytic chiral synthesis. The artificial photoenzyme CMP is constructed by inserting an organic photosensitizer into a natural protein backbone using site-directed protein modification technology. Then, CMP is used as a photoenzyme catalyst under visible light as the reaction driving force to catalyze a [2+2] asymmetric photocycloaddition reaction of a 2-functionalized alkenylindole derivative to generate a chiral cyclobutane-fused tetracyclic indole spirocyclic compound. The site-directed protein modification technology provided by this invention can be used to develop diverse artificial photoenzymes. The CMP artificial photoenzyme constructed in this invention can achieve highly efficient chiral photocatalysis through an energy transfer pathway under visible light as the driving force, exhibiting characteristics such as being environmentally friendly, operating under mild conditions, and possessing high reactivity.

[0005] According to a first aspect of the present invention, a method for preparing an artificial photoenzyme is provided, wherein a cysteine ​​containing a thiol functional group is introduced into a protein backbone containing a chiral cavity by site-directed mutagenesis, and then an iodinated photosensitizer molecule is subjected to a nucleophilic addition reaction with the cysteine ​​to obtain the artificial photoenzyme.

[0006] Preferably, the photosensitizer small molecule is an iodoacetaminated photosensitizer.

[0007] Preferably, the iodoacetaminated photosensitizer is iodoacetaminated thioxanone, iodoacetaminated benzophenone, iodoacetaminated thioxanone, or iodoacetaminated 9-fluorenone.

[0008] Preferably, the protein backbone is the lactobacillus multidrug resistance regulator LmrR protein.

[0009] Preferably, after introducing cysteine ​​containing a thiol functional group, the method further includes optimizing the cavity structure of the lactobacillus multidrug resistance regulator LmrR protein; the amino acid sequence of the optimized lactobacillus multidrug resistance regulator LmrR protein is shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4.

[0010] According to another aspect of the present invention, an artificial photoenzyme prepared by any one of the methods is provided.

[0011] According to another aspect of the present invention, the application of the aforementioned artificial photoenzyme as a photosensitive catalyst in photobiocatalytic chiral synthesis is provided.

[0012] Preferably, the application specifically involves: under light irradiation conditions, the artificial photoenzyme catalyzes a [2+2] asymmetric photocycloaddition reaction of the 2-functionalized alkenylindole derivative to obtain a chiral cyclobutane-fused tetracyclic indole spirocyclic compound;

[0013] The reaction formula is as follows:

[0014]

[0015] Wherein: R1 is 5-H, 5-F, 5-CH3, 5-Br, 5-Cl, 5-OCH3, 4-OCH3, 6-Br or 6-Cl;

[0016] R2 is -CH3 or -benzyl.

[0017] Preferably, the solvent for the reaction is a mixture of protein buffer and organic solvent, wherein the volume of the organic solvent is 5-10% of the volume of the mixture; the initial concentration of the substrate 2-functionalized alkenylindole derivative is 0.1-0.5 mM, and the concentration of the artificial photoenzyme is 2.5-5 mol%.

[0018] Preferably, the organic solvent is dimethyl sulfoxide.

[0019] Preferably, the reaction temperature is 4–6°C; and the wavelength range of the illumination is 400–415 nm.

[0020] In summary, compared with the prior art, the above-described technical solutions conceived by this invention mainly possess the following technical advantages:

[0021] (1) The protein site-directed chemical modification method of this invention can introduce organic molecules containing different groups into specific positions in the protein backbone. This technology can solve the problem that the gene codon expansion method is difficult to construct artificial enzymes with multiple photosensitive centers. However, there are currently no reports on the construction of artificial photoenzymes based on protein site-directed chemical modification for photobiological chiral catalysis. This is mainly because the traditional protein site-directed chemical modification method is difficult to combine with high-throughput screening processes for artificial enzyme structure optimization, thus making the application of photobiological chiral catalysis very challenging. The protein site-directed modification technology provided by this invention can develop a variety of artificial photoenzymes. The constructed CMP artificial photoenzyme can achieve efficient chiral photocatalysis through energy transfer pathways under visible light as the driving force, and has the characteristics of being green and environmentally friendly, having mild conditions, and high reactivity.

[0022] (2) The results of this invention show that introducing excellent photosensitive catalysts and photocatalytic mechanisms developed in synthetic chemistry into natural proteases through site-directed protein chemical modification can fundamentally expand the types of photoenzymes, thereby broadening the types of photobiocatalytic reactions. The developed artificial photoenzymes not only possess the high reactivity of small organic molecule photosensitizers but also the highly selective reaction characteristics of biomacromolecules. Furthermore, site-directed protein chemical modification technology can introduce a variety of photosensitizer molecules with different structures and properties. Therefore, artificial photoenzymes constructed using this technology can adapt to substrates with different chemical properties and reaction requirements, making it a universal solution for asymmetric photochemical synthesis.

[0023] (3) Preferably, this invention is the first to use site-directed chemical modification technology to introduce iodoacetamide-modified thioxanthone 3c, an organic photosensitive center, into a natural protein backbone to construct an artificial photoenzyme. This is the first time that a photobiological chiral catalysis has been achieved using the artificial photoenzyme CMP. Taking the [2+2] asymmetric photocycloaddition reaction of 2-functionalized alkenylindole derivatives as an example, photocatalytic [2+2] photocycloaddition reactions are the most direct and convenient method for constructing molecules containing cyclobutane structures. However, due to the high activity of the intermediate in the reaction process, small-molecule chiral catalysts often have difficulty controlling their stereoselectivity. In this invention, small-molecule organic photocatalysts developed in organic chemistry are introduced into a natural protein backbone to obtain the CMP artificial photoenzyme. The secondary coordination layer of the protein backbone can be used to provide multiple hydrogen bonds and weak interactions with the substrate, thereby achieving the [2+2] asymmetric photocycloaddition reaction. The artificial photoenzyme constructed in this invention can achieve yields greater than 90% and ee values ​​of 81-92% for 12 substrates, exhibiting high stereoselectivity. Attached Figure Description

[0024] Figure 1 This invention provides the structural formula and specific construction method of the photosensitizer.

[0025] Figure 2 This is a schematic diagram of the artificial photoenzyme constructed and a reaction formula for a specific catalytic reaction.

[0026] Figure 3 This is a flowchart of the artificial photoenzyme combined with directed evolution optimization obtained from the examples.

[0027] Figure 4 This is the first-order mass spectrum of CMP4.0 obtained in the example without chemical modification.

[0028] Figure 5 This is the first-order mass spectrum of CMP4.0 obtained in the example after 3c protein site-directed modification.

[0029] Figure 6The image shows the secondary mass spectrum of CMP4.0 obtained in the example after site-directed protein modification (3c). LC-MS / MS analysis was performed on the CMP4.0 polypeptide fragment digested by trypsin.

[0030] Figure 7 The figures (A) and (B) show the kinetic curves (B) of the [2+2] asymmetric photocycloaddition reaction of the 2-functionalized alkenyl indole derivative catalyzed by CMP4.0 obtained in the examples. Detailed Implementation

[0031] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention. Furthermore, the technical features involved in the various embodiments of this invention described below can be combined with each other as long as they do not conflict with each other.

[0032] This invention discloses the application of an artificial photoenzyme CMP as a catalyst in photobiocatalytic chiral synthesis. The artificial photoenzyme CMP is characterized by the introduction of a cysteine ​​residue containing a thiol functional group into a protein backbone with a chiral cavity via site-directed mutagenesis. Preferably, the protein backbone is the lactobacillus multidrug resistance regulator LmrR protein, followed by a site-directed bioconjugation reaction with an iodinated photosensitizer small molecule to insert the organic photosensitizer into the cysteine ​​residue of the protein backbone, thereby undergoing a nucleophilic addition reaction with cysteine. Preferably, the organic photosensitizing center is an iodoacetamidized thioxanone 3c. The photosensitizer structural formula and specific construction method are as follows: Figure 1 As shown, the application of artificial photoenzyme (CMP) as a photosensitive catalyst in photobiocatalytic synthesis in this invention specifically refers to the application of CMP as a photosensitive catalyst in the synthesis of chiral cyclobutane-fused tetracyclic indole spirocyclic compounds in photobiocatalysis. Figure 2 This is a schematic diagram of the constructed artificial photoenzyme and a specific catalytic reaction formula.

[0033] In some embodiments, the chiral cyclobutane-fused tetracyclic indole spirocyclic compound has the structure shown in Formula 2, and the reaction substrate has the structure shown in Formula 1. The chiral catalytic reaction is shown below:

[0034]

[0035] Among them, R1 is independently selected from: 5-H, 5-F, 5-CH3, 5-Br, 5-Cl, 5-OCH3, 4-OCH3, 6-Br, 6-Cl;

[0036] R2 is independently selected from: -CH3, -benzyl.

[0037] In summary, the artificial photoenzyme CMP in this invention is constructed by utilizing and developing protein site-directed chemical modification technology to insert organic photosensitizers into the natural protein backbone. This artificial photoenzyme can catalyze the [2+2] asymmetric photocycloaddition reaction of 2-functionalized alkenyl indole derivatives under visible light to generate chiral cyclobutane-fused tetracyclic indole spirocyclic compounds.

[0038] Based on this invention, taking the LmrR protein backbone, a multidrug resistance regulator of lactobacilli, as an example, a cysteine ​​residue containing a thiol functional group can be introduced into the cavity of the target protein backbone through site-directed mutagenesis. Then, a site-directed bioconjugation reaction with an iodinated photosensitizer molecule inserts the organic photosensitizer into a specific position on the protein backbone, constructing an artificial photoenzyme CMP. At the dimer interface, LmrR creates a large hydrophobic binding pocket due to the π-π stacking of its two tryptophan residues. From the perspective of artificial enzyme construction, this naturally formed pocket is not only suitable for introducing new catalytic active sites but also facilitates the binding of reaction substrates to the active site. Currently, the technology of expressing LmrR using *E. coli* as a heterologous expression system is quite mature. Each 1L of culture expression system can ultimately yield approximately 30mg of wild-type target protein. Furthermore, this protein backbone has made some progress in the construction and application of artificial metalloenzymes. Therefore, LmrR is a relatively ideal choice as a natural backbone carrier.

[0039] Specifically:

[0040] (1) Introduction of organic photosensitizers into protein cavities based on site-directed chemical modification: Protein chemical modification based on the chemical reactivity of active functional groups on the side chains of amino acid residues is a relatively mature technology in the biomedical field. Among the 20 natural amino acids, cysteine ​​is the primary choice for researchers studying site-directed chemical modification due to its high nucleophilicity of the thiol functional group, low redox potential, and low abundance in the natural protein backbone. Based on this, this invention introduces cysteine ​​containing a specific reactive functional group into the cavity of the LmrR protein backbone through site-directed mutagenesis, followed by a site-directed biocoupler reaction with an iodinated photosensitizer small molecule to achieve the site-directed introduction of an organic small molecule photosensitizer into the LmrR protein backbone.

[0041] (2) Chemical Modification / Directed Evolution Integration Technology: Although existing directed evolution technology is quite mature, it is mainly used in conjunction with heritable gene engineering techniques such as gene codon expansion (APGreen et al, Nature, 2022, 611, 709-714; Y. Wu et al, Nature, 2022, 611, 715-720). The current challenge in combining chemical modification with directed evolution technology lies in the fact that site-specific chemical modification of proteins requires additional cumbersome processes such as protein purification and removal of redundant modification molecules. Combining this with a high-throughput screening process like directed evolution results in a huge workload. In this invention, the site-specific chemical modification process is optimized, enabling this reaction to be achieved in cell lysis buffer. Specifically, the dosage and reaction time of the small molecule photosensitizer in the bioconjugation reaction with the protein backbone are optimized. The optimized coupling conditions are as follows: 0.9 equivalents (each equivalent based on a protein monomer) of a small molecule photosensitizer are incubated with cell lysis buffer containing the target protein in the dark for 1 hour, followed by centrifugation at 4°C and 12000 rpm for 10 minutes to remove insoluble precipitates, yielding the crude enzyme solution containing CMP-containing artificial photoenzyme. The optimized chemical modification process can then be combined with directed evolution technology (the specific process and details of this technology can be found in existing technical literature: Chem. Sci., 2015, 6, 770; Chem. Sci., 2013, 4, 3578; Nature Chem., 2018, 10, 946-952). The main purpose of this combined process is to optimize the amino acid residues surrounding the active site of the first-generation artificial photoenzyme through multiple rounds of iterative evolution, thereby completing the enzyme's structural optimization (the main process is shown in the appendix). Figure 3As shown in the figure, the optimal mutant capable of achieving chiral catalysis of 2-functionalized alkenylindole derivatives was screened. In each round of evolutionary screening, the mutant from the previous generation that best controlled the chiral response was used as the evolutionary template. In this embodiment, four rounds of evolution were performed. Taking the first round as an example, V15, N19, M89, A92, and F93 were selected as the introduction sites for the photosensitizer molecule. The F93 position was mutated to a cysteine ​​residue, introducing iodoacetamide-modified thioxanthone 3c into the organic photosensitizing center. Reaction analysis showed that this mutant exhibited the best chiral results (a 5% ee value in reaction with the 2-functionalized alkenylindole model substrate). Therefore, the corresponding mutant photoenzyme was named CMP1.0 and used as the template for the next round of evolution. Then, using CMP1.0 as the template, further evolution was performed to obtain the mutant CMP2.0 (CMP1.0_W96L). A third round of mutation was then performed using CMP2.0 as a template to obtain the optimal mutant CMP3.0 (CMP2.0_M8L). A fourth round of evolution was then performed using CMP3.0 as a template, ultimately yielding the optimal mutant artificial photoenzyme CMP4.0 (CMP3.0_N88H). Unless otherwise specified, all CMP photoenzymes in this paper were created by mutating the F93 position in the LmrR protein backbone to C93 to insert the organic photosensitizer iodoacetamylthioxanthone 3c. Furthermore, besides inserting iodoacetamylthioxanthone 3c, other iodoacetamyl organic photosensitizer molecules of suitable structural size can also be inserted.

[0042] The obtained nucleic acid and amino acid sequences of CMP1.0 to CMP4.0 are as follows (the bolded values ​​in the nucleic acid and amino acid sequences below represent the mutation sites compared to the wild-type LmrR; in addition, the parentheses in the amino acid sequences indicate the introduction of the photosensitizing center by modifying the organic photosensitizer molecule in the parentheses to the cysteine ​​residue before the parentheses). The mass spectrometry characterization is attached. Figure 4 , Figure 5 , Figure 6 As shown.

[0043] (i)CMP_F93C_3c(CMP1.0)

[0044] nucleotide sequence

[0045] ATGGGTGCCGAAATCCCGAAAGAAATGCTGCGTGCTCAAACCAATGTCATCCTGCTGAATGTCC

[0046] TGAAACAAGGCGATAACTATGTGTATGGCATTATCAAACAGGTGAAAGAAGCGAGCAACGGTG

[0047] AAATGGAACTGAATGAAGCCACCCTGTATACGATTTTTGATCGTCTGGAACAGGACGGCATTAT

[0048] CAGCTCTTACTGGGGTGATGAAAGTCAAGGCGGTCGTCGCAAATATTACCGTCTGACCGAAATC

[0049] GGCCATGAAAACATGCGCCTGGCGTGCGAATCCTGGAGTCGTGTGGACAAAATCATTGAAAAT

[0050] CTGGAAGCAAACAAAAAATCTGAAGCGATCAAATCTAGAGGTGGCAGCGGTGGCTGGAGCCA

[0051] CCCGCAGTTCGAAAAATAA

[0052] Amino acid sequence

[0053] MGAEIPKEMLRAQTNVILLNVLKQGDNYVYGIIKQVKEASNGEMELNEATLYTIFDRLEQDGIISSYWGDESQGGRRKYYRLTEIGHENMRLAC(3c)ESWSRVDKIIENLEANKKSEAIKSRGGSGGWSHPQF EK.

[0054] (ii)CMP1.0_W96L_3c(CMP2.0)

[0055] Nucleotide sequence

[0056] ATGGGTGCCGAAATCCCGAAAGAAATGCTGCGTGCTCAAACCAATGTCATCCTGCTGAATGTCC

[0057] TGAAACAAGGCGATAACTATGTGTATGGCATTATCAAACAGGTGAAAGAAGCGAGCAACGGTG

[0058] AAATGGAACTGAATGAAGCCACCCTGTATACGATTTTTGATCGTCTGGAACAGGACGGCATTAT

[0059] CAGCTCTTACTGGGGTGATGAAAGTCAAGGCGGTCGTCGCAAATATTACCGTCTGACCGAAATC

[0060] GGCCATGAAAACATGCGCCTGGCGTGCGAATCCTTGAGTCGTGTGGACAAAATCATTGAAAAT

[0061] CTGGAAGCAAACAAAAAATCTGAAGCGATCAAATCTAGAGGTGGCAGCGGTGGCTGGAGCCA

[0062] CCCGCAGTTCGAAAAATAA

[0063] Amino acid sequence (SEQ ID NO:1)

[0064] MGAEIPKEMLRAQTNVILLNVLKQGDNYVYGIIKQVKEASNGEMELNEATLYTIFDRLEQDGIISSYWGDESQGGRRKYYRLTEIGHENMRLAC(3c)ESLSRVDKIIENLEANKKSEAIKSRGGSGGWSHPQF EK.

[0065] (iii)CMP2.0_M8L_3c(CMP3.0)

[0066] Nucleotide sequence

[0067] ATGGGTGCCGAAATCCCGAAAGAATTGCTGCGTGCTCAAACCAATGTCATCCTGCTGAATGTC

[0068] CTGAAACAAGGCGATAACTATGTGTATGGCATTATCAAACAGGTGAAAGAAGCGAGCAACGGT

[0069] GAAATGGAACTGAATGAAGCCACCCTGTATACGATTTTTGATCGTCTGGAACAGGACGGCATTA

[0070] TCAGCTCTTACTGGGGTGATGAAAGTCAAGGCGGTCGTCGCAAATATTACCGTCTGACCGAAAT

[0071] CGGCCATGAAAACATGCGCCTGGCGTGCGAATCCTTGAGTCGTGTGGACAAAATCATTGAAAA

[0072] TCTGGAAGCAAACAAAAAATCTGAAGCGATCAAATCTAGAGGTGGCAGCGGTGGCTGGAGCC

[0073] ACCCGCAGTTCGAAAAATAA

[0074] Amino acid sequence (SEQ ID NO:2)

[0075] MGAEIPKELLRAQTNVILLNVLKQGDNYVYGIIKQVKEASNGEMELNEATLYTIFDRLEQDGIISSYWGDESQGGRRKYYRLTEIGHENMRLAC(3c)ESLSRVDKIIENLEANKKSEAIKSRGGSGGWSHPQF EK.

[0076] (iv)CMP3.0_N88H_3c(CMP4.0)

[0077] Nucleotide sequence

[0078] ATGGGTGCCGAAATCCCGAAAGAATTGCTGCGTGCTCAAACCAATGTCATCCTGCTGAATGTC

[0079] CTGAAACAAGGCGATAACTATGTGTATGGCATTATCAAACAGGTGAAAGAAGCGAGCAACGGT

[0080] GAAATGGAACTGAATGAAGCCACCCTGTATACGATTTTTGATCGTCTGGAACAGGACGGCATTA<...>​​​​​​​TCTGGAAGCAAACAAAAAATCTGAAGCGATCAAATCTAGAGGTGGCAGCGGTGGCTGGAGCC

[0084] ACCCGCAGTTCGAAAAATAA

[0085] Amino acid sequence (SEQ ID NO:3)

[0086] MGAEIPKELLRAQTNVILLNVLKQGDNYVYGIIKQVKEASNGEMELNEATLYTIFDRLEQDGIISSYWGDESQGGRRKYYRLTEIGHEHMRLAC(3c)ESLSRVDKIIENLEANKKSEAIKSRGGSGGWSHPQF EK.

[0087] In addition, other mutations can be introduced into the artificial photoenzyme protein backbone, for example:

[0088] (v)CMP4.0_A11N_A92I_3c

[0089] nucleotide sequence

[0090] ATGGGTGCCGAAATCCCGAAAGAATTGCTGCGTAATCAAACCAATGTCATCCTGCTGAATGTC

[0091] CTGAAACAAGGCGATAACTATGTGTATGGCATTATCAAACAGGTGAAAGAAGCGAGCAACGGT

[0092] GAAATGGAACTGAATGAAGCCACCCTGTATACGATTTTTGATCGTCTGGAACAGGACGGCATTA

[0093] TCAGCTCTTACTGGGGGTGATGAAAGTCAAGGCGGTCGTCGCAAATATTACCGTCTGACCGAAAT

[0094] CGGCCATGAACATATGCGCCTGATCTGCGAATCCTTGAGTCGTGTGGACAAAATCATTGAAAA

[0095] TCTGGAAGCAAACAAAAAATCTGAAGCGATCAAATCTAGAGGTGGCAGCGGTGGCTGGAGCC

[0096] ACCCGCAGTTCGAAAAATAA

[0097] Amino acid sequence (SEQ ID NO:4)

[0098] MGAEIPKELLRNQTNVILLNVLKQGDNYVYGIIKQVKEASNGEMELNEATLYTIFDRLEQDGIISSYWGDESQGGRRKYYRLTEIGHEHMRLIC(3c)ESLSRVDKIIENLEANKKSEAIKSRGGSGGWSHPQF EK.

[0099] (3) Enzyme kinetics studies

[0100] Taking the catalytic performance of CMP4.0 as an example, we explored the kinetic parameters of CMP4.0 for model substrate 1a. The optimal mutant CMP4.0 (4.5 μM) and substrate 1a (final concentration range from 0.02 mM to 0.5 mM) were added to a MOPS buffer solution containing 5% (v / v) DMSO (20 mM MOPS, 150 mM NaCl, 1% Triton X-100, pH 7.0), and the reaction was carried out at 4 °C under a 405 nm lamp. A certain volume of the reaction solution was taken at reaction times of 5 s, 10 s, 15 s, 20 s, and 30 s for liquid chromatography analysis. The results were fitted using OriginPro 2021. The fitting results are attached. Figure 7 As shown, CMP4.0 follows the Michaelis-Menten equation kinetics, demonstrating that the catalytic process of substrate 1a by CMP4.0 is a typical single-molecule enzymatic reaction. Furthermore, by comparing the time-response progression of substrate 1a catalyzed by the CMP4.0 mutant and the small-molecule photosensitizer 3c, the reaction time-progress curves show that, compared to organic small-molecule photosensitizers, the artificial photoenzyme exhibits highly efficient catalytic performance, enabling almost complete substrate conversion within 3 hours. These findings demonstrate the high reactivity of the artificial photoenzyme.

[0101] The following are specific examples:

[0102] Example 1: Expression and preparation of CMP artificial photoenzyme

[0103] First, using the wild-type LmrR recombinant plasmid as a template, LmrR mutant plasmids containing cysteine ​​residues at the above-mentioned sites were constructed through site-directed mutagenesis. The specific operation process is as follows: Pre- and post-mutation primers synthesized by Shanghai Sangon Biotech Co., Ltd., template, and deionized water were added, and a polymerization reaction (PCR) was performed under the action of high-fidelity DNA polymerase. The PCR amplification program was as follows: pre-denaturation at 95℃ for 5 min, followed by 18 cycles, each cycle consisting of denaturation at 95℃ for 30 s, annealing at 58℃ for 30 s, extension at 72℃ for 1 min, and holding at 16℃ for 10 min after the cycle. After the PCR reaction, the reaction mixture was incubated with Dpn I restriction endonuclease at 37℃ for 1 h, and transformed into *E. coli* DH5α clone strain. Recombinants were obtained by screening in ampicillin-resistant LB agar plates. After sequencing confirmed successful mutation of the recombinant plasmid, it was then transformed into *E. coli* BL21(DE3) expression strain.

[0104] Select an appropriate amount of BL21(DE3) strain containing the recombinant LmrR protein gene and inoculate it into 5 mL of LB liquid medium (containing 100 μg / L ampicillin antibiotic). Incubate overnight at 37°C and 200 rpm, then transfer to 400 mL of fresh liquid medium and expand culture at 37°C and 200 rpm for 3–4 hours. When the cell OD 600nm value reaches 0.8–1.0, add 400 μL of 1 MIPTG (isopropyl β-D-thiogalactoside) and induce protein expression for 18–20 hours at 30°C and 200 rpm. After expression, centrifuge at 4°C and 12000 rpm for 20 minutes, discard the supernatant, and collect the bacterial pellet. The bacterial cells were resuspended in 10 mL of cell lysis buffer (50 mM Tris·HCl, 100 mM sodium chloride, 5 mL Triton-100, pH 8), and an appropriate amount of DNase and PMSF (a protease inhibitor) were added. The mixture was placed on ice and the cells were lysed using a cell sonicator. The sonicator parameters were set as follows: power 350 W, single sonication time 1.5 s, interval time 2.5 s, and total sonication time 20 min. After centrifugation, the supernatant was purified using a Strep-Tactin purification column. The purified protease was incubated with 10 equivalents (each equivalent based on a protein monomer) of a small molecule photosensitizer for 1 h in the dark, followed by ultrafiltration to remove excess free photosensitizer molecules. Finally, the artificial photosensitizer CMP was collected.

[0105] Example 2: CMP photoenzyme-catalyzed [2+2] asymmetric photocycloaddition reaction of 2-functionalized alkenylindole derivatives

[0106] The CMP-catalyzed reaction was as follows: The mutant CMP4.0 (4.5 μM) and substrate 1a (final concentration range from 0.02 mM to 0.5 mM) were mixed with a MOPS buffer solution containing 5% (v / v) DMSO (20 mM MOPS, 150 mM NaCl, 1% Triton X-100, pH 7.0). The reaction solution was then placed in a quartz glass tube and reacted at 4°C under a 405 nm lamp for 3-4 h. The results are shown below (where 2a, 2b, 2c, 2d, 2e, 2f, 2g, 2h, 2i, 2j, 2k, and 2l are products obtained from the artificial photoenzyme CMP catalyzed by different 2-functionalized alkenyl indole derivative substrates in the examples).

[0107]

[0108] 2h and 2i were obtained by catalysis with CMP4.0_A11N_A92I under the same reaction conditions. All other products, unless otherwise specified, were obtained by catalysis with CMP4.0.

[0109] Based on existing technologies, in the field of photobiocatalysis, the reported [2+2] asymmetric photocycloaddition reactions of indole olefin derivatives have all been achieved at 365 nm using artificial photoenzymes constructed using gene codon expansion technology. The example presented in this invention is the first photobiocatalytic [2+2] asymmetric photocycloaddition reaction of indole olefin derivatives driven by visible light, offering advantages such as being environmentally friendly, highly reactive, and operating under mild conditions. The protein site-directed chemical modification / directed evolution integration technology provided in this paper can construct artificial photoenzymes containing photosensitive centers with significant structural and chemical differences, fundamentally expanding the types of photoenzymes to meet the energy matching requirements of different photoreaction substrates and enriching the types of photobiological chiral catalysis reactions—something that is difficult to achieve with currently reported gene codon expansion technologies.

[0110] The above embodiments are merely illustrative examples. For instance, the structure of the photosensitizing center can be flexibly altered (specifically, based on substrate energy matching requirements and catalyst triplet lifetime requirements, a suitable small organic photosensitizer molecule structure can be selected as the main body, linked to an iodoacetamide group, and then biocoupled with the thiol group on protein cysteine ​​to construct a rich variety of artificial photoenzymes). Furthermore, the substrate concentration in photobiological chiral catalysis can also be adjusted, for example, by increasing or decreasing the substrate concentration (final concentration less than 0.5 mM, such as 0.1–0.3 mM; or the final concentration can be increased to 5 mM; if necessary, a high concentration of substrate reaction can be achieved by adding a mild surfactant to promote substrate dissolution, such as adding 1% Triton X-100); and the introduction position of the photosensitizer center, the selection of the natural protein backbone, and the optimization of the amino acid residues in the active cavity can all be flexibly adjusted according to actual conditions to adapt to different photobiological chiral catalytic reactions.

[0111] Those skilled in the art will readily understand that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for preparing an artificial photoenzyme, characterized in that, A cysteine ​​residue containing a thiol functional group is introduced into a protein backbone containing a chiral cavity using site-directed mutagenesis. The protein backbone is the LmrR protein, a multidrug resistance regulator of the lactobacillus. The cavity structure of the LmrR protein is optimized. The optimized amino acid sequence of the LmrR protein is shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:

4. Then, an iodinated photosensitizer molecule undergoes a nucleophilic addition reaction with the cysteine ​​residue to obtain the artificial photoenzyme. The iodinated photosensitizer molecule is iodoacetaminated thioxanone, iodoacetaminated benzophenone, iodoacetaminated thioxanone, or iodoacetaminated 9-fluorenone.

2. The artificial photoenzyme prepared by the method of claim 1.

3. The application of the artificial photoenzyme as described in claim 2 as a photosensitive catalyst in photobiocatalytic chiral synthesis, characterized in that, The specific application is as follows: under light conditions, the artificial photoenzyme catalyzes the [2+2] asymmetric photocycloaddition reaction of the 2-functionalized alkenylindole derivative to obtain a chiral cyclobutane-fused tetracyclic indole spirocyclic compound; The reaction formula is as follows: ； Wherein: R1 is 5-H, 5-F, 5-CH3, 5-Br, 5-Cl, 5-OCH3, 4-OCH3, 6-Br or 6-Cl; R2 is -CH3 or -benzyl.

4. The application as described in claim 3, characterized in that, The solvent for the reaction is a mixture of protein buffer and organic solvent, wherein the volume of organic solvent is 5-10% of the volume of the mixture; the initial concentration of the substrate 2-functionalized alkenylindole derivative is 0.1-0.5 mM, and the concentration of the artificial photoenzyme is 2.5-5 mol.

5. The application as described in claim 4, characterized in that, The organic solvent is dimethyl sulfoxide.

6. The application as described in any one of claims 3-5, characterized in that, The reaction temperature is 4~6℃; the wavelength range of the illumination is 400~415 nm.

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