CN-118045630-B - Artificial photo-enzyme and preparation thereof and application thereof in photo-biocatalysis chiral synthesis

Abstract

The invention belongs to the field of photo-biological chiral catalytic synthesis, and relates to an artificial photo-enzyme and preparation thereof and application thereof in photo-biological catalytic chiral synthesis. The artificial light enzyme is constructed by inserting an organic photosensitizer into a natural protein skeleton by utilizing a protein site-directed chemical modification technology, and can catalyze 2-functionalized alkenyl indole derivatives to generate [2+2] asymmetric light cycloaddition reaction under the driving force of visible light to generate chiral cyclobutane condensed tetracyclic indole spiro compounds. The artificial light enzyme constructed by the protein fixed-point chemical modification method can realize high-efficiency asymmetric photocatalytic reaction through an energy transfer way, and has the characteristics of environment friendliness, mild conditions, high reaction activity and the like.

Inventors

• WU YUZHOU
• ZHONG FANGRUI
• GUO JUAN

Assignees

• 华中科技大学

Dates

Publication Date

20260505

Application Date

20240117

Claims (6)

1. 1. A preparation method of artificial photo-enzyme is characterized in that cysteine containing sulfhydryl functional groups is introduced into a protein skeleton containing chiral cavities through a site-directed mutagenesis technology, the protein skeleton is lactococcus multi-drug resistance regulating factor LmrR protein, the cavity structure of the lactococcus multi-drug resistance regulating factor LmrR protein is optimized, the amino acid sequence of the optimized lactococcus multi-drug resistance regulating factor LmrR protein is shown as SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3 or SEQ ID NO. 4, and then nucleophilic addition reaction is carried out on iodinated photosensitizer small molecules and the cysteine to obtain the artificial photo-enzyme; the iodinated photosensitizer small molecule is iodinated ethyl amidated thioxanthone, iodinated ethyl amidated diphenyl ketone, iodinated ethyl amidated thioxanthone or iodinated ethyl amidated 9-fluorenone.
2. 2. An artificial light enzyme prepared by the method of claim 1.
3. 3. The application of the artificial photo-enzyme as a photosensitive catalyst in photocatalysis chiral synthesis is characterized in that the application is that the artificial photo-enzyme catalyzes 2-functionalized alkenyl indole derivatives to generate [2+2] asymmetric light cycloaddition reaction under the illumination condition to obtain chiral cyclobutane condensed tetracyclic indole spiro compounds; The reaction formula is as follows: ; Wherein R 1 is 5-H, 5-F, 5-CH 3 , 5-Br, 5-Cl, 5-OCH 3 , 4-OCH 3 , 6-Br or 6-Cl; r 2 is-CH 3 or-benzyl.
4. 4. The method according to claim 3, wherein the solvent for the reaction is a mixed solution of a protein buffer solution and an organic solvent, the volume of the organic solvent is 5-10% of the volume of the mixed solution, the initial concentration of the substrate 2-functionalized alkenylindole derivative for the reaction is 0.1-0.5 mM, and the addition concentration of the artificial photo-enzyme is 2.5-5 mol%.
5. 5. The use according to claim 4, wherein the organic solvent is dimethyl sulfoxide.
6. 6. The use according to any one of claims 3 to 5, wherein the reaction temperature is 4 to 6 ℃ and the wavelength of the illumination is 400 to 415 nm.

Description

Artificial photo-enzyme and preparation thereof and application thereof in photo-biocatalysis chiral synthesis Technical Field The invention belongs to the field of photo-biological chiral catalytic synthesis, and in particular relates to an artificial photo-enzyme and preparation thereof and application thereof in photo-biological catalytic chiral synthesis. Background Photocatalytic synthesis has been developed rapidly in recent years as an efficient and green synthesis method, and has been widely used for oxidation, reduction, free radical atom transfer, cycloaddition and other different types of organic reactions, and is an important branch of current organic synthesis chemistry. For example, the [2+2] cycloaddition of olefins is an important-studied photocatalytic asymmetric synthesis reaction, which is the most efficient and direct way to prepare the molecular structure of cyclobutane, which is an important intermediate for synthesizing many chiral drugs, but due to the higher activity of the intermediate in the reaction process, the stereoselectivity of small molecule chiral catalysts is often difficult to control, and the three-dimensional chiral environment of protein macromolecules is expected to provide an effective means for solving the problem, so asymmetric photocatalysis is increasingly attracting attention of researchers. Asymmetric photo-biocatalysis is used as the front field of intersection of organic chemistry and chemical biology, integrates the respective advantages of photochemical reaction and biological enzyme, and is an ideal driving force for organic reaction. However, naturally occurring photosensitizers are rare in species, have strong specificity in catalytic performance, and are difficult to meet the requirements of unnatural organic reactions. Therefore, the development of artificial photosensitive enzymes with unnatural active centers has important research significance for expanding the type and application range of photocatalytic reactions. Recently, the Green professor and our task group have reported research (a.p. Green et al, nature,2022,611,709-714;Y.Wu et al,Nature,2022,611,715-720) for constructing artificial photosensitive enzymes by introducing benzophenone unnatural amino acids (BpA) with excellent photocatalytic properties into natural protein frameworks by using gene codon extension technology, which demonstrates that the artificial photosensitive enzymes encoded by the genes can further optimize their cavity microenvironment in combination with directed evolution technology, thereby providing hydrogen bond and multiple weak bond interactions with substrates, and realizing catalytic synthesis of unnatural chiral compounds. Although the research proves that the artificial photosensitive enzyme has great potential in widening the photo-biocatalysis reaction type, the asymmetric photo-biocatalysis is still in a starting stage, mainly because the available photo-cofactor in nature is limited in variety, and the gene codon expansion technology is only suitable for introducing specific photosensitive groups into a protein skeleton although the chemical synthesis direction develops various chemical photosensitizers, and is difficult to be used for efficiently screening a large number of different photosensitive groups to adapt to the catalysis requirements of different substrates, and particularly the photosensitizer which can be introduced by the current gene codon expansion technology is still limited to taking ultraviolet light as a reaction driving force, so that abundant visible light resources are difficult to develop and utilize. Disclosure of Invention Aiming at the defects or improvement demands of the prior art, the invention mainly aims to provide application of artificial photo-enzyme CMP in photo-biocatalytic chiral synthesis, wherein an organic photosensitizer is inserted into a natural protein skeleton by utilizing a protein site-directed chemical modification technology to construct the artificial photo-enzyme CMP, and then the CMP is used as a photo-enzyme catalyst to catalyze 2-functionalized alkenyl indole derivatives to generate [2+2] asymmetric light cycloaddition reaction under the condition that visible light is used as reaction driving force to generate chiral cyclobutane condensed tetracyclic indole spiro compounds. The protein site-directed modification technology provided by the invention can develop various artificial photo-enzymes, wherein the CMP artificial photo-enzyme constructed herein can realize efficient chiral photocatalysis through an energy transfer way under the driving force of visible light, and has the characteristics of environmental protection, mild conditions, high reactivity and the like. According to a first aspect of the present invention, there is provided a method for preparing an artificial photo-enzyme, wherein cysteine containing a thiol functional group is introduced into a protein skeleton containing a chiral cav