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CN-121975878-A - Preparation of cycloheptatriene method for preparing ketene derivatives

CN121975878ACN 121975878 ACN121975878 ACN 121975878ACN-121975878-A

Abstract

The invention relates to a method for preparing a tropone derivative, belonging to the technical field of bioengineering. The invention provides a method for preparing a tropone derivative, which is based on a biocatalytic skeleton editing strategy and comprises the steps of taking a phenol derivative as a substrate, taking nitroalkane as an auxiliary substrate, and biocatalyzing the substrate and the auxiliary substrate by using a multi-copper oxidase and/or a microorganism producing the multi-copper oxidase as a catalyst, so that the auxiliary substrate is inserted into the skeleton editing of an aromatic ring substrate as a C1 unit to generate the tropone derivative. The method uses the multi-copper oxidase as a catalyst, uses the simple, easily-obtained, safe and highly stable nitroalkane as a C1 unit, and successfully converts the phenol derivative into the tropone derivative. The method constructs a single-carbon insertion-mediated framework editing biocatalysis strategy, which breaks through the limitation of the traditional carbene insertion reaction, and has great application prospect in framework modification in the fields of chemistry and biocatalysis.

Inventors

  • WANG YAJIE
  • JIANG BO
  • LIU XIAO
  • GAO HONGXUN
  • HUANG JIAXIN

Assignees

  • 西湖大学

Dates

Publication Date
20260505
Application Date
20251217

Claims (10)

  1. 1. A method for preparing a tropone derivative is characterized by comprising the steps of taking a phenol derivative as a substrate, taking nitroalkane as a cosubstrate, and biocatalyzing the substrate and the cosubstrate by using a multi-copper oxidase and/or a microorganism producing the multi-copper oxidase as a catalyst, so that the cosubstrate is inserted into a skeleton of an aromatic ring substrate as a C1 unit for editing, thereby generating the tropone derivative.
  2. 2. The method of claim 1, wherein the phenol derivative has the structure: ; the nitroalkane has the following structure: ; the tropone derivative has the following structure: ; Wherein R 1 is a substituted sulfonamide compound, R 2 is a hydrogen atom, a single halogen atom, a double halogen atom or a phenyl group, and R 3 is a hydrogen atom, a methyl group, a benzyl group or a hydroxymethyl group; Preferably, the substituent of the sulfonamide compound is a substituted phenyl group, an unsubstituted naphthyl group, a thiophene or a methyl group, and the substituent of the phenyl group is a halogen atom, a methyl group or a trifluoromethyl group.
  3. 3. The method according to claim 1 or 2, wherein the multicopper oxidase is a wild-type multicopper oxidase and/or a multicopper oxidase mutant, wherein the amino acid sequence of the wild-type multicopper oxidase is shown in SEQ ID NO.1, and wherein the multicopper oxidase mutant has a mutation of lysine at position 474 to phenylalanine and/or a mutation of isoleucine at position 500 to leucine compared to the wild-type multicopper oxidase having the amino acid sequence shown in SEQ ID NO. 1.
  4. 4. A method according to any one of claims 1 to 3, wherein the method comprises mixing a substrate and a co-substrate with a co-solvent to obtain a mixed substrate system, suspending the catalyst in a buffer to obtain a catalyst system, mixing the mixed substrate system and the catalyst system to obtain a biocatalysis system, and reacting the biocatalysis system at a pH of 7 to 9 and a temperature of 20 to 25 ℃ for 12 to 36 hours to obtain the tropone derivative.
  5. 5. The method of claim 4, wherein the concentration of the substrate in the biocatalysis system is 10-20 mmol/L, the concentration of the cosubstrate in the biocatalysis system is 50-100 mmol/L, and the mixing volume ratio of the mixed substrate system and the biocatalysis system is 1-2:9; when the catalyst is a microorganism producing the multi-copper oxidase, the density OD value of the catalyst in the biocatalysis system is 5-40.
  6. 6. A multi-copper oxidase mutant is characterized in that compared with a wild-type multi-copper oxidase with an amino acid sequence shown as SEQ ID NO.1, the multi-copper oxidase mutant has a lysine mutation at 474 as phenylalanine and/or an isoleucine mutation at 500 as leucine.
  7. 7. A nucleic acid molecule encoding the multiple copper oxidase mutant of claim 8.
  8. 8. A recombinant plasmid expressing the multicopper oxidase mutant of claim 8, or carrying the nucleic acid molecule of claim 9.
  9. 9. A host cell expressing the multicopper oxidase mutant of claim 8, or carrying the nucleic acid molecule of claim 9, or carrying the recombinant plasmid of claim 10.
  10. 10. Use of the method of any one of claims 1 to 5 or the multicopper oxidase mutant of claim 6 or the nucleic acid molecule of claim 7 or the recombinant plasmid of claim 8 or the host cell of claim 9 or the multicopper oxidase in the preparation of a tropone derivative.

Description

Preparation of cycloheptatriene method for preparing ketene derivatives Technical Field The invention relates to a method for preparing a tropone derivative, belonging to the technical field of bioengineering. Background The molecular skeleton editing technology is used as an important tool for developing modern medicines, can directionally reform the structure of a compound through accurate modification (insertion, deletion or replacement) on an atomic level, realizes molecular post-modification, optimizes physicochemical properties and biological activity of the compound while maintaining core skeleton characteristics, and provides an efficient and sustainable alternative path for constructing a compound library with diversified structures. Among them, single atom insertion technology (especially single carbon insertion) for ring compound molecules has been attracting attention because of the ability to rapidly construct potentially bioactive ring-expanding molecules. The origin of this field can be traced back to classical transformations such as CIAMICIAN-DENNSTEDT and the Buchner rearrangement reaction, which effect carbon atom insertion of pyrrole and benzene rings by a carbene insertion mechanism. In recent years, chemists have developed a variety of more valuable and widely applicable carbene precursor reagents (such as α -chlorobisaziridine, trifluoromethyl N-trifluoromethanesulfonyl hydrazone, α -iodonium diazonium compound, etc.) as C1 units based on the above reaction mechanism, and have precisely achieved single-carbon insertion framework editing of ring compounds. While the development of these carbene precursor reagents plays a critical role in the development of framework editing of ring compounds, the conventional carbene insertion strategy has obvious limitations, such as the fact that the carbene precursor needs to be prepared in advance and has poor stability, harsh reaction conditions, complex operation and other problems seriously limit the practical application of the technology. Thus, exploring new non-carbene insertion mechanisms, replacing traditional carbene precursors with simple and readily available bulk materials as new C1 units to achieve single carbon insertion of ring compounds is a very challenging and valuable study. Meanwhile, the enzyme engineering technology has made great progress in the past ten years, and through means of directed evolution and the like, the modification of enzymes to catalyze non-natural chemical transformation is realized, and the gap between chemical catalysis and biological catalysis is effectively closed. For example, cytochrome P450 mediated carbene and nitrene transfer reactions, non-heme iron enzyme catalyzed olefin difunctional, and enzyme catalyzed visible light driven radical conversion depending on nicotinamide, flavin, thiamine and pyridoxal-5' -phosphate, and the like. Although biosynthetic strategies provide a powerful platform for achieving certain complex chemical transformations, there is a significant lack of research in molecular scaffold editing. Current research has focused mainly on the insertion of a single oxygen atom into the cyclic backbone catalyzed by Baeyer-Villiger monooxygenase (BVMO). In contrast, single carbon insertion strategies remain in the initiation phase, with only sporadic reports of natural ring-expansion by intramolecular carbon radical rearrangement. However, biocatalytically mediated intermolecular single carbon insertion to achieve skeletal remodeling has not been explored so far. In conclusion, the inherent limitation of the traditional carbene insertion is broken through, a novel intermolecular single carbon insertion method based on a biocatalysis system is developed, the boundary of a synthetic chemical tool can be expanded, an innovative strategy can be provided for the directional optimization of a drug lead compound, and important theoretical and application values are achieved. The breakthrough in the direction will promote the skeleton editing technology to develop in the direction of high efficiency, accuracy and sustainability. Disclosure of Invention In order to solve the above problems, the present invention provides a method for preparing a tropone derivative, comprising biocatalyzing a substrate and a cosubstrate with a phenol derivative as a substrate and nitroalkane (non-carbene C1 synthon) as a cosubstrate using a multi-copper oxidase (MCOs) and/or a multi-copper oxidase-producing microorganism as a catalyst, such that the cosubstrate is inserted as a C1 unit into the backbone editing of an aromatic ring substrate to produce the tropone derivative. In one embodiment of the invention, the phenol derivative has the structure shown below: ; the nitroalkane has the following structure: ; the tropone derivative has the following structure: ; Wherein R 1 is a substituted sulfonamide compound, R 2 is a hydrogen atom, a single halogen atom, a double halogen atom or a phenyl group, and R 3 is a