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Biological enzyme catalysis synthesis method for 6-bromoindanone

A synthesis method and technology of bromindanone are applied in the field of preparing medicines by using enzyme mutation technology, and the effects of high product rate, high yield and high purity are achieved

Pending Publication Date: 2020-12-04
JIANGXI BONTAC GREEN BIOCATALYSIS ECOIND PARK CO LTD
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  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The work of transpeptidase catalyzing pluripotency has been widely reported, including aldol reaction, Michael reaction, Mannich reaction, Markov addition reaction and Henry reaction, etc. The present invention intends to utilize biological enzyme mutation technology to obtain a kind of enzyme that can effectively catalyze Fu The gram reaction obtains the mutant type transpeptidase of 6-bromoindanone, and the technical report of obtaining 6-bromoindanone by about the transpeptidase mutation as yet in the prior art

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  • Biological enzyme catalysis synthesis method for 6-bromoindanone
  • Biological enzyme catalysis synthesis method for 6-bromoindanone

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Embodiment 1

[0033] Preparation of mutants of SEQ ID NO.1: mutant library construction and high-throughput screening methods:

[0034] Construction of mutant library:

[0035] In order to improve the activity of transpeptidase, using the recombinant expression vector PET-28a(+)-BS as the DNA template, a random mutant library was constructed by error-prone PCR, and the concentration of Mg2+ and Mn2+ in the error-prone PCR reaction system was adjusted And the concentration of dCTP and dTTP oligonucleotides, so that the base mismatch rate of the mutant library is 10 per thousand, that is, to ensure that a mutant has 2 to 6 amino acid mutations. The specific process of constructing the mutant library is as follows. Error-prone PCR reaction system and conditions:

[0036] Error-prone PCR reaction system:

[0037] 10×Buffer 8μL 2mmol / L dNTPS 3μL 100mmol / L dCTP 0.7μL 100mmol / L dTTP 0.7μL 10mmol / L MnCl 2μL 50mmol / L MgSO4 2μL Primer T7 promoter ...

Embodiment 2

[0043] Add 229g of p-bromophenylpropionic acid (1.0mol) into 1000ml of methanol, stir to fully dissolve, then add 200ml of 5mM potassium dihydrogen phosphate buffer solution (pH=2.0), 0.95KU of mutated transpeptidase, room temperature Stir under low pressure for 20 hours, evaporate the organic solvent to dryness, filter to obtain the crude product, and then recrystallize with methanol / water to obtain 197.50 g of fine 6-bromoindanone, with a conversion rate of 93.83%.

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Abstract

The invention relates to a biological enzyme catalysis synthesis method for 6-bromoindanone, and belongs to the field of biological pharmacy. Transpeptidase site-specific mutagenesis type transpeptidase with a SEQ ID NO.2 amino acid sequence has an amino acid sequence disclosed by SEQ ID NO.1, then, propionic acid is added into methyl alcohol to be stirred and fully dissolved, monopotassium phosphate buffer solution of which the pH is 2.0 and 0.95KU site-specific mutagenesis type transpeptidase are added and are stirred for 20h at a room temperature, and after solvent is dried by distillation,filtering is carried out to obtain a crude product; and then, through methyl alcohol / water recrystallization, a 6-bromoindanone competitive product is obtained. An active transpeptidase enzyme prepared by an enzymatic mutation technology is adopted to replace a traditional chemical synthesis process. A reaction condition is moderate and simple, and extremely few three wastes are generated. Compared with a traditional chemical synthesis method, the biological enzyme catalysis synthesis method has higher synthesis efficiency, high yield and high purity.

Description

technical field [0001] The invention belongs to the field of biopharmaceuticals, and in particular relates to a method for preparing medicine by adopting enzyme mutation technology. [0002] The method of intermediates. Background technique [0003] The indene ring structure exists in molecules such as natural products, synthetic drugs, and pesticides. Indenone as a raw material for the synthesis of biologically active compounds has strong industrial application prospects. It is also used in the synthesis of organic light-emitting materials and dyes. It is also used as a Organic protecting groups for removal by visible light. [0004] There are more than 100 naturally occurring indanone compounds. Molecules with indanone structure show various biological activities, such as smooth muscle relaxation activity, cyclooxygenase inhibitor, etc. The growth activity of vascular endothelin has strong applications in the regulation of tumor angiogenesis. However, the natural extrac...

Claims

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Application Information

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IPC IPC(8): C12N9/10C12N15/10C12P7/38
CPCC12N9/104C12N15/1031C12P7/38C12Q2531/113
Inventor 谢志军
Owner JIANGXI BONTAC GREEN BIOCATALYSIS ECOIND PARK CO LTD
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