Wild oxidase mutant and its preparation method and application
A technology of wild oxidase and mutant is applied in the preparation of pharmaceutical intermediates and in the field of medicinal chemistry, and can solve the problems of complex synthesis process, difficult to obtain raw materials, unsuitable for industrial production, etc., and achieve the effect of an efficient preparation method and mild reaction conditions.
Active Publication Date: 2022-05-31
JIANGSU ALPHA PHARM CO LTD
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Problems solved by technology
[0008] Although this method simplifies the process, the raw materials are not easy to obtain, and the synthesis process is also complicated, which is not suitable for industrial production.
Method used
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Embodiment 1
2
0.2mmol / L 20μL, F Primer 1μL, R Primer 1μL, pUC18 template 1pUC18,
Taq enzyme 2U, ddH
2
O make up to 100 μL.
[0038] The PCR amplification program is: 95°C pre-denaturation for 5min; 94°C denaturation for 45s, 55°C denaturation for 45s, and 72°C extension for 45s
30 cycles; continued extension at 72°C for 10 min, and stored at 4°C.
1.3 engineering bacteria are prepared
Select BamH I and XhoI enzyme cleavage site and import above-mentioned PCR amplification product in plasmid pET-30a, primer sequence
[0042] R: ccgctcgagtcaaaatgtcaggacagtgc.
F: gcgggatccatgttgctcaagcaaataaaatatctgt,
[0042] R: ccgctcgagtcaaaatgtcaggacagtgc.
Embodiment 2
Embodiment 3
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The present invention relates to the field of medicinal chemistry, in particular to the field of preparation of pharmaceutical intermediates, more specifically to wild oxidase mutants and their preparation methods and applications. The wild oxidase mutant is an NADH-dependent oxidase, and its amino acid sequence is shown in SEQ ID NO.1. The wild oxidase mutant disclosed by the present invention can efficiently oxidize (4R-cis)-6-formaldehyde-2,2-dimethyl-1,3-dioxane-4-tert-butyl acetate. Not only the reaction conditions are mild, but also the effect of no by-products can be achieved with the participation of the coenzyme system, which is environmentally friendly and is an economical and efficient preparation method.
Description
Wild oxidase mutants and preparation methods and applications thereof technical field The present invention relates to the field of medicinal chemistry, particularly relate to the preparation field of pharmaceutical intermediates, more specifically It relates to a wild oxidase mutant and its preparation method and application. Background technique 2-((4R, 6S)-6-formaldehyde group-2,2-dimethyl-1,3 dioxane-4-base)-methyl acetate is a kind of important Pharmaceutical intermediate compounds. Currently, 2-((4R,6S)-6-carbaldehyde-2,2-dimethyl-1,3dioxane-4-yl)-ethyl The commonly used method of methyl ester is chemical synthesis, mainly including two kinds: Method one: [0004] The method needs to use oxygen oxidation to break the double bond, and the process operation is complicated, the preparation cost is high, and the effective conversion rate is low, The obtained product was not of high purity. At the same time, because the raw materials are not easy to obtain, th...
Claims
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IPC IPC(8): C12N9/02C12N15/53C12N15/70C12N1/21C12P17/06C12R1/19
CPCC12N9/0036C12N15/70C12P17/06C12Y106/99003
Inventor 陈本顺李大伟何伟石利平徐春涛钱若灿叶金星张维冰万新强尹斌徐秋斌
Owner JIANGSU ALPHA PHARM CO LTD