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Rapamycin structural analogue and preparation method thereof

A technology of methyl rapamycin and crude extract, applied in the field of combined biosynthesis, can solve problems such as stereoisomerism, and achieve the effects of good antifungal effect, simple process and low cost

Active Publication Date: 2016-12-07
国药集团健康产业研究院有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] At present, the chemical synthesis or group structure modification of rapamycin structural analogs is faced with many technical difficulties such as stereoisomerism and selectivity. Therefore, there is an urgent need for a simple, low-cost, and environmentally friendly synthetic method to Synthesis of Structural Analogs of Rapamycin

Method used

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  • Rapamycin structural analogue and preparation method thereof
  • Rapamycin structural analogue and preparation method thereof
  • Rapamycin structural analogue and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Structural analysis of functional domains of polyketide synthase rapA from Actinomycetes molluscs.

[0042]Using http: / / nrps.igs.umaryland.edu / nrps / _ to predict the functional domain of polyketide synthase rapA, the results are shown in Table 1:

[0043] Table 1

[0044]

[0045] The rapA gene contains a loading module and four chain extension modules, and the layout of each module is shown in figure 1 shown. Act_rapA refers to Actinomycetes rapA gene, CL is carboxylic acid ligase, AT is acyltransferase domain, KS is ketosynthase domain, DH is dehydrogenase domain, ER is enoyl reductase domain , KR is the ketoreductase functional domain, and ACP is the acyl carrier protein functional domain.

Embodiment 2

[0047] Obtaining a homologous double-crossover strain with a point mutation in the ER functional domain of Actinomycetes mobilis rapA.

[0048] The schematic diagram of the point mutation of the ER functional domain of Actinomycetes rapA is shown in figure 1 . Two pairs of primers ERIAUs-ERIAUas and ERIADAs-ERIADAas were used to amplify the two fragments upstream and downstream of the mutation site of the ER functional domain of Actinomyces mobilis rapA gene, respectively, and then the restriction endonuclease contained in the introduced mutation site NheI joins the two fragments.

[0049] A) Extraction of Actinomycetes mobilis N902-109 genome:

[0050] Take a small amount of mycelium of Actinomycetes mobilis N902-109 and inoculate it into a test tube containing 5 mL of YEME medium, and culture it with shaking at 30°C for about 72 hours. The mycelium was collected by centrifugation at 3500 rpm, washed twice with TES (10 mM Tris-HCl pH 7.5, EDTA 1 mM, NaCl 50 mM). Add 1mL T...

Embodiment 3

[0085] Preparation and Identification of Structural Analogues of Rapamycin by Fermentation

[0086] A) Fermentation of Actinomycetes N902-109ERIA

[0087] Put the actinomycetes N902-109ERIA droplet preserved in the glycerin tube into the slant of the actinomycetes fermentation slant medium, spread it evenly with an inoculation rod, and culture at a constant temperature of 28°C for 7-9 days. Use the inoculation shovel to scrape the slant mycelia and insert it into the 30ml first-level seed culture medium, and at 28° C., 220 revolutions for constant temperature cultivation for 4-5 days, then receive the second-level seed culture medium by 10% of the inoculum size, and the percentage is accounted for. The volume percentage of secondary seed culture medium volume, 28 ℃ 220 rpm constant temperature culture 3-4 days, then receive in the fermentation bottle that secondary seed culture medium is housed according to inoculum size 7.5%-10%, described percentage is Accounting for the vo...

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Abstract

The invention provides a rapamycin structural analogue and a preparation method thereof. The rapamycin structural analogue is as shown in a formula I. The preparation method comprises the following steps of performing fermentation culture on actinoplanes containing polyketide synthase rapA mutant genes, and obtaining the rapamycin structural analogue from fermentation liquor, wherein the polyketide synthase rapA mutant genes are as shown in a sequence table SEQ ID No.1. Compared with a conventional chemical synthesis method, the preparation method of the rapamycin structural analogue provided by the invention is simpler to operate, lower in cost, and smaller in environmental pollution; and besides, the rapamycin structural analogue prepared by the preparation method has good anti-fungal effects.

Description

technical field [0001] The invention relates to the field of combinatorial biosynthesis, in particular to a structural analog of rapamycin and a preparation method thereof. Background technique [0002] Rapamycin is a 31-membered ring nitrogen-containing triene macrolide compound, which is an important clinically used immunosuppressant and an intermediate in the development of anti-tumor drugs. Temsirolimus, everolimus and zotarolimus are all semi-synthetic structural analogs of rapamycin, which were approved by the FDA in 2004, 2005, and 2007 as anti-tumor drugs and coating drugs for cardiovascular stents. [0003] Among natural products, except for some compounds that can be used as drugs themselves, most of them are used as lead compounds through group modification to modify their drug-making properties and become semi-synthetic drugs for clinical application. For the chemical synthesis or group structure modification of most natural compounds with complex structures, th...

Claims

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Application Information

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IPC IPC(8): C07D498/18C12P17/18A61P31/10A61P31/02
Inventor 胡海峰黄鹤高苹赵琪
Owner 国药集团健康产业研究院有限公司
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