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Novel fluoronosiheptide and preparation method and application thereof

A technology of flunoxetin and gnostomycin, which is applied in the field of precursor feeding to obtain fluorine-substituted gnostomycin, which can solve the problems of complex synthetic pathways and the failure to develop active gnostomycin.

Active Publication Date: 2012-05-16
SHANGHAI INST OF ORGANIC CHEM CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Structurally, in addition to the characteristic macrocyclic skeleton of thiopeptide antibiotics, Knnostoricin also has a special indole acid side ring, so the synthetic route is very complicated
[0005] In order to improve the antibacterial activity of gnosmycin, the field has been trying to develop various derivatives of gnosmycin, but no derivatives with higher activity have been successfully developed so far.

Method used

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  • Novel fluoronosiheptide and preparation method and application thereof
  • Novel fluoronosiheptide and preparation method and application thereof
  • Novel fluoronosiheptide and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0087] Example 1 The acquisition of Escherichia coli recombinant strain SL4101 and the preparation of MIA

[0088] In this example, the gene sequence encoding NosL in Streptomyces actuosus was amplified by PCR and ligated into the pET28a expression vector, and then the successfully constructed plasmid was introduced into E.coliBL21(DE3) to obtain the recombinant strain SL4101. The expression of NosL in SL4101 was induced for more than 5 hours, and a large amount of MIA (greater than 40 mg / l) was detected in the supernatant of the fermentation broth and bacteria.

[0089] 1. Construction of NosL expression strain SL4101

[0090] The primer sequences for cloning NosL are as follows:

[0091] NosL-forward: 5'-TTGAATTCATGACGCAGAACTCCCAGG-3' (SEQ ID NO: 1)

[0092] NosL-reverse: 5'-TTTAAGCTTTCAGACCGCCCGGGACGCCTC-3' (SEQ ID NO: 2)

[0093] Using the extracted total DNA of active Streptomyces as a template, PCR amplification was performed with the above-mentioned specific primers ...

Embodiment 2

[0109] The acquisition of embodiment 2 fluorinated MIA

[0110] In this example, the SL4101 bacteria expressed for 5 hours were resuspended in a mixed medium formed by mixing conventional LB medium and M9 medium at a ratio of 1:1, and fed with fluorine-substituted tryptophan (5- fluorotryptophan or 6-fluorotryptophan), cultivated for more than 5 hours, can obtain about 10 mg / l of corresponding fluorine-substituted MIA, and the compound is detected by LC / MS ( Figure 4 ), and after separation, purification and characterization, the structure was confirmed.

[0111] In addition, using a method similar to that of Example 1, the SL4101 bacterial solution stored at -80°C was streaked on an LB (kanamycin 50 μg / ml) plate and cultured overnight. Pick single colonies and inoculate them in 3ml LB medium (Kanamycin 50μg / ml) and incubate at 37°C for more than 8 hours, then inoculate in 800ml LB medium (Kanamycin 50μg / ml), and incubate at 37°C for 2.5 hours (A 600nm 0.5-0.7), transferred...

Embodiment 3

[0123] Embodiment 3 Fermentation, detection, separation and purification and activity determination of fludenoxetin

[0124] In this example, the fluorine-substituted tryptophan (5-fluorotryptophan or 6-fluorotryptophan) was fed to Nosiheptide-producing bacteria Streptomyces actuosus to obtain the corresponding fluorine-substituted Nosiella (5'-flunoxetin or 6'-flunoxetin). Methods as below:

[0125]Inoculate a single colony of S.actuosus, a gnosmycin-producing bacterium, into 3ml of conventional TSB medium, culture for 36-48 hours, and inoculate the bacterial liquid into 50ml of TSB medium until the logarithmic growth phase. At this time, inoculate 3-4ml of bacterial liquid into 50ml of pre-fermentation medium (glucose 5g, corn steep liquor 15ml, peptone 5g, yeast extract 1g, malt extract 1g, magnesium sulfate heptahydrate 0.2g, ammonium sulfate 1.5g, calcium carbonate 2.5g, pH=6.8), cultivated at 30°C for 28-36 hours until the cells grow dense. Inoculate 10ml of pre-ferme...

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Abstract

The invention relates to a novel fluoronosiheptide and a preparation method and application thereof, and particularly discloses a method for preparing 3-methyl-2- indolic acid (MIA) and a fluorine-substituted analogue thereof by fermenting on the basis of escherichia coli. The invention further relates to a type of novel fluorine-substituted nosiheptides (5'-fluoronosiheptide and 6'-fluoronosiheptide) and a preparation method and application thereof. Compounds of the type can be obtained by adding fluorine-substituted tryptophan in the fermenting process of a nosiheptide producing bacterium, i.e. streptomycesactuosus. Compared with a nosiheptide, the 5'-fluoronosiheptide has higher anti-bacteria activity, which is one time higher on Gram-negative bacteria bacillus subtilis.

Description

technical field [0001] The invention belongs to the field of biotechnology engineering, and in particular relates to the use of Escherichia coli recombinant strain SL4101 to ferment and prepare 3-methyl-2-indolic acid and its fluorine-substituted analogs, and to obtain fluorine-substituted nosstactin through precursor feeding method. Background technique [0002] Thiopeptide antibiotics are a class of cyclic peptide compounds rich in sulfur and highly modified amino acid residues. Most of them are produced by actinomycetes and have good antibacterial activity. These compounds all have a three- or four-substituted pyridine core, multiple nitrogen heterocycles such as thiazole, oxazole or thiazoline, and dehydrated amino acids, and some have highly modified aromatic ring side chains. [0003] Studies have shown that most thiopeptide antibiotics can inhibit the growth of Gram-positive bacteria, and have a strong killing effect on multi-drug-resistant opportunistic pathogens. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K5/027C12P21/02A61K38/08A61P31/04A61P31/10
CPCY02A50/30
Inventor 刘文张琪陈单丹
Owner SHANGHAI INST OF ORGANIC CHEM CHINESE ACAD OF SCI
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