Novel thiostrepton analogue and its preparation method and use
A technology of thiostrepton and analogs, which is applied in the field of medicine and biotechnology engineering, and can solve the problems of low yield and cumbersome operation
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[0120] Preparation of active ingredients
[0121] The present invention also provides a method for preparing a compound of formula I, comprising the steps of:
[0122] Feed the modified engineering strain (recombinant strain) with the compound of formula II;
[0123] extracting the compound of formula I from the culture;
[0124] Discard the supernatant by centrifugation, soak the cells in acetone, remove insoluble matter by filtration, and dry the filtrate by distillation under reduced pressure. After further purification, the effluent of the compound of formula I was collected to finally obtain the target product.
[0125] The inventor not only confirmed the structure of the compound through mass fermentation and separation and purification of the compound of formula I, but also proved that the antibacterial activity of the active product of the present invention was significantly improved compared with thiostrepton through antibacterial activity experiments.
[0126] Pha...
Embodiment 1
[0147] Synthesis of mutant synthetic element 4-acetyl-6-fluoro-quinoline-2-carboxylic acid
[0148] 1. Synthesis of methyl 6-fluoro-quinoline-2-carboxylate
[0149] Dissolve 10 mmol of 6-fluoro-quinoline-2-carboxylic acid in 10 ml of methanol, slowly drop into 0.92 ml of thionyl chloride in an ice-water bath, slowly warm up to room temperature, and heat to reflux for 12 hours. The reaction solution was cooled to room temperature, poured carefully into a saturated solution of sodium bicarbonate, and then extracted several times with dichloromethane. The extracts were combined, dried with anhydrous sodium sulfate, filtered, concentrated under reduced pressure and separated by column to obtain 1.25 g of the product with a yield of 61%.
[0150] Carry out NMR identification to 6-fluoro-quinoline-2-carboxylic acid methyl ester, identification result is as follows:
[0151] 1 H NMR (400MHz, CDCl 3 )δ8.32 (dd, J=9.2, 5.6, 1H), 8.27 (d, J=8.8, 1H), 8.22 (d, J=8.8, 1H), 7.57 (dt, J...
Embodiment 2
[0171] Construction of Streptomyces Recombinant Strain SL1102
[0172] 1. Construction of tsrT in-frame deletion plasmid
[0173] The primer sequences for cloning the left arm of the in-frame deletion of tsrT are as follows:
[0174] tsrT left arm forward primer (SEQ ID NO: 1)
[0175] 5’-AAG CTT TCC ACG TCT TCC CTC CCA TGG-3’
[0176] tsrT left arm reverse primer (SEQ ID NO: 2)
[0177] 5’-AGA TCT GTC GAT GAC CGC CTT GTA CCC-3’
[0178] The primer sequences for cloning the right arm of the tsrT in-frame deletion are as follows:
[0179] tsrT right arm forward primer (SEQ ID NO: 3)
[0180] 5’-AGA TCT GTC GAC CGG ATG ATG GAA CTC-3’
[0181] tsrT right arm reverse primer (SEQ ID NO: 4)
[0182] 5’-TCT AGA TTC TTG TCC TGG TAC GAG ACC-3’
[0183] Using pS L1001 (cosmid based on pOJ446, including the entire thiostrepton biosynthetic gene cluster) as a template, a PCR reaction system was composed of dNTP, DMSO, enzyme-free water, high-fidelity Primestar DNA polymerase and b...
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