Polycyclic macrocyclic lactam compound as well as preparation method and application thereof

A technology of macrocyclic lactams and compounds, applied in the field of natural medicinal chemistry and microbiology, can solve the problems of weak cytotoxic activity, no report on the activity of inhibiting bacterial virulence protein secretion, and no anti-bacterial involvement, etc., and achieve the effect of strong inhibitory activity

Active Publication Date: 2021-07-16
SHANDONG UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the functions of the compounds provided by these two patents are anti-tumor and anti-fungal, and there is no reference to anti-bacterial aspects
[0005] The published article "Polycyclic Tetramic Acid Macrocyclic Lactam Produced by Streptomyces S001 Metabolism" accidentally activated the host's own PoTeMs gene cluster mtm (GenBank No. MN817126) when expressing a recombinant plasmid in a heterologous host, thereby Obtained the reported compound montamide A, which has weak cytotoxic activity, but no activity of inhibiting bacterial virulence protein secretion
[0006] At present, there is only one literature report on PoTeMs compounds with antibacterial virulence and the preparation of such compounds, namely "Targeted discovery and combinatorial biosynthesis of polycyclic tetramate macrolactam combamides A–E", although combinatorial biosynthesis is also used to biosynthesize combamides The gene cluster cbm (GenBank No. MH167394) was transformed and expressed heterologously, but the cytochrome P450 enzyme gene contained in the engineering bacteria constructed in this paper is cbmD

Method used

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  • Polycyclic macrocyclic lactam compound as well as preparation method and application thereof
  • Polycyclic macrocyclic lactam compound as well as preparation method and application thereof
  • Polycyclic macrocyclic lactam compound as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Construction of embodiment 1 plasmid pUC-cbmA

[0058] Using the fosmid XIV-12H containing the polyketide-nonribosomal peptide synthase gene cbmA as a template, the cbmA-part1 fragment was amplified by PCR, and the primers were designed using bioinformatics software to introduce NdeI and SpeI single enzyme cleavage sites at both ends of the sequence , the primer sequences are as follows:

[0059] NdeI-cbmA-LF:5'-cggacatatgtcggacctatgcaaggtc-3'

[0060] SpeI-cbmA-LR:5'-attactagtctacgccgcatgcgagattgatgtggggtggga-3'

[0061] The PCR amplification system was prepared according to the kit instructions;

[0062] PCR amplification conditions: pre-denaturation, 95°C 5min; denaturation, 95°C 30sec, annealing, 65°C 30sec, extension, 72°C 2min, 30 cycles; final extension, 72°C 5min.

[0063]The obtained PCR product was recovered by a gel recovery kit, and the PCR product and the vector plasmid pUC-kasOp* / ermEp* were digested with NdeI and SpeI respectively, and the obtained cbm...

Embodiment 2

[0064] Example 2 Construction of plasmids pUC-cbmB, pUC-cbmC, pUC-OX4, pUC-sgr810 and pUC-sshg_05717

[0065] Streptomyces sp.S10 genomic DNA was used as a template to amplify the cbmB fragment by PCR, and bioinformatics software was used to design primers to introduce NdeI and SpeI single enzyme cleavage sites at both ends of the sequence respectively. The primer sequences are as follows:

[0066] NdeI-cbmB-F:5'-cggacatatgcggcgtagtaacagggtg-3'

[0067] SpeI-cbmB-R:5'-attactagttcacctcatgctcttgtcg-3'

[0068] Streptomyces sp.S10 genomic DNA was used as a template to amplify the cbmC fragment by PCR, and bioinformatics software was used to design primers to introduce NdeI and SpeI single enzyme cleavage sites at both ends of the sequence respectively. The primer sequences are as follows:

[0069] NdeI-cbmC-F:5'-cggacatatgcctcgcaagccccg-3'

[0070] SpeI-cbmC-R:5'-attactagttcagcggacgtgaacgg-3'

[0071] The OX4 fragment was amplified by PCR using Lysobacter enzymogenes C3 genomi...

Embodiment 3

[0081] Example 3: Construction of expression vectors pSET5035-cbm-OX4-sgr810 and pSET5035-cbm-OX4-sshg_05717

[0082] Plasmids pUC-cbmA, pUC-cbmB, pUC-OX4 and vector plasmid pSET5035-KT were digested with MfeI and SpeI respectively to obtain cbmA-MfeI / SpeI, cbmB-MfeI / SpeI, OX4-MfeI / SpeI fragments and vector fragment pSET5035 -MfeI / PstI.

[0083] The cbmC-XbaI / PstI, sgr810-XbaI / PstI and sshg_05717-XbaI / PstI fragments were obtained from the plasmids pUC-cbmC, pUC-sgr810 and pUC-sshg_05717 respectively after double digestion with XbaI and PstI.

[0084] cbmB-MfeI / SpeI, cbmC-XbaI / PstI and vector fragment pSET5035-MfeI / PstI were ligated by ligase to obtain plasmid pSET5035-cbmB-C.

[0085] Plasmid pSET5035-cbmB-C was digested with XbaI and PstI to obtain cbmB-C-XbaI / PstI fragment; cbmA-MfeI / SpeI, cbmB-C-XbaI / PstI and vector fragment pSET5035-MfeI / PstI were ligated by ligase, Plasmid pSET5035-cbmA-B-C was obtained.

[0086] OX4-MfeI / SpeI, sgr810-XbaI / PstI and carrier plasmid pSET...

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Abstract

The invention relates to a polycyclic macrocyclic lactam compound as well as a preparation method and application thereof. According to the invention, the engineering bacteria for producing the polycyclic macrocyclic lactam compound are constructed, i.e. S001-cbm-OX4-sgr810 and S001-cbm-OX4-sshg_05717, and thereby the preparation of the polycyclic macrocyclic lactam compound is realized.- The polycyclic macrocyclic lactam compound provided by the invention has relatively strong inhibitory activity on secretion of virulence protein of a bacterial type III secretion system and has concentration gradient dependence, and the type III secretion system widely exists in gram-negative pathogenic bacteria and has a highly conservative structure; therefore, the polycyclic macrocyclic lactam compound can be used for preparing drugs for resisting gram-negative pathogenic bacterium virulence protein secretion.

Description

technical field [0001] The invention relates to polycyclic macrocyclic lactam compounds, a preparation method and application thereof, and belongs to the technical field of natural medicine chemistry and microorganisms. Background technique [0002] The emergence of new drug-resistant bacteria and pathogenic bacteria continues to threaten human health and economic development, and the research and development of new active and new mechanism of action antibiotics is imminent. Natural products are important resources for the development of new antibiotics. Polycyclic tetramate macrolactam (PoTeM) is a macrocyclic lactam with tetramic acid structural units and a polycyclic system. compound. The structural diversity of PoTeMs is mainly reflected in the different polycyclic systems, such as 5 / 6 / 5 tricyclic, 5 / 5 / 6 tricyclic and 5 / 5 bicyclic, etc. In addition, the hydroxylation and carbonylation catalyzed by cytochrome P450 enzymes Post-modifications such as epoxidation and epoxi...

Claims

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Application Information

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IPC IPC(8): C07D487/08A61K31/407A61P31/04C12N1/21C12N15/53C12N15/52C12N15/70C12P17/18C12R1/19
CPCC07D487/08C12N9/93C12N9/0004C12N9/0042C12N15/70C12P17/182C12Y603/02C12Y106/02004A61P31/04
Inventor 李瑶瑶高翔颜雅倩
Owner SHANDONG UNIV
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