CN115028518A - Preparation and application of 2,2' -oxo-bis-1, 4-di-tert-butylbenzene - Google Patents
Preparation and application of 2,2' -oxo-bis-1, 4-di-tert-butylbenzene Download PDFInfo
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- CN115028518A CN115028518A CN202210757021.2A CN202210757021A CN115028518A CN 115028518 A CN115028518 A CN 115028518A CN 202210757021 A CN202210757021 A CN 202210757021A CN 115028518 A CN115028518 A CN 115028518A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C43/00—Ethers; Compounds having groups, groups or groups
- C07C43/02—Ethers
- C07C43/257—Ethers having an ether-oxygen atom bound to carbon atoms both belonging to six-membered aromatic rings
- C07C43/263—Ethers having an ether-oxygen atom bound to carbon atoms both belonging to six-membered aromatic rings the aromatic rings being non-condensed
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C41/00—Preparation of ethers; Preparation of compounds having groups, groups or groups
- C07C41/01—Preparation of ethers
- C07C41/34—Separation; Purification; Stabilisation; Use of additives
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C41/00—Preparation of ethers; Preparation of compounds having groups, groups or groups
- C07C41/01—Preparation of ethers
- C07C41/34—Separation; Purification; Stabilisation; Use of additives
- C07C41/36—Separation; Purification; Stabilisation; Use of additives by solid-liquid treatment; by chemisorption
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C41/00—Preparation of ethers; Preparation of compounds having groups, groups or groups
- C07C41/01—Preparation of ethers
- C07C41/34—Separation; Purification; Stabilisation; Use of additives
- C07C41/38—Separation; Purification; Stabilisation; Use of additives by liquid-liquid treatment
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention provides a preparation method and application of 2,2' -oxo-bis-1, 4-di-tert-butylbenzene. The preparation method of the 2,2 '-oxo-bis-1, 4-di-tert-butylbenzene comprises the step of extracting 2,2' -oxo-bis-1, 4-di-tert-butylbenzene from marine Prorocentrum micans (Prorocentrum minimum). The 2,2' -oxo-bis-1, 4-di-tert-butylbenzene can be applied to inhibition of toxic protein secretion of gram-negative pathogenic bacteria.
Description
Technical Field
The invention relates to the technical field of biological pharmacy, in particular to preparation and application of 2,2' -oxo-bis-1, 4-di-tert-butylbenzene.
Background
The rise of bacterial drug resistance, the decrease of the discovery amount of new antibiotics, the evolution of drug-resistant bacteria and the horizontal transfer of drug-resistant genes cause the global public health crisis. Although the viability of recent antibiotic research has been re-stimulated, the mechanism of action of most new antibiotics is similar to that of traditional antibiotics. Therefore, there is an urgent need to develop novel antibiotics having different mechanisms of action. The search for an anti-virus drug which does not affect the growth of pathogenic bacteria and can effectively reduce the pathogenicity of pathogenic bacteria is lower than that of a bactericide, so that the research of the current antibacterial drug is the front line. Gram-negative pathogenic bacteria are currently believed to form a specific secretory system that invades host cells during long-term evolution, of which the most prominent is the type iii secretory system (T3SS), which secretes toxic proteins that are the most important virulence factors for many gram-negative pathogenic bacteria. The T3SS inhibitor does not affect the survival of pathogenic bacteria, and has great advantage in drug resistance compared with the traditional antibacterial drugs.
Therefore, finding a T3SS inhibitor that works against a specific bacterium and how to obtain the T3SS inhibitor stably at low cost have become a problem to which wide researchers are now interested.
Disclosure of Invention
The invention aims to provide a preparation method and an application of 2,2' -oxo-bis-1, 4-di-tert-butylbenzene, so as to solve one or more technical problems in the prior art and provide at least one beneficial choice or creation condition.
The first aspect of the present invention is to provide a method for biosynthesizing and extracting 2,2' -oxybis-1, 4-di-tert-butylbenzene. In particular to 2,2' -oxo-bis-1, 4-di-tert-butylbenzene extracted from oceanic Prorocentrum minimum.
Further, the method specifically comprises the following steps:
expanding and culturing marine Prorocentrum micans: aseptically inoculating 5mL of the algal solution into a conical flask containing the culture medium, and performing static culture at 20 ℃ for 11 days under 12-hour light and 12-hour dark every day.
Extraction: centrifuging the cultured marine Prorocentrum micrum micans by using a high-speed centrifuge, and filtering by using a filter membrane of 10 mu m to respectively obtain algae cells. Soaking algae cells in ethyl acetate, concentrating the ethyl acetate crude extract at a temperature of below 50 ℃ under reduced pressure, separating the ethyl acetate crude extract by silica gel column chromatography, collecting 10-50% ethyl acetate/petroleum ether eluent, and equally dividing the eluent into 10 components according to the collection sequence.
Separation: and (3) purifying the component by silica gel column chromatography, and eluting by using methanol-dichloromethane with the volume ratio of 1:100 as an eluent to obtain the 2,2' -oxybis-1, 4-di-tert-butylbenzene.
The compound obtained by separation is 2,2 '-oxybis-1, 4-di-tert-butylbenzene [2, 2' -oxybis (1,4-ditert-butylbenzene)]The molecular formula is C 28 H 42 O, molecular weight 394.32, structural formula as follows:
the conventional method for obtaining 2,2' -oxybis-1, 4-di-tert-butylbenzene is synthesis by a solid-phase photolysis method. The preparation method provides a brand-new preparation method, has low cost and can be applied to industrial production in a large scale.
The second aspect of the invention provides the application of 2,2' -oxybis-1, 4-di-tert-butylbenzene in inhibiting toxic protein secretion of gram-negative pathogenic bacteria.
Further, the gram-negative pathogenic bacterium mentioned in the application includes Vibrio parahaemolyticus.
Further, toxic proteins mentioned in said use are expressed by a type III secretion system.
Further, the type iii secretion system includes a transporter gene VopD1 and/or an effector gene VopS.
The 2,2' -oxo-bis-1, 4-di-tert-butylbenzene can obviously inhibit the expression of a transport protein gene VopD1 and an effector protein gene VopS of marine pathogen vibrio parahaemolyticus under the concentration of 50-100 mu M. Meanwhile, the growth of the marine pathogenic vibrio parahaemolyticus is not influenced, and the toxicity is extremely low.
The inventor unexpectedly finds that the 2,2' -oxybis-1, 4-di-tert-butylbenzene compound has a remarkable inhibiting effect on the expression of T3SS related genes of marine pathogenic vibrio parahaemolyticus, such as a transporter gene VopD1 and an effector gene VopS. T3SS is highly conserved in gram-negative pathogenic bacteria, but is usually deleted in nonpathogenic bacteria, and the research on the T3SS inhibitor has important significance for developing broad-spectrum medicaments for resisting the gram-negative pathogenic bacteria. The development of novel T3SS inhibitors that do not inhibit bacterial growth but inhibit pathogenic bacterial invasion into host cells is the leading edge in the development of novel antibacterial agents. Therefore, the verification test of the invention selects the vibrio gramicus T3SS as a novel antibacterial drug target and has important representative significance.
T3SS is one of the main virulence factors of Vibrio parahaemolyticus, and can transmit virulence factors related to pathogenicity to exert the virulence of pathogenic bacteria. The strategy of the T3SS inhibitor is to inhibit the virulence proteins secreted by the bacteria and prevent the bacteria from infecting host cells, wherein the regulation and control of the gene expression of T3SS is one of effective means for limiting the synthesis and secretion of the virulence proteins of the bacteria. The transporter gene VopD1 and the effector gene VopS are related functional genes of Vibrio parahaemolyticus T3 SS. Therefore, the verification test of the invention selects the VopD1 and the VopS gene of T3SS as the action screening target of the antibacterial drug, and has scientific reasonability and representative significance.
Compared with the prior art, the invention has the following beneficial effects:
1. the 2,2' -oxo-bis-1, 4-di-tert-butylbenzene disclosed by the invention has a remarkable inhibition effect on the expression of T3SS gene of marine pathogenic vibrio parahaemolyticus.
2. The 2,2' -oxo-bis-1, 4-di-tert-butylbenzene disclosed by the invention has no inhibition on the growth of pathogenic bacteria while effectively inhibiting the expression of T3SS gene of marine pathogenic vibrio parahaemolyticus, the compound does not influence the growth of the pathogenic bacteria, but can effectively reduce the pathogenicity of the pathogenic bacteria, the possibility of causing drug resistance is lower than that of a bactericide, and the compound is an antibacterial drug lead compound with a prospect.
3. The 2,2' -oxo-bis-1, 4-di-tert-butylbenzene disclosed by the invention is a natural product, can be obtained by large-scale culture and extraction of marine dinoflagellate, can also be obtained by an artificially mature synthesis process, has reliable and stable sources, has large popularization and application potentials, and has good application prospects in antibacterial drugs.
Drawings
FIG. 1 is a structural diagram of 2,2' -oxybis-1, 4-di-tert-butylbenzene;
FIG. 2 is a bar graph showing the results of inhibition of the expression of Vibrio parahaemolyticus T3SS gene by 2,2' -oxybis-1, 4-di-tert-butylbenzene.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to specific embodiments, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
The 2,2' -oxo-bis-1, 4-di-tert-butylbenzene compound can be separated from algae cells of marine Prorocentrum minimum. The marine Prorocentrum minimum is CCMA-039, purchased from the university of Xiamen. The method comprises the following specific steps:
1.1 preparation of culture medium: 3g of ferric chloride, 4g of sodium ethylene diamine tetracetate, 1mL of 5g/L sodium molybdate, 1mL of 10g/L copper sulfate, 1mL of 20g/L zinc sulfate, 1mL of 10g/L cobalt chloride, 1mL of 180g/L manganese chloride and 1L of deionized water, evenly distributing into 2 1L conical flasks, and sterilizing at 121 ℃ for 30 minutes.
1.2 culturing: sterile procedure 5mL of CCMA-039 algal solution was inoculated into a conical flask containing the medium prepared in step 1.1, and subjected to static culture at 20 ℃ for 11 days with 12 hours of light and 12 hours of darkness each day.
1.3, extraction: and (3) centrifuging the bacterial liquid obtained by culturing in the step 1.2 by using a high-speed centrifuge (3000rpm/min), and filtering by using a 10-micron filter membrane to respectively obtain the algae cells. Soaking algae cells in ethyl acetate, concentrating the ethyl acetate crude extract at the temperature of below 50 ℃ under reduced pressure, separating the ethyl acetate crude extract by silica gel column chromatography, collecting 10-50% ethyl acetate/petroleum ether eluent, and evenly dividing the eluent into 10 components according to the elution sequence.
1.4, separation: and (2) selecting the component 3 extracted in the step 1.3, purifying by adopting 200-300-mesh silica gel column chromatography, eluting by using methanol-dichloromethane as an eluent in a volume ratio of 1:100 to obtain 2,2' -oxo-bis-1, 4-di-tert-butylbenzene, wherein nuclear magnetic data of the component is shown in table 1, and a chemical structure diagram is 1.
TABLE 1 preparation of 2,2' -oxybis-1, 4-di-tert-butylbenzene compound 1 H and 13 CNMR data
Example 2
The 2,2' -oxybis-1, 4-di-tert-butylbenzene in example 1 was subjected to Vibrio parahaemolyticus T3SS inhibitory activity test.
Separating Vibrio parahaemolyticus from sea shrimp, detecting, inoculating into 2216E culture medium, and shaking at 28 deg.C for overnight culture. 2,2' -oxybis-1, 4-di-tert-butylbenzene was dissolved in dimethyl sulfoxide (DMSO) to prepare a solution having a concentration of 10 mM. The solution containing 2,2' -oxybis-1, 4-di-tert-butylbenzene was added to a culture medium containing Vibrio parahaemolyticus as an experimental group, and co-incubated at 28 ℃. The final concentration of 2,2' -oxybis-1, 4-di-tert-butylbenzene was controlled to be 100. mu.M for test group 1 and 50. mu.M for test group 2.
An equal volume of DMSO was added to the control group and an equal volume of 2216E medium was added to the blank group.
FIG. 2 shows the relative expression levels of Vibrio parahaemolyticus T3SS genes in the experimental group (labeled 100. mu.M and 50. mu.M), the control group (labeled DMSO) and the blank group (labeled black). Can be seen in OD 600 When the relative expression quantity reaches 0.8, the relative expression quantity of the VopD1 gene and the VopS of the control group is 1.00 and 0.87 respectively, the relative expression quantity of the blank group is 1.00, and the relative expression quantity of the VopD1 gene and the VopS of the control group and the blank group is not obviously different (p)>0.05), indicating that DMSO as a solvent is not a harmful substance affecting the expression of the Vibrio parahaemolyticus T3SS gene. The relative expression amounts of the VopD1 gene and the VopS of the experimental group treated by the 2,2' -oxybis-1, 4-di-tert-butylbenzene compound are obviously lower than those of the control group, and the difference is obvious (p)<0.05), indicating that the 2,2' -oxybis-1, 4-di-tert-butylbenzene can effectively inhibit the expression of the vibrio parahaemolyticus T3SS gene. Therefore, the 2,2' -oxo-bis-1, 4-di-tert-butylbenzene has good application prospect in the application of novel antibacterial drugs.
It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.
Claims (7)
- A process for producing 2,2' -oxybis-1, 4-di-tert-butylbenzene is characterized by separating it from Prorocentrum micans.
- 2. The method for preparing according to claim 1, characterized by comprising the steps of:soaking Prorocentrum micranthum in ethyl acetate;concentrating the crude extract with ethyl acetate at 50 deg.C under reduced pressure;separating the crude extract by silica gel column chromatography, collecting 10-50% ethyl acetate/petroleum ether eluent, and equally dividing into 10 components in sequence;and (3) purifying the component by silica gel column chromatography, and eluting by using methanol-dichloromethane with the volume ratio of 1:100 as an eluent to obtain the 2,2' -oxo-bis-1, 4-di-tert-butylbenzene.
- Application of 2,2' -oxo-bis-1, 4-di-tert-butylbenzene in inhibiting toxic protein secretion of gram-negative pathogenic bacteria.
- 4. The use according to claim 3, wherein the gram-negative pathogenic bacterium comprises Vibrio parahaemolyticus.
- 5. The use according to claim 3, wherein the toxic protein is expressed by a type III secretion system.
- 6. Use according to claim 5, characterized in that the type III secretion system comprises a transporter gene VopD1 and/or an effector gene VopS.
- 7. The use according to any one of claims 3 to 6, wherein the concentration of the 2,2' -oxybis-1, 4-di-tert-butylbenzene is 50 to 100. mu.M.
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| CN1775723A (en) * | 2005-11-24 | 2006-05-24 | 四川大学 | Bromo-2, 4'-dihydroxy diphenyl ether compound and its synthesizing method |
| CN110283055A (en) * | 2019-07-12 | 2019-09-27 | 浙江大学自贡创新中心 | A kind of compound and preparation method thereof and purposes |
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| Publication number | Priority date | Publication date | Assignee | Title |
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