CN114788872A - Compound for preventing, preventing or treating microbial infection and preparation and application thereof - Google Patents

Compound for preventing, preventing or treating microbial infection and preparation and application thereof Download PDF

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Publication number
CN114788872A
CN114788872A CN202210484471.9A CN202210484471A CN114788872A CN 114788872 A CN114788872 A CN 114788872A CN 202210484471 A CN202210484471 A CN 202210484471A CN 114788872 A CN114788872 A CN 114788872A
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acid
soluble
group
water
virus
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赵一麟
周媛媛
刘凤武
周旭
陈炜斌
刘弘毅
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Sun Rainforest Beijing Biomedical Co ltd
Zhongshan Hospital Xiamen University
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Sun Rainforest Beijing Biomedical Co ltd
Zhongshan Hospital Xiamen University
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Priority to CN202210484471.9A priority Critical patent/CN114788872A/en
Publication of CN114788872A publication Critical patent/CN114788872A/en
Priority to PCT/CN2023/092349 priority patent/WO2023213310A1/en
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Abstract

The present invention relates to a complex for the prevention, prevention or treatment of microbial infection, comprising an acting portion, a binding portion and a water soluble portion; the active part is fat-soluble saturated and/or unsaturated carbon chains with branched chains, cyclic structures and/or straight-chain structures, and can be inserted into/fused into a lipid membrane of a microorganism to destroy the lipid membrane or wrap non-enveloped viruses to be isolated by hydrophobicity; the binding part can be combined with the lipid membrane component or the virus surface protein structure domain, so that the compound is connected to the surface of the lipid membrane or the virus, and can also specifically target the lipid membrane component or the virus surface protein structure domain to endow targeting; the water-soluble portion allows the complex to be uniformly dispersed in an aqueous solution and prevents the active portion from aggregating into a mass. The compound can be made into various dosage forms, can be used before infection to prevent or prevent the infection of microorganisms such as virus, bacteria and fungi, can be used after infection to kill in vivo microorganisms, and can also be used for killing environment to prevent the spread of virus, bacteria and fungi.

Description

Compound for preventing, preventing or treating microbial infection and preparation and application thereof
Technical Field
The invention relates to the field of pharmacy, in particular to a group of compounds capable of preventing and treating viral, bacterial and fungal infections, a preparation method of the compounds, and application of the prepared compounds in preventing and treating viral, bacterial and fungal infectious diseases.
Background
Among the numerous microorganisms, the pathogenic microorganisms that can directly cause human diseases are generally viruses, bacteria, and fungi. Except a few non-enveloped viruses, most microorganisms have lipid membranes, the lipid membranes of the microorganisms have the same functions with cell membranes of other organisms, the microorganisms structurally form a basic scaffold of membranes by phospholipid bilayers, and proteins penetrate, are inserted and are attached to the surfaces of the phospholipid bilayers. The outer surface of the membrane has protein, glycoprotein composed of a small amount of polysaccharide, and a part of carbohydrate combined with lipid to form glycolipid. The lipid membrane of the microorganism is usually 7-8 nm, has certain fluidity, not only serves as a barrier to create a stable internal environment for the life activities of the microorganism, but also has semi-permeability or selective permeability, namely substances are selectively allowed to enter cells through modes such as diffusion, permeation and active transportation, and therefore the normal metabolism of the cells is guaranteed. The invention relates to a group of complexes and preparations thereof for destroying and influencing the structure and function of microbial lipid membrane or non-enveloped virus nucleocapsid in a targeted way.
1. Virus
1.1 enveloped and non-enveloped viruses
Viruses (viruses) are a minimal class of infectious particles in which one or more nucleic acid (DNA or RNA) molecules are encapsulated by a protein coat, and are non-cellular microorganisms that must replicate themselves in susceptible living cells. Outside the cell, the virus exists in particulate form, and structurally intact infectious viral particles are called virions. The viral genome is surrounded by a protein coat, which is called the nucleocapsid, and the protein coat is called the capsid. The basic structure of a virion is the nucleocapsid, but some viruses have a double lipid envelope outside the nucleocapsid, and such viruses are called enveloped viruses, while viruses without an envelope are correspondingly called naked viruses.
1.2 viral envelope Structure and function
The envelope is the cytoplasmic membrane that is obtained when the virus is released from the host cell, and may also be the membrane of the intracellular apparatus or nucleus, and thus the viral envelope possesses certain properties of the host cell membrane that make the virus exhibit a specific "tropism" for the host cell membrane. The envelope contains double-layer lipid, and some proteins synthesized by viral gene coding, called envelope proteins, have virus specificity, usually form glycoprotein subunits with polysaccharides, are embedded in lipid layer, and have Spike-shaped protrusions on the surface, called Spike (Spike) or capsule microparticles. They are located on the surface of the virion, are highly antigenic, and selectively bind to host cell receptors, facilitating fusion of the viral envelope with the host cell membrane, and intracellular entry of the infectious nucleocapsid leading to infection. Thus, the envelope proteins of enveloped viruses determine the infectivity of the virus, whereas the nucleocapsid of an enveloped virus is the viral core and is present alone without infectivity even if the envelope is lost.
1.3 Structure and function of non-enveloped viruses
Because the naked virus has no envelope, the nucleocapsid of the naked virus is mature virus, and the infectivity of the naked virus is determined by capsid protein. Capsid protein is a viral gene product that confers the inherent shape of the virus and protects internal nucleic acids from the destruction by nucleases in the external environment (e.g., blood); meanwhile, capsid protein has the function of auxiliary infection, and the binding protein of virus surface specific receptor side has special affinity with the corresponding receptor on the cell surface, which is the first step of selectively adsorbing host cells and establishing infection focus by virus; the capsid protein also presents virus-specific antigenicity and can stimulate the body to generate antigen virus immune response.
1.4 types of enveloped viruses
The enveloped virus includes influenza virus, coronavirus, AIDS virus, hepatitis B virus, hepatitis C virus, rabies virus, herpesvirus, Ebola virus, hantavirus, dengue virus, encephalitis B virus, Secat virus, etc.
The immune system relies on proteins on the cell membrane to recognize enemies, and enveloped viruses are recognized as own by the host immune system due to the addition of a lipid membrane. The glycosylation modification of envelope protein, on one hand, plays the role of antigen shielding, which leads to more difficult vaccine development; on the other hand, modified glycans also have a spatial remodeling effect on the antigen epitope structure.
1.5 coronavirus
Coronavirus: a total of 7 human-infectable coronaviruses are currently found, HCoV-229E, HCoV-OC43, SARS-CoV, HCoV-NL63, HCoV-HKU1, MERS-CoV and SARS-CoV-2, respectively. The coronavirus diameter is about 60-220 nm. Viruses have an envelope structure with three proteins: spike glycoproteins (S proteins), small envelope glycoproteins and membrane glycoproteins (M proteins), and a few classes of hemagglutinin glycoproteins (HE proteins). The S protein plays a key role in identifying and combining with a host cell surface receptor and mediating the fusion process of a virus envelope and a cell membrane; the M protein participates in the formation and budding process of the virus envelope; HE proteins are short bulges that make up the envelope and may be involved in early attachment of coronaviruses, and some of these can cause agglutination and attachment of red blood cells.
1.6 treatment of coronaviruses
The currently clinically researched new coronary pneumonia vaccines and treatment medicines are mainly divided into the following four types:
the first is small molecule antiviral drug: including Molnupiravir from mesua, Paxlovid from fei rui, Ensitrelvir from yaje, and the marketed drugs rituxivir, lopinavir/ritonavir, favipiravir, and the like. Although small-molecule drugs such as lopinavir/ritonavir and the like are widely used for antiviral therapy, the small-molecule drugs are not specific drugs for treating neocoronary pneumonia.
Secondly, anti-inflammatory drugs: various biological agents are used to inhibit inflammatory factor storm, such as tositumumab (Tocilizumab), cetuximab (Siltuximab), etc.; there are also some clinical trials of small molecule anti-inflammatory drugs, such as Baricitinib (Baricitinib), ruxotinib (Ruxolitinib), etc.
Thirdly, neutralizing antibody: refers to an antibody that is capable of abolishing the ability of a virus to infect upon binding to the virus. The action mechanism is to change the surface configuration of the virus, prevent the virus from being adsorbed on susceptible cells and prevent the virus from penetrating into the cells to proliferate; the immune complex formed by the virus and the neutralizing antibody is easy to be phagocytized and eliminated by macrophages.
Fourthly, vaccine: comprises five kinds of recombinant protein vaccines, nucleic acid vaccines, virus vector vaccines, inactivated vaccines, attenuated live vaccines and the like.
1.7 non-enveloped viruses
Non-enveloped naked viruses include hepatitis A virus, human papilloma virus, adenovirus, poliovirus, coxsackievirus, and the like.
Human Papillomavirus (HPV) belongs to the papillomavirus genus of the papillomaviraceae family, is a spherical double-stranded DNA virus without an envelope, and has the diameter of 52-55 nm. The viral genome is double-stranded circular DNA with a length of about 7.8-8.0 kb and is divided into an early region, a late region and a regulatory region. The early region encodes proteins involved in viral replication, transcriptional regulation and cellular transformation (e.g., E5, E6, E7), and the late region encodes major capsid protein L1 and minor capsid protein L2. At present, more than 130 types are separated, different types cause different clinical manifestations, and the types can be classified into skin low-risk type, skin high-risk type, mucous membrane low-risk type and mucous membrane high-risk type according to different invading tissue parts. The infection of the HPV population of the skin type is very common, such as common warts, toe warts, flat warts and the like, but specific infection rate cannot be obtained. Of comparative interest are genital warts and cervical cancer caused by high risk HPV infection and low risk HPV infection of the external genitalia.
1.8 HPV prevention and treatment
At present, prophylactic nine-valent vaccines, tetravalent vaccines and the like exist internationally, can prevent the four virus types from being infected, including 16 and 18 types capable of causing cervical cancer lesions, so that most of cervical cancer can be reduced, and some scientific researches show that the vaccine has certain protection on other types. However, there is no prophylactic vaccine effect on already infected persons, and no effective therapeutic vaccine is currently available.
2 bacteria
The basic structure of bacteria includes cell wall, cell membrane, cytoplasm, and nucleoplasm.
2.1 bacterial cell membranes
The cell membrane of the bacteria is an elastic semipermeable membrane consisting of phospholipid bilayers and mosaic proteins. The thickness of the membrane is 8-10 nm, and the outer side of the membrane is tightly attached to the cell wall. The absence of cholesterol in bacterial cell membranes is a point of distinction from eukaryotic cell membranes. Bacterial cell membranes contain a rich array of enzymes that perform many important metabolic functions. The multifunctionality of bacterial cell membranes is a very remarkable feature different from other cell membranes, such as enzyme systems containing electron transfer and oxidative phosphorylation on the inner side of cell membranes and having partial functions of executing mitochondria of eukaryotic cells.
Structural characteristics of bacterial cell membranes: the membrane has main body of lipid bilayer, lipid bilayer has fluidity, integrin is soluble in hydrophobic inner layer of lipid bilayer due to hydrophobic surface, peripheral protein surface contains hydrophilic group, lipid molecule or lipid and protein molecule are not covalently combined together, lipid bilayer is just like sea, peripheral protein can float on it, integrin is immersed in ice mountain and moves transversely.
Physiological function of bacterial cell membrane: the membrane contains enzyme system related to oxidative phosphorylation or photosynthetic phosphorylation energy metabolism, so that the energy production base of the cell is the implantation part of flagellar body and can provide energy required by flagellar rotation movement.
2.2 bacterial cell walls
The major component of the cell wall is peptidoglycan, also known as mucin. The peptidoglycan is a polysaccharide scaffold formed by connecting two amino sugars of N-acetylglucosamine and N-acetylcytosine at intervals through beta-1, 4 glycosidic bonds. The N-acetylmuramic acid molecule is connected with a tetrapeptide side chain, and peptide chains are connected by a peptide bridge or a peptide chain to form a mechanically strong net structure.
2.2.1 gram-Positive bacteria
The cell wall of the gram-positive bacteria is thick and about 20-80 mm. The content of peptidoglycan is rich, 15-50 layers are provided, and each layer is 1nm thick and accounts for 50-80% of the dry weight of cell walls. In addition, there are a number of specific components, teichoic acids. Teichoic acid has strong antigenicity and is an important surface antigen of gram-positive bacteria; plays a role in regulating the passage of ions through the mucin layer; may also be associated with the activity of certain enzymes; teichoic acids of certain bacteria, which adhere to the surface of human cells, act like fimbriae and may be associated with pathogenicity.
2.2.2 gram-negative bacteria
Gram-negative bacteria have a cell wall with multiple structures. The cell wall is thin, about 10-15 nm, and 1-2 layers of peptidoglycan are contained, which accounts for about 5-20% of the dry weight of the cell wall; the cell wall is surrounded by a bacterial outer membrane formed of proteins, phospholipids and lipopolysaccharides. The outer membrane has a lower phospholipid content than the cytoplasmic membrane, but a higher lipopolysaccharide content. The outer membrane proteins are different from cytoplasmic membranes in that the proteins on the outer membrane are covalently linked at one end to the tetrapeptide side chains of peptidoglycan with a protein moiety and at the other end to the phosphate of the outer membrane with a lipid moiety via a covalent bond. Its function is to stabilize the outer membrane and to immobilize it to the peptidoglycan layer. Lipopolysaccharides, known as bacterial endotoxins, are present in the outermost layer of the outer membrane. The outer membrane is the main structure of the cell wall of gram-negative bacteria, has barrier function besides transferring nutrient substances, can prevent various substances from permeating and resist the action of a plurality of chemical drugs.
2.3 antibiotics
Antibiotics are mainly secondary metabolites or artificially synthesized analogues produced by bacteria, molds or other microorganisms, and are mainly used for treating various bacterial infections or diseases caused by pathogenic microorganisms, and generally do not have serious side effects on the hosts. The action mechanism of antibiotics is generally to block the synthesis of bacterial cell walls, so that bacteria swell, rupture and die in a low-osmotic environment; interacting with the bacterial cell membrane, enhancing the permeability of the bacterial cell membrane, opening an ion channel on the membrane, and leading useful substances in the bacteria to leak out of thalli or leading electrolyte to be imbalanced and killed; used in conjunction with bacterial ribosomes or their reaction substrates (e.g., tRNA, mRNA), inhibit protein synthesis, resulting in the inability to synthesize structural proteins and enzymes necessary for cell survival; the replication and transcription of bacterial DNA are hindered, and the processes of bacterial cell division, propagation and transcription and translation into protein are hindered.
2.4 antibiotic resistance
It is well known that extended, high dose, prolonged use of antibiotic drugs can lead to the development of drug resistance. In order to deal with pathogenic microorganisms, human beings have been developing new antibiotics, and microorganisms such as bacteria gradually adapt to the drug environment for survival, and continuously generate variation to form new and stronger bacteria, so that the cycle is repeated. Even multiple resistant bacteria appear, i.e. one bacteria is resistant to three or more antibiotics simultaneously. Further studies have found that the development of drug resistance in bacteria is due to the presence of drug resistance genes in the body. NDM-1 is a new super drug-resistant gene discovered by scientists, encodes a new drug-resistant enzyme NDM-1, is totally called 'New Delhi metallo-beta-lactamase 1', is a high-efficiency enzyme, and can decompose most antibiotics to cause the antibiotics to lose efficacy. The drug-resistant gene can not only enable the bacteria to generate drug resistance, but also can be spread in the environment and transferred to other bacteria to become drug-resistant bacteria resistant antibiotics. Because few new antibiotics are available at present, the existing antibiotics can not effectively kill drug-resistant bacteria, once the drug-resistant bacteria are infected, the death risk of patients is greatly increased, and the death rate of patients infected with drug-resistant bacteria is about 2 times that of patients infected with non-drug-resistant bacteria. Infection and transmission problems of drug-resistant bacteria thus become a major challenge in contemporary medical fields.
3. Fungi, chlamydia and mycoplasma
The basic structure of fungal cells is the cell wall, cell membrane, nucleus, endoplasmic reticulum, mitochondria, and the like. The main component of the fungal cell wall is chitin, and the fungal cell membrane is also composed of phospholipid bilayers, but sterol is contained in the plasma membrane, and the ergosterol plays an important role in keeping the permeability, the fluidity and the like of the membrane. Antifungal drugs are of three classes: polyenes (amphotericin B formulations), triazoles (voriconazole, itraconazole, posaconazole) and echinocandins (caspofungin, micafungin, anidulafungin).
Chlamydia is a gram-negative pathogen, has cell walls and cell membranes, but does not contain peptidoglycan, and is a polypeptide linked by disulfide bonds as a scaffold. Mycoplasma has no cell wall, only cell membrane, consists of phospholipid bilayer, and has certain effect on maintaining the integrity of cell membrane.
4. Targeting lipid membrane components of microorganisms
The main structure of the microbial lipid membrane is a phospholipid bilayer, the main components are phospholipid, protein and polysaccharide, and the microbial lipid membrane can cause the change of the permeability of the membrane by destroying the continuity and stability of the microbial lipid membrane, enhances the permeability and plays a role in resisting microorganisms.
4.1 Structure and composition of microbial cell membranes
The thickness of the microbial lipid membrane is generally 7-8 nm. The lipid membrane is mainly composed of lipids and proteins. Lipid accounts for 50%, protein accounts for 40%, and polysaccharide accounts for about 1-10%. The membrane lipid mainly comprises phospholipid and glycolipid, wherein the phospholipid accounts for more than 50% of the membrane lipid. The phospholipid is mainly glycerophospholipid, glycerol is used as a skeleton, two fatty acid chains and a phosphate group are combined on the skeleton, and molecules such as choline, ethanolamine, serine or inositol are connected to lipid molecules through the phosphate group. The hydrophilic end of the phospholipid molecule is a phosphate group, called the head; the hydrophobic ends of the phospholipid molecules are two hydrocarbon chains with different lengths, called tails, and generally contain 14-24 even-numbered carbon atoms; one of the hydrocarbon chains often contains one or several double bonds, the presence of which causes an angular twist of this unsaturated chain. The glycolipid content is less than about 5% of the membrane lipid; the simplest glycolipid is galactocerebroside, which has only one galactose as the polar head; glycolipids function as integral membrane proteins.
4.2 microbial Membrane lipids
Is a basic skeleton for forming the membrane, and removes membrane lipid to disintegrate the membrane; the membrane lipid is a solvent of membrane protein, and some proteins are embedded on the membrane through the action of hydrophobic ends and the membrane lipid so as to execute special functions; the membrane lipids provide an environment for certain membrane proteases to maintain conformation and exhibit activity, and the activity of many enzymes on the membrane depends on the existence of the membrane lipids.
4.3 microbial Membrane proteins
The content of protein in the membrane is more than 40 to 50 percent of the membrane, and the more complex the function of the membrane is. Membrane proteins are classified into: integrins, peripherins, lipocalins. Proteins in which some or all of the integrin is embedded in or on both the inside and outside of the cell membrane are tightly bound to the membrane and can be washed off the membrane only with detergents, SDS and Triton-X100 are commonly used. The peripherin, also called the exoprotein, is water-soluble and is distributed on the surface of the cell membrane, and is bound to the hydrophilic part of the protein molecules or lipid molecules on the membrane surface by ionic bonds or other weaker bonds, so that the membrane can be separated from the membrane by changing the ionic strength of the solution even at elevated temperatures. Lipocalin: also known as adiponectin, there are two ways of associating homolipids: one way is to indirectly bind to the lipid in the lipid bilayer via one sugar molecule; one is that the protein binds directly to the lipid in the lipid bilayer. The lipocalins are covalently bound by phospholipid or fatty acid anchoring. The membrane protein has the functions of transportation, catalysis of related metabolic reaction, connexin and receptor.
4.4 microbial Membrane sugars
2-10% of the film component; mainly on the outer surface of the lipid membrane. The carbohydrates present in the membrane are mainly 7 types in the animal cell membrane: d-glucose, D-galactose, D-mannose, L-fucose, N-acetylgalactosamine, and N-acetylglucosamine. There are two main forms of linkage of sugars to amino acids: n-connection: namely, the sugar chain is connected with the asparagine residue in the peptide chain; o-connection: it is the sugar chain linked to a serine or threonine residue in the peptide chain.
4.5 asymmetry of microbial lipid membranes
The composition and function of the inner and outer layers of the lipid membrane are obviously different, which is called the asymmetry of the membrane. Membrane lipids, membrane proteins and membrane sugars are all asymmetrically distributed on the membrane, resulting in asymmetry and directionality of membrane functions, i.e., the fluidity of the inner and outer layers of the membrane is different, so that the substance transmission has a certain direction, and the signal receiving and transmission also has a certain direction. The asymmetry and directionality of membrane function ensures a high degree of order in the life activities. Cell-cell recognition, movement, substance transport, signal transmission, and the like are directional. These directional maintenance is provided by asymmetric distribution of membrane proteins, membrane lipids and membrane sugars.
4.6 fluidity of microbial lipid film
The microbial lipid membrane has fluidity, and membrane lipid molecules on the lipid membrane can laterally diffuse, rotate, swing, flex and vibrate, turn over and rotate to be heterogeneous. Several forms of membrane protein movement are mainly lateral diffusion and rotational diffusion. The fluidity of the lipid membrane is a necessary condition for ensuring its normal function. When the fluidity of the lipid membrane is below a certain threshold, the activity of many enzymes and transport across the membrane will cease, whereas if the fluidity is too high, this will cause the dissolution of the lipid membrane.
5. Method and formulation for disrupting microbial cell membranes
5.1 physical disruption
The simplest in vitro method is to put the microorganism in distilled water, and utilize the osmosis principle to make the cell absorb water and burst, and the low temperature and high temperature can destroy the microbial lipid membrane. Direct differential centrifugation can also disrupt the structure of the lipid membrane.
5.2 protease and phospholipase disruption
The protease can catalyze the hydrolysis of protein in the lipid membrane so as to destroy the lipid membrane; phospholipase enzymes also disrupt the lipid membrane by hydrolyzing phospholipids in the membrane.
5.3 disruption of cell membranes by Ionic, nonionic and zwitterionic detergents
Detergents are amphiphilic molecules, containing both hydrophilic and hydrophobic regions, that are capable of disrupting protein, protein lipid and lipid binding, denaturing proteins and other macromolecules. Ionic detergents commonly used in experiments such as Sodium Dodecyl Sulfate (SDS), deoxycholate, cholate, sarcosinate; commonly used nonionic detergents include: triton X-100, DDM, digitonin, tween 20, tween 80. Detergents are amphiphilic organic compounds consisting of a hydrophobic, non-polar hydrocarbon moiety and a hydrophilic, polar group. This molecular structure is very similar to the amphiphilic phospholipids that make up the lipid membrane. Phospholipids have two fatty acid hydrophobic tails each attached to a hydrophilic group. When at high concentrations, the amphipathic molecules self-assemble into structures with their hydrophilic head groups held outside and the hydrophobic tails held inside away from the water. Due to their molecular differences, detergent molecules form spherical micelles. Due to the similarity of molecular structures, detergents are able to penetrate the phospholipid bilayer membrane, thus disrupting the lipid membrane.
5.4. In vitro bactericidal action of fatty acids
Fatty acids are a class of compounds consisting of three elements, carbon, hydrogen, and oxygen, and are the main components of neutral fat, phospholipid, and glycolipid. Fatty acid metabolism fatty acid radicals can be further classified into: short chain fatty acids, also known as volatile fatty acids, having less than 6 carbon atoms in the carbon chain; medium-chain fatty acid refers to fatty acid with 6-12 carbon atoms on the carbon chain; long chain fatty acids having a carbon chain with greater than 12 carbon atoms. Fatty acids can be classified into 3 groups, depending on the difference between saturated and unsaturated hydrocarbon chains, namely: saturated fatty acids, having no unsaturated bonds in the carbon to hydrogen; monounsaturated fatty acids having one unsaturated bond in a hydrocarbon chain; polyunsaturated fatty acids having two or more unsaturated bonds in the carbon-hydrogen chain.
The fatty acid in food is re-esterified in intestinal cell, mixed with bile salt and monoglyceride to form 4-6 nm fat particle, which is absorbed directly by intestinal epithelial cell via swallowing and coated with lecithin and protein membrane to form chylomicron entering lymph system, and via lymph duct and thoracic duct, the chylomicron is refluxed into blood circulation in the form of oil-in-water emulsion. Medium chain fatty acids, except for small amounts that are present in the peripheral blood for a short period of time, are mostly non-covalently bound to serum proteins and reach the liver relatively quickly through the portal system. In the liver, medium-chain fatty acids rapidly pass through the mitochondrial bilayer membrane, are rapidly acylated by octanoyl CoA, and are hardly synthesized into fat. The excess acetyl CoA produced by acylation undergoes various metabolic actions in the mitochondrial cytosol, most of which tend to synthesize ketone bodies.
Studies have shown that the antimicrobial action of fatty acids is generally broad-spectrum. Although the mechanism of their antibacterial action is still poorly understood, many studies speculate that the primary target of action of fatty acids is the cell membrane, where they disrupt the electron transport chain and oxidatively phosphorylate. In addition to interfering with cellular energy production, the effects of fatty acids may be due to inhibition of enzyme activity, impairment of nutrient uptake, production of peroxidation and autoxidative degradation products, or direct breakdown of bacterial cells.
It has potential for development due to its mechanism of action, which is different from most traditional antibiotics. However, there are still some problems that have heretofore hindered progress. First, some free fatty acids do not taste good. Secondly, free fatty acids are unstable and they also have a tendency to bind non-specifically to proteins, and more fatty acids are transported in the body in the form of stable lipids (e.g. triglycerides). Third, the fat solubility and rapid metabolization of fatty acids make them not directly available in the body. Free fatty acids are insoluble in water or have low water solubility and cannot be directly injected into the blood circulation, and direct injection into a vein can cause pulmonary embolism and injection into an artery can cause arterial embolic tissue necrosis.
The concentration of the fatty acid for bacteriostasis and sterilization in vitro used in research is often very high, and the high concentration can destroy the cell membrane of human cells. Human cells are also composed of phospholipid bilayers and are also targets for high-concentration fatty acid attack. The invention relates to a group of stable water-soluble carbon chain compounds which can be injected into veins and arteries and can be orally taken, and fat-soluble hydrophobic carbon chains are changed into water-soluble compounds which can kill microorganisms in vivo in a targeted way through hydrophobic carbon chains which are covalently combined with large, medium and small water-soluble molecules and binding molecules. At the treatment concentration, the compound has the advantages of targeted combination and killing of pathogenic microorganisms, no influence and damage on human cell tissues, and short-term difficult elimination and metabolism by liver. The high water solubility and high affinity compound has the function of resisting microbial infection, and can be used for intravenous injection and oral dosage forms besides nasal spray and dry powder inhalant.
Disclosure of Invention
The invention provides a compound capable of preventing, preventing or treating microbial infection, aiming at the defect of no toxic or side effect and no agent capable of widely killing and preventing, preventing or treating microbial infection in the prior art.
Specifically, in order to solve the problem of lack of a reagent which has no toxic or side effect and particularly cannot widely kill and prevent, prevent or treat microbial infection in the prior art, the invention provides a first set of technical scheme as follows:
(1) the present invention provides a complex for the prevention, prevention and/or treatment of a microbial infection, comprising an active moiety, a binding moiety and a water soluble moiety,
said effect is partly fat-soluble saturated and/or-
Or an unsaturated carbon chain, wherein the carbon chain is a molecule or a residue of a molecule, and the carbon chain is a carbon chain with 3-100 carbon atoms;
the water-soluble moiety is a water-soluble molecule containing one or two or more functional group groups selected from an amide group, a phosphoryloxy group, a carboxylic acid group, a phosphoric acid group, a sulfonic acid group, a sulfonyloxy group, a hydroxyl group, a quaternary ammonium group, a sulfide group, a disulfide group, an ether group, a mercapto group, an aldehyde group, an ester group, an amine group, an amino group, a urea group, and a guanidino group, or a residue of a molecule, and the water-soluble moiety may be one or two or more functional group groups connected to a carbon chain as an acting portion and/or a binding portion;
the binding moiety is a molecule or residue of a molecule capable of binding to a microbial lipid membrane, a microbial surface protein, a microbial surface polysaccharide or cell wall component or capable of binding to a polysaccharide or protein or polypeptide in a microbe, which may be the same as the water-soluble moiety, i.e. a protein, polypeptide, amino acid, oligopeptide, oligosaccharide, mono-and/or polysaccharide molecule or residue thereof capable of binding to a microbial lipid membrane, surface domain;
Wherein the number of any one of the acting portion, the water-soluble portion and the binding portion may be 1 or 2 or more.
(2) The complex according to claim 1, wherein the acting moiety is selected from the group consisting of saturated and/or unsaturated aliphatic hydrocarbons, saturated and/or unsaturated aliphatic alcohols or oxoaliphatic alcohols, saturated and/or unsaturated fatty acids, hydrophobic amino acids, fat-soluble vitamins, steroid lipids, phospholipids, sphingomyelin, glycolipids, and surfactants forming a carbon chain or a carbon chain residue with a number of carbon atoms ranging from 3 to 100, preferably from 3 to 48, and more preferably from 3 to 26; wherein the number of carbon atoms is preferably 3 to 26;
the water-soluble part is a water-soluble molecule or a residue of a molecule containing one or more groups selected from mercapto, amino, phosphate, carboxylate, sulfonate, hydroxyl, amino, ureido, guanidino and disulfide groups;
the binding moiety has a binding group capable of binding to a microbial lipid membrane, a microbial surface protein, a microbial surface polysaccharide or a cell wall component or to a polysaccharide or protein or polypeptide in a microbe, the group being derived from a water-soluble moiety or from one or more than two groups selected from thiol, amino, phosphate, carboxylate, sulfonate, hydroxyl, amine, ureido, guanidino and disulfide groups independently as a binding moiety or from one or more than two groups selected from thiol, amino, phosphate, carboxylate, sulfonate, hydroxyl, amine, ureido, guanidino and disulfide groups providing attachment of the carbon chain to the carbon chain, such that the complex has one or more than two groups selected from thiol, amino, phosphate, carboxylate, sulfonate, hydroxyl, amine, ureido, guanidino and disulfide groups;
That is, for the complexes of the invention, the binding moiety may in some cases be the same as the water-soluble moiety, and may also function to provide the carbon chain function of the functional moiety.
Preferably, the binding part is selected from one or more than two of dibasic fatty acid or polybasic fatty acid, amino acid, targeting protein, targeting polypeptide and targeting polysaccharide;
more preferably, the complex is a complex formed by connecting a fatty acid with 3-100 carbon atoms, preferably 3-50 carbon atoms and a water-soluble amino acid; or the compound is formed by connecting a targeting polypeptide selected from fatty acid with 3-50 carbon atoms;
or the compound is formed by the reaction of fatty acid with 3-100 carbon atoms, preferably 3-50 carbon atoms, targeting polypeptide and PEG;
or the compound is formed by the reaction of a surfactant and one or more than two selected from dibasic fatty acid or polybasic fatty acid, amino acid, targeting protein, targeting polypeptide and targeting polysaccharide, wherein the surfactant is preferably one or more than two selected from fatty alcohol polyoxyethylene ether, fatty acid polyoxyethylene ester, alkyl glycoside, fatty acid sucrose ester, sorbitan fatty acid ester, sorbitan polyoxyethylene fatty acid ester, N-fatty acyl-N-methylglucamine and mannose erythritol ester.
(3) The compound according to claim 2, wherein the saturated and/or unsaturated fatty acid is selected from saturated or unsaturated fatty acids with 3-100 carbon atoms, the fatty acid is a fatty acid or amino acid containing double bond, triple bond, hydroxyl, amino group and/or being oxo-substituted, and the fatty acid can be a monobasic acid, a dibasic acid or a polybasic acid.
(4) The compound according to claim 3, wherein the saturated and/or unsaturated fatty acid is selected from saturated fatty acids having 3 to 46 carbon atoms, monooleic acids having 3 to 34 carbon atoms, dienoic acids having 5 to 30 carbon atoms, trienoic acids having 7 to 30 carbon atoms, arachidonic acids having 12 to 38 carbon atoms, pentaenoic acids having 12 to 38 carbon atoms, hexaenoic acids having 22 to 38 carbon atoms, alkynoic acids having 6 to 22 carbon atoms, diynoic acids having 10 to 22 carbon atoms, trialkynoic acids having 12 to 22 carbon atoms, alkynoic acids having 8 to 20 carbon atoms (preferably acids containing one or two C ═ C double bonds and one or two or three triple bonds), fatty acids having 3 to 30 carbon atoms in the main chain and having 1 to 10 alkyl groups and/or 1 to 3 hydroxyl groups in the side chain (preferably having 1 carbon atom in the main chain), and the compound of the present invention is useful for the treatment of diabetes -saturated fatty acids with 3 methyl groups or fatty acids with C ═ C double bonds), saturated linear and branched dicarboxylic and tricarboxylic acids with 3 to 38 carbon atoms and unsaturated linear or branched dicarboxylic and tricarboxylic acids with 4 to 18 carbon atoms which may be substituted with hydroxyl groups, carboxylic acids with 3 to 18 carbon atoms substituted with amino, hydroxyl, oxo and/or methyl groups, N-acyl amino acids with 6 to 30 carbon atoms, amino acids containing 2 or more acyl groups, polycarboxylic acids linked by thioether bonds and amide bonds;
The saturated and/or unsaturated fatty alcohol is a saturated fatty straight-chain or branched-chain alcohol with the carbon atom number of 3-33; and/or unsaturated aliphatic straight chain or branched chain alcohol containing 1-3 hydroxyl groups and containing 1-5 double bonds and 1-5 triple bonds and having 3-33 carbon atoms; the oxo-fatty alcohol is C8-31 alcohol ketone containing 1-3 double or triple bonds and 1-3 hydroxyl groups, and the ketone is monoketone or diketone.
(5) The compound according to claim 4, wherein the saturated or unsaturated fatty acid is one or more selected from fumaric acid, octanoic acid, glutaconic acid, hexanoic acid, nonanoic acid, dodecanoic acid, tetradecanoic acid, hexadecanoic acid, octadecanoic acid, eicosanoic acid, oleic acid, linoleic acid, linolenic acid, eicosapentaenoic acid, docosapentaenoic acid, docosahexaenoic acid, pentacosanoic acid, heptanoic acid, decanoic acid, dodecenoic acid, tetradecanoic acid, docosahexaenoic acid, octacosanoic acid, and carbon chain residues formed therefrom.
(6) The complex according to any one of claims 1 to 5, wherein the water-soluble moiety is a molecule or a residue of a molecule containing one or more groups selected from a mercapto group, an amino group, a carboxylic acid group, a hydroxyl group and a disulfide group; the molecule is selected from one or more than two water-soluble macromolecules selected from protein, polysaccharide, nucleic acid and artificially synthesized water-soluble polymers or residues thereof;
And/or, one or more than two medium molecules selected from polypeptide, oligopeptide, oligosaccharide, oligonucleotide and synthetic water-soluble medium molecular weight polymer or residues thereof;
and/or, water-soluble small molecules selected from one or more than two of amino acid, monosaccharide, disaccharide, nucleotide, water-soluble vitamin and deoxynucleotide or residues thereof;
and/or a molecule or residue of a molecule attached to the carbon chain as the active moiety, said molecule or residue of a molecule comprising one or more groups selected from thiol, amino, carboxylic acid, hydroxyl, disulphide groups.
(7) The compound according to claim 6, wherein the protein as the water-soluble macromolecule is one or more water-soluble macromolecules selected from the group consisting of serum albumin, immunoglobulin, water-soluble collagen, chaperonin, water-soluble glycoprotein, and CD 14; the polysaccharide as the macromolecule is one or more than two water-soluble macromolecules selected from dextran, hyaluronic acid, sialic acid, heparin sulfate, heparan sulfate, chondroitin sulfate, dermatan sulfate, keratan sulfate, acetyl water-soluble cellulose derivatives, beta-cyclodextrin and derivatives thereof and water-soluble chitosan derivatives; the water-soluble polymer as the macromolecule is one or more than two water-soluble macromolecules selected from polyethylene glycol, carboxylated or aminated polyethylene glycol, polyvinyl alcohol, carboxylated or quaternized polyvinyl alcohol, polyacrylic acid and ammonium polyacrylate;
The water-soluble medium molecular weight polymer is selected from targeting polypeptide, oligopeptide, oligosaccharide, oligonucleotide and/or water-soluble polyamino acid; preferably the targeting polypeptide comprises a protein or neutralizing antibody fragment (including e.g. taurocholate transit peptide, SBP1) that specifically targets microbial lipid membranes, bacterial and fungal cell walls, viral surface protein domains; preferably the water-soluble polyamino acid is selected from polyglutamic acid, polylysine and/or polyaspartic acid; and oligopeptides, oligosaccharides, oligonucleotides;
the monosaccharide and/or disaccharide of the water-soluble micromolecule is one or more than two of glucose, fructose, rhamnose, sorbose, sucrose, maltose, lactose and trehalose; the nucleotide and/or deoxynucleotide as water-soluble small molecule is selected from adenylic acid, guanylic acid, uridylic acid, cytidylic acid, thymidylic acid, inosinic acid, deoxyadenylic acid, deoxyguanylic acid, deoxycytidylic acid, deoxythymidylic acid; amino acids such as one or more of serine, threonine, cysteine, asparagine, glutamine, tyrosine, lysine, arginine, histidine, aspartic acid, glutamic acid, citrulline, ornithine, taurine and aminobutyric acid; the vitamin as the water-soluble small molecule is one or more of vitamin B1, pantothenic acid, vitamin B6 and vitamin C.
(8) The complex according to any of claims 1-7, wherein the binding moiety and the water-soluble moiety are the same, i.e. are proteins, polypeptides, oligopeptides, oligosaccharides, monosaccharides, disaccharides, amino acids, nucleotides, vitamins, water-soluble polymers, water-soluble polyamino acids and/or polysaccharide molecules or residues of these molecules, which molecules or residues of molecules comprise one or more groups selected from thiol, amino, carboxylic acid, hydroxyl, disulfide groups.
(9) The compound according to any one of claims 1 to 8, which is a compound obtained by reacting a substance having a carbon chain having 3 to 100 carbon atoms as an active moiety with one or more members selected from the group consisting of proteins, polypeptides, oligopeptides, oligosaccharides, monosaccharides, disaccharides, amino acids, nucleotides, vitamins, water-soluble polymers, water-soluble polyamines and/or polysaccharides; or a mixture of a substance containing a carbon chain having 3 to 100 carbon atoms as an active moiety and one or more members selected from the group consisting of proteins, polypeptides, oligopeptides, oligosaccharides, monosaccharides, disaccharides, amino acids, nucleotides, vitamins, water-soluble polymers, water-soluble polyamines and/or polysaccharide molecules, and unreacted substances as an active moiety and/or unreacted molecules of the proteins, polypeptides, oligopeptides, oligosaccharides, monosaccharides, disaccharides, amino acids, nucleotides, vitamins, water-soluble polymers, water-soluble polyamines and/or polysaccharides; wherein preferably, the substance as the acting portion is one or more substances selected from the group consisting of saturated and/or unsaturated aliphatic hydrocarbons, saturated and/or unsaturated aliphatic alcohols or oxoaliphatic alcohols, saturated and/or unsaturated fatty acids, hydrophobic amino acids, fat-soluble vitamins, steroid lipids, phospholipids, sphingomyelin, glycolipids and surfactants, which provide or have a carbon chain or a residue forming a carbon chain having a carbon number of 3 to 100, preferably 3 to 48, more preferably 3 to 26.
That is, the complex for preventing, preventing or treating a microbial infection includes the compound obtained by the reaction, and also includes a reaction mixture (also referred to as "reaction product" or "reaction mixture", "reaction product solution", "reaction mixture solution") containing the compound obtained by the reaction, and a purified product obtained by purifying the reaction mixture to separate unreacted reaction starting materials, a catalyst, and the like. )
(10) The complex according to any one of claims 1 to 8, which is a mixture obtained by complexing a substance having a carbon chain having 3 to 100 carbon atoms as an active moiety with one or more compounds selected from proteins, polypeptides, oligopeptides, oligosaccharides, monosaccharides, disaccharides, amino acids, nucleotides, vitamins, water-soluble polymers, water-soluble polyamines and/or polysaccharide molecules by physicochemical action including hydrogen bonding or van der waals force or a combination of both actions, or by direct physical mixing; wherein the substance as an active portion is preferably one or two or more substances selected from the group consisting of saturated and/or unsaturated aliphatic hydrocarbons, saturated and/or unsaturated aliphatic alcohols or oxoaliphatic alcohols, saturated and/or unsaturated fatty acids, hydrophobic amino acids, fat-soluble vitamins, steroid lipids, phospholipids, sphingomyelins, glycolipids and surfactants, which provide or have a carbon chain or a residue forming a carbon chain having a carbon number of 3 to 100, preferably 3 to 48, more preferably 3 to 26.
(11) The compound according to claim 9, which is a compound obtained by reacting a substance having a carbon chain of 3 to 100 carbon atoms as an active moiety with at least one member selected from the group consisting of proteins, polypeptides, oligopeptides and amino acids; or a mixture of a compound obtained by reacting a substance having a carbon chain of 3 to 100 carbon atoms as an active moiety with at least one member selected from the group consisting of proteins, polypeptides, oligopeptides and amino acids, and an unreacted substance as an active moiety and/or an unreacted substance selected from the group consisting of at least one member selected from the group consisting of proteins, polypeptides, oligopeptides and amino acids.
(12) The compound according to claim 9, which is a compound obtained by reacting a substance having a carbon chain of 3 to 100 carbon atoms as an active moiety, PEG, and at least one selected from the group consisting of proteins, polypeptides, oligopeptides, and amino acids; or a compound obtained by reacting PEG with at least one selected from the group consisting of proteins, polypeptides, oligopeptides and amino acids, and a mixture of unreacted substances acting as the acting portion, unreacted PEG and/or unreacted at least one selected from the group consisting of proteins, polypeptides, oligopeptides and amino acids.
(13) The compound according to claim 9, which is a compound obtained by reacting a substance having a carbon chain having 3 to 100 carbon atoms as an active moiety with one or more than two kinds selected from polysaccharides, monosaccharides, disaccharides and/or oligosaccharides; or a compound obtained by reacting a substance having a carbon chain having 3 to 100 carbon atoms as an active moiety with one or more than two kinds selected from polysaccharides, monosaccharides, disaccharides and/or oligosaccharides, and a mixture of the unreacted substance serving as the active moiety and/or the unreacted polysaccharides, monosaccharides, disaccharides and/or oligosaccharides.
(14) The compound according to claim 9, which is a compound obtained by reacting a substance having a carbon chain with 3 to 100 carbon atoms as an active moiety, PEG, and one or more selected from polysaccharides, monosaccharides, disaccharides, and/or oligosaccharides; or a compound obtained by reacting a saturated and/or unsaturated fatty acid having 3 to 100 carbon atoms, PEG and one or more selected from polysaccharides, monosaccharides, disaccharides and/or oligosaccharides, and a mixture of the unreacted PEG and/or unreacted polysaccharides, monosaccharides, disaccharides and/or oligosaccharides.
(15) The complex according to any one of claims 6 to 12, wherein the protein is selected from one or more of serum albumin, immunoglobulin, water-soluble collagen, chaperonin, water-soluble glycoprotein and CD 14.
(16) The complex according to claim 13, wherein the polysaccharide is one or more selected from dextran and/or hyaluronic acid sialic acid, heparin sulfate, heparan sulfate, chondroitin sulfate, dermatan sulfate, keratan sulfate, acetyl water-soluble cellulose derivatives, β -cyclodextrin and its derivatives, and water-soluble chitosan derivatives.
(17) The complex according to claim 11 or 12, which is a compound obtained by reacting a substance having a carbon chain of 3 to 100 carbon atoms as an active moiety, a linker and a thiol-group-containing protein; or a mixture of the compound obtained by the above reaction, the unreacted acting substance, the unreacted linker, and/or the unreacted thiol-group-containing protein; wherein the connecting object is one or more than two of amino acid, succinic acid, butadiene acid, glutaconic acid, hexylamine diacid, carbamate, short peptide, N-hydroxyl crotonoimide, polyethylene glycol and derivatives of the compounds; preferably, the compound is a substance containing a carbon chain with 3-100 carbon atoms as an active part and a compound obtained by reacting N-hydroxy crotonoimide with protein containing sulfydryl; or a mixture of the compound obtained by the above reaction, the unreacted substance as the acting portion, the unreacted N-hydroxybutylimide and/or the unreacted thiol-group-containing protein.
(18) The compound according to claim 13, which is a compound obtained by reacting a substance containing a carbon chain having 3 to 100 carbon atoms as an active moiety, cystamine, and one or more selected from polysaccharides, monosaccharides, disaccharides, and/or oligosaccharides; or a compound obtained by reacting a substance having a carbon chain with 3 to 100 carbon atoms as an active moiety with cystamine and one or more than two substances selected from polysaccharides, monosaccharides, disaccharides and/or oligosaccharides, and a mixture of the unreacted substance having a carbon chain with 3 to 100 carbon atoms as an active moiety and unreacted polysaccharides, monosaccharides, disaccharides, oligosaccharides and/or unreacted cystamine.
(19) The compound according to any one of claims 8 to 17, wherein the compound obtained by the reaction contains one or more of an amide group, an ester group, a thioether group or an ether group as a linking moiety between the water-soluble moiety and the acting moiety.
(20) The complex according to any one of claims 3 to 18, wherein the substance which provides a carbon chain or a residue of a carbon chain as an active moiety is selected from one or more of saturated and/or unsaturated fatty hydrocarbons, saturated and/or unsaturated fatty alcohols or oxofatty alcohols, saturated and/or unsaturated fatty acids, hydrophobic amino acids, fat-soluble vitamins, steroid lipids, phospholipids, sphingomyelin, glycolipids and surfactants, and the carbon chain has 3 to 100, preferably 3 to 50, more preferably 3 to 48, and still more preferably 3 to 26 carbon atoms.
(21) The complex according to any one of claims 3 to 18, wherein the substance which provides a carbon chain or a residue of a carbon chain as an active moiety is selected from one or more of saturated and/or unsaturated fatty hydrocarbons, saturated and/or unsaturated fatty alcohols or oxofatty alcohols, saturated and/or unsaturated fatty acids, hydrophobic amino acids, fat-soluble vitamins, steroid lipids, phospholipids, sphingomyelin, glycolipids and surfactants, and preferably saturated and/or unsaturated fatty acids; more preferably, the saturated and/or unsaturated fatty acid is a fatty acid having 3 to 100 carbon atoms, preferably 3 to 50 carbon atoms, still more preferably 3 to 48 carbon atoms, still more preferably 3 to 40 carbon atoms, and may be a fatty acid having 1 to 7 carbon double bonds, a fatty acid having 1 to 6 carbon double bonds, a fatty acid having 1 to 5 carbon double bonds, a fatty acid having 1 to 4 carbon double bonds, a fatty acid having 1 to 3 carbon double bonds, or a fatty acid having 1 to 2 carbon double bonds.
(22) The complex according to any of claims 3 to 18, wherein the substance which provides a carbon chain or a residue of a carbon chain as an active moiety is a saturated and/or unsaturated fatty acid, which may have 1 to 6 double bonds and a carbon number of 2 to 30, preferably 2 to 26, more preferably 2 to 22.
(23) The complex according to any of claims 3 to 18, wherein the saturated and/or unsaturated fatty acid has 3 to 30, preferably 3 to 26, preferably 8 to 22, preferably 8 to 20, preferably 8 to 18 carbon atoms.
(24) The composite according to any one of claims 3 to 18, wherein the saturated and/or unsaturated fatty acid is one or more fatty acids selected from fumaric acid, octanoic acid, glutaconic acid, hexanoic acid, nonanoic acid, dodecanoic acid, tetradecanoic acid, hexadecanoic acid, octadecanoic acid, eicosanoic acid, oleic acid, linoleic acid, linolenic acid, eicosapentaenoic acid, docosapentaenoic acid, docosahexaenoic acid, pentacosanoic acid, heptanoic acid, decanoic acid, dodecenoic acid, tetradecanoic acid, docosahexaenoic acid, and octacosanoic acid.
(25) The complex according to any one of claims 8-23, wherein the protein is human serum albumin or bovine serum albumin, or CD 14; or the polysaccharide is dextran and/or hyaluronic acid.
(26) The complex according to claim 11, wherein the compound obtained by the reaction is any one or more compounds having the following structural formula, obtained by reacting fatty acid with Albumin (Albumin) or SBP 1:
Figure BDA0003628692260000191
(27) According to the compound of claim 12, the compound obtained by the reaction is a compound obtained by reacting a C3-10 monobasic fatty acid, PEG and an amino acid, or a compound obtained by reacting a C3-10 monobasic fatty acid, a C5-8 saturated dibasic fatty acid, PEG and taurine; preferred are compounds having at least one of the following structural formulae:
Figure BDA0003628692260000192
Figure BDA0003628692260000201
wherein n is an integer of 1 to 200.
(28) The compound according to claim 13, wherein the compound obtained by the reaction is any one or more compounds having the following structural formula, which are obtained by reacting fatty acid with dextran:
Figure BDA0003628692260000202
Figure BDA0003628692260000211
(29) the complex according to claim 13, wherein the compound obtained by the reaction is any one or more compounds having the following structural formula, which are obtained by reacting fatty acid with hyaluronic acid:
Figure BDA0003628692260000221
Figure BDA0003628692260000231
n is an integer of 1 to 2000.
(30) The complex according to claim 14, wherein the compound obtained by the reaction is a compound obtained by reacting a fatty acid having 3 to 10 carbon atoms with PEG and glucose, and preferably a compound having the following structural formula:
Figure BDA0003628692260000241
n is an integer from 1 to 200.
(31) According to the compound of claim 17, the compound obtained by the reaction is any one or more than two compounds with thioether bond having the following structural formula and obtained by the reaction of fatty acid, N-hydroxy crotonoimide and albumin (albumin):
Figure BDA0003628692260000242
Figure BDA0003628692260000251
Figure BDA0003628692260000261
(32) According to the compound of claim 18, the compound obtained by the reaction is any one or more compounds having the following structural formula obtained by reacting fatty acid, cystamine and dextran:
Figure BDA0003628692260000262
Figure BDA0003628692260000271
(33) the complex according to claim 18, wherein the compound obtained by the reaction is any one or more compounds obtained by reacting fatty acid, cystamine and hyaluronic acid, and having the following structural formula:
Figure BDA0003628692260000272
Figure BDA0003628692260000281
(34) the compound according to any one of claims 1 to 8, which is a compound obtained by reacting a surfactant containing a carbon chain with 3 to 30 carbon atoms with a dibasic or polybasic fatty acid, an amino acid, a targeted protein, a targeted polypeptide, a targeted polysaccharide and/or a targeted polysaccharide; or the compound obtained by the reaction, the unreacted surfactant and/or the unreacted dibasic or polybasic fatty acid, the amino acid, the targeting protein, the targeting polypeptide, the targeting polysaccharide and/or the mixture of the targeting polysaccharide.
(35) According to the composition of claim 34, the surfactant is one or more selected from fatty alcohol-polyoxyethylene ether, fatty acid-polyoxyethylene ester, alkyl glycoside, fatty acid sucrose ester, sorbitan fatty acid ester, sorbitan polyoxyethylene fatty acid ester, mannosylerythritol ester, and N-fatty acyl-N-methylglucamine.
(36) The complex according to any of claims 1-35, wherein the microbial infection comprises a viral, induced infection.
(37) A preparation for preventing, preventing or treating a microbial infection, which is prepared by using the complex according to any one of claims 1 to 35,
(38) the formulation of claim 37, which is a pharmaceutical formulation or an environmental disinfectant formulation.
(39) The formulation of claim 38, wherein the pharmaceutical formulation is one selected from the group consisting of an inhalant, a nasal spray, an injection, an oral formulation, and a topical formulation for skin.
(40) Use of a complex according to any one of claims 1 to 35 in the preparation of a pharmaceutical preparation for the prevention, prevention and/or treatment of a microbial infection.
(41) The use according to claim 40, wherein the microorganism is any one or more selected from the group consisting of viruses, bacteria, fungi, chlamydia and mycoplasma.
(42) The use of claim 41, wherein the virus is an enveloped virus; and/or non-enveloped viruses.
(43) The use according to claim 42, wherein the enveloped virus is one or more of coronavirus, influenza virus, HIV, hepatitis B virus, hepatitis C virus, herpes virus, Secat virus, dengue fever virus, encephalitis B virus, Ebola virus, rabies virus, and/or Hantaan virus; the non-enveloped virus is more than two of hepatitis A virus, human papilloma virus, poliovirus and/or coxsackievirus.
(44) The use according to claim 43, wherein the virus is any one or more of coronavirus, HIV, HBV, HCV, herpesvirus, encephalitis B virus, rabies virus, human papilloma virus and Ebola virus.
(45) The use according to claim 41, wherein the bacterium is a gram-positive and/or gram-negative bacterium and the fungus is a pathogenic and/or conditionally pathogenic fungus; the chlamydia is chlamydia trachomatis, chlamydia pneumoniae and/or chlamydia psittaci; the mycoplasma includes mycoplasma pneumoniae, ureaplasma urealyticum, mycoplasma hominis, and/or mycoplasma genitalium.
(46) The use according to claim 41 wherein the bacteria are selected from one or more of Escherichia coli, Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, Streptococcus pneumoniae, Klebsiella pneumoniae and Pseudomonas aeruginosa; the fungi is selected from one or more of Candida albicans, Aspergillus niger, Actinomyces viscosus, Chaetomium globosum, Aspergillus wart and Microsporum canis.
(47) The use according to claim 41 wherein the virus is selected from one or more of the group consisting of influenza H7N9, influenza H5N1, HIV, neocoronavirus, HPV and rabies.
(48) The method for preparing a complex according to any one of claims 1 to 35, wherein the complex is obtained by reacting a compound having a fat-soluble saturated and/or unsaturated carbon chain having a branched, cyclic and/or linear structure with a water-soluble molecule, and optionally a protein, polypeptide, amino acid, oligopeptide, oligosaccharide, monosaccharide and/or polysaccharide molecule capable of binding to a lipid membrane, a viral surface domain or a cell wall of a microorganism, and optionally a linker molecule, in the presence of a catalyst.
(49) The method for producing a complex according to claim 48, wherein the complex is a product obtained by purifying a compound obtained by the reaction.
(50) The method for preparing a complex according to any one of claims 1 to 35, wherein the complex is obtained by physically mixing a compound having a fat-soluble saturated and/or unsaturated carbon chain having a branched, cyclic and/or linear structure with a water-soluble molecule, and optionally a protein, polypeptide, amino acid, oligopeptide, oligosaccharide, monosaccharide and/or polysaccharide molecule capable of binding to a microbial lipid membrane, a microbial surface domain or a cell wall.
(51) The method of preparing a complex according to any one of claims 1 to 35, wherein the complex is obtained by reacting a saturated and/or unsaturated fatty acid having 3 to 100 carbon atoms with any one of a protein, a polypeptide, an oligopeptide, an oligosaccharide, a monosaccharide, a disaccharide, a nucleotide, a vitamin, a water-soluble polymer, a water-soluble polyamino acid, and/or a polysaccharide in the presence of a catalyst.
(52) The method of claim 51, wherein the compound is a purified compound obtained from the reaction.
(53) The method for preparing a complex according to any one of claims 1 to 35, wherein the complex is obtained by a physicochemical complex of a saturated and/or unsaturated fatty acid having 3 to 100 carbon atoms with a protein, a polypeptide, an oligopeptide, an oligosaccharide, a monosaccharide, a disaccharide, a nucleotide, a vitamin, a water-soluble polymer, a water-soluble polyamino acid and/or a polysaccharide molecule, or by direct physical mixing.
Specifically, in order to solve the problem of lack of a reagent which has no toxic or side effect and particularly cannot widely kill and prevent, prevent or treat microbial infection in the prior art, the invention provides the following second technical scheme:
[ MEANS FOR SOLVING PROBLEMS ] A complex capable of preventing, preventing and/or treating a microbial infection, which comprises an acting portion, a binding portion and a water-soluble portion,
the acting part is a fat-soluble saturated and/or unsaturated carbon chain with a branched chain, a cyclic structure and/or a straight chain structure, and the carbon chain is a molecule or a residue of the molecule; wherein the action part is a carbon chain or residue of a carbon chain with 3-100 carbon atoms formed by one or more than two substances selected from hydrophobic amino acid, fat-soluble vitamin, steroid lipid, phospholipid, sphingomyelin, glycolipid, surfactant, saturated and/or unsaturated aliphatic hydrocarbon and saturated and/or unsaturated aliphatic alcohol or oxo-aliphatic alcohol;
The water-soluble portion is a water-soluble molecule containing one or two or more functional group groups selected from an amide group, a phosphoryloxy group, a carboxylic acid group, a phosphoric acid group, a sulfonic acid group, a sulfonyloxy group, a hydroxyl group, a quaternary ammonium group, a sulfide group, a disulfide group, an ether group, a mercapto group, an aldehyde group, an ester group, an amine group, an amino group, a urea group, and a guanidino group, or a residue of a molecule, and may be one or two or more functional group groups described above attached to a carbon chain as an acting portion;
the binding moiety is a molecule or residue of a molecule capable of binding to a microbial lipid membrane, a microbial surface protein, a microbial surface polysaccharide or cell wall component or capable of binding to a polysaccharide or protein or polypeptide in a microbe, which may be the same as the water-soluble moiety, i.e. a protein, polypeptide, amino acid, oligopeptide, oligosaccharide, mono-and/or polysaccharide molecule or residue thereof capable of binding to a microbial lipid membrane, surface domain;
wherein the number of any one of the acting portion, the water-soluble portion and the binding portion may be 1 or 2 or more.
The carbon atom number of the compound is 3-48 according to the technical scheme 1.
③ according to the compound of the technical proposal 1, the carbon atom number is 3 to 26.
(iv) the complex according to any one of claims 1 to 3, wherein the water-soluble moiety is a water-soluble molecule or a residue of a molecule containing one or two or more groups selected from a mercapto group, an amino group, a phosphate group, a carboxylate group, a sulfonate group, a hydroxyl group, an amine group, a urea group, a guanidine group and a disulfide group;
the binding moiety has a group capable of binding, i.e., capable of binding to a microbial lipid membrane, a microbial surface protein, a microbial surface polysaccharide or a cell wall component, or capable of binding to a polysaccharide or protein or polypeptide in a microbe, the group being derived from a water-soluble moiety or from two or more groups selected from thiol, amino, phosphate, carboxylate, sulfonate, hydroxyl, amine, urea, guanidine and disulfide groups independently as binding moieties, or from one or two or more groups selected from thiol, amino, phosphate, carboxylate, sulfonate, hydroxyl, amine, urea, guanidine and disulfide groups providing attachment of the carbon chain to the carbon chain, such that the complex has one or two or more groups selected from thiol, amino, phosphate, carboxylate, sulfonate, hydroxyl, amine, urea, guanidine and disulfide groups.
The compound according to the technical scheme 4, wherein the binding part is one or more than two selected from dibasic fatty acid or polybasic fatty acid, amino acid, targeting protein, targeting polypeptide and targeting polysaccharide.
The compound is formed by the reaction of a surfactant and one or more than two of dibasic fatty acid or polybasic fatty acid, amino acid, targeted protein, targeted polypeptide and targeted polysaccharide according to any one of technical schemes 1 to 4.
The compound according to any one of technical schemes 1-6, wherein the saturated and/or unsaturated fatty alcohol is alcohol with a cyclic structure, a linear chain or a branched chain of saturated fat with 3-33 carbon atoms; and/or unsaturated aliphatic straight chain or branched chain alcohol containing 1-3 hydroxyl groups and containing 1-5 double bonds and 1-5 triple bonds and having 3-33 carbon atoms; the oxo-fatty alcohol is C8-31 alcohol ketone containing 1-3 double or triple bonds and 1-3 hydroxyl groups, and the ketone is monoketone or diketone.
(iii) the complex according to any one of claims 1 to 5 and 7, wherein the water-soluble moiety is a molecule or a residue of a molecule containing one or two or more groups selected from a mercapto group, an amino group, a carboxylic acid group, a hydroxyl group and a disulfide group; the molecule is selected from one or more than two water-soluble macromolecules or residues thereof in protein, polysaccharide, nucleic acid and artificially synthesized water-soluble high polymer;
And/or one or more than two medium molecules selected from polypeptide, oligopeptide, oligosaccharide, oligonucleotide and artificially synthesized water-soluble medium molecular weight polymer or residues thereof;
and/or, water-soluble small molecules selected from one or more of amino acids, monosaccharides, disaccharides, nucleotides, water-soluble vitamins, and deoxynucleotides, or residues thereof;
and/or a molecule or a residue of a molecule linked to the carbon chain as the acting moiety, said molecule or residue of a molecule comprising one or more groups selected from thiol, amino, carboxylic acid, hydroxyl, disulfide groups.
Ninthly, according to the complex of claim 8, wherein the protein serving as the water-soluble macromolecule is one or more water-soluble macromolecules selected from serum albumin, immunoglobulin, water-soluble collagen, chaperonin, water-soluble glycoprotein and CD 14; the polysaccharide as the macromolecule is one or more than two water-soluble macromolecules selected from dextran, hyaluronic acid, sialic acid, heparin sulfate, heparan sulfate, chondroitin sulfate, dermatan sulfate, keratan sulfate, acetyl water-soluble cellulose derivatives, beta-cyclodextrin and derivatives thereof and water-soluble chitosan derivatives; the water-soluble polymer as the macromolecule is one or more than two water-soluble macromolecules selected from polyethylene glycol, carboxylated or aminated polyethylene glycol, polyvinyl alcohol, carboxylated or quaternized polyvinyl alcohol, polyacrylic acid and ammonium polyacrylate;
The water-soluble polymer with medium molecular weight is selected from one or more than two of targeting polypeptide, oligopeptide, oligosaccharide, oligonucleotide and/or water-soluble polyamino acid;
the monosaccharide and/or disaccharide of the water-soluble micromolecule is selected from one or more of glucose, fructose, rhamnose, sorbose, sucrose, maltose, lactose and trehalose; the nucleotide and/or deoxynucleotide as water soluble small molecule is selected from adenylic acid, guanylic acid, uridylic acid, cytidylic acid, thymidylic acid, inosinic acid, deoxyadenylic acid, deoxyguanylic acid, deoxycytidylic acid, deoxythymidylic acid; amino acids such as one or more of serine, threonine, cysteine, asparagine, glutamine, tyrosine, lysine, arginine, histidine, aspartic acid, glutamic acid, citrulline, ornithine, taurine and aminobutyric acid; the vitamin as the water-soluble small molecule is one or more of vitamin B1, pantothenic acid, vitamin B6 and vitamin C.
The complex according to any of claims 5-9, wherein said targeting polypeptide comprises any of a protein or neutralizing antibody fragment specifically targeting microbial lipid membranes, bacterial and fungal cell walls, viral surface protein domains.
Figure BDA0003628692260000332
The compound according to claim 9, wherein the water-soluble polyamino acid is selected from polyglutamic acid, polylysine and/or polyaspartic acid.
Figure BDA0003628692260000333
The complex according to any of claims 1-11, wherein the binding moiety and the water-soluble moiety are the same, i.e. are proteins, polypeptides, oligopeptides, oligosaccharides, monosaccharides, disaccharides, amino acids, nucleotides, vitamins, water-soluble polymers, water-soluble polyamino acids and/or polysaccharide molecules or residues of these molecules, which molecules or residues of molecules comprise one or more groups selected from thiol, amino, carboxylic acid, hydroxyl, disulfide groups.
Figure BDA0003628692260000334
The compound according to claim 1 to 12, which is a compound obtained by reacting a substance having a carbon chain of 3 to 100 carbon atoms as an active moiety with any one or two or more members selected from the group consisting of proteins, polypeptides, oligopeptides, oligosaccharides, amino acids, monosaccharides, disaccharides, nucleotides, vitamins, water-soluble polymers, water-soluble polyamines and/or polysaccharide molecules; or a compound obtained by reacting a substance having a carbon chain having 3 to 100 carbon atoms as an active moiety with any one or two or more members selected from the group consisting of proteins, polypeptides, oligopeptides, oligosaccharides, amino acids, monosaccharides, disaccharides, nucleotides, vitamins, water-soluble polymers, water-soluble polyamines and/or polysaccharide molecules, and a mixture of unreacted substances as an active moiety and/or unreacted molecules of the proteins, polypeptides, oligopeptides, oligosaccharides, amino acids, monosaccharides, disaccharides, oligonucleotides, vitamins, water-soluble polymers, water-soluble polyamines and/or polysaccharide molecules;
Wherein the substance having a carbon chain of 3 to 100 carbon atoms as an active moiety is selected from the group consisting of saturated and/or unsaturated aliphatic hydrocarbons, saturated and/or unsaturated aliphatic alcohols or oxoaliphatic alcohols, hydrophobic amino acids, fat-soluble vitamins, steroid lipids, phospholipids, sphingomyelin, glycolipids and/or surfactants.
Figure BDA0003628692260000341
The complex according to any one of claims 1 to 12, which is a mixture obtained by compounding a substance having a carbon chain with 3 to 100 carbon atoms as an acting moiety with a physicochemical action including a combination of hydrogen bond or van der waals force or both actions selected from any one or two of proteins, polypeptides, oligopeptides, oligosaccharides, amino acids, monosaccharides, disaccharides, nucleotides, vitamins, water-soluble polymers, water-soluble polyamines and polysaccharide molecules, or by direct physical mixing;
wherein the substance having a carbon chain with 3 to 100 carbon atoms as an active moiety is selected from the group consisting of saturated and/or unsaturated aliphatic hydrocarbons, saturated and/or unsaturated aliphatic alcohols or oxoaliphatic alcohols, hydrophobic amino acids, fat-soluble vitamins, steroid lipids, phospholipids, sphingomyelin, glycolipids, and/or surfactants.
Figure BDA0003628692260000342
The compound according to claim 13, which is a compound obtained by reacting a substance having a carbon chain of 3 to 100 carbon atoms as an active moiety with at least one selected from the group consisting of a protein, a polypeptide, an oligopeptide and an amino acid; or a mixture of a compound obtained by reacting a substance having a carbon chain of 3 to 100 carbon atoms as an active moiety with at least one member selected from the group consisting of proteins, polypeptides, oligopeptides and amino acids, and an unreacted substance as an active moiety and/or an unreacted substance selected from the group consisting of at least one member selected from the group consisting of proteins, polypeptides, oligopeptides and amino acids.
Figure BDA0003628692260000343
The complex according to claim 13, which is a compound obtained by reacting a substance having a carbon chain of 3 to 100 carbon atoms as an active moiety with PEG and at least one selected from the group consisting of proteins, polypeptides, oligopeptides and amino acids; or a compound obtained by reacting PEG with at least one selected from the group consisting of proteins, polypeptides, oligopeptides and amino acids, and a mixture of unreacted substances acting as the acting portion, unreacted PEG and/or unreacted at least one selected from the group consisting of proteins, polypeptides, oligopeptides and amino acids.
Figure BDA0003628692260000351
The compound according to claim 13, which is a compound obtained by reacting a substance having a carbon chain of 3 to 100 carbon atoms as an active moiety with at least one selected from the group consisting of polysaccharides, monosaccharides, disaccharides and oligosaccharides; or a compound obtained by reacting a substance having a carbon chain of 3 to 100 carbon atoms as an active moiety with at least one selected from the group consisting of polysaccharides, monosaccharides, disaccharides and oligosaccharides, and an unreacted substance as an active moiety and/or a mixture of unreacted polysaccharides, monosaccharides, disaccharides and/or oligosaccharides.
Figure BDA0003628692260000352
The compound according to claim 13, which is a compound obtained by reacting a substance having a carbon chain with 3 to 100 carbon atoms as an active moiety, PEG, and at least one selected from the group consisting of polysaccharides, monosaccharides, disaccharides, and oligosaccharides; or a compound obtained by reacting PEG with at least one selected from polysaccharide, monosaccharide, disaccharide and oligosaccharide, and a mixture of unreacted carbon chain of 3-100 carbon atoms as an acting part, unreacted PEG and/or unreacted polysaccharide, monosaccharide, disaccharide and/or oligosaccharide.
Figure BDA0003628692260000353
The complex according to claim 13, wherein the protein is selected from one or more of serum albumin, immunoglobulin, water-soluble collagen, chaperonin, water-soluble glycoprotein, and CD 14.
Figure BDA0003628692260000354
The complex according to any one of claims 13 to 19, wherein the polysaccharide is selected from one or more of dextran and/or hyaluronic acid, sialic acid, heparin sulfate, heparan sulfate, chondroitin sulfate, dermatan sulfate, keratan sulfate, acetyl water-soluble cellulose derivatives, β -cyclodextrin and its derivatives, and water-soluble chitosan derivatives.
Figure BDA0003628692260000355
The complex according to claim 13, which is a compound obtained by reacting a substance having a carbon chain with 3 to 100 carbon atoms as an active moiety, a linker and a thiol-group-containing protein; or a mixture of the compound obtained by the above reaction, the unreacted substance as the acting portion, the unreacted linker, and/or the unreacted thiol-group-containing protein; wherein the connecting matter is one or more than two of amino acid, succinic acid, butadiene acid, glutaconic acid, hexylamine diacid, carbamate, short peptide, N-hydroxyl crotonoimide, polyethylene glycol and derivatives of the compounds.
Figure BDA0003628692260000361
The compound according to claim 21, which is a compound obtained by reacting a substance containing a carbon chain having 3 to 100 carbon atoms as an active moiety with N-hydroxybuteneimide with a protein containing a thiol group; or a compound obtained by the above reaction, the unreacted substance as the acting part, the unreacted N-hydroxybutylimide and Or unreacted thiol-containing proteins.
Figure BDA0003628692260000362
The compound according to claim 13, which contains 3 to 100 carbon atoms
A compound obtained by reacting a substance having a chain as an active moiety, cystamine, and at least one selected from the group consisting of polysaccharides, monosaccharides, disaccharides, and oligosaccharides; or a compound obtained by reacting cystamine with at least one selected from the group consisting of polysaccharides, monosaccharides, disaccharides and oligosaccharides, and a mixture of unreacted substances acting as a part and unreacted polysaccharides, monosaccharides, disaccharides, oligosaccharides and/or unreacted cystamine.
Figure BDA0003628692260000363
The compound according to any one of claims 13 to 23, wherein the compound obtained by the reaction contains one or more of an amide group, an ester group, a thioether group or an ether group as a linking moiety between the water-soluble moiety and the acting moiety.
Figure BDA0003628692260000364
The compound according to any one of claims 4 to 23, wherein the saturated and/or unsaturated aliphatic hydrocarbon, the saturated and/or unsaturated aliphatic alcohol or the oxoaliphatic alcohol has 3 to 50 carbon atoms.
Figure BDA0003628692260000365
The composite of claim 25, wherein the number of carbon atoms is from 3 to 48.
Figure BDA0003628692260000366
The compound of claim 26, wherein the carbon atom is from 3 to 26.
Figure BDA0003628692260000367
The complex according to any of claims 4-23, wherein the protein is human serum albumin or bovine serum albumin, or CD 14; or the polysaccharide is dextran, heparin and/or hyaluronic acid.
Figure BDA0003628692260000368
The compound according to any one of claims 4 to 12, which is a compound obtained by reacting a surfactant having a carbon chain of 3 to 30 carbon atoms with at least one selected from the group consisting of a dibasic or polybasic fatty acid, an amino acid, a targeting protein, a targeting polypeptide and a targeting polysaccharide; or a mixture of the compound obtained by the reaction, the unreacted surfactant and/or the unreacted dibasic or polybasic fatty acid, the amino acid, the targeting protein, the targeting polypeptide and/or the targeting polysaccharide.
Figure BDA0003628692260000369
The compound according to any one of claims 1 to 29, wherein the surfactant is one or more selected from fatty alcohol polyoxyethylene ether, fatty acid polyoxyethylene ester, alkyl glycoside, sucrose fatty acid ester, sorbitan polyoxyethylene fatty acid ester, mannosylerythritol ester, and N-acyl-N-methylglucamine.
Figure BDA0003628692260000371
The composition of any of claims 1-30, wherein the microbial infection comprises an infection by any one or more of a virus, a bacterium, a fungus, a chlamydia, or a mycoplasma.
Figure BDA0003628692260000372
Prevention, prevention or treatment of disease made with the complexes of any of claims 1-31A preparation for treating microbial infection is provided,
Figure BDA0003628692260000373
the formulation of claim 32, which is a pharmaceutical formulation or an environmental disinfectant formulation.
Figure BDA0003628692260000374
The formulation of claim 33, wherein the pharmaceutical formulation is one selected from the group consisting of an inhalant, a nasal spray, an injection, an oral formulation, and a topical formulation for skin.
Figure BDA0003628692260000375
Use of a complex according to any one of claims 1 to 31 in the preparation of a pharmaceutical preparation or an environmentally acceptable microbicidal agent for the prevention, prevention and/or treatment of microbial infections.
Figure BDA0003628692260000376
The use according to claim 35 wherein the microorganism is any one or more selected from the group consisting of viruses, bacteria, fungi, chlamydia and mycoplasma.
Figure BDA0003628692260000377
The use of claim 36, wherein the virus is an enveloped virus; and/or non-enveloped viruses.
Figure BDA0003628692260000378
The use according to claim 37, wherein the enveloped virus is one or more of coronavirus, influenza virus, aids virus, hepatitis b virus, hepatitis c virus, herpes virus, tecavirus, dengue virus, encephalitis b virus, ebola virus, rabies virus, and hantavirus; the non-envelope The virus is one or more of hepatitis A virus, human papilloma virus, poliovirus and coxsackievirus.
Figure BDA0003628692260000379
The use according to claim 36, wherein the virus is any one or more of coronavirus, hiv, hepatitis b virus, hepatitis c virus, herpes virus, encephalitis b virus, rabies virus, human papilloma virus and ebola virus.
Figure BDA00036286922600003710
The use according to claim 36, wherein the bacterium is a gram-positive and/or gram-negative bacterium and the fungus is a pathogenic and/or conditionally pathogenic fungus; the chlamydia is chlamydia trachomatis, chlamydia pneumoniae and/or chlamydia psittaci; the mycoplasma includes mycoplasma pneumoniae, ureaplasma urealyticum, mycoplasma hominis, and/or mycoplasma genitalium.
Figure BDA00036286922600003711
The use according to claim 36, wherein the bacteria are selected from one or more of escherichia coli, staphylococcus aureus, methicillin-resistant staphylococcus aureus, streptococcus pneumoniae, klebsiella pneumoniae, and pseudomonas aeruginosa; the fungi is selected from one or more of Candida albicans, Aspergillus niger, Actinomyces viscosus, Chaetomium globosum, Aspergillus wart and Microsporum canis.
Figure BDA0003628692260000381
The use according to claim 36, wherein the virus is selected from one or more of the group consisting of H7N9 influenza virus, H5N1 influenza virus, HIV virus, neocoronavirus, HPV virus and rabies virus.
Figure BDA0003628692260000382
The method for preparing a complex according to any one of claims 1 to 32, wherein the complex is obtained by reacting a compound having a fat-soluble saturated and/or unsaturated carbon chain having a branched, cyclic and/or linear structure with a water-soluble molecule, and optionally a protein, polypeptide, amino acid, oligopeptide, oligosaccharide, monosaccharide and/or polysaccharide molecule capable of binding to a microbial lipid membrane, a microbial surface domain or a cell wall, and optionally a linker molecule in the presence of a catalyst.
Figure BDA0003628692260000383
The method of claim 44, wherein the compound is a purified compound obtained by the reaction.
Figure BDA0003628692260000384
The method for preparing a complex according to any one of claims 1 to 31, wherein the complex is obtained by physically mixing a compound having a fat-soluble saturated and/or unsaturated carbon chain having a branched, cyclic and/or linear structure with a water-soluble molecule, and optionally at least one molecule selected from the group consisting of proteins, polypeptides, amino acids, oligopeptides, oligosaccharides, monosaccharides, and polysaccharides capable of binding to a lipid membrane, a viral surface domain, and a cell wall of a microorganism.
Figure BDA0003628692260000385
The method for producing a complex according to any one of claims 1 to 31, wherein the complex is obtained by reacting a substance having a carbon chain with 3 to 100 carbon atoms as an active moiety with at least one substance selected from the group consisting of proteins, polypeptides, oligopeptides, oligosaccharides, amino acids, monosaccharides, disaccharides, nucleotides, vitamins, water-soluble polymers, water-soluble polyamines and polysaccharides in the presence of a catalyst.
Wherein, the catalyst can be one or more of EDC, DCC, NHS, DMAP, HoBt and derivatives and analogues thereof, the catalyst is preferably carbodiimide and succinimide, and the molar ratio of the carbodiimide to the succinimide is 0.1:1-10:1, and is preferably 1:1-1: 10.
Figure BDA0003628692260000386
The method for producing a complex according to claim 46, wherein the complex is a product obtained by purifying a compound obtained by the reaction.
Figure BDA0003628692260000387
The method for producing a complex according to any one of claims 1 to 31, wherein the complex is obtained by physically mixing a substance having a carbon chain with 3 to 100 carbon atoms as an active moiety with at least one member selected from the group consisting of proteins, polypeptides, oligopeptides, polysaccharides, oligosaccharides, amino acids, monosaccharides, disaccharides, nucleotides, vitamins, water-soluble polymers, water-soluble polyamines and polysaccharide molecules in a physicochemical manner or directly.
Specifically, in order to solve the problem of lack of reagents which have no toxic or side effect and particularly cannot widely kill and prevent, prevent or treat microbial infection in the prior art, the invention provides the following third technical scheme:
a complex capable of preventing, arresting and/or treating viral or bacterial infection comprising an active moiety, a binding moiety and a water soluble moiety;
the virus is one or more than two viruses selected from neocoronavirus, influenza virus, AIDS virus, hepatitis B virus, human herpesvirus, Ebola virus, rabies virus and human papilloma virus, and the bacteria is one or more than two bacteria selected from escherichia coli, staphylococcus aureus, methicillin-resistant staphylococcus aureus, streptococcus pneumoniae, klebsiella pneumoniae and pseudomonas aeruginosa;
the action part is a fat-soluble saturated and/or unsaturated carbon chain with a branched chain, a cyclic structure and/or a straight chain structure, the carbon chain is a molecule or a residue of the molecule, and the carbon chain is a carbon chain with 3-100 carbon atoms; wherein the acting part is a carbon chain or residue of a carbon chain with 3-100 carbon atoms formed by saturated and/or unsaturated fatty acid;
The water-soluble moiety is a water-soluble molecule containing one or two or more functional group groups selected from an amide group, a phosphoryloxy group, a carboxylic acid group, a phosphoric acid group, a sulfonic acid group, a sulfonyloxy group, a hydroxyl group, a quaternary ammonium group, a sulfide group, a disulfide group, an ether group, a mercapto group, an aldehyde group, an ester group, an amine group, an amino group, a urea group, and a guanidino group, or a residue of a molecule, and the water-soluble moiety may be the above one or two or more functional group groups linked to a carbon chain as an acting moiety;
the binding moiety is a molecule or residue of a molecule capable of binding to a microbial lipid membrane, a microbial surface protein, a microbial surface polysaccharide or cell wall component or capable of binding to a polysaccharide or protein or polypeptide in a microbe, and the binding moiety may be water soluble
Protein, polypeptide, amino acid, oligopeptide, oligosaccharide, monosaccharide and/or polysaccharide molecules or residues thereof which are partially identical and can bind to the lipid membrane and surface domain of the microorganism;
wherein the number of any one of the acting portion, the water-soluble portion and the binding portion may be 1 or more.
The carbon atom number of the compound is 3-48 according to the technical scheme 1.
③ according to the compound of the technical proposal 1, the carbon atom number is 3 to 26.
The compound according to claim 1, wherein the water-soluble moiety is a water-soluble molecule or a residue of a molecule containing one or more groups selected from a mercapto group, an amino group, a phosphate group, a carboxylate group, a sulfonate group, a hydroxyl group, an amino group, a ureido group, a guanidino group, and a disulfide group;
the binding moiety has a group capable of binding to a microbial lipid membrane, a microbial surface protein, a microbial surface polysaccharide or a cell wall component or to a polysaccharide or a protein or a polypeptide in a microorganism, the group being derived from a water-soluble moiety or from two or more groups selected from mercapto groups, amino groups, phosphate groups, carboxylic acid groups, sulfonic acid groups, hydroxyl groups, amine groups, urea groups, guanidine groups and disulfide groups independently serving as a binding moiety or from one or two or more groups selected from mercapto groups, amino groups, phosphate groups, carboxylic acid groups, sulfonic acid groups, hydroxyl groups, amine groups, urea groups, guanidine groups and disulfide groups providing a carbon chain linkage to the carbon chain, so that the complex has one or two or more groups selected from mercapto groups, amino groups, phosphate groups, carboxylic acid groups, sulfonic acid groups, hydroxyl groups, amine groups, urea groups, guanidine groups and disulfide groups.
The compound according to the technical scheme 4, wherein the binding part is one or more than two selected from dibasic fatty acid or polybasic fatty acid, amino acid, targeting protein, targeting polypeptide and targeting polysaccharide.
Sixthly, the compound is a compound formed by connecting fatty acid with 3-50 carbon atoms and water-soluble amino acid according to the technical scheme 4; or the compound is formed by connecting fatty acid with 3-50 carbon atoms with targeting polypeptide; or the compound is formed by the reaction of fatty acid with 3-50 carbon atoms, targeting polypeptide and PEG; or the compound is formed by the reaction of a surfactant and one or more than two of dibasic fatty acid or polybasic fatty acid, amino acid, targeting protein, targeting polypeptide and targeting polysaccharide.
The compound of claim 4, wherein the saturated and/or unsaturated fatty acid is selected from saturated or unsaturated fatty acids with 3-50 carbon atoms, which is a fatty acid or amino acid containing double bond, triple bond, hydroxyl, amino and/or oxo-substituted, and is a mono-, di-or poly-acid.
-the compound according to claim 4, wherein the saturated and/or unsaturated fatty acids are selected from saturated fatty acids with 3-46 carbon atoms, monooleic acids with 3-34 carbon atoms, dienoic acids with 5-30 carbon atoms, trienoic acids with 7-30 carbon atoms, arachidonic acids with 12-38 carbon atoms, pentaenoic acids with 12-38 carbon atoms, hexaenoic acids with 22-38 carbon atoms, alkynoic acids with 6-22 carbon atoms, diynoic acids with 10-22 carbon atoms, trialkynoic acids with 12-22 carbon atoms, alkynoic acids with 8-20 carbon atoms, fatty acids with 3-30 carbon atoms in the main chain and 1-10 alkyl and/or 1-3 hydroxyl groups in the side chain, saturated straight and branched dicarboxylic and tricarboxylic acids with 3-38 carbon atoms, and unsaturated straight or branched chain dicarboxylic or tricarboxylic acids with 4-18 carbon atoms Dicarboxylic acids and tricarboxylic acids which are branched and may be substituted with a hydroxyl group, carboxylic acids substituted with an amino group having 3 to 18 carbon atoms, a hydroxyl group, an oxo group and/or a methyl group, N-acyl amino acids having 6 to 30 carbon atoms, amino acids containing 2 or more acyl groups, and polycarboxylic acids which are linked by a thioether bond and an amide bond.
Ninthly, according to the complex of claim 4, the saturated/unsaturated fatty acid is one or more selected from fumaric acid, octanoic acid, glutaconic acid, hexanoic acid, nonanoic acid, dodecanoic acid, tetradecanoic acid, hexadecanoic acid, octadecanoic acid, eicosanoic acid, oleic acid, linoleic acid, linolenic acid, eicosapentaenoic acid, docosapentaenoic acid, docosahexaenoic acid, pentacosanoic acid, heptanoic acid, decanoic acid, dodecenoic acid, arachidonic acid, triacontahexaenoic acid, octacosanoic acid, or a carbon chain residue formed therefrom.
The complex of claim 4, wherein the water-soluble moiety is a molecule or residue of a molecule containing one or more groups selected from thiol, amino, carboxylate, hydroxyl, and disulfide groups; the molecule is selected from one or more than two water-soluble macromolecules or residues thereof in protein, polysaccharide, nucleic acid and artificially synthesized water-soluble polymers;
and/or one or more than two medium molecules selected from polypeptide, oligopeptide, oligosaccharide, oligonucleotide and artificially synthesized water-soluble medium molecular weight polymer or residues thereof;
and/or, water-soluble small molecules selected from one or more than two of amino acids, monosaccharides, disaccharides, nucleotides, water-soluble vitamins and deoxynucleotides, or residues thereof;
And/or a molecule or a residue of a molecule linked to the carbon chain as the acting moiety, said molecule or residue of a molecule comprising one or more groups selected from thiol, amino, carboxylic acid, hydroxyl, disulfide groups.
Figure BDA0003628692260000411
The complex according to claim 10, wherein the protein as the water-soluble macromolecule is one or more water-soluble macromolecules selected from the group consisting of serum albumin, immunoglobulin, water-soluble collagen, chaperonin, water-soluble glycoprotein, and CD 14; the polysaccharide as the macromolecule is one or more than two water-soluble macromolecules selected from dextran, hyaluronic acid, sialic acid, heparin sulfate, heparan sulfate, chondroitin sulfate, dermatan sulfate, keratan sulfate, acetyl water-soluble cellulose derivatives, beta-cyclodextrin and derivatives thereof and water-soluble chitosan derivatives; the water-soluble polymer as the macromolecule is one or more than two water-soluble macromolecules selected from polyethylene glycol, carboxylated or aminated polyethylene glycol, polyvinyl alcohol, carboxylated or quaternized polyvinyl alcohol, polyacrylic acid and ammonium polyacrylate;
the water-soluble polymer with medium molecular weight is selected from one or more than two of targeting polypeptide, oligopeptide, oligosaccharide, oligonucleotide and/or water-soluble polyamino acid;
The monosaccharide and/or disaccharide of the water-soluble micromolecule is selected from one or more of glucose, fructose, rhamnose, sorbose, sucrose, maltose, lactose and trehalose; the nucleotide and/or deoxynucleotide as the water-soluble small molecule is selected from adenylic acid, guanylic acid, uridylic acid, cytidylic acid, thymidylic acid, inosinic acid, deoxyadenylic acid, deoxyguanylic acid, deoxycytidylic acid and deoxythymidylic acid; amino acids such as one or more of serine, threonine, cysteine, asparagine, glutamine, tyrosine, lysine, arginine, histidine, aspartic acid, glutamic acid, citrulline, ornithine, taurine and aminobutyric acid; the vitamins as the water-soluble small molecules are selected from one or more of vitamin B1, pantothenic acid, vitamin B6 and vitamin C.
Figure BDA0003628692260000421
The complex according to claim 11, wherein the targeting polypeptide comprises any one of a protein or neutralizing antibody fragment that specifically targets microbial lipid membranes, bacterial and fungal cell walls, viral surface protein domains.
Figure BDA0003628692260000422
The complex according to claim 11, wherein the water-soluble polyamino acid is selected from polyglutamic acid, polylysine and/or polyaspartic acid.
Figure BDA0003628692260000423
The compound according to claim 1, wherein the binding moiety and the water-soluble moiety are the same, i.e. a protein, a polypeptide, an oligopeptide, an oligosaccharide, a monosaccharide, a disaccharide, an amino acid, a nucleotide, a vitamin, a water-soluble polymer, a water-soluble polyamino acid and/or a polysaccharide molecule capable of binding to a microbial lipid membrane, a surface domain, or a residue of such a molecule, and the molecule or residue of the molecule comprises one or more groups selected from thiol, amino, carboxylic acid, hydroxyl, and disulfide groups.
Figure BDA0003628692260000424
The compound according to claim 1, which is a compound obtained by reacting a saturated and/or unsaturated fatty acid having 3 to 100 carbon atoms with a protein, a polypeptide, an oligopeptide, an oligosaccharide, a monosaccharide, a disaccharide, a nucleotide, a vitamin, an amino acid, a water-soluble polymer, a water-soluble polyamino acid and/or a polysaccharide; or a compound obtained by reacting saturated and/or unsaturated fatty acid containing 3-50 carbon atoms with protein, polypeptide, oligopeptide, oligosaccharide, monosaccharide, disaccharide, nucleotide, vitamin, amino acid, water-soluble polymer, water-soluble polyamino acid and/or polysaccharide moleculeAnd a mixture of unreacted said fatty acids and/or unreacted said proteins, polypeptides, oligopeptides, oligosaccharides, monosaccharides, disaccharides, nucleotides, vitamins, amino acids, water-soluble polymers, water-soluble polyamines and/or polysaccharide molecules.
Figure BDA0003628692260000431
The compound according to claim 1, which is a compound comprising saturated and/or unsaturated fatty acids having 3 to 100 carbon atoms and proteins, polypeptides, oligopeptides, oligosaccharides, monosaccharides, disaccharides, nucleotides, vitamins, amino acids, water-soluble polymers, water-soluble polyamines and/or polysaccharide molecules, complexed by physicochemical interactions including hydrogen bonding or van der waals forces or a combination of both interactions, or a mixture obtained by direct physical mixing.
Figure BDA0003628692260000432
The compound according to claim 15, which is a compound obtained by reacting a saturated and/or unsaturated fatty acid having 3 to 100 carbon atoms with at least one selected from the group consisting of a protein, a polypeptide, an oligopeptide and an amino acid; or a mixture of a compound obtained by reacting a saturated and/or unsaturated fatty acid having 3 to 100 carbon atoms with at least one selected from the group consisting of proteins, polypeptides, oligopeptides and amino acids, and an unreacted fatty acid and/or an unreacted at least one selected from the group consisting of proteins, polypeptides, oligopeptides and amino acids.
Figure BDA0003628692260000433
The compound according to claim 15, which is a compound obtained by reacting a saturated and/or unsaturated fatty acid having 3 to 100 carbon atoms, PEG, and at least one selected from the group consisting of a protein, a polypeptide, an oligopeptide, and an amino acid; or a compound obtained by reacting saturated and/or unsaturated fatty acid having 3-100 carbon atoms, PEG and at least one selected from protein, polypeptide, oligopeptide and amino acid And unreacted fatty acids, unreacted PEG, and/or unreacted at least one member selected from the group consisting of proteins, polypeptides, oligopeptides, and amino acids.
Figure BDA0003628692260000434
The compound according to claim 15, which is a compound obtained by reacting a saturated and/or unsaturated fatty acid having 3 to 100 carbon atoms with a polysaccharide, a monosaccharide, a disaccharide and/or an oligosaccharide; or a compound obtained by reacting a saturated and/or unsaturated fatty acid having 3 to 100 carbon atoms with a polysaccharide, a monosaccharide, a disaccharide and/or an oligosaccharide, and a mixture of an unreacted fatty acid and/or an unreacted polysaccharide, monosaccharide, disaccharide and/or oligosaccharide.
Figure BDA0003628692260000435
The compound according to claim 15, which is a compound obtained by reacting a saturated and/or unsaturated fatty acid having 3 to 100 carbon atoms, PEG, and at least one selected from the group consisting of polysaccharides, monosaccharides, disaccharides, and oligosaccharides; or a compound obtained by reacting a saturated and/or unsaturated fatty acid having 3 to 100 carbon atoms, PEG and at least one member selected from the group consisting of polysaccharides, monosaccharides, disaccharides and oligosaccharides, and a mixture of unreacted fatty acid, unreacted PEG and/or unreacted polysaccharides, monosaccharides, disaccharides and/or oligosaccharides.
Figure BDA0003628692260000441
The complex according to claim 15, wherein the protein is selected from one or more of serum albumin, immunoglobulin, water-soluble collagen, chaperonin, water-soluble glycoprotein, and CD 14.
Figure BDA0003628692260000442
The complex according to claim 15, wherein the polysaccharide is selected from the group consisting of dextran and/or hyaluronic acid, sialic acid, heparin sulfate, heparan sulfate, heparin sulfate, and heparin sulfate, and heparin sulfate, wherein the conjugate is a conjugate,Chondroitin sulfate, dermatan sulfate, keratan sulfate, acetyl water-soluble cellulose derivative, beta-cyclodextrin and its derivative, and water-soluble chitosan derivative.
Figure BDA0003628692260000443
The complex according to claim 15, which is a compound obtained by reacting a saturated and/or unsaturated fatty acid having 3 to 100 carbon atoms, a linker and a thiol-group-containing protein; or a mixture of compounds obtained by the above reaction, unreacted fatty acids, unreacted linkers, and/or unreacted thiol-containing proteins; wherein the connecting matter is one or more than two of amino acid, succinic acid, butadiene acid, glutaconic acid, hexylamine diacid, carbamate, short peptide, N-hydroxyl crotonoimide, polyethylene glycol and derivatives of the compounds.
Figure BDA0003628692260000444
The compound according to claim 23, which is a compound obtained by reacting saturated and/or unsaturated fatty acid containing 3-100 carbon atoms, N-hydroxy crotonoimide and protein containing sulfydryl; or a mixture of compounds obtained by the above reaction, unreacted fatty acids, unreacted N-hydroxybuteneimide and/or unreacted thiol-containing proteins.
Figure BDA0003628692260000445
The compound according to claim 15, which is a compound obtained by reacting saturated and/or unsaturated fatty acids having 3 to 100 carbon atoms, cystamine, and at least one selected from polysaccharides, monosaccharides, disaccharides, and oligosaccharides; or a compound obtained by reacting saturated and/or unsaturated fatty acid having 3-50 carbon atoms, cystamine and at least one selected from polysaccharide, monosaccharide, disaccharide and oligosaccharide, and unreacted fatty acid selected from polysaccharide, monosaccharide, disaccharide and oligosaccharideAnd/or a mixture of unreacted cystamines.
Figure BDA0003628692260000446
The compound according to any one of claims 15 to 25, wherein the compound obtained by the reaction contains one or more groups selected from amide groups, ester groups, thioether groups and ether groups, as a linking moiety between the water-soluble moiety and the acting moiety.
Figure BDA0003628692260000447
The complex according to any one of claims 4 to 25, wherein the saturated and/or unsaturated fatty acid has 3 to 50 carbon atoms.
Figure BDA0003628692260000448
The compound of claim 27, wherein the number of carbon atoms is from 3 to 48.
Figure BDA0003628692260000451
The compound of claim 27, wherein the carbon atom is from 3 to 26.
Figure BDA0003628692260000452
The complex according to any one of claims 4 to 25, wherein the saturated and/or unsaturated fatty acid is a fatty acid having 3 to 40 carbon atoms and containing 1 to 8C ═ C double bonds, a fatty acid having 1 to 7C ═ C double bonds, a fatty acid having 1 to 6 double bonds, a fatty acid having 1 to 5 double bonds, a fatty acid having 1 to 4 double bonds, a fatty acid having 1 to 3 double bonds, or a fatty acid having 1 to 2 double bonds.
Figure BDA0003628692260000453
The compound according to any one of claims 4 to 25, which is saturated and/or unsaturatedThe saturated fatty acid has 1-6 double bonds and 3-30 carbon atoms.
Figure BDA0003628692260000454
The complex according to any one of claims 4 to 25, wherein the saturated and/or unsaturated fatty acid has 3 to 30 carbon atoms.
Figure BDA0003628692260000455
The complex according to any one of claims 4 to 25, wherein the saturated and/or unsaturated fatty acid is selected from one or more fatty acids selected from fumaric acid, octanoic acid, glutaconic acid, hexanoic acid, nonanoic acid, dodecanoic acid, tetradecanoic acid, hexadecanoic acid, octadecanoic acid, eicosanoic acid, oleic acid, linoleic acid, linolenic acid, eicosapentaenoic acid, docosapentaenoic acid, docosahexaenoic acid, pentacosanoic acid, heptanoic acid, decanoic acid, dodecenoic acid, arachidonic acid, triacontahexaenoic acid, and octacosanoic acid.
Figure BDA0003628692260000456
The complex according to any one of claims 4-25, wherein the protein is human serum protein or bovine serum albumin, or CD 14; or the polysaccharide is dextran and/or hyaluronic acid.
Figure BDA0003628692260000457
The invention also provides a preparation prepared from the compound for preventing, preventing or treating microbial infection.
Figure BDA0003628692260000458
The preparation is a pharmaceutical preparation or an environmental disinfectant preparation.
Figure BDA0003628692260000459
According to the preparation, the pharmaceutical preparation is one selected from inhalant, nasal spray, injection, oral preparation and external preparation for skin.
Figure BDA00036286922600004510
The use of a complex according to the invention for the preparation of a pharmaceutical preparation or an environmentally sound microbicide for the prevention, prevention and/or treatment of microbial infections.
The use according to the present invention, wherein the microorganism is any one or two selected from viruses and bacteria.
The use according to the invention, wherein the virus is an enveloped virus; and/or non-enveloped viruses.
According to the application of the invention, the virus is one or more than two viruses selected from new corona virus, influenza virus, Human Immunodeficiency Virus (HIV), hepatitis B virus, human herpesvirus, Ebola virus, rabies virus and Human Papilloma Virus (HPV), and the bacteria is one or more than two bacteria selected from escherichia coli, staphylococcus aureus, methicillin-resistant staphylococcus aureus, streptococcus pneumoniae, klebsiella pneumoniae and pseudomonas aeruginosa.
The application of the invention, wherein the virus is one or more than two of H7N9 influenza virus, H5N1 influenza virus, HIV virus, new corona virus, HPV virus and rabies virus.
Figure BDA0003628692260000461
The invention also provides a preparation method of the compound, which comprises the steps of carrying out reverse reaction on the fatty acid and the water-soluble molecule which have fat-soluble saturated and/or unsaturated carbon chains with branched chains, cyclic structures and/or linear structures, and protein, polypeptide, amino acid, oligopeptide, oligosaccharide, monosaccharide and/or polysaccharide molecules which can be combined with microbial lipid membranes, microbial surface domains or cell walls and linker molecules which are added according to needs in the presence of a catalystThe complex should be obtained.
Further preferably, according to the preparation method of the complex of the present invention, the complex is a product obtained by purifying a compound obtained by the reaction.
Figure BDA0003628692260000462
The invention also provides a preparation method of the compound, which is obtained by physically mixing the fatty acid with fat-soluble saturated and/or unsaturated carbon chains with branched chains, cyclic structures and/or linear structures, the water-soluble molecule and protein, polypeptide, amino acid, oligopeptide, oligosaccharide, monosaccharide and/or polysaccharide molecules which can be combined with microbial lipid membranes, virus surface domains or cell walls and are added according to needs.
Figure BDA0003628692260000463
The invention also provides a preparation method of the compound, which comprises the step of reacting saturated and/or unsaturated fatty acid with 3-100 carbon atoms with any one of protein, polypeptide, oligopeptide, polysaccharide, oligosaccharide, monosaccharide, disaccharide, nucleotide, vitamin, amino acid, water-soluble polymer, water-soluble polyamino acid and/or polysaccharide in the presence of a catalyst to obtain the compound.
Further preferably, the complex is a product obtained by purifying a compound obtained by the reaction.
Figure BDA0003628692260000464
The invention also provides a preparation method of the compound, which is obtained by compounding the compound which contains saturated and/or unsaturated fatty acid with 3-100 carbon atoms and protein, polypeptide, oligopeptide, polysaccharide, oligosaccharide, monosaccharide, disaccharide, nucleotide, vitamin, amino acid, water-soluble polymer, water-soluble polyamino acid and/or polysaccharide molecules by physicochemical action or direct physical mixing.
The compound of the invention can prevent and treat the application of the compound with the effects of virus, bacteria and fungal infection and the preparation thereof in preventing or treating various virus, bacteria and fungal infectious diseases; the specific application mode comprises the following steps:
can be used before infection to prevent viral, bacterial and fungal infection;
can be used for killing virus, bacteria and fungi in vivo after infection; and
the environment of the article may be sanitized to prevent the spread of viruses, bacteria, and fungi.
Compared with the prior art, the invention has the following beneficial effects:
(1) the effect of the complex provided by the invention on viruses is not influenced by virus variation
The action target of the compound provided by the invention is the basic structure of the virus, namely, the envelope and the nucleocapsid, and for enveloped viruses, the compound destroys the virus envelope, so that the virus loses the capability of infecting cells; for non-enveloped viruses, the complex directly wraps the virus nucleocapsid for hydrophobic isolation, so that the virus cannot infect cells; the complex is not rendered ineffective by viral variation.
(2) The compound provided by the invention can kill drug-resistant microorganisms without causing the microorganisms to generate drug resistance.
The bacteria show drug resistance because of the existence of drug resistance gene in the body, which can express enzyme for decomposing antibiotic to make the antibiotic lose efficacy. The mechanism of killing the microbes by the compound is different from that of antibiotics, the compound directly acts on a basic structure of microbes, namely a lipid membrane, and the compound is partially fused into the lipid membrane by action to influence the steady state of the lipid membrane and destroy cell walls and cell membranes so as to achieve the killing effect. Therefore, the enzyme is not affected by the enzyme decomposing the antibiotic in the drug-resistant bacteria.
(3) The complex provided by the invention is safe to human cells
The cells of the virus particles and bacteria and fungi are far smaller than those of human body, and the compound with therapeutic dose is preferentially combined with the viruses, bacteria and fungi to produce the effect. Cell experiments prove that the compound has no obvious influence on cell membranes under the treatment dosage. The complex is safe to human cells.
(4) The complex provided by the invention can play a role in different areas according to the size of molecules; the macromolecular complexes can be retained on the surface of the respiratory mucosa or in the blood circulation, and can inactivate viruses, bacteria or fungi for the first time and prevent the viruses, bacteria or fungi from diffusing in the body. The macromolecular compound can not enter normal tissues and can only enter inflammatory parts after virus, bacteria or fungi infection; and the small molecular compound can penetrate through the blood vessel wall to enter the interstitial space and interstitial fluid of the tissue, and target viruses, bacteria or fungi to kill.
(5) Fatty acid, fatty alcohol, fat-soluble vitamin, steroid and the like are insoluble in water or have extremely low water solubility, cannot be directly injected into a human body, and can cause pulmonary embolism when being directly injected into a vein; fatty acid absorption in food is absorbed by human body in the form of emulsion, flows back into blood circulation in the form of chylomicron through lymphatic system via lymphatic vessels and thoracic ducts, and is encapsulated in the emulsion in the form of non-covalent binding with protein. The hydrophobic group in this form is encapsulated inside and does not contact infected viruses and bacteria, and thus has no bactericidal and antiviral effects. The above problems can be avoided by converting the fat-soluble hydrophobic compound into an aqueous solution compound having a high affinity for pathogenic microorganisms.
Drawings
FIG. 1 is a graph comparing the infrared spectra of linolenic acid-serum albumin produced in example 1;
FIG. 2 is a chart showing the results of Coomassie blue staining of fumaric acid, linolenic acid, eicosapentaenoic acid, and docosahexaenoic acid-serum albumin obtained in example 1;
FIG. 3A is a chart of mass spectrometry of linolenic acid-serum albumin obtained in example 1;
FIG. 3B is a graph showing the site analysis of linolenic acid-modified albumin prepared in example 1;
FIG. 4A is a graph of mass spectrometry of docosahexaenoic acid-serum albumin prepared in example 1;
FIG. 4B is a graph showing the site analysis of docosahexaenoic acid-modified albumin obtained in example 1;
FIG. 5 is a mass spectrum of oleic acid-serum albumin obtained in example 2;
FIG. 6 is a graph showing the site analysis of oleic acid-modified serum albumin in the compound prepared in example 2;
FIG. 7 is a graph comparing the infrared spectra of eicosapentaenoic acid-serum albumin obtained in example 3;
FIG. 8 is a mass spectrum of eicosapentaenoic acid-serum albumin obtained in example 4;
FIG. 9 is a graph showing the site analysis of eicosapentaenoic acid-modified serum albumin in the compound produced in example 4;
FIG. 10 is a mass spectrum of linoleic acid-serum albumin obtained in example 5;
FIG. 11 is a graph showing the site analysis of linoleic acid modified serum albumin in the compound prepared in example 5;
FIG. 12 is a graph comparing the infrared spectra of docosahexaenoic acid-serum albumin obtained in example 6;
FIG. 13 is a mass spectrum of docosahexaenoic acid-serum albumin obtained in example 6;
FIG. 14 is a graph showing the site analysis of docosahexaenoic acid-modified serum albumin in the compound prepared in example 6;
FIG. 15 is a graph comparing infrared spectra of linoleic acid and hyaluronic acid obtained in example 7;
FIG. 16 is a graph comparing infrared spectra of docosahexaenoic acid-hyaluronic acid obtained in example 8;
FIG. 17 is a comparison of the IR spectrum of fatty acid-SBP 1 obtained in example 9;
FIG. 18 is a comparative chart showing the infrared spectrum of 9-decatetraenoic acid-SBP 1 obtained in example 10;
FIG. 19 is an infrared spectrum of an eight carbon saturated carbon chain-glucose complex prepared in example 14;
FIG. 20 is a graph showing the bacteriostatic results of the eight-carbon saturated carbon chain-glucose complex prepared in example 14;
FIG. 21 is an infrared spectrum of an eight carbon saturated carbon chain-sucrose complex prepared in example 15;
FIG. 22 is a graph showing the bacteriostatic results of the eight-carbon saturated carbon chain-sucrose complex prepared in example 15;
FIG. 23 is an infrared spectrum of the fatty acid-adenosine monophosphate complex prepared in example 16;
FIG. 24 is a graph showing the bacteriostatic results of the eight-carbon saturated carbon chain-adenosine monophosphate complex prepared in example 16;
FIG. 25 is an infrared spectrum of an eight carbon saturated carbon chain-ascorbic acid complex prepared in example 17;
FIG. 26 is a graph showing the bacteriostatic results of the eight-carbon saturated carbon chain-ascorbic acid complex prepared in example 17;
FIG. 27 is an IR spectrum of an eight carbon saturated carbon chain-polyethylene glycol 400-COOH complex prepared in example 18;
FIG. 28 is a graph showing the bacteriostatic results of the eight-carbon saturated carbon chain-polyethylene glycol 400-COOH complex prepared in example 18;
FIG. 29 is a transmission electron microscope image of the liposome of ethyl oleate obtained in (1) of example 19;
FIG. 30 is a transmission electron microscope image of linoleic acid liposome prepared in (2) of example 19;
FIG. 31 shows an injection of linolenic acid-serum albumin prepared in example 20;
FIG. 32 is a graph showing the particle size distribution measured by a linolenic acid-serum albumin Malvern sizer prepared in example 20;
FIG. 33 is a transmission electron microscope image of linolenic acid-serum albumin obtained in example 20;
FIG. 34 is a lyophilized powder injection of linoleic acid-hyaluronic acid prepared in example 21;
FIG. 35 is a lyophilized powder injection of docosahexaenoic acid-hyaluronic acid prepared in example 21;
FIG. 36 is a transmission electron microscope image of a lyophilized linoleic acid-hyaluronic acid powder prepared in example 21 redissolved in water;
FIG. 37 is a graph showing the particle size distribution measured by a Malvern particle sizer after the linoleic acid-hyaluronic acid lyophilized powder prepared in example 21 was redissolved in water;
FIG. 38 is a liposome-lyophilized powder of the dodecanoic acid aspartic acid complex prepared in example 22;
FIG. 39 is the SEM image of the dodecanoic acid aspartic acid complex liposome lyophilized powder preparation prepared in example 22;
FIG. 40 is a graph showing the distribution of particle size of the dodecanoic acid aspartic acid complex liposome lyophilized powder preparation prepared in example 22 after being reconstituted in water;
FIG. 41 is a graph showing the potential distribution of a dodecanoic acid aspartic acid complex liposome lyophilized powder prepared in example 22;
FIG. 42 is a transmission electron micrograph of ethyl eicosapentaenoate injection prepared according to example 23;
figure 43 is a particle size distribution plot of an ethyl eicosapentaenoate injection prepared in example 23;
figure 44 is a graph of the potential profile of ethyl eicosapentaenoate injection prepared in example 23;
FIG. 45 is a transmission electron micrograph of docosahexaenoic acid-SBP 1 obtained in example 24;
FIG. 46 is a graph showing the particle size distribution measured by a docosahexaenoic acid-SBP 1 Malvern particle sizer obtained in example 24;
FIG. 47 is a graph showing the products of grafting SBP1 polypeptide prepared in example 24 with different omega-3 fatty acids (ALA: linolenic acid, EPA: eicosapentaenoic acid, DHA: docosahexaenoic acid);
FIG. 48 is a TEM image of a reconstituted solution of the lyophilized powder injection containing CD14 protein grafted dodecenoic acid prepared in example 25;
FIG. 49 is a TEM image of a reconstituted solution of CD14 protein grafted DOA lyophilized powder for injection prepared in example 25;
FIG. 50 is a TEM image of a reconstituted CD14 protein-grafted eicosapentaenoic acid lyophilized powder for injection prepared in example 25;
FIG. 51 is a chart of the results of the VERO E6 cell safety experiment for the eight carbon saturated carbon chain-threonine prepared in example 29;
FIG. 52 is a graph of the results of a VERO E6 cell safety experiment for the eight carbon saturated carbon chain-serine prepared in example 29;
FIG. 53 is a chart of VERO E6 cell safety assay results for the octadecane monounsaturated carbon chain-serine prepared in example 29;
FIG. 54 is a chart of VERO E6 cell safety assay results for the octadecane monounsaturated carbon chain-threonine prepared in example 29;
FIG. 55 is a graph of the results of a VERO E6 cell safety experiment for the docosane polyunsaturated carbon chain-threonine made in example 29;
FIG. 56 is a chart of VERO E6 cell safety assay results for the eicosa polyunsaturated carbon chain-serine prepared in example 29;
FIG. 57 is a chart of VERO E6 cell safety assay results for the octadecane monounsaturated carbon chain-lysine prepared in example 29;
FIG. 58 is a graph of the results of a VERO E6 cell safety experiment for the docosane polyunsaturated carbon chain-lysine prepared in example 29;
FIG. 59 is a chart of VERO E6 cell safety assay results for the octadecane polyunsaturated carbon chain-threonine prepared in example 29;
FIG. 60 is a graph of the results of a VERO E6 cell safety experiment with an eight carbon saturated carbon chain-5' -monophosphate adenosine-four carbon unsaturated carbon chain-carboxy group prepared in example 29;
FIG. 61 is a graph of the results of the VERO E6 cell safety experiments for N-octyl-N-methylglucamine from example 29;
FIG. 62 is a graph of the results of the VERO E6 cell safety experiment for N-nonyl-N-methylglucamine of example 29;
FIG. 63 is a graph showing the bacteriostatic results of Staphylococcus aureus using the eight-carbon saturated carbon chain-threonine prepared in example 30;
FIG. 64 is a graph showing the bacteriostatic results of Staphylococcus aureus using the eight-carbon saturated carbon chain-serine prepared in example 30;
FIG. 65 is a graph showing the bacteriostatic results of Staphylococcus aureus using the octadecane monounsaturated carbon chain-serine prepared in example 30;
FIG. 66 is a graph showing the bacteriostatic results of Staphylococcus aureus with an eighteen carbon monounsaturated carbon chain-threonine prepared in example 30;
FIG. 67 is a graph of Staphylococcus aureus inhibition results of the docosane polyunsaturated carbon chain-threonine made in example 30;
FIG. 68 is a graph of Staphylococcus aureus inhibition results of the docosane polyunsaturated carbon chain-serine prepared in example 30;
FIG. 69 is a graph showing the bacteriostatic results of Staphylococcus aureus with the eighteen carbon monounsaturated carbon chain-lysine prepared in example 30;
FIG. 70 is a graph showing the bacteriostatic results of Staphylococcus aureus using the docosane polyunsaturated carbon chain-lysine prepared in example 30;
FIG. 71 is a graph of Staphylococcus aureus bacteriostatic results of the eighteen polyunsaturated carbon chain-threonine made in example 30;
FIG. 72 is a graph showing the bacteriostatic effect of Staphylococcus aureus with eight carbon saturated carbon chain-5' -monophosphoadenosine-four carbon unsaturated carbon chain-carboxyl group prepared in example 30;
FIG. 73 is a graph showing the bacteriostatic results for Staphylococcus aureus of N-octyl-N-methylglucamine in example 30;
FIG. 74 is a graph showing the bacteriostatic results for Staphylococcus aureus of N-nonyl-N-methylglucamine of example 30;
FIG. 75 is a graph of the cytotoxicity of omega-3 fatty acid-serum albumin complex against VERO E6, as determined in example 32;
FIG. 76 shows the results of the toxicity test of fatty acid-serum albumin complex for hepatocytes in example 33;
FIG. 77 is the cytotoxicity assay results of carboxy-eight carbon unsaturated carbon chain-taurocholic acid against VERO-E6 in example 34;
FIG. 78 is a graph showing the results of the hepatorenal function in the animal safety test in (1) of example 35;
FIG. 79 is a graph showing the results of the liver and kidney functions in the animal safety test in (2) of example 35;
FIG. 80 is a graph showing the results of the liver and kidney functions in the animal safety test in (3) of example 35;
FIG. 81 is a graph showing the results of the liver and kidney functions in the animal safety test in (4) of example 35;
FIG. 82 is a graph showing the results of the hepatorenal function in the animal safety test in (5) of example 35;
FIG. 83 is a graph showing the result of a hemolysis experiment in the animal safety test in (6) of example 35;
FIG. 84 is the neutralization inhibition ratio of fatty acid (omega-3 fatty acid) -serum albumin complex against neocoronavirus in example 36;
FIG. 85 shows the neutralization inhibition rate of the docosahexaenoic acid-cyclodextrin inclusion complex for rabies pseudoviruses in example 37;
FIG. 86 is the neutralization inhibition rate of the docosahexaenoic acid-SBP 1 complex of example 38 against the novel coronaviruses;
FIG. 87 is a graph showing the neutralization inhibition rate of hexanoic acid-hyaluronic acid complex against HIV pseudovirus HIV18A-41 in example 39;
FIG. 88 is the neutralization inhibition rate of the influenza pseudovirus H7N9-Fluc by the N-nonanoic acid-hyaluronic acid complex in example 40;
FIG. 89 is the neutralization inhibition of HIV pseudovirus by octadecanoic acid-serum albumin complex in example 41;
FIG. 90 is the neutralization inhibition ratio of H7N9-Fluc pseudovirus by the eicosanoic acid-hyaluronic acid complex in example 42;
FIG. 91 is a graph of the neutralization inhibition rate of octacosanoic acid-serum albumin complexes against H5N1-Fluc pseudovirus in example 43;
FIG. 92 shows the results of HIV-derived lentiviruses transfected by hepatocytes pretreated with fatty acid-serum albumin complex of example 44;
FIG. 93 is a transmission electron micrograph of the novel coronavirus of example 45 after treatment;
FIG. 94 is a schematic representation of the process of hydrophobic sequestration of human papillomavirus by the "carbon chain interacting moiety + small molecule water soluble moiety/binding moiety" complex of example 46;
FIG. 95 is a graph that shows a simulation of the process of encapsulating protein particles loaded with L1 protein with N-octyl-N-methylglucamine in vitro in example 46;
FIG. 96 is the neutralization inhibition ratio of docosahexaenoic acid-conjugated serine on HPV pseudovirus in example 47;
FIG. 97 is a graph showing the results of different concentrations of the docosahexaenoic acid-serum albumin complex prepared in example 48 against bacteria (methicillin-resistant Staphylococcus aureus);
FIG. 98 is a graph showing bacteriostatic (E.coli) results for different concentrations of docosahexaenoic acid-serum albumin complex prepared in example 48;
FIG. 99 is a structural change of Escherichia coli observed under a scanning electron microscope after the docosahexaenoic acid-serum albumin complex prepared in example 48 acts thereon;
FIG. 100 is a graph showing the structural changes of Staphylococcus aureus observed under a scanning electron microscope after the action of the docosahexaenoic acid-serum albumin complex prepared in example 48;
FIG. 101 is a graph showing the detachment of a membrane of Staphylococcus aureus, which was observed under a transmission electron microscope, after the action of the docosahexaenoic acid-serum albumin complex prepared in example 48;
FIG. 102 is a graph showing the comparison of binding rates of docosahexaenoic acid-serum albumin complex by the neocoronavirus, Staphylococcus aureus, Escherichia coli, hepatic stellate cell and the serum albumin complex in example 49;
FIG. 103 is a graph showing the results of the retention time of docosahexaenoic acid-serum albumin complex in the lung in example 50;
FIG. 104 is a graph showing the results of the retention time of eicosapentaenoic acid-hyaluronic acid complex in example 50 in the lung;
FIG. 105 is a graph showing the results of an animal experiment using docosahexaenoic acid-serum albumin complex in example 51;
FIG. 106 is a graph showing the results of the lung administration test of the eicosapentaenoic acid-hyaluronic acid complex animal in example 51;
FIG. 107 is a graph showing the fluorescence results of lungs obtained by the oral administration of the small molecule complex to the animal in example 52;
FIG. 108 is a graph showing the result of analysis of mean lung fluorescence value ImageJ in the animal subjected to oral administration of the small molecule complex in example 52;
FIG. 109 is a graph showing the fluorescence results of the lungs of the animal subjected to the gavage administration of the small molecule complex in example 52;
FIG. 110 is an infrared spectrum of a heparin-oleic acid complex prepared in example 53;
FIG. 111 is a graph showing the results of the neutralization inhibition rate of the heparin-oleic acid complex against hepatitis B pseudovirus in example 53;
FIG. 112 is a graph showing the results of HE staining of a mucosal section of mouse sinus in example 54;
FIG. 113 is a graph showing the results of measuring the myeloperoxidase activity in the nasal mucosa tissues of the mouse in example 54;
FIG. 114 is a graph of an infrared spectrum of a Tween 80-threonine complex prepared in example 55;
FIG. 115 is an infrared spectrum of a cholesterol-PEG 400-fumaric acid complex obtained in example 56;
FIG. 116 is an infrared spectrum of a phosphatidylethanolamine-PEG 1000-suberic acid complex prepared in example 57;
FIG. 117 is an infrared spectrum of an α -tocopherol-hyaluronic acid complex prepared in example 58;
FIG. 118 is an infrared spectrum of a sodium cholate-hyaluronic acid complex prepared in example 59;
FIG. 119 is an infrared spectrum of a composite obtained in example 60.
Detailed Description
It is an object of the present invention to provide a complex for the prevention, prevention or treatment of microbial infections.
In particular, the complexes of the invention comprise an active moiety, a binding moiety and a water-soluble moiety.
The active part is a fat-soluble hydrophobic carbon chain which can exist in a molecular form or a molecular residue form and is a saturated and/or unsaturated carbon chain with a branched and/or linear structure, and the carbon chain can be inserted into/fused into a lipid membrane of a microorganism so as to break the lipid membrane structure or wrap non-enveloped viruses to make the viruses isolated by hydrophobicity;
the binding moiety may be a molecule or residue of a molecule that binds to a microbial lipid membrane, a microbial surface protein, a microbial surface polysaccharide or cell wall component (including a polysaccharide or protein) or is capable of binding to a polysaccharide or protein or polypeptide in a microbe such that the complex is attached to the microbial lipid membrane or viral surface.
The water-soluble part is water-soluble molecules or residues of the molecules, contains water-soluble groups, can endow the compound with water solubility, so that the compound can be uniformly dispersed in an aqueous solution and the fat-soluble hydrophobic groups are prevented from aggregating into groups, and the fat-soluble hydrophobic groups of the compound are prevented from aggregating into groups in the aqueous solution or blood to form lipid droplets.
In further detail, the acting part is a fat-soluble hydrophobic saturated and/or unsaturated carbon chain with a branched chain, a cyclic structure and/or a straight chain, the carbon chain is a molecule or a residue of the molecule, and the carbon chain is a carbon chain with 3-100 carbon atoms;
the water-soluble portion is a water-soluble molecule containing one or two or more groups selected from an amide group, a phosphoryloxy group, a carboxylic acid group, a phosphoric acid group, a sulfonic acid group, a sulfonyloxy group, a hydroxyl group, a quaternary ammonium group, a sulfide group, a disulfide group, an ether group, a mercapto group, an amine group, an amino group, a ureido group, a guanidino group, or a residue of a molecule, and may be a group connected to a carbon chain as an acting portion;
the binding moiety is a molecule or residue of a molecule that binds to a microbial lipid membrane, a microbial surface protein, a microbial surface polysaccharide or cell wall component, or to a polysaccharide or protein or polypeptide in a microorganism. The binding moiety may be the same as the water soluble moiety, i.e. a protein, polypeptide, oligopeptide, oligosaccharide, monosaccharide and/or polysaccharide molecule or residue thereof that is capable of binding to a microbial lipid membrane, surface domain; the binding moiety may also be the same as the water-soluble moiety, i.e., one or more than two of free carboxyl, phosphate, sulfonate, hydroxyl, thiol, amine, amino, ureido, guanidino groups remain after the complex is formed; in some cases, the binding moiety can be a di-or poly-fatty acid, a fat-soluble amino acid molecule, or a residue thereof that binds to a microbial lipid membrane, a surface domain, in which case the di-or poly-fatty acid, or the carboxylic acid groups and amino acid groups of the fat-soluble amino acid molecule or residue substantially bind.
Specifically, the active part is a fat-soluble carbon chain, including a saturated or unsaturated carbon chain with a branched chain and a cyclic structure; preferably, the active moiety is selected from the group consisting of saturated and/or unsaturated fatty hydrocarbons, saturated and/or unsaturated fatty alcohols or oxofatty alcohols, saturated and/or unsaturated fatty acids, hydrophobic amino acids, fat-soluble vitamins, steroid lipids, phospholipids, sphingomyelin, glycolipids, surfactants forming a carbon chain or carbon chain residue with a number of carbon atoms ranging from 3 to 48, more preferably from 3 to 26; wherein the number of carbon atoms is preferably 3 to 26;
the water-soluble moiety is a water-soluble molecule or a residue of a molecule containing one or two or more groups selected from a mercapto group, an amino group, a phosphoric acid group, a carboxylic acid group, a sulfonic acid group, a hydroxyl group, an amine group, a ureido group, a guanidino group, and a disulfide group; the group for binding by the binding moiety (capable of binding to a microbial lipid membrane, a microbial surface protein, a microbial surface polysaccharide or cell wall component or capable of binding to a polysaccharide or protein or polypeptide in a microorganism) is one or more groups selected from thiol, amino, phosphate, carboxylic acid, sulfonic acid, hydroxyl, amine, urea, guanidine and disulfide groups from the water-soluble moiety or one or more groups selected from thiol, amino, phosphate, carboxylic acid, sulfonic acid, hydroxyl, amine, urea, guanidine and disulfide groups providing a connection of the carbon chain to the carbon chain.
Specifically, for the complexes of the invention, the water-soluble moiety is a water-soluble molecule or residue of a molecule, including macromolecular proteins, polysaccharides, nucleic acids, synthetic water-soluble polymers, intermediate-molecular polypeptides, oligopeptides, oligosaccharides, oligonucleotides, synthetic water-soluble intermediate-degree polymers, small molecules including amino acids, mono-or disaccharides, nucleotides, water-soluble vitamins; or functional groups capable of increasing water solubility, such as amide groups, phosphoryloxy groups, carboxylic acid groups, phosphoric acid groups, sulfonic acid groups, sulfonyloxy groups, hydroxyl groups, quaternary ammonium groups, thioether groups, disulfide bonds, ether groups, mercapto groups, aldehyde groups, ester groups, amine groups, amino groups, urea groups, guanidine groups and the like, which are directly bonded to the carbon chain; the water soluble moiety may be a group attached to a carbon chain as an acting and/or binding moiety;
the binding moiety is a molecule or residue of a molecule (including a functional group on the molecule) that binds to a microbial lipid membrane, a microbial surface protein, a microbial surface polysaccharide, or a cell wall component, and may be the third moiety that forms the complex, or may be the same as the water-soluble moiety, or may be linked to the acting moiety. When the binding moiety is the same moiety as the water-soluble moiety such as a protein, polypeptide, polysaccharide, etc. that can bind to a microbial lipid membrane, microbial surface protein, microbial surface polysaccharide, the water-soluble moiety contains one selected from the group consisting of a thiol group, an amino group, a ureido group, a guanidino group, a carboxylic acid group, a hydroxyl group, and a dithiosulfide group. When the binding moiety is in some cases, it may bind to a microbial lipid membrane, a microbial surface protein, a microbial surface polysaccharide, a dibasic or polybasic fatty acid, a fat-soluble amino acid, or the like.
Without being limited by the reaction mechanism, the functional group in the binding moiety which plays a binding role may be a carboxyl group, a sulfonic acid group, a phosphoric acid group, a hydroxyl group, an aldehyde group or a hydroxyl group (saccharide) of a hemiacetal, an amino group, a ureido group, a guanidino group, a mercapto group or the like.
1. The following is further illustrated by the example of fatty acids as carbon chain donors (i.e., as part of the donor):
(1) the fatty acid is insoluble in water or has extremely low water solubility, so that the fatty acid cannot be directly injected into blood circulation, and the direct injection into veins can cause pulmonary embolism and the injection into arteries can cause arterial embolism tissue necrosis;
(2) the fatty acid is re-esterified in intestinal cells, mixed with bile salt and monoglyceride to form 4-6 nm fat particles, which are directly absorbed by intestinal epithelial cells through swallowing action and coated with a layer of lecithin and protein membrane to form chylomicron which enters lymphatic system and flows back to blood circulation in the form of oil-in-water emulsion through lymphatic vessels and thoracic ducts. Medium chain fatty acids, except for small amounts that are present in the peripheral blood for a short period of time, are mostly non-covalently bound to serum proteins and reach the liver relatively quickly through the portal system. In the liver, medium-chain fatty acids rapidly pass through the mitochondrial bilayer membrane, are rapidly acylated by octanoyl CoA, and are hardly synthesized into fat. The excess acetyl CoA produced by acylation undergoes various metabolic actions in the mitochondrial cytosol, most of which tend to synthesize ketone bodies.
(3) The fatty acid covalently combined with the large, medium and small molecules changes fat-soluble fatty acid into water-soluble fatty acid which is not easy to be cleared and metabolized by liver,
(4) the high water solubility and high affinity compound has the function of resisting microbial infection, and can be used as a nasal spray, a dry powder inhalant, an intravenous injection and an oral preparation besides a skin external preparation.
2. Composite formed by fatty acid and water-soluble amino acid, monosaccharide or disaccharide, nucleotide and water-soluble vitamin
At this time, the carbon chain of the fatty acid is an active part, the water-soluble amino acid, monosaccharide or disaccharide, nucleotide and water-soluble vitamin are water-soluble parts, and then the composite with the function of resisting microbial infection is formed by connecting the upper binding part. The binding moiety may be selected from the group consisting of a di-or poly-fatty acid, an amino acid, a targeting protein, a targeting polypeptide, a targeting polysaccharide. Wherein, the dibasic fatty acid or the polybasic fatty acid and the fat-soluble amino acid are not only a combination part but also an action part; the water-soluble amino acid, the target protein, the target polypeptide and the target polysaccharide are both binding parts and water-soluble parts.
In one embodiment, the complex of the present invention is selected from complexes formed by connecting fatty trienoic acid with 3-50 carbon atoms and water-soluble amino acid, and may be, for example, a complex formed by connecting octadecatrienoic acid with aspartyl lysine as shown in the following compounds:
Figure BDA0003628692260000581
wherein the carbon chain of the octadecatrienoic acid is the acting moiety and the aspartyl lysine is both the water soluble moiety and the binding moiety.
3. Complexes of fatty acids with proteins, polypeptides and polysaccharides
In this case, the carbon chain of the fatty acid is the active moiety, and the protein, polypeptide or polysaccharide that targets the surface domain, lipid membrane or cell wall of the virus is the binding moiety, as well as the water-soluble moiety.
In one embodiment, the complex of the present invention is selected from complexes formed by connecting fatty olefine acid with 3-50 carbon atoms to a targeting polypeptide, and can be, for example, a schematic structural formula of a complex formed by connecting octadecenoic acid to a targeting polypeptide as shown below, wherein the octadecenoic acid is connected with lysine residue in the polypeptide by amide bond.
Figure BDA0003628692260000591
Wherein the carbon chain of octadecenoic acid is an action part, and the targeting polypeptide is both a water-soluble part and a binding part.
4. In the three cases, if the water solubility of the complex is poor or the complex molecule needs to be enlarged, a water-soluble high molecular polymer, for example
The fatty acid + targeting polypeptide + PEG is the compound formed by the reaction of the fatty acid with 3-50 carbon atoms, the targeting polypeptide and the PEG.
At this point the carbon chain of the fatty acid is the active moiety, the targeting polypeptide is the binding moiety, and the PEG is the water soluble moiety.
5. Fatty alcohol-polyoxyethylene ether, fatty acid-polyoxyethylene ester, alkyl glycoside, fatty acid sucrose ester, sorbitan fatty acid ester, sorbitan polyoxyethylene fatty acid ester, mannosylerythritol ester, N-fatty acyl-N-methylglucamine and other compounds have the advantages that fatty alcohol or fatty acid is a carbon chain donor, and the compounds have good water solubility, but the combination effect with virus surface structural domain, lipid membrane or cell wall components is weak, and the microorganisms can be killed only by high concentration, and under the concentration, the compounds can also damage human cells, so that the compounds are not suitable for being used inside human bodies. When the compound is connected with a combining part to form a new compound, the compound has the effect of killing microorganisms at a lower concentration in a human body and playing a role in resisting microbial infection; furthermore, at this therapeutic concentration, the novel complexes having an active moiety + water-soluble moiety + binding moiety have no effect on human tissue cells and organs. The binding moiety may be selected from the group consisting of a di-or poly-fatty acid, an amino acid, a targeting protein, a targeting polypeptide, a targeting polysaccharide. That is, in this case, the complex of the present invention is a complex formed by reacting a surfactant with one or more selected from the group consisting of a dibasic or polybasic fatty acid, an amino acid, a targeting protein, a targeting polypeptide and a targeting polysaccharide.
At the moment, the carbon chain of fatty alcohol or fatty acid is an acting part, Polyoxyethylene Ether (PEG), glucan, sucrose, sorbitan, mannitol erythritol and glucosamine are water-soluble parts, the connected dibasic fatty acid or polybasic fatty acid and fat-soluble amino acid are a combining part and an acting part, and the water-soluble amino acid, the targeted protein, the targeted polypeptide and the targeted polysaccharide are a combining part and a water-soluble part.
Specifically, in one embodiment of the present invention, the present invention provides a group of compounds for preventing and treating viral, bacterial and fungal infections, which have a main structure formed by coupling an acting moiety, a binding moiety, and a water-soluble moiety through covalent bonds, hydrogen bonds or van der waals forces;
the acting moiety imparts to the complex an effect of disrupting a microbial lipid membrane or hydrophobically sequestering non-enveloped viruses; the binding moiety confers the complex with the function of binding to a microbial lipid membrane or viral surface domain, and the specific binding moiety may also confer the complex with the function of specifically targeting a microbe; the water-soluble moiety imparts water solubility to the complex, allowing the complex to disperse uniformly in aqueous solutions and avoiding agglomeration of hydrophobic groups, forming lipid droplets.
The lipid membrane is an envelope formed by phospholipid bimolecular layers of microorganisms;
the acting part, the combining part and the water-soluble part in the compound can be natural compounds, artificially synthesized compounds or natural compounds coupled with artificially synthesized compounds;
the number of groups of the cognate moiety in the complex may be 1 or more than 1; the arrangement and sequence of the groups are not fixed; groups of the same or different types can be coupled linearly or in a side chain manner.
The compound can be used for preventing and treating infectious diseases caused by viruses, bacteria, fungi, chlamydia and mycoplasma.
Further, the acting part is a natural or artificial hydrophobic group, and comprises a straight chain carbon chain, a branched chain carbon chain and a cyclic structure carbon chain; the carbon chain may be a saturated/unsaturated carbon chain, and the unsaturated carbon chain may carry one or more unsaturated bonds, wherein the unsaturated bond may be a double or triple bond;
for microorganisms with lipid membrane structures, such as enveloped viruses, bacteria, fungi, chlamydia and mycoplasma, the action part can penetrate, insert and fuse into the lipid membrane to destroy the structural stability of the lipid membrane, further destroy the integrity of the lipid membrane and cell walls, and achieve the effect of killing the microorganisms;
In the case of non-enveloped viruses, the binding part is combined with a virus surface protein domain, and the acting part wraps the surface of the non-enveloped virus, so that the non-enveloped virus is isolated by hydrophobicity and then is eliminated by immune cells, thereby achieving the effect of preventing and treating non-enveloped virus infection.
Further, the binding moiety has 1 or more than 1 functional group that can bind to a protein, polysaccharide or bindable domain, such as carboxyl, hydroxyl, amino, sulfhydryl, ureido, guanidino, and can bind to a protein, polysaccharide or bindable domain on a lipid membrane or a viral surface, such that the complex is attached to the lipid membrane or viral surface;
the binding moieties may also be designed to have a molecular structure that specifically targets lipid membranes, bacterial and fungal cell wall components, viral surface protein domains, thereby conferring on the complex the function of targeting viruses, bacteria and fungi;
the binding moiety and the hydrophilic group may be the same group, either binding to a lipid membrane, cell wall or viral surface protein domain, or conferring water solubility to the complex.
Still further, the binding moiety that can specifically target microbial lipid membranes, bacterial and fungal cell wall components, viral surface protein domains is a protein, polypeptide or polysaccharide comprising:
(1) Targeting coronavirus envelope proteins include: neutralizing antibodies of spike glycoprotein (S), small envelope glycoprotein (E), membrane glycoprotein (M) and hemagglutinin glycoprotein (HE), and amino acid sequences and small molecule polypeptides capable of specifically binding to the protein domains;
(2) proteins targeting the human immunodeficiency virus envelope include: neutralizing antibodies of gp120 and gp41 proteins, and an amino acid sequence and a small molecule polypeptide which can be specifically combined with the protein structural domain;
(3) targeting hepatitis b virus envelope proteins include: neutralizing antibodies of SHBs protein, MHBs protein and LHBs protein, and amino acid sequences and small molecular polypeptides capable of specifically binding to the protein domains;
(4) targeting hepatitis c virus envelope proteins include: neutralizing antibodies of E1 and E2 proteins, and amino acid sequences and small molecule polypeptides capable of specifically binding to the protein domains;
(5) the targeted rabies virus envelope proteins include: neutralizing antibody of envelope glycoprotein, and amino acid sequence and small molecule polypeptide capable of combining with the protein structure domain specifically;
(6) targeting herpes virus envelope proteins include: gB. Neutralizing antibodies of gC, gD, gE, gG and gH glycoproteins, and amino acid sequences and small molecule polypeptides capable of specifically binding to the protein domains;
(7) The targeted Ebola virus envelope protein comprises a neutralizing antibody of envelope glycoprotein on a virus outer membrane, an amino acid sequence capable of being specifically combined with the protein structural domain and a small molecular polypeptide;
(8) targeting hantavirus envelope proteins include: neutralizing antibodies of G1 and G2 glycoprotein, and amino acid sequences and small molecule polypeptides capable of specifically binding to the above protein domains;
(9) targeting dengue virus envelope proteins include: neutralizing antibodies of protein E and protein M, and amino acid sequences and small molecular polypeptides capable of being specifically combined with the protein structural domains;
(10) targeting encephalitis b virus envelope proteins include: neutralizing antibodies to glycoprotein E (i.e., viral hemagglutinin) and protein M, as well as amino acid sequences and small polypeptides that specifically bind to the aforementioned protein domains;
(11) targeting influenza virus envelope proteins include: neutralizing antibodies of hemagglutinin and neuraminidase, and amino acid sequences and small molecule polypeptides capable of specifically binding with the protein domains;
(12) targeting hepatitis a virus capsid proteins include: neutralizing antibodies of VP1, VP2, VP3 and VP4 proteins, and an amino acid sequence and a small molecule polypeptide which can be specifically combined with the protein domains;
(13) Targeting human papillomavirus capsid proteins include: neutralizing antibodies of L1 and L2 proteins, and amino acid sequences and small molecule polypeptides capable of specifically binding to the protein domains;
(14) targeting adenoviral capsid proteins includes: p II, P III a, P IV, P VI, P VIII, P IX protein neutralizing antibody, and the protein domain can be specifically combined with the amino acid sequence and small molecular polypeptide;
(15) targeting poliovirus capsid proteins include: neutralizing antibodies of VP1, VP2, VP3 and VP4 proteins, and an amino acid sequence and a small molecule polypeptide which can be specifically combined with the protein domains;
(16) targeting coxsackievirus capsid proteins include: neutralizing antibodies of VP1, VP2, VP3 and VP4 proteins, and an amino acid sequence and a small molecule polypeptide which can be specifically combined with the protein domains;
(17) proteins targeting bacterial or fungal cell walls include: CD14 and amino acid sequences and small molecule polypeptides that specifically bind to its domains;
(18) also included are ligands designed to target the cell wall of enveloped viruses, bacteria or fungi that are directed against low affinity receptors such as heparan sulfate, proteoglycans, etc.
That is, for the purpose of the present invention, in order to prevent, prevent or treat microbial infectious diseases, the basic structure of the complex of the present invention includes any one of the following:
Binding moiety + water soluble moiety + acting moiety;
binding moiety + acting moiety + water soluble moiety;
water soluble + active + binding moieties;
water soluble + binding + acting moieties;
binding moiety + water soluble moiety + binding moiety + acting moiety + … … + XX moiety;
binding moiety + water soluble moiety + effect moiety + water soluble moiety + … … + XX moiety;
binding moiety + water soluble moiety + effect moiety + binding moiety + … … + XX moiety;
water soluble + binding + acting + binding + … … + XX moieties; and
water soluble + active + binding + active + … … + XX moiety.
Wherein, the term "XX moiety" refers to any one or more of "water-soluble moiety", "binding moiety", "acting moiety".
The number of homogeneous parts in the compound can be one or more, and the arrangement and the sequence of the parts are not fixed.
Still further, in a more specific embodiment, the structure of the composite of the present invention includes the structure described below.
The small molecule water soluble part/binding part is covalently bound with the functional part of the medium and short carbon chain, the medium and short carbon chain comprises saturated or unsaturated straight chain carbon chain, branched carbon chain and carbon chain with cyclic structure, and can be carbon chain with 3-10 carbon atoms, and the small molecule water soluble part/binding part can be small molecule, medium molecule and macromolecule with carboxyl/hydroxyl, such as:
The compound formed by amino acid or monosaccharide with carboxyl/hydroxyl, nucleotide and carbon chain with 3-10 carbon atoms has obviously improved water solubility, good dispersion and no aggregation.
The macromolecule water soluble part + the binding part + the long carbon chain (more than 10 carbon atoms) acting part forms a compound, wherein the long carbon chain comprises a saturated or unsaturated linear carbon chain, a branched carbon chain and a carbon chain with a cyclic structure, such as the compound formed by the following structures:
protein/targeting protein + binding moiety + linear, branched or cyclic saturated/unsaturated carbon chain,
targeting small molecules + proteins + binding moieties + linear, branched or cyclic structures of saturated/unsaturated carbon chains,
polysaccharide + binding moiety + linear, branched or cyclic saturated/unsaturated carbon chains,
targeting micromolecules, polysaccharides, a binding part, a straight chain, a branched chain or a saturated/unsaturated carbon chain with a cyclic structure,
water-soluble high molecular polymer, a binding part, a straight chain, a branched chain or a saturated/unsaturated carbon chain with a cyclic structure,
targeting micromolecules, water-soluble high molecular polymers, binding parts, straight chains, branched chains or saturated/unsaturated carbon chains with cyclic structures.
Further, the complex is a derivative of a water-soluble moiety, a binding moiety and a lipid covalently coupled, and the construction mode comprises:
a macromolecular water-soluble moiety/binding moiety/targeting binding moiety + a lipid,
a macromolecular water-soluble part, a binding part, a targeting binding part and lipid,
2 or more than 2 small molecule water-soluble parts/binding parts/targeting binding parts + lipid,
2 or more than 2 small molecule water-soluble parts + binding parts/targeting binding parts + lipid.
Still further, the lipids include fatty alcohols, fatty acids, phospholipids, lipid-soluble vitamins and steroid lipids;
the fatty alcohol comprises saturated fatty alcohol, monounsaturated fatty alcohol, polyunsaturated fatty alcohol and one or more of the fatty alcohol derivatives; the unsaturated bond in the carbon chain of the unsaturated fatty alcohol is a double bond or a triple bond; the carbon chain of the fatty alcohol can be a straight chain, a branched chain or a cyclic structure;
the fatty acids comprise saturated fatty acids, monounsaturated fatty acids, dibasic or polybasic unsaturated fatty acids and one or more of the fatty acid derivatives; the unsaturated bond in the unsaturated fatty acid carbon chain is a double bond or a triple bond; the carbon chain of the fatty acid can be a straight chain, a branched chain, a cyclic structure and a carbon chain with hydroxyl; the number of carboxyl groups of the fatty acid may be 1 or more;
The phospholipids comprise glycerophospholipids and sphingomyelins, wherein the glycerophospholipids comprise phosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine, phosphatidylinositol, and one or more of the above phospholipid derivatives;
the fat-soluble vitamins comprise vitamin A, vitamin D, vitamin E and vitamin D, and one or more of the fat-soluble vitamin derivatives.
The steroid comprises cholesterol, lanosterol, sitosterol, stigmasterol, ergosterol, bile acid, bile alcohol, and one or more of the steroid lipid derivatives.
Further, the composite is ensured to have a molecular weight ratio of the hydrophobic group to the hydrophilic group suitable, if the molecular weight of the hydrophilic group is much larger than that of the hydrophobic group, the hydrophilic group will prevent the hydrophobic group from being inserted into and fused into the biological membrane to weaken the damage capability of the hydrophobic group on the biological membrane, and if the molecular weight of the hydrophilic group is significantly smaller than that of the hydrophobic group, the hydrophobic group will aggregate to form an oil-in-water structure, and the hydrophobic group will not contact the biological membrane and can not exert the damage function.
Further, in the present invention, the acting moiety may be a carbon chain or a carbon chain residue selected from the group consisting of saturated and/or unsaturated aliphatic hydrocarbons, saturated and/or unsaturated aliphatic alcohols, and saturated and/or unsaturated fatty acids having 3 to 48 carbon atoms, more preferably 3 to 26 carbon atoms; among them, the number of carbon atoms is preferably 3 to 26.
Among them, the saturated and/or unsaturated fatty acids used to provide the above carbon chains or carbon chain residues specifically include the fatty acids shown below.
The saturated fatty acid having 3 to 46 carbon atoms includes:
propionic acid, butyric acid, valeric acid, caproic acid, enanthic acid, caprylic acid, pelargonic acid, capric acid, undecanoic acid, dodecanoic acid, tridecanoic acid, tetradecanoic acid, pentadecanoic acid, hexadecanoic acid, heptadecanoic acid, octadecanoic acid, nonadecanoic acid, eicosanoic acid, heneicosanoic acid, docosanoic acid, tricosanoic acid, tetracosanoic acid, pentacosanoic acid, hexacosanoic acid, heptacosanoic acid, octacosanoic acid, nonacosanoic acid, triacontanoic acid, hentriacontanoic acid, dodecacosanoic acid, tricosanoic acid, tetradecanoic acid, pentadecanoic acid, hexadecanoic acid, heptatriacontanoic acid, octatriacontanoic acid, and forty-hexaanoic acid.
The C3-34 monoenoic acid includes:
2-acrylic acid, 2-butenoic acid, 3-butenoic acid, 2-pentenoic acid, 3-pentenoic acid, 4-pentenoic acid, 2-hexenoic acid, 3-hexenoic acid, 4-hexenoic acid, 5-hexenoic acid, 2-heptenoic acid, 3-heptenoic acid, 4-heptenoic acid, 5-heptenoic acid, 6-heptenoic acid, 2-octenoic acid, 3-octenoic acid, 4-octenoic acid, 5-octenoic acid, 6-octenoic acid, 7-octenoic acid, 2-nonenoic acid, 3-nonenoic acid, 4-nonenoic acid, 6-nonenoic acid, 8-nonenoic acid, 2-decenoic acid, 3-decenoic acid, 4-decenoic acid, 5-decenoic acid, 6-decenoic acid, 8-decenoic acid, 9-decenoic acid, 2-undecylenic acid, 3-undecylenic acid, 4-undecylenic acid, 5-undecylenic acid, 6-undecylenic acid, 7-undecylenic acid, 8-undecylenic acid, 9-undecylenic acid, 10-undecylenic acid, 2-dodecenoic acid, 3-dodecenoic acid, 4-dodecenoic acid, 5-dodecenoic acid, 6-dodecenoic acid, 7-dodecenoic acid, 9-dodecenoic acid, 10-dodecenoic acid, 11-dodecenoic acid, 2-tridecenoic acid, 7-tridecenoic acid, 8-tridecenoic acid, 11-tridecenoic acid, 12-tridecenoic acid, 2-tetradecenoic acid, 3-tetradecenoic acid, 4-tetradecenoic acid, 5-tetradecenoic acid, 7-tetradecenoic acid, 8-tetradecenoic acid, 9-decatetraenoic acid, 11-decatetraenoic acid, 2-pentadecenoic acid, 6-pentadecenoic acid, 7-pentadecenoic acid, 9-pentadecenoic acid, 10-pentadecenoic acid, 14-pentadecenoic acid, 2-hexadecenoic acid, 3-hexadecenoic acid, 4-hexadecenoic acid, 5-hexadecenoic acid, 6-hexadecenoic acid, 7-hexadecenoic acid, 9-hexadecenoic acid, 10-hexadecenoic acid, 11-hexadecenoic acid, 13-hexadecenoic acid, 2-heptadecenoic acid, 3-heptadecenoic acid, 7-heptadecenoic acid, 8-heptadecenoic acid, 9-heptadecenoic acid, 10-heptadecenoic acid, 11-heptadecenoic acid, 12-heptadecenoic acid, 16-heptadecenoic acid, 2-octadecenoic acid, 3-octadecenoic acid, 4-octadecenoic acid, 5-octadecenoic acid, 6-octadecenoic acid, 7-octadecenoic acid, 8-octadecenoic acid, 9-octadecenoic acid, 10-octadecenoic acid, 11-octadecenoic acid, 12-octadecenoic acid, 13-octadecenoic acid, 14-octadecenoic acid, 15-octadecenoic acid, 16-octadecenoic acid, 17-octadecenoic acid, 2-nonadecenoic acid, 5-nonadecenoic acid, 6-nonadecenoic acid, 7-nonadecenoic acid, 9-nonadecenoic acid, 10-nonadecenoic acid, 11-nonadecenoic acid, 12-nonadecenoic acid, 13-nonadecenoic acid, 16-nonadecenoic acid, 3-eicosenoic acid, 5-eicosenoic acid, 6-eicosenoic acid, 7-eicosenoic acid, 8-eicosenoic acid, 9-eicosenoic acid, 10-eicosenoic acid, 11-eicosenoic acid, 13-eicosenoic acid, 14-eicosenoic acid, 15-eicosenoic acid, 16-eicosenoic acid, 7-heneicosenic acid, 12-heneicosenic acid, 5-docosenoic acid, 7-docosenoic acid, 9-docosenoic acid, 11-docosenoic acid, 13-docosenoic acid, 15-docosenoic acid, 19-docosenoic acid, 9-eicosenoic acid, 14-eicosenoic acid, 16-eicosenoic acid, 17-eicosenoic acid, 18-eicosenoic acid, 22-eicosenoic acid, 11-eicosatetraenoic acid, 15-eicosatetraenoic acid, 17-eicosatetraenoic acid, 5-eicosapentaenoic acid, 16-eicosapentaenoic acid, 17-eicosapentaenoic acid, 18-eicosapentaenoic acid, 19-eicosapentaenoic acid, 5-docosahexaenoic acid, 9-docosahexaenoic acid, 11-docosahexaenoic acid, 14-docosahexaenoic acid, 17-docosahexaenoic acid, 19-docosahexaenoic acid, 21-docosahexaenoic acid, 18-heptacosenoic acid, 20-heptacosenoic acid, 9-octacosenoic acid, 11-octacosenoic acid, 19-octacosenoic acid, 21-octacosenoic acid, 23-octacosenoic acid, 20-nonacosenoic acid, 21-triacontenoic acid, 22-hendecenoic acid, 23-triacontenoic acid, 25-triacontanoic acid.
The C5-30 dienoic acid includes:
2, 4-pentadienoic acid, 2, 4-hexadienoic acid, 3, 5-hexadienoic acid, 2, 7-octadienoic acid, 4, 7-octadienoic acid, 5, 7-octadienoic acid, 2, 4-nonadienoic acid, 2, 6-nonadienoic acid, 5, 7-nonadienoic acid, 2, 4-decadienoic acid, 2, 5-decadienoic acid, 2, 6-decadienoic acid, 2, 7-decadienoic acid, 3, 5-decadienoic acid, 4, 6-decadienoic acid, 4, 8-decadienoic acid, 4, 9-decadienoic acid, 5, 9-decadienoic acid, 6, 8-decadienoic acid, 7, 9-decadienoic acid, 2, 4-undecadienoic acid, 2, 4-dodecenoic dienoic acid, 2, 6-dodecenoic acid, 2, 8-dodecenoic acid, 3, 6-dodecenoic acid, 5, 7-dodecenoic acid, 7, 9-dodecenoic acid, 8, 10-dodecenoic acid, 3, 5-tridecadienoic acid, 2, 4-tetradecadienoic acid, 3, 5-tetradecadienoic acid, 5, 8-tetradecadienoic acid, 6, 9-tetradecadienoic acid, 10, 12-tetradecadienoic acid, 2, 4-hexadecadienoic acid, 3, 9-hexadecadienoic acid, 4, 7-hexadecadienoic acid, 5, 9-hexadecadienoic acid, 6, 9-hexadecadienoic acid, 7, 10-hexadecadienoic acid, 8, 10-hexadecadienoic acid, 9, 12-hexadecadienoic acid, 10, 12-hexadecadienoic acid, 8, 11-heptadecadienoic acid, 9, 12-heptadecadienoic acid, 2, 4-octadecadienoic acid, 2, 5-octadecadienoic acid, 2, 6-octadecadienoic acid, 3, 7-octadecadienoic acid, 3, 12-octadecadienoic acid, 4, 7-octadecadienoic acid, 4, 8-octadecadienoic acid, 4, 9-octadecadienoic acid, 5, 8-octadecadienoic acid, 5, 9-octadecadienoic acid, 5, 10-octadecadienoic acid, 5, 11-octadecadienoic acid, 5, 12-octadecadienoic acid, 6, 8-octadecadienoic acid, 6, 9-octadecadienoic acid, 6, 10-octadecadienoic acid, 6, 11-octadecadienoic acid, 6, 12-octadecadienoic acid, 7, 9-octadecadienoic acid, 7, 10-octadecadienoic acid, 7, 11-octadecadienoic acid, 7, 12-octadecadienoic acid, 8, 10-octadecadienoic acid, 8, 11-octadecadienoic acid, 8, 12-octadecadienoic acid, 9-11-octadecadienoic acid, 9, 12-octadecadienoic acid, 9, 13-octadecadienoic acid, 10, 12-octadecadienoic acid, 10, 14-octadecadienoic acid, 5, 9-nonadecenoic acid, 10, 13-nonadecenoic acid, 5, 9-eicosadienoic acid, 5, 11-eicosadienoic acid, 5, 13-eicosadienoic acid, 5, 15-eicosadienoic acid, 6, 9-eicosadienoic acid, 6, 11-eicosadienoic acid, 7, 13-eicosadienoic acid, 7, 14-eicosadienoic acid, 8, 11-eicosadienoic acid, 11, 13-eicosadienoic acid, 11, 14-eicosadienoic acid, 11, 15-eicosadienoic acid, 5, 14-heneicosenoic acid, 5, 16-heneicosenoic acid, 12, 15-heneicosenoic acid, 5, 13-docosenoic acid, 7, 15-docosenoic acid, 13, 16-docosenoic acid, 5, 9-tetracosenoic acid, 15, 18-tetracosenoic acid, 5, 9-hexacosenoic acid, 17, 20-hexacosenoic acid, 17, 21-hexacosadienoic acid, 9, 21-octadienoic acid, 19, 23-octadienoic acid, 5, 9-nonacosadienoic acid, 5, 9-triacontadienoic acid, 9, 23-triacontadienoic acid.
The trienoic acid with carbon number of 7-30 comprises:
2,4, 6-heptatriene, 2,6, 8-decatriene, 4,7, 10-hexadecatrienoic acid, 5,8, 11-hexadecatrienoic acid, 6,9, 12-hexadecatrienoic acid, 6,10, 14-hexadecatrienoic acid, 7,10, 13-hexadecatrienoic acid, 7,11, 14-hexadecatrienoic acid, 9,12, 15-hexadecatrienoic acid, 5,9, 12-heptadecenoic acid, 2,9, 12-octadecatrienoic acid, 3,9, 12-octadecatrienoic acid, 5,8, 11-octadecatrienoic acid, 5,9, 12-octadecatrienoic acid, 6,10, 14-octadecatrienoic acid, 7,9, 12-octadecatrienoic acid, 8,10, 12-octadecatrienoic acid, 9,11, 13-octadecatrienoic acid, 9,11, 14-octadecatrienoic acid, 9,12, 15-octadecatrienoic acid, 10,12, 14-octadecatrienoic acid, 10,12, 15-octadecatrienoic acid, 11,13, 15-octadecatrienoic acid, 2,4, 8-eicosatrienoic acid, 3,6, 9-eicosatrienoic acid, 5,8, 11-eicosatrienoic acid, 5,8, 14-eicosatrienoic acid, 5,9, 12-eicosatrienoic acid, 5,11, 14-eicosatrienoic acid, 5,13, 16-eicosatrienoic acid, 7,10, 13-eicosatrienoic acid, 7,11, 14-eicosatrienoic acid, 8,11, 14-eicosatrienoic acid, 8,12, 14-eicosatrienoic acid, 9,11, 14-eicosatrienoic acid, 11,14, 17-eicosatrienoic acid, 5,14, 17-heneicosanoic acid, 3,9, 15-docosatrienoic acid, 5,11, 17-docosatrienoic acid, 7,10, 13-docosatrienoic acid, 8,11, 14-docosatrienoic acid, 13,16, 19-docosatrienoic acid, 15,18, 21-tetracosatrienoic acid, 5,9, 17-hexacosatrienoic acid, 5,9, 19-hexacosatrienoic acid, 5,9, 21-hexacosatrienoic acid, 5,9, 20-heptacosatrienoic acid, 5,9, 21-octacosatrienoic acid, 5,9, 23-nonacosatrienoic acid, 5,9, 23-triacontatrienoic acid, 5,9, 25-triacontatrienoic acid
The C12-38 arachidonic acid includes:
2,4,8, 10-dodecatetraenoic acid, 2,6,8, 12-hexadecatetraenoic acid, 4,7,10, 13-hexadecatetraenoic acid, 4,7,11, 14-hexadecatetraenoic acid, 4,8,12, 16-hexadecatetraenoic acid, 6,9,12, 15-hexadecatetraenoic acid, 2,4,6, 11-octadecatetraenoic acid, 3,9,12, 15-octadecatetraenoic acid, 5,8,11, 14-octadecatetraenoic acid, 5,9,12, 15-octadecatetraenoic acid, 6,9,12, 15-octadecatetraenoic acid, 9,11,13, 15-octadecatetraenoic acid, 9,12,15, 17-octadecatetraenoic acid, 2,8,11, 14-eicosatetraenoic acid, 4,7,10, 13-eicosatetraenoic acid, 4,8,11, 14-eicosatetraenoic acid, 4,8,12, 16-eicosatetraenoic acid, 5,8,11, 14-eicosatetraenoic acid, 5,11,14, 17-eicosatetraenoic acid, 5,13,16, 19-eicosatetraenoic acid, 6,10,14, 18-eicosatetraenoic acid, 7,11,14, 17-eicosatetraenoic acid, 8,11,14, 18-eicosatetraenoic acid, 4,7,10, 13-docosatetraenoic acid, 7,10,13, 16-docosatetraenoic acid, 8,12,16, 19-docosatetraenoic acid, 2,4,6, 8-tetracosatetraenoic acid, 9,12,15, 18-tetracosatetraenoic acid, 11,14,17, 20-hexacosatetraenoic acid, 13,16,19, 22-dioctadecylic acid, 15,18,21, 24-triacontatetraenoic acid, 17,20,23, 26-triacontatetraenoic acid, 19,22,25, 28-thirty-four-carbon-tetraenoic acid, 21,24,27, 30-tricecatetraenoic acid, 23,26,29, 32-trioctadecatetraenoic acid.
The C12-38 pentaenoic acid comprises:
3,5,7,9, 11-eicosapentaenoic acid, 5,7,9,11, 13-tetradecapentaenoic acid, 3,6,9,12, 15-octadeceneoic acid, 2,5,8,11, 14-eicosapentaenoic acid, 4,8,12,15, 18-eicosapentaenoic acid, 5,7,9,14, 17-eicosapentaenoic acid, 5,8,11,14, 16-eicosapentaenoic acid, 5,8,11,14, 17-eicosapentaenoic acid (EPA), 4,7,10,13, 16-docosapentaenoic acid, 4,8,12,15, 19-docosapentaenoic acid, 7,10,13,16, 19-docosapentaenoic acid, 6,9,12,15, 18-tetracosapentaenoic acid, 9,12,15,18, 21-tetracosapentaenoic acid, 8,11,14,17, 20-hexacosapentaenoic acid, 11,14,17,20, 23-hexacosapentaenoic acid, 10,13,16,19, 22-octacospentaenoic acid, 13,16,19,22, 25-dioctadepentaenoic acid, 12,15,18,21, 24-triacontopentenic acid, 15,18,21,24, 27-triacontentaenoic acid, 14,17,20,23, 26-triacontentaenoic acid, 17,20,23,26, 29-triacontentaenoic acid, 16,19,22,25, 28-trimyripentaenoic acid, 19,22,25,28, 31-triacontentaenoic acid, 18,21,24,27, 30-tripalmentaenoic acid, 21,24,27,30, 32-tripalmentaenoic acid, 20,23,26,29, 32-trioctapentaenoic acid, 23,26,29,32, 35-thirty-eight carbon pentaenoic acid.
The hexaenoic acid with carbon atoms of 22-38 comprises:
4,7,10,13,16, 19-docosahexaenoic acid, 2,4,6,8,10, 12-tetracosahexanoic acid, 4,8,12,15,19, 22-tetracosahexanoic acid, 6,9,12,15,18, 21-tetracosahexanoic acid (THA), 8,11,14,17,20, 23-hexacosahexanoic acid, 10,13,16,19,22, 25-dioctadecylohexanoic acid, 12,15,18,21,24, 27-triacontahexaenoic acid, 14,17,20,23,26, 29-triacontahexahexanoic acid, 16,19,22,25,28, 31-trimarahexaenoic acid, 18,21,24,27,30, 32-triacontahexahexanoic acid, 20,23,26,29,32, 35-triacontahexahexanoic acid.
The acetylenic acid with 6-22 carbon atoms comprises:
3-hexynoic acid, 4-hexynoic acid, 5-hexynoic acid, 3-heptynoic acid, 4-heptynoic acid, 6-heptynoic acid, 2-octynoic acid, 7-octynoic acid, 2-nonynoic acid, 4-nonynoic acid, 5-nonynoic acid, 6-nonynoic acid, 7-nonynoic acid, 8-nonynoic acid, 3-decynoic acid, 4-decynoic acid, 5-decynoic acid, 6-decynoic acid, 7-decynoic acid, 8-decynoic acid, 9-decynoic acid, 2-undecylenic acid, 3-undecylenic acid, 4-undecylenic acid, 5-undecylenic acid, 6-undecylenic acid, 7-undecylenic acid, 8-undecylenic acid, 9-undecylenic acid, 10-undecylenic acid, 3-decaacetylenic acid, 4-dodecaynoic acid, 5-dodecaynoic acid, 6-dodecaynoic acid, 7-dodecaynoic acid, 8-dodecaynoic acid, 9-dodecaynoic acid, 10-dodecaynoic acid, 11-dodecaynoic acid, 3-tridecynoic acid, 4-tridecynoic acid, 5-tridecynoic acid, 6-tridecynoic acid, 7-tridecynoic acid, 8-tridecynoic acid, 9-tridecynoic acid, 10-tridecynoic acid, 11-tridecynoic acid, 12-tridecynoic acid, 13-tridecynoic acid, 3-tetradecynoic acid, 4-tetradecynoic acid, 5-tetradecynoic acid, 6-tetradecynoic acid, 7-tetradecynoic acid, 8-tetradecynoic acid, 9-tetradecynoic acid, 10-tetradecynoic acid, 11-tetradecynoic acid, 12-tetradecynoic acid, 13-tetradecynoic acid, 3-pentadecynoic acid, 14-pentadecynoic acid, 2-hexadecynoic acid, 4-hexadecynoic acid, 7-hexadecynoic acid, 10-hexadecynoic acid, 7-heptadecylic acid, 8-heptadecylic acid, 9-heptadecylic acid, 12-heptadecylic acid, 16-heptadecylic acid, 2-octadecynoic acid, 3-octadecynoic acid, 4-octadecynoic acid, 5-octadecynoic acid, 6-octadecynoic acid, 7-octadecynoic acid, 8-octadecynoic acid, 9-octadecynoic acid, 10-octadecynoic acid, 11-octadecynoic acid, 12-octadecynoic acid, 13-octadecynoic acid, 14-octadecynoic acid, 15-octadecynoic acid, 16-octadecynoic acid, 17-octadecynoic acid, 18-nonadecylic acid, 13-docosaynoic acid.
The C10-22 diacetylenic acid comprises:
2, 4-decadiynoic acid, 5, 11-dodecadiynoic acid, 3, 9-hexadecadiynoic acid, 7, 10-hexadecadiynoic acid, 8, 10-hexadecadiynoic acid, 5, 8-heptadecadiynoic acid, 6, 9-heptadecadiynoic acid, 7, 10-heptadecadiynoic acid, 10, 16-heptadecadiynoic acid, 2, 5-octadecadiynoic acid, 2, 6-octadecadiynoic acid, 2, 7-octadecadiynoic acid, 3, 6-octadecadiynoic acid, 3, 7-octadecadiynoic acid, 3, 8-octadecadiynoic acid, 4, 6-octadecadiynoic acid, 4, 7-octadecadiynoic acid, 4, 8-octadecadiynoic acid, 4, 9-octadecadiynoic acid, 5, 7-octadecadiynoic acid, 5, 8-octadecadienoic acid, 5, 9-octadecadienoic acid, 5, 10-octadecadienoic acid, 5, 12-octadecadienoic acid, 6, 8-octadecadienoic acid, 6, 9-octadecadienoic acid, 6, 10-octadecadienoic acid, 6, 11-octadecadienoic acid, 6, 12-octadecadienoic acid, 7, 9-octadecadienoic acid, 7, 10-octadecadienoic acid, 7, 11-octadecadienoic acid, 7, 12-octadecadienoic acid, 8, 10-octadecadienoic acid, 8, 11-octadecadienoic acid, 8, 12-octadecadienoic acid, 9, 11-octadecadienoic acid, 9, 12-octadecadienoic acid, 9, 13-octadecadienoic acid, 10, 12-octadecadienoic acid, 10, 13-octadecadienoic acid, 10, 14-octadecadienoic acid, 11, 15-octadecadienoic acid, 12, 14-octadecadienoic acid, 12, 15-octadecadienoic acid, 12, 16-octadecadienoic acid, 13, 17-octadecadienoic acid, 14, 17-octadecadienoic acid, 10, 13-nonadecadienoic acid, 7, 3-eicosadienoic acid, 8, 11-eicosadienoic acid, 10, 13-eicosadienoic acid, 12, 14-pentacosadienoic acid, 12, 14-heptacosadienoic acid.
The C12-22 alkynoic acid comprises:
5,8, 11-dodecatrienoic acid, 9,11, 13-pentadecatrienoic acid, 5,8, 11-heptadecatrienoic acid, 5,8, 11-octadecatrienoic acid, 6,9, 12-octadecatrienoic acid, 8,11, 14-nonadecanoic acid, 5,8, 11-eicosatrienoic acid, 6,9, 12-eicosatrienoic acid, 7,10, 13-eicosatrienoic acid, 8,11, 14-eicosatrienoic acid, 9,12, 15-eicosatrienoic acid, 3,9, 15-docosatrienoic acid, 8,11, 14-docosatrienoic acid, 10,13, 16-docosatrienoic acid.
Alkenoic acids having 8 to 20 carbon atoms, preferably acids containing one or two C ═ C double bonds and one or two or three triple bonds, include:
10-ene-8-heptadecylic acid, 9-ene-12-octadecynoic acid, 11-ene-9-octadecynoic acid, 17-ene-9-octadecynoic acid, 9, 12-diene-6-octadecynoic acid, 9, 14-diene-12-octadecynoic acid, 11, 13-diene-9-octadecynoic acid, 5,8, 14-triene-11-eicosynoic acid, 5,11, 14-triene-8-eicosynoic acid, 8,11, 14-triene-5-eicosynoic acid, 6-ene-2, 4-octadiynoic acid, 8-ene-4, 6-decadiynoic acid, 2, 8-diene-4, 6-decadiynoic acid, 8-diene-4, 6-undecadienoic acid, 10, 12-diene-4, 6-tetradecadienoic acid, 5-ene-7, 9-octadecadienoic acid, 9-ene-12, 14-octadecadienoic acid, 13-ene-9, 11-octadecadienoic acid, 17-ene-9, 11-octadecadienoic acid, 13, 17-diene-9, 11-octadecadienoic acid, 3-ene-5, 7, 10-undecadienoic acid, 4-ene-6, 8, 10-undecadienoic acid.
Saturated fatty acids having 3 to 30 carbon atoms in the main chain, 1 to 10 alkyl groups and/or 1 to 3 hydroxyl groups in the side chain, preferably 1 to 3 methyl groups, or fatty acids having a C ═ C double bond, include:
2-methylpropanoic acid, 2-methylbutanoic acid, 3-methylbutanoic acid, 2, 2-dimethylpropionic acid, 2-methyl-2-butenoic acid, 3-methyl-3-butenoic acid, 2-ethyl-2-acrylic acid, 2-methylpentanoic acid, 3, 5-dihydroxy-3-methylpentanoic acid, 2-hydroxy-3-methylpentanoic acid, 2-ethylbutanoic acid, 2, 2-dimethylbutanoic acid, 3, 3-dimethylbutanoic acid, 3-methyl-2-pentenoic acid, 3-methyl-3-pentenoic acid, 3-methyl-4-pentenoic acid, 4-methyl-2-pentenoic acid, 4-methyl-3-pentenoic acid, 4-methyl-4-pentenoic acid, 2, 2-dimethyl-3-butenoic acid, 2, 3-dimethyl-2-butenoic acid, 2-methylhexanoic acid, 3-methylhexanoic acid, 4-methylhexanoic acid, 5-methylhexanoic acid, 2-ethylpentanoic acid, 2, 2-dimethylpentanoic acid, 3, 3-dimethylpentanoic acid, 3, 4-dimethylpentanoic acid, 4, 4-dimethylpentanoic acid, 2-ethyl-methylbutyric acid, 2-methyl-2-hexenoic acid, 5-methyl-5-hexenoic acid, 2-butyl-2-acrylic acid, 2-isopropyl-2-butenoic acid, 3-isopropyl-3-butenoic acid, 2, 2-dimethyl-4-pentenoic acid, 4, 4-dimethyl-2-pentenoic acid, 2-methylheptanoic acid, 3-methylheptanoic acid, 5-methylheptanoic acid, 6-methylheptanoic acid, 2-ethylhexanoic acid, 2, 2-dimethyl-hexanoic acid, 2-ethyl-2-methyl-pentanoic acid, 2-methyl-2-heptenoic acid, 5-hydroxy-4-methyl-2-heptenoic acid, 6-methyl-5-heptenoic acid, 2-ethyl-3-hexenoic acid, 3-tert-butyl-3-butenoic acid, 2-methyloctanoic acid, 3-methyloctanoic acid, 4-methyloctanoic acid, 5-methyloctanoic acid, 6-methyloctanoic acid, 7-methyloctanoic acid, 2-isopropylhexanoic acid, 6, 6-dimethyl-heptanoic acid, 3,5, 5-trimethylhexanoic acid, 6-methyl-5-octenoic acid, 2-pentyl-3-butenoic acid, 6-methyl-2, 4-octadienoic acid, 8-hydroxy-6-methyl-2, 4-octadienoic acid, 2-methylnonanoic acid, 3-methylnonanoic acid, 4-methylnonanoic acid, 7-methylnonanoic acid, 8-methylnonanoic acid, 3-methyl-2-nonenoic acid, 2, 7-dimethyl-6-octenoic acid, 3, 7-dimethyl-2-octenoic acid, 3, 7-dimethyl-6-octenoic acid, 3, 7-dimethyl-2, 6-suberic acid, 2-methyldecanoic acid, 3-methyldecanoic acid, 4-methyldecanoic acid, 5-methyldecanoic acid, 6-methyldecanoic acid, 7-methyldecanoic acid, 8-methyldecanoic acid, 9-methyldecanoic acid, 3, 3-dimethylnonanoic acid, 4, 8-dimethylnonanoic acid, 8, 8-dimethylnonanoic acid, 2, 7-dimethyl-6-nonenoic acid, 4-ethyl-2-methyl-2-octenoic acid, 2-methylundecanoic acid, 3-methylundecanoic acid, 4-methylundecanoic acid, 5-methylundecanoic acid, 6-methylundecanoic acid, 8-methylundecanoic acid, 9-methylundecanoic acid, 10-methylundecanoic acid, 4, 9-dimethyldecanoic acid, 5-methyl-2-undecanoic acid, 2-methyldodecanoic acid, 3-methyldodecanoic acid, 4-methyldodecanoic acid, 5-methyldodecanoic acid, 6-methyldodecanoic acid, 7-methyldodecanoic acid, 8-methyldodecanoic acid, 9-methyldodecanoic acid, 10-methyldodecanoic acid, 11-methyldodecanoic acid, 2, 6-dimethylundecanoic acid, 10, 10-dimethylundecanoic acid, 2-methyl-2-dodecenoic acid, 11-methyl-2-dodecenoic acid, 2-decyl-2-acrylic acid, 2-methyldodecanedioic acid, 3-methyldodecanedioic acid, 4-methyldodecanedioic acid, 6-methyldodecanedioic acid, 11-methyl-2, 5-dodecanedioic acid, 11-hydroxy-4-methyl-2, 4, 6-tridecanoic acid, 2-methyltridecanoic acid, 3-methyltridecanoic acid, 4-methyltridecanoic acid, 6-methyltridecanoic acid, 9-methyltridecanoic acid, 12-methyltridecanoic acid, 2, 4-dimethyldodecanoic acid, 2, 5-dimethyldodecanoic acid, 2, 6-dimethyldodecanoic acid, 4, 8-dimethyldodecanoic acid, 4, 10-dimethyldodecanoic acid, 4, 11-dimethyldodecanoic acid, 2,6, 10-trimethylundecanoic acid, 5-methyl-2-tridecenoic acid, 2, 4-dimethyl-2-dodecanoic acid, 2-methyl-tridecanedioic acid, 3-methyl-tridecanedioic acid, 4-methyl-tridecanedioic acid, 2-methyltetradecanedioic acid, 3-methyltetradecanoic acid, 11-methyltetradecanoic acid, 12-methyltetradecanoic acid, 13-methyltetradecanoic acid, 4, 12-dimethyltridecanoic acid, 12, 12-dimethyltridecanoic acid, 3,7, 11-trimethyldodecanoic acid, 2, 5-dimethyl-2-tridecenoic acid, 3-methyltetradecanedioic acid, 5-methyltetradecanedioic acid, 3-methylpentadecanoic acid, 13-methylpentadecanoic acid, 14-methylpentadecanoic acid, 2-propyltridecanoic acid, 2-heptylnonanoic acid, 4-hexyldodecanoic acid, 6-ethyltetradecanoic acid, 2, 4-dimethyltetradecanoic acid, 2, 6-dimethyltetradecanoic acid, 2, 8-dimethyltetradecanoic acid, 2, 12-dimethyltetradecanoic acid, 2, 13-dimethyltetradecanoic acid, 3, 5-dimethyltetradecanoic acid, 4, 12-dimethyltetradecanoic acid, 4, 13-dimethyltetradecanoic acid, 10, 13-dimethyltetradecanoic acid, 13, 13-dimethyltetradecanoic acid, 2-ethyl-2-butyldecanoic acid, 3-ethyl-3-methyltridecanoic acid, 4,8, 12-trimethyltridecanoic acid, 13-methyl-4-pentadecenoic acid, 14-methyl-4-pentadecenoic acid, 2-hexyl-2-decenoic acid, 2, 4-dimethyl-2-tetradecanoic acid, 6-isopentyl-9-methyl-5-decenoic acid, 2-methylhexadecanoic acid, 3-methylhexadecanoic acid, 4-methylhexadecanoic acid, 5-methylhexadecanoic acid, 6-methylhexadecanoic acid, 7-methylhexadecanoic acid, 8-methylhexadecanoic acid, 9-methylhexadecanoic acid, 10-methylhexadecanoic acid, 11-methylhexadecanoic acid, 12-methylhexadecanoic acid, 13-methylhexadecanoic acid, 14-methylhexadecanoic acid, 15-methylhexadecanoic acid, 2, 6-dimethyl-pentadecanoic acid, 4, 8-dimethyl-pentadecanoic acid, 8, 14-dimethyl-pentadecanoic acid, 9, 14-dimethyl-pentadecanoic acid, 7-methyl-6-hexadecenoic acid, 9-methyl-10-hexadecenoic acid, 10-methyl-9-hexadecenoic acid, 14-methyl-8-hexadecenoic acid, 15-methyl-6-hexadecenoic acid, 15-methyl-8-hexadecenoic acid, 15-methyl-9-hexadecenoic acid, 15-methyl-10-hexadecenoic acid, 15-methyl-11-hexadecenoic acid, 2-methylhexadecanoic acid, 3-methylhexadecanoic acid, 4-methylhexadecanoic acid, 5-methylhexadecanoic acid, 8-methylhexadecanoic acid, 2-methylheptadecanoic acid, 10-methylheptadecanoic acid, 14-methylheptadecanoic acid, 15-methylheptadecanoic acid, 16-methylheptadecanoic acid, 3-hydroxy-16-methylheptadecanoic acid, 2, 6-dimethyl-hexadecanoic acid, 2, 14-dimethyl-hexadecanoic acid, 4, 8-dimethyl-hexadecanoic acid, 4, 14-dimethyl-hexadecanoic acid, 6, 14-dimethyl-hexadecanoic acid, 10, 15-dimethyl-hexadecanoic acid, 11, 15-dimethyl-hexadecanoic acid, 12, 15-dimethyl-hexadecanoic acid, 15, 15-dimethyl-hexadecanoic acid, 2-methyl-16-heptadecenoic acid, 7-methyl-12-heptadecenoic acid, 9-methyl-6-heptadecenoic acid, 15-methyl-4-heptadecenoic acid, 16-methyl-6-heptadecenoic acid, 16-methyl-8-heptadecenoic acid, 8, 9-methylene-8-heptadecenoic acid, 16-methyl-6, 9-heptadecenoic acid, 16-methyl-9, 12-heptadecenoic acid, 4, 6-dimethyl-2, 4-hexadecadienoic acid, 5, 7-dimethyl-2, 4-hexadecadienoic acid, 16-methyl-6, 9, 12-heptadecatrienoic acid, 2-methyloctadecanoic acid, 3-methyloctadecanoic acid, 4-methyloctadecanoic acid, 5-methyloctadecanoic acid, 6-methyloctadecanoic acid, 7-methyloctadecanoic acid, 8-methyloctadecanoic acid, 9-methyloctadecanoic acid, 10-methyloctadecanoic acid, 11-methyloctadecanoic acid, 3-hydroxy-11-methyloctadecanoic acid, 11, 12-methylene-octadecanoic acid, 12-methyloctadecanoic acid, 13-methyloctadecanoic acid, 14-methyloctadecanoic acid, 15-methyloctadecanoic acid, 16-methyloctadecanoic acid, 17-methyloctadecanoic acid, 4, 14-dimethyl-heptadecanoic acid. 10, 16-dimethyl-heptadecanoic acid, 12, 16-dimethyl-heptadecanoic acid, 10-methyl-9-octadecenoic acid, 11-methyl-12-octadecenoic acid, 17-methyl-6-octadecenoic acid, 17-methyl-7-octadecenoic acid, 17-methyl-13-octadecenoic acid, 2, 5-dimethyl-2-heptadecenoic acid, 4-heptyl-2-methyl-2-undecenoic acid, 9, 10-methylene-9-octadecenoic acid, 16-methyl-5, 9-octadecadienoic acid, 17-methyl-5, 9-octadecadienoic acid, 16-methyl-5, 9, 12-octadecatrienoic acid, 11-methylnonadecanoic acid, 17-methylnonadecanoic acid, 18-methylnonadecanoic acid, 2, 11-dimethyl-octadecanoic acid, 2, 14-dimethyl-octadecanoic acid, 4, 14-dimethyl-octadecanoic acid, 6, 14-dimethyl-octadecanoic acid, 4, 16-dimethyl-octadecanoic acid, 6, 16-dimethyl-octadecanoic acid, 12, 17-dimethyl-octadecanoic acid, 6-methyl-9-nonadecenoic acid, 18-methyl-8, 11, 14-nonadecenoic acid, 18-methyl-5, 8,11, 14-nonadecatetraenoic acid, 18-methyleicosanoic acid, 19-methyleicosanoic acid, 2, 6-dimethylnonadecanoic acid, 12, 18-dimethylnonadecanoic acid, 2-methyl-2-eicosenoic acid, 2-propyl-9-octadecenoic acid, 18-methyl-5, 9-eicosadienoic acid, 19-methyl-5, 9-eicosadienoic acid, 3-methylheneicosanoic acid, 19-methylheneicosanoic acid, 20-methyleicosanoic acid, 14, 19-dimethyleicosanoic acid, 2, 4-dimethyl-2-eicosenoic acid, 7, 7-dimethyl-5, 8-eicosadienoic acid, 7, 7-dimethyl-5, 8, 11-eicosatrienoic acid, 10, 10-dimethyl-5, 8, 11-eicosatrienoic acid, 20-methyldocosanoic acid, 21-methyldocosanoic acid, 22-methyltricosanoic acid, 21-methyltricosanoic acid, 2, 4-dimethyldocosanoic acid, 3, 15-dimethyldocosanoic acid, 23-methyltetracosanoic acid, 2, 4-dimethyltetracosanoic acid, 23-methyl-5, 9-tetracosanoic acid, 3,7, 11-trimethyl-2, 6-docosanoic acid, 23-methylpentacosanoic acid, 24-methylpentacosanoic acid, 2, 4-dimethyltetracosanoic acid, 3,13, 19-trimethyltetracosanoic acid, 24-methylhexanoic acid, 2-methyl-2-hexacosanoic acid, 2, 4-dimethyl-2-pentacosanoic acid, 2,4, 6-trimethyl-2-eicosatetraenoic acid, 9, 10-dimethyl-octacosanoic acid, 28-methyltrisitriacontanoic acid, 2,4, 6-trimethyloctacosanoic acid, 15, 16-dimethyl tridecanedioic acid.
Saturated straight and branched chain dicarboxylic and tricarboxylic acids having 3 to 38 carbon atoms include:
malonic acid, succinic acid, 2-methylmalonic acid, glutaric acid, 2, 2-dimethylmalonic acid, 2-methylsuccinic acid, 2-ethylmalonic acid, adipic acid, 2, 2-dimethylsuccinic acid, 2-methyl-glutaric acid, 3-methyl-glutaric acid, 2-hydroxyadipic acid, 2,3,4, 5-tetrahydroxyadipic acid, 3-hydroxymethyl-glutaric acid, pimelic acid, 3, 3-dimethylglutaric acid, 3-methyl-adipic acid, suberic acid, azelaic acid, sebacic acid, undecanedioic acid, dodecanedioic acid, tridecanedioic acid, tetradecanedioic acid, pentadecanedioic acid, hexadecanedioic acid, heptadecanedioic acid, octadecanedioic acid, 9, 10-dihydroxy-octadecanedioic acid, nonadecanedioic acid, eicosanedioic acid, heneicosanedioic acid, docosanedioic acid, tricosanedioic acid, tetracosanedioic acid, hexacosanedioic acid, heptacosanedioic acid, nonacosanedioic acid, tridecanedioic acid, 13, 14-dimethyl-octacosanedioic acid.
Unsaturated straight-chain or branched dicarboxylic acids and tricarboxylic acids having 4 to 18 carbon atoms (which may be hydroxyl-or amino-containing dicarboxylic acids or tricarboxylic acids) include:
butenedioic acid, 2-hydroxy-2-butenedioic acid, 2-methyl-2-pentenedioic acid, 3-hexenedioic acid, 3-hydroxy-2, 4-hexadiene diacid, 2-pentyl-4-tridecenedioic acid, 2- (2-octenyl) -1, 10-decanedioic acid, 2- (2, 5-octadienyl) -1, 10-decanedioic acid, 2- (2-pentenyl) -4-tridecene-1, 13-dioic acid, 2-en-4-octadecyne-dioic acid;
The tricarboxylic acid having 4 carbon atoms and substituted with a hydroxyl group includes:
3-hydroxypropane-1, 2, 3-tricarboxylic acid, 2-hydroxybutane-1, 2, 3-tricarboxylic acid, 3-hydroxybutane-1, 2, 3-tricarboxylic acid, 1-butene-1, 2, 4-tricarboxylic acid, 1, 3-butadiene-1, 2, 4-tricarboxylic acid.
Saturated and/or unsaturated fatty acids useful in the present invention also include amino, hydroxyl, oxo, and/or alkyl substituted fatty acids as described below. Specifically, it may be:
1) amino fatty acids and fatty acyl amino fatty acids: c3-18, amino, hydroxyl, oxo and/or methyl substituted carboxylic acid, which comprises one or more than two amino fatty acids selected from the following substances:
2-amino-3-hydroxy-propionic acid, 2, 3-diamino-propionic acid, 2-amino-butyric acid, 4-amino-butyric acid, 2-amino-3, 4-dihydroxybutyric acid, 2-methyl-2-amino-propionic acid, 2-methyl-3-amino-propionic acid, 3-methyl-3-amino-propionic acid, 2, 4-diamino-butyric acid, 2-amino-3-oxo-butyric acid, 2-amino-valeric acid, 4-amino-valeric acid, 5-amino-valeric acid, 2-amino-3-methyl-butyric acid, 2, 5-diamino-valeric acid, 2-amino-4-hydroxy-3-methylpentanoic acid, 2-amino-4-oxo-pentanoic acid, 2-oxo-5-amino-pentanoic acid, 4-oxo-5-amino-pentanoic acid, 2-amino-hexanoic acid, 6-amino-hexanoic acid, 2-amino-3-methyl-pentanoic acid, 2-amino-4-methyl-pentanoic acid, 3-oxo-5-amino-hexanoic acid, 2-amino-adipic acid, 2-amino-3-oxo-adipic acid, 2-amino-6-oxo-2, 4-hexadienoic acid, 2-amino-heptanoic acid, 2, 6-diamino-pimelic acid, 2-amino-4, 5-dihydroxy-6-oxo-heptanoic acid, 2-amino-octanoic acid, 3-amino-octanoic acid, 8-amino-octanoic acid, 3-amino-nonanoic acid, 9-amino-nonanoic acid, 7, 8-diamino-nonanoic acid, 7-oxo-8-amino-nonanoic acid, 2-amino-decanoic acid, 3-amino-decanoic acid, 9-amino-decanoic acid, 10-amino-decanoic acid, 11-amino-undecanoic acid, 12-amino-dodecanoic acid, 2-amino-tridecanoic acid, 13-amino-tridecanoic acid, 2-amino-tetradecanoic acid, 2-amino-hexadecanoic acid, 2-amino-octadecanoic acid, 12-amino-octadecanoic acid.
2) An N-acyl amino acid, which comprises the following N-acyl amino acids with 6-30 carbon atoms: n-hexadecanoyl-gamma-aminobutyric acid, N-octadecanoyl-gamma-aminobutyric acid, N- (9-octadecenoyl) -gamma-aminobutyric acid, N- (5,8,11, 14-eicosatetraenoyl) -gamma-aminobutyric acid, N- (12-hydroxy-5, 8,10, 14-eicosatetraenoyl) -gamma-aminobutyric acid, N- (15-hydroxy-5, 8,11, 13-eicosatetraenoyl) -gamma-aminobutyric acid, N- (4,7,10,12,16, 19-docosahexaenoyl) -gamma-aminobutyric acid; n- (6-aminocaproyl) -6-aminocaproic acid; n-hexadecanoylalanine, N-octadecanoylalanine, N- (9-octadecenoyl) alanine, N- (5,8,10, 14-eicosatetraenoyl) alanine, N- (12-hydroxy-5, 8,10, 14-eicosatetraenoyl) alanine, N- (15-hydroxy-5, 8,11, 13-eicosatetraenoyl) alanine; n-octadecanoyl arginine; n-octadecanoylasparagine, N- (9-octadecenoyl) asparagine, N-tetradecanoylglutamine, N-hexadecanoylglutamine, N- (9-hexadecenoyl) glutamine, N-octadecanoylglutamine, N- (9-octadecenoyl) glutamine, N- (9, 12-octadecadienoyl) glutamine, N- (17-hydroxy-9, 12-octadecadienoyl) glutamine, N- (9,12, 15-octadecatrienoyl) glutamine, N- (17-hydroxy-9, 12, 15-octadecatrienoyl) glutamine, N- (5,8,11, 14-eicosatetraenoyl) glutamine, n- (4,7,10,12,16, 19-docosahexaenoyl) glutamine; n-hexadecanoyl glutamic acid, N-octadecanoyl glutamic acid, N- (9-octadecenoyl) glutamic acid, N- (9, 12-octadecadienoyl) glutamic acid, N- (9,12, 15-octadecatrienoyl) glutamic acid, N- (5,8,11, 14-eicosatetraenoyl) glutamic acid, N- (4,7,10,12,16, 19-docosahexaenoyl) glutamic acid;
N-lauroyl glycine, N- (3-hydroxy-tetradecanoyl) glycine, N- (14-methyl-3- (13-methyl-4-tetradecanoyloxy) -pentadecanoyl) -glycine, N-hexadecanoyl glycine, N- (3-hydroxy-hexadecanoyl) glycine, N- (3-hydroxy-9-hexadecanoyl) glycine, N- (15-methylhexanoyl) glycine, N- (3-hydroxy-15-methylhexanoyl) glycine, N- (2,3, 4-trihydroxy-15-methylhexanoyl) glycine, N-octadecanoyl glycine, N- (9-octadecenoyl) glycine, n- (3-hydroxy-9-octadecenoyl) glycine, N- (5,8,11, 14-eicosatetraenoyl) glycine, N- (12-hydroxy-5, 8,10, 14-eicosatetraenoyl) glycine, N- (15-hydroxy-5, 8,11, 13-eicosatetraenoyl) glycine;
n-hexanoylhistidine, N-octanoylhistidine, N-decanoylhistidine, N- (3, 4-methylene-decanoyl) histidine, N-hexadecanoylhistidine, N-octadecanoylhistidine, N- (9-octadecenoyl) histidine, N- (4,7,10,12,16, 19-docosahexaenoyl) histidine; n-hexadecanoyl isoleucine, N- (9-octadecenoyl) isoleucine, N- (5,8,11, 14-eicosatetraenoyl) isoleucine;
n-hexadecanoyl leucine, N- (9-octadecenoyl) leucine, N- (5,8,11, 14-eicosatetraenoyl) leucine; alpha-N-lauroyl lysine, 6-N-lauroyl lysine, alpha-N-tetradecanoyl lysine, 6-N-tetradecanoyl lysine, alpha-N- (5-tetradecenoyl) lysine, 6-N- (5-tetradecenoyl) lysine, alpha-N- (5, 8-tetradecadienoyl) lysine, 6-N- (5, 8-tetradecadienoyl) lysine; n-hexadecanoyl methionine, N- (9-octadecenoyl) methionine; α -N- (3-hydroxy-13-methyl-tetradecanoyl) ornithine, α -N- (3-hydroxy-hexadecanoyl) ornithine, α -N- (3-hydroxy-14-methyl-pentadecanoyl) ornithine, α -N- (3-hydroxyoctadecanoyl) ornithine; n-hexadecanoylphenylalanine, N-octadecanoylphenylalanine, N- (9-octadecenoyl) phenylalanine, N- (4,7,10,12,16, 19-docosahexaenoyl) phenylalanine; n-hexadecanoyl proline, N-octadecanoyl proline, N- (9-octadecenoyl) proline;
N-hexadecanoylserine, N-octadecanoylserine, N- (9-octadecenoyl) serine, N- (5,8,11, 14-eicosatetraenoyl) serine; n-hexadecanoyl taurine, N-heptadecanoyl taurine, N-octadecanoyl taurine, N- (9-octadecenoyl) taurine, N- (9, 12-octadecadienoyl) taurine, N-nonadecanoyl taurine, N- (9-nonadecenoyl) taurine, N-eicosanoyl taurine, N- (11-eicosenoyl) taurine, N- (5,8,11, 14-eicosatetraenoyl) taurine, N- (12-hydroxy-5, 8,10, 14-eicosatetraenoyl) taurine, N- (15-hydroxy-5, 8,11, 13-eicosatetraenoyl) taurine, N-heneicosyl taurine, N-docosanoyl taurine, n- (13-eicosanoyl) taurine, N-tricosyl taurine, N- (14-eicosatrioyl) taurine, N-tetracosanoyl taurine, N- (15-tetracosanoyl) taurine, N-pentacosanoyl taurine, N-hexacosanoyl taurine; n-hexadecanoyl threonine, N- (9-octadecenoyl) threonine; n-hexadecanoyl tryptophan, N-octadecanoyl tryptophan, N- (9-octadecenoyl) tryptophan; n-lauroyl-6-methyl-tyrosine, N-hexadecanoyl-alpha, O-dimethyltyrosine, N-octadecanoyl-tyrosine, N- (9-octadecenoyl) tyrosine, N- (5,8,11, 14-eicosatetraenoyl) tyrosine, N-nonanoyl-2, 3-dehydro-tyrosine, N-decanoyl-2, 3-dehydro-tyrosine, N- (8-methylnonanoyl) -2, 3-dehydro-tyrosine, N-dodecanoyl-6-methyl-2, 3-dehydro-tyrosine, N- (2-dodecenoyl) -6-methyl-2, 3-dehydro-tyrosine, N-hexadecanoyl-alpha, O-dimethyltyrosine, N-octadecanoyl-O-methyl-2, 3-dehydro-tyrosine, N- (9-octadecenoyl) -O-methyl-2, 3-dehydro-tyrosine; n-hexadecanoylvaline, N-octadecanoylvaline, N- (9, 12-octadecadienoyl) valine.
3) Amino acids containing 2 or more than 2 acyl groups, which include the following:
n- (15-methyl-3- (12-methyltridecanyloxy) -hexadecanoyl) -glycine, N- (15-methyl-3- (13-methyltetradecanoyloxy) -hexadecanoyl) -glycine, N- (15-methyl-3- (13-methyl-4-tetradecenoyloxy) -hexadecanoyl) -glycine, N- (15-methyl-3- (13-methyl-tetradecanoyloxy) -hexadecanoyl) glycine;
α -N- (3-hexadecanoyloxy-hexadecanoyl) ornithine, α -N- (3-octadecanoyloxy-octadecanoyl) ornithine, α -N- (3- (3-hydroxyoctadecanoyloxy) -octadecanoyl) ornithine, α -N- (3- (11, 12-methylene) octadecanoyloxy-hexadecanoyl) ornithine, α -N- (3- (11, 12-methylene) octadecanoyloxy-octadecanoyl) ornithine, α -N- (3- (3-hydroxy- (11, 12-methylene) octadecanoyloxy) -octadecanoyl) ornithine;
n- (3-oxo-decanoyl) -leucyl) alanine, N- ((3- (13-methyl-tetradecyloxy) -13-methyl-hexadecanoyl) glycyl) serine, N- ((15-methyl-3- (13-methyltetradecyloxy) -hexadecanoyl) -glycyl) -serine, N- (((3-hydroxy-15-methyl-hexadecanoyl) -glycyl) -seryl) -ornithine, N- ((3-hydroxy-13-methyl-hexadecanoyl) -glycyl) -serine, N- ((9-octadecenoyl) - β -alanyl) -L-histidine;
4) A plurality of acids linked by thioether linkages and amide linkages comprising the following:
11, 15-dihydroxy-14- (S-cysteinyl-glycyl) -5,8, 12-eicosatrienoic acid, 11, 15-dihydroxy-14- (S-glutathione) -5,8, 12-eicosatrienoic acid.
Among them, the saturated and/or unsaturated fatty alcohols used to provide the above carbon chains or carbon chain residues specifically include the fatty alcohols shown below.
Saturated aliphatic straight-chain or branched-chain alcohol with 3-33 carbon atoms and 1-3 hydroxyl groups, which comprises:
propane-1-ol, butane-1-ol, 2-methylpropan-1-ol, pentane-1-ol, 2-methylbutane-1-ol, 3-methylbutane-1-ol, hexane-2-ol, hexane-3-ol, hexane-1, 5-diol, 3-methylpentane-1-ol, 3-methylpentane-3-ol, 4-methylpentane-1-ol, 1-methyl-cyclopentane-1-ol, heptane-2-ol, heptane-3-ol, heptane-4-ol, 3-methylhexan-2-ol, 4-methylhexan-3-ol, 5-methylhexan-3-ol, heptane-1, 2, 3-triol, octane-1-ol, octane-2-ol, octane-3-ol, octane-1, 2-diol, octane-1, 3-diol, octane-1, 8-diol, 2-methylheptan-4-ol, 3-methylheptan-2-ol, 4-methylheptan-3-ol, nonane-1-ol, nonane-2-ol, nonane-3-ol, nonane-5-ol, 2-methyloctane-4-ol, 3-methyloctane-4-ol, 4-methyloctan-1-ol, 5-methyloctan-4-ol, 6-methyloctan-3-ol, 3,5, 5-trimethylhexane-1-ol, decan-2-ol, decan-3-ol, 4-methylnonan-1-ol, 4-methylnonan-5-ol, 6-methylnonan-3-ol, 3, 7-dimethyloctan-1, 7-diol, undecan-1-ol, undecan-2-ol, undecan-3-ol, dodecane-1-ol, tridecan-1-ol, tridecan-2-ol, 4-methyl-dodecane-7-ol, 10-methyl-dodecane-1-ol, tetradecan-1-ol, 4-methyl-tridecan-7-ol, 3, 9-dimethyl-dodecane-6-ol, 2,2, 10-trimethyl-undecane-1, 10-diol, pentadecan-1-ol, pentadecan-2-ol, 4-methyl-tetradecan-7-ol, 3, 7-dimethyl-tridecan-2-ol, 4, 10-dimethyl-tridecan-7-ol, hexadecan-1-ol, 14-methyl-pentadecan-1-ol, 3, 7-dimethyltetradecan-2-ol, heptadecane-2-ol, 4-methyl-hexadecan-7-ol, 3, 7-dimethylpentadecane-2-ol, 6,10, 13-trimethyl-tetradecan-1-ol, 2-methyl-hexadeca-1, 2-diol, heptadecane-1, 17-diol, octadecan-1-ol, 3, 7-dimethylhexadeca-2-ol, 2-methyl-heptadecane-1, 2-diol, 3-methyl-heptadecane-1, 2-diol, 11-methyl-heptadecane-1, 2-diol, nonadecane-1, 2-diol, nonadecane-1, 2, 4-triol, 2-methyl-octadecane-1, 2-diol, eicosan-1-ol, eicosan-1, 2-diol, eicosan-1, 3-diol, eicosan-1, 20-diol, 13-methyl-eicosan-1, 2-diol, heneicosane-1, 21-diol, 15-methyl-heneicosane-1, 2-diol, docosan-1-ol, docosane-1, 2-diol, docosane-1, 3-diol, 15-methyl-docosane-1, 2-diol, tricosane-12-ol, tricosane-1, 2-diol, tetracosan-1-ol, tetracosan-1, 2-diol, tetracosan-1, 3-diol, tetracosan-1, 24-diol, hexacosan-1-ol, hexacosan-1, 26-diol, 23-hexacosan-1-ol, heptacosan-14-ol, heptacosan-6, 8-diol, octacosan-1-ol, octacosan-1, 28-diol, nonacosan-1-ol, nonacosan-10-ol, nonacosan-15-ol, nonacosan-6, 8-diol, triacontan-1-ol, triacontane-1, 11-diol, triacontane-1, 14-diol, dotriacontane-1-ol, triacontriacontane-1-ol, tetratriacontane-1-ol.
The unsaturated aliphatic straight-chain or branched-chain alcohol with 3-33 carbon atoms, 1-5 double bonds and 1-5 triple bonds and 1-3 hydroxyl groups comprises:
2-penten-1-ol, 2-methylenebutan-1-ol, 2-methyl-2-buten-1-ol, 2-methyl-3-buten-1-ol, 2-hexen-1-ol, 3-hexen-3-ol, 4-hexen-1-ol, 4-hepten-2-ol, 2, 4-heptadien-1-ol, 6-methylheptan-3-ol, 2, 4-dimethyl-hexan-1-ol, 2-ethylhexan-1-ol, 1-octen-3-ol, 2-octen-1-ol, 3-octen-2-ol, 5-octen-1-ol, 7-octen-2-ol, 4-methyl-4-hepten-3-ol, 6-methyl-2-hepten-4-ol, 6-methyl-5-hepten-2-ol, 5-octen-1, 3-diol, 1, 5-octadien-3-ol, 9, 12-octadien-1-ol, 2, 4-dimethyl-2, 4-hexadien-1-ol, 2,4, 6-octatriyn-1-ol, 1-nonen-3-ol, 2-nonen-1-ol, 3-nonen-1-ol, 6-nonen-2-ol, 2, 4-dimethyl-5-hepten-1-ol, 2, 6-dimethyl-6-hepten-1-ol, 2, 4-nonadien-1-ol, 3, 6-nonadien-1-ol, 6, 8-nonadien-2-ol, 2, 4-dimethyl-2, 4-heptadien-1-ol, 1-decen-3-ol, 2-decen-1-ol, 3-decen-1-ol, 4-decen-1-ol, 5-decen-1-ol, 7-methyl-6-nonen-3-ol, 2, 6-dimethyl-6-octen-2-ol, 2, 6-dimethyl-7-octen-2, 3, 6-triol, 7-methyl-3-methylene-octane-1, 6, 7-triol, 2, 4-decadien-1-ol, 7, 9-decadien-1-ol, 3, 7-dimethyl-3, 6-octadien-1-ol, 4, 6-decadiyn-1-ol, 2, 6-dimethyl-2, 7-octadien-1, 6-diol, 2-methyl-6-methylene-2, 7-octadien-1-ol, 2-ene-4, 6, 8-decyltriakin-1-ol, 1-undecen-3-ol, 2-undecen-1-ol, 6-undecen-2-ol, 10-undecen-1-ol, 3-methyl-4-decen-1-ol, 3,4, 7-trimethyl-2, 6-octadien-1-ol, dodecane-2-ol, 2-butyloctan-1-ol, 3-dodecen-1-ol, 5-dodecen-1-ol, 6-dodecen-1-ol, 7-dodecen-1-ol, 8-dodecen-1-ol, 9-dodecen-1-ol, 10-dodecen-1-ol, 11-dodecen-1-ol, 3, 5-dodecen-1-ol, 3, 6-dodecen-1-ol, 5, 7-dodecen-1-ol, 7, 9-dodecen-1-ol, 8, 10-dodecen-1-ol, 9, 11-dodecen-1-ol, 3,4, 7-trimethyl-2, 6-nonadien-1-ol, 3,6, 8-dodecatrien-1-ol, 3,6, 9-dodecatrien-1-ol, 6-tridecen-2-ol, 10-tridecen-2-ol, 11-en-3, 5,7, 9-tridecatetrayn-1-ol, 3-tetradecen-1-ol, 5-tetradecen-1-ol, 7-tetradecen-1-ol, 8-tetradecen-1-ol, 9-tetradecen-1-ol, 11-tetradecen-1-ol, 9- (2-cyclopentenyl) -1-ol, 8, 10-tetradecene-1-ol, 9, 11-tetradecene-1-ol, 9, 12-tetradecene-1-ol, 10, 12-tetradecadien-1-ol, 11, 13-tetradecadien-1-ol, 3-methyl-6- (1-methyl-ethyl) -3, 9-decadien-1-ol, 13-en-2, 4-tetradecadien-1-ol, 13-en-1, 3-tetradecediyne-6, 7-diol, 9-pentadecen-1-ol, 5, 10-pentadecen-1-ol, 8, 10-pentadecen-1-ol, 3,7, 11-trimethyl-6, 10-dodecadien-1-ol, 7-hexadecen-1-ol, 9-hexadecen-1-ol, 11-hexadecen-1-ol, 4, 6-hexadecadien-1-ol, 6, 11-hexadecadien-1-ol, 7, 11-hexadecadien-1-ol, 10, 12-hexadecadien-1-ol, 11, 13-hexadecadien-1-ol, 10-propyl-5, 9-tridecadien-1-ol, 13-en-11-hexadecyn-1-ol, 4,6, 10-hexadecatrien-1-ol, 8-heptadecen-2-ol, 11-heptadecen-1-ol, 14-methyl-8-hexadecen-1-ol, 16-heptadecen-1, 2, 4-triol, 16-heptadecayne-1, 2, 4-triol, 2,6,8, 12-tetramethyl-2, 4-tridecadiene-1-ol, 4, 6-heptadecadiyne-3, 9, 10-triol, 1-ene-4, 6-heptadecadiyne-3, 9-diol, 1-ene-4, 6-heptadecadiyne-3, 9, 10-triol, 1, 9-diene-4, 6-heptadecadiyne-3-ol, 1, 8-diene-4, 6-heptadecadiyne-3, 10-diol, 1, 9-diene-4, 6-heptadecadiyne-3, 8-diol, 2, 9-diene-4, 6-heptadecadiyne-1, 8-diol, 1, 16-diene-4, 6-heptadecadiyne-3, 9, 10-triol, 1,9, 16-triene-4, 6-heptadecadiyne-3, 8-diol, 9-octadecen-1-ol, 11-octadecen-1-ol, 13-octadecen-1-ol, 2, 13-octadecadien-1-ol, 3, 13-octadecadien-1-ol, 9,12, 15-octadecatrien-1-ol, 11-eicosen-1-ol, 15-eicosen-1-ol, 6, 9-eicosadien-11-ol, 3,7,11, 15-tetramethyl-6, 10, 14-eicosatrien-1-ol, 6-heneicosene-11-ol, 6, 9-heneicosene-11-ol, 3,7,11,15, 19-pentamethyl-2, 6,10,14, 18-eicosapentaen-1-ol.
The oxo-fatty alcohol (C8-31 alcoholic ketone containing 1-3 double or triple bonds and 1-3 hydroxyl groups, the ketone being a mono-ketone or a diketone) comprises:
1-hydroxyoctan-3-one, 3-hydroxymethylheptan-2-one, 6-methyl-7-hydroxy-3, 5-heptadien-1-one, 6-methyl-7-hydroxy-3, 5-heptadien-2-one, 1-hydroxy-nonan-3-one, 1-hydroxy-nonan-6-one, 3-hydroxymethyloctan-2-one, 1, 3-dihydroxy-8-decen-5-one, 1-hydroxy-5-phenyl-pentan-3-one, 3-hydroxypentadecan-4-one, 1- (furan-3-yl) -6-hydroxy-4, 8-dimethyl-1-one, 1-hydroxy-2, 12, 15-heneicosatrien-4-one, 2- (12-hydroxy-5, 10-dodecadiyn-1-yl) -3,5, 6-trimethyl-2, 5-cyclohexadiene-1, 4-dione, 25-hydroxy-hentriacontane-14, 16-dione.
Further, the water-soluble part is a natural or synthetic compound having a structure of carboxyl, sulfonic acid group, sulfonyloxy group, phosphate group, hydroxyl, amino, ureido, guanidino, quaternary ammonium group, and sulfhydryl, including proteins, polypeptides, nucleic acids, polysaccharides, and high molecular compounds having the above structure, such as:
water-soluble macromolecules: water-soluble proteins such as serum albumin, immunoglobulins, water-soluble collagens, chaperones, water-soluble glycoproteins, dextran (dextran), hyaluronic acid, sialic acid, heparin sulfate, heparan sulfate, chondroitin sulfate, dermatan sulfate, keratan sulfate, water-soluble cellulose derivatives, beta-cyclodextrin and its derivatives, water-soluble chitosan derivatives, polyethylene glycol and carboxylated or aminated polyethylene glycol, polyvinyl alcohol and carboxylated or quaternized polyvinyl alcohol, polyacrylic acid, ammonium polyacrylate;
Water-soluble medium molecules: polypeptides, oligopeptides, water-soluble polyamines (polymers of the same amino acid), oligosaccharides, oligonucleotides and artificially synthesized water-soluble polymers of moderate degree.
Water-soluble small molecules: comprises monosaccharide or disaccharide, amino acid, nucleotide, and vitamin;
the above water-soluble molecules can impart the property of dissolving and uniformly dispersing the hydrophobic functional moiety in an aqueous solution and prevent the hydrophobic structure from aggregating into a mass.
Further in a preferred embodiment, the water-soluble macromolecule may be a water-soluble protein such as serum albumin, immunoglobulin, water-soluble collagen, chaperone protein, water-soluble glycoprotein, CD 14;
further in a preferred embodiment, the water-soluble macromolecule may also be a water-soluble polysaccharide such as dextran (dextran), hyaluronic acid, sialic acid, heparin sulfate, heparan sulfate, chondroitin sulfate, dermatan sulfate, keratan sulfate, acetyl water-soluble cellulose derivatives, β -cyclodextrin and its derivatives, water-soluble chitosan derivatives;
in addition, the water-soluble macromolecule can also be water-soluble macromolecule polymers such as polyethylene glycol and carboxylated or aminated polyethylene glycol, polyvinyl alcohol and carboxylated or quaternized polyvinyl alcohol, polyacrylic acid, and ammonium polyacrylate.
Further in a preferred embodiment, the medium size water soluble molecule (referred to as "water soluble medium molecule" for short) may be; targeting polypeptides include proteins or neutralizing antibody fragments that specifically target microbial lipid membranes, bacterial and fungal cell walls, viral surface protein domains, such as taurine transporter peptide, SBP 1; water-soluble polyamino acids such as polyglutamic acid, polylysine, polyaspartic acid; and oligopeptides, oligosaccharides, oligonucleotides.
Further in a preferred embodiment, as water-soluble small molecules, there may be mentioned monosaccharides and disaccharides such as glucose, fructose, rhamnose, sorbose, sucrose, maltose, lactose, trehalose;
nucleotides and deoxynucleotides such as adenylic acid, guanylic acid, uridylic acid, cytidylic acid, thymidylic acid, inosinic acid, deoxyadenylic acid, deoxyguanylic acid, deoxycytidylic acid, deoxythymidylic acid;
amino acids such as serine, threonine, cysteine, asparagine, glutamine, tyrosine, lysine, arginine, histidine, aspartic acid, glutamic acid, citrulline, ornithine, taurine, aminobutyric acid;
vitamins such as vitamin B1, pantothenic acid, vitamin B6, and vitamin C.
Further, for the complexes of the invention, the mode of coupling between the acting, binding and water-soluble moieties is
(1) Coupling by hydrogen bond and intermolecular force;
(2) amide bond, ester bond, hydrazone bond and thioether bond;
more specifically, the compound for preventing, preventing or treating microbial infection of the present invention is a compound obtained by reacting a saturated and/or unsaturated fatty acid having 3 to 100 carbon atoms with a protein, polypeptide, oligopeptide, oligosaccharide, monosaccharide and/or polysaccharide molecule; or a mixture of compounds obtained by reacting saturated and/or unsaturated fatty acids having 3 to 50 carbon atoms with proteins, polypeptides, oligopeptides, oligosaccharides, monosaccharides, disaccharides, oligonucleotides, vitamins, water-soluble polymers, water-soluble polyamines and/or polysaccharide molecules, and unreacted fatty acids and/or unreacted proteins, polypeptides, oligopeptides, oligosaccharides, monosaccharides, disaccharides, oligonucleotides, vitamins, water-soluble polymers, water-soluble polyamines and/or polysaccharide molecules.
More specifically, the complex for preventing, preventing or treating microbial infection of the present invention is a complex or a mixture obtained by direct physical mixing of saturated and/or unsaturated fatty acids having 3 to 100 carbon atoms with proteins, polypeptides, oligopeptides, oligosaccharides, monosaccharides, disaccharides, oligonucleotides, vitamins, water-soluble polymers, water-soluble polyamines and/or polysaccharide molecules, with physicochemical action including hydrogen bonding or van der waals force or a combination of both actions.
For example, the carboxyl group in the acting group reacts with the amino group in the lysine residue in the water-soluble protein or polypeptide to generate amide, and the reaction formula is shown in the following equation;
Figure BDA0003628692260000831
wherein R is a saturated or unsaturated fatty acid with a direct or branched chain of 3-100 carbon atoms.
The terminal amino group in the water-soluble protein and the polypeptide reacts with the carboxyl group of the fatty acid to generate an amido bond:
Figure BDA0003628692260000832
wherein R is a saturated or unsaturated fatty acid with a direct or branched chain of 3-100 carbon atoms.
Specifically, in a preferred embodiment, the protein is Human Serum Albumin (HSA), the polypeptide is SBP1, and the carbon chain donor is docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), octadecatrienoic acid (linolenic acid), octadecadienoic acid (linoleic acid), octadecamonooleic acid (oleic acid), octanoic acid, and butenedioic acid (fumaric acid) for forming the functional moiety.
The following illustrates preferred complexes of the invention as follows:
(1) the lysine side chain in the albumin (HSA) or SBP1 molecules reacts with the fatty acid-amidated complex:
Figure BDA0003628692260000841
Figure BDA0003628692260000842
the reaction product is schematically shown below:
Figure BDA0003628692260000843
Figure BDA0003628692260000851
Figure BDA0003628692260000861
Figure BDA0003628692260000871
in the above products, the carbon chain of the fatty acid is the active moiety, and albumin and polypeptide are both the binding and water-soluble moieties.
(2) And (2) performing a linking reaction of the fatty acid and the protein through a linker, wherein the linker comprises one or more of amino acid, succinic acid, butadiene acid, glutaconic acid, hexylaminedicarboxylic acid, carbamate, short peptide, polyethylene glycol and derivatives of the above compounds.
For example, albumin (HSA) has 34-Cys with a free thiol group as a thioetherification product with fatty acids and N-hydroxymaleimide:
Figure BDA0003628692260000872
Figure BDA0003628692260000881
the structural formula of the product is shown as follows:
Figure BDA0003628692260000882
Figure BDA0003628692260000891
the fatty acids in the above products together with the linker molecule are the active moiety, and albumin (HSA) is both the binding moiety and the water soluble moiety.
In another embodiment of the present invention, the complex for preventing, preventing or treating microbial infection according to the present invention may be an ester of a hydroxyl group of a water-soluble polysaccharide (dextran, hyaluronic acid, a water-soluble cellulose derivative, cyclodextrin, etc.) and a carboxyl group of a fatty acid.
Specifically, the polysaccharide is dextran or hyaluronic acid, and the carbon chain donor is docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), octadecatrienoic acid (linolenic acid), octadecadienoic acid (linoleic acid), octadecamonooleic acid (oleic acid), octanoic acid, or butenedioic acid (fumaric acid). In a more specific embodiment, the composite of the present invention includes a composite of the structure.
(1) Complex obtained by reaction of dextran with fatty acid:
Figure BDA0003628692260000892
Figure BDA0003628692260000901
the resulting complex has one or more of the following compounds of the formula:
Figure BDA0003628692260000902
Figure BDA0003628692260000911
the carbon chain of fatty acid in the above product is used as the active part, and dextran is used as both binding part and water soluble part
(2) Complex obtained by reacting hyaluronic acid with fatty acid:
Figure BDA0003628692260000912
n is an integer from 1 to 200.
Figure BDA0003628692260000921
The transparent acid esterification product is one or two compounds shown in the following structural formula:
Figure BDA0003628692260000922
Figure BDA0003628692260000931
n is an integer from 1 to 200.
The fatty acid carbon chain in the above product is the active part, and the hyaluronic acid is both the binding part and the water-soluble part.
(3) The compound obtained by the reaction of the end of the polysaccharide with a hemiacetal structure and the reductive amination reaction of cystamine and the amidation reaction of the other amino group of cystamine with fatty acid is used as the compound for preventing, preventing or treating microbial infection, and the compound obtained by the reaction of dextran, cystamine and fatty acid and the compound obtained by the reaction of hyaluronic acid, cystamine and fatty acid are examples.
Wherein, the reaction process of the glucan (DEX), the cystamine and the fatty acid is simply shown as follows:
Figure BDA0003628692260000932
Figure BDA0003628692260000941
the structure of the complex of Dextran (DEX) and cystamine with fatty acids as the invention is shown in the following formula:
Figure BDA0003628692260000942
Figure BDA0003628692260000951
The fatty acids and cystamine in the above products are the active moieties and dextran is both the binding moiety and the water soluble moiety.
The reaction of Hyaluronic Acid (HA) with cystamine and fatty acids is briefly as follows:
Figure BDA0003628692260000952
Figure BDA0003628692260000961
(when too much fatty acid is bonded, the water solubility of the product may be affected, and thus the reaction may be performed using hemiacetal hydroxyl group existing at the chain end of hyaluronic acid, so that 1 molecule of hyaluronic acid may be controlled to bond 1 molecule of fatty acid).
Wherein the compound structural formula of the compound is shown as follows, wherein the product obtained by reacting Hyaluronic Acid (HA) with cystamine and fatty acid is used as the compound of the invention:
Figure BDA0003628692260000962
Figure BDA0003628692260000971
the fatty acid and cystamine in the above products are the active moieties, and hyaluronic acid is both the binding moiety and the water-soluble moiety.
In another embodiment of the present invention, the complex for preventing, preventing or treating microbial infection of the present invention may be a compound formed by reacting a fatty acid with an oligosaccharide, for example, a compound formed by reacting a fatty acid with fondaparinux sodium
Figure BDA0003628692260000981
Figure BDA0003628692260000982
In the above product, the carbon chain of the fatty acid is the active moiety, and fondaparinux is both the binding moiety and the water-soluble moiety.
In another embodiment of the present invention, the complex for preventing, preventing or treating microbial infection according to the present invention may be a compound formed by reacting fatty acid with a water-soluble small molecule, wherein the water-soluble small molecule comprises monosaccharide or polysaccharide, amino acid, nucleotide or deoxynucleotide, vitamin; for example, the compound may have a structure shown below, wherein R below denotes a carbon chain having an integer of 1 to 99 carbon atoms:
(1) Compounds formed from fatty acids and glucose
Figure BDA0003628692260000991
The carbon chain of the fatty acid in the above product is the active moiety and glucose is both the binding moiety and the water soluble moiety.
It can also be further connected with a binding part, and the connected binding part can be selected from dibasic fatty acid or polybasic fatty acid, amino acid, targeted protein, targeted polypeptide, and targeted polysaccharide, wherein the glutamic acid is taken as an example, the reaction is as follows
Figure BDA0003628692260000992
At this time, the carbon chain of fatty acid in the product is the active part, glucose is the water-soluble part (the binding effect of glucose is weakened and the effect of increasing water solubility is retained), and glutamic acid is both the binding part and the water-soluble part.
(2) Compounds of fatty acids with sucrose
Figure BDA0003628692260000993
The carbon chain of the fatty acid in the above product is the active moiety and sucrose is both the binding moiety and the water soluble moiety.
It can also be linked with a binding part, and the linked binding part can be selected from dibasic fatty acid or polybasic fatty acid, amino acid, targeted protein, targeted polypeptide, and targeted polysaccharide, wherein the linking of the binding part with the butenedioic acid is taken as an example and is reacted as follows
Figure BDA0003628692260001001
At this time, the carbon chain of fatty acid in the product is an active part, sucrose is a water-soluble part (the combination effect of sucrose is weakened and the effect of increasing water solubility is reserved), and the butenedioic acid is both the combination part and the active part.
(3) Compounds of fatty acids with aminoethanesulfonic acid
Figure BDA0003628692260001002
The carbon chain of the fatty acid in the product is an acting part, and the taurine is a combining part and a water-soluble part.
(4) Compounds formed from fatty acids and lysine
Figure BDA0003628692260001003
The carbon chain of the fatty acid in the above product is the active moiety and lysine is both the binding moiety and the water soluble moiety.
(5) Compounds formed from fatty acids and serine
Figure BDA0003628692260001004
The carbon chain of the fatty acid in the above product is the active moiety and serine is both the binding moiety and the water soluble moiety.
(6) Compounds formed from fatty acids and threonine
Figure BDA0003628692260001011
The carbon chain of the fatty acid in the above product is the active moiety and threonine is both the binding moiety and the water soluble moiety.
(7) Compounds formed from fatty acids with adenosine monophosphate and aspartic acid
Figure BDA0003628692260001012
Wherein the carbon chain of the fatty acid is the active moiety, the adenylate is the water-soluble moiety, and the aspartate is both the binding moiety and the water-soluble moiety.
(8) Compounds formed by fatty acid, ascorbic acid and glutaric acid
Figure BDA0003628692260001013
Figure BDA0003628692260001021
Wherein the carbon chain of the fatty acid is the active moiety, the ascorbic acid is the water soluble moiety, and the glutaric acid is both the binding moiety and the active moiety.
In the above examples, R is an integer of 1 to 100 carbon atoms, and preferably R is as follows.
Figure BDA0003628692260001022
In another embodiment of the present invention, the complex for preventing, preventing or treating a microbial infection of the present invention may be a compound obtained by linking a water-soluble moiety and a binding moiety with a fat-soluble vitamin as an active moiety. The fat-soluble vitamins include vitamin a, vitamin E, vitamin K, and vitamin D, and retinoic acid in the vitamin a group and α -tocopherol in the vitamin E group will be exemplified below.
(1) The composition of the compound is retinoic acid + PEG + succinic acid + alanine, wherein retinoic acid, succinic acid and alanine are used as active parts, PEG is a water-soluble part, and alanine is a binding part.
Figure BDA0003628692260001031
n is an integer of 1 to 200.
(2) The composition of the compound is alpha-tocopherol succinate + PEG + butenedioic acid, wherein the alpha-tocopherol succinate and the butenedioic acid are active parts, the PEG is a water-soluble part, and the butenedioic acid is a binding part.
Figure BDA0003628692260001032
n is an integer of 1 to 200.
In another embodiment of the present invention, the complex for preventing, preventing or treating a microbial infection of the present invention may be a compound having a steroid lipid as an active moiety, and a water-soluble moiety and a binding moiety linked thereto. The steroid lipid comprises cholesterol, lanosterol, sitosterol, stigmasterol, ergosterol, bile acid, bile alcohol, and one or more of the above steroid lipid derivatives. Next, cholesterol and glycocholic acid among bile acids will be described as an example.
(1) The compound consists of cholesterol succinate (carbon chain with cyclic structure) + PEG + glutamic acid, wherein the cholesterol succinate is an active part, PEG and glutamic acid are water-soluble parts, and glutamic acid is a binding part
Figure BDA0003628692260001041
n is an integer of 1 to 200.
(2) The composition of the compound is glycocholic acid, succinic acid, PEG and octadecatrienoic acid, wherein a cholestane skeleton, succinic acid and octadecatrienoic acid in the glycocholic acid are used as action parts, an acyl glycine part and PEG in the glycocholic acid are used as water-soluble parts, and glycine is used as a binding part.
Figure BDA0003628692260001051
n is an integer of 1 to 200.
In the complex of the present invention, PEG unit is contained to various degrees or PEG (polyethylene glycol) is added in the formation of the complex, and specifically, in order to form the complex of the present invention, the complex contains PEG unit, i.e., -CH, as required 2 -CH 2 The number of repetitions of-O- (ethoxy) or the degree of polymerization n is an integer of 1 to 200. Further preferably, first, when the PEG unit is used as a backbone for the complex to link other acting, binding, water-soluble moieties, and also to render the complex water-soluble, n is an integer from 4 to 200; second, in the case where the PEG unit functions as a water-soluble moiety to solubilize, n is an integer of 4 to 20; thirdly, when the PEG unit is used as a connecting arm, the distance between the macromolecule and the carbon chain is extended to expand the action space, and n is an integer of 1-10.
In another embodiment of the present invention, the compound for preventing, preventing or treating microbial infection according to the present invention may be fatty alcohol-polyoxyethylene ether, fatty acid-polyoxyethylene ester, alkyl glycoside, sucrose fatty acid ester, sorbitan polyoxyethylene fatty acid ester, mannosylerythritol ester, N-fatty acyl-N-methylglucamine. The compound has fatty alcohol or fatty acid as a carbon chain donor, has good water solubility, has weak binding effect with virus surface structural domain, lipid membrane or cell wall components, can kill microorganisms only by high concentration, can also damage human cells at the concentration, and is not suitable for being used inside human bodies. When the compound is connected with the binding part to form a new compound, the compound has the effects of killing microorganisms at a lower concentration in a human body and resisting microbial infection, and the new compound with the action part, the water-soluble part and the binding part has no influence on tissue cells and organs of the human body at the treatment concentration. The binding moiety may be selected from the group consisting of a di-or poly-fatty acid, an amino acid, a targeting protein, a targeting polypeptide, a targeting polysaccharide. For example, the compound may have the structure shown below.
(1) The fatty alcohol-polyoxyethylene ether is connected with the butenedioic acid to obtain the compound, the carbon chain part of the fatty alcohol in the fatty alcohol-polyoxyethylene ether and the connected butenedioic acid carbon chain part are acting parts, a Polyoxyethylene (PEG) unit is a water-soluble part, and the butenedioic acid is a binding part.
Figure BDA0003628692260001061
Wherein n is an integer of 1 to 200.
(2) The compound is obtained by connecting fatty acid polyoxyethylene ester with aspartic acid, wherein the carbon chain part of fatty acid in the fatty acid polyoxyethylene ester is an acting group, a Polyoxyethylene (PEG) unit and the aspartic acid are water-soluble groups, and the aspartic acid is a binding group.
Figure BDA0003628692260001062
Wherein n is an integer of 1 to 200.
(3) The compound is obtained by connecting polyoxyethylene sorbitan fatty acid ester with glutamic acid, wherein the carbon chain part of fatty acid in the polyoxyethylene sorbitan fatty acid ester is an active part, sorbitan and Polyoxyethylene (PEG) units and the connected glutamic acid are water-soluble parts, and the glutamic acid is a binding part.
Figure BDA0003628692260001071
Wherein n is an integer of 1 to 200.
In yet another embodiment, the present invention also provides a technical solution for a method of preparing the complex for preventing, preventing or treating a microbial infection of the present invention.
The compound of the invention is prepared by reacting a compound such as fatty acid which provides a carbon chain with a protein, a polypeptide, an oligopeptide, an oligosaccharide, a monosaccharide, a disaccharide, an oligonucleotide, a vitamin, a water-soluble polymer, a water-soluble polyamino acid and/or a polysaccharide molecule, or adding any one or more than two of a linker such as PEG, N-hydroxy crotonoimide, an amino acid, succinic acid, butadiene acid, glutaconic acid, hexanedicarboxylic acid, carbamate, short peptide and derivatives thereof according to the needs to react to obtain a reaction mixture; in a preferred embodiment, the reaction mixture is further purified to isolate a purified reaction product (the "purified reaction product" is also referred to herein as a "reaction-derived compound", and the term "reaction-derived compound" refers to a substance remaining after the reaction mixture is separated by a purification means to remove unreacted substances, and such remaining substance is referred to as a "reaction-derived compound").
For the specific purification means, the classification is described below.
1. In the case of fatty acid-coupled proteins or polysaccharides
Such as fatty acid-coupled proteins, polypeptides, and/or polysaccharides, to give a reaction mixture, which is purified by either dialysis or ultrafiltration. Wherein the unreacted fatty acid and the catalyst added in the process are small molecules, and can be removed by dialysis (laboratory miniprep) or ultrafiltration (mass production after conversion). The specific purification process is to perform dialysis after the reaction is finished, and to select dialysis bags (the cut-off molecular weight can be 500-1000, 1000-1500, 1500-3000) for dialysis, and to change water every 4h for dialysis for 24 h. The molecular weight of the small molecular compound is less than 500, and the catalyst and unreacted fatty acid in the reaction liquid can be removed.
2. Case of fatty acid-coupled small molecules
The fatty acid coupled small molecule can be purified by molecular sieve chromatography, the molecular weight of the coupled product is different from that of the reaction substrate, the molecular weight of the product is also larger than that of the catalyst molecule, and the product can be separated by molecular exclusion method, and the filler can be selected from filler made of dextran, agarose, polypropylene, etc., such as commercial Shephadex, Sephacryl, Shepharose, etc,
Figure BDA0003628692260001081
EMD SEC, Bio-Gel P, Bio-Gel A and the like, obtaining eluent collected step by step after chromatography purification, detecting the eluent by any one of a phenol-sulfuric acid method, an indantrione method, an ultraviolet spectrophotometry method, a barium chloride-iodine solution method, a formaldehyde sulfate color development method and the like, and merging the first elution peak to obtain the product obtained by reaction. Taking sheephadex G10 as an example, the size of the column and the elution flow rate can be optimally adjusted according to different production scales. The specific description is as follows:
2.1. (fatty acid coupled mono-or disaccharides)
And (3) after the reaction is finished, carrying out chromatographic purification, adding the reaction solution into a Shephadex G10 chromatographic column, eluting with normal saline, washing out the reaction product, collecting the eluent step by step, detecting by using a phenol-sulfuric acid method, and combining the 1 st elution peak to obtain the reaction product.
And (3) detecting by a phenol-sulfuric acid method:
taking 100ul of a sample in a collecting tube, placing the sample in a test tube with a plug, taking deionized water as a blank, adding 100ul of 5% phenol solution, shaking and mixing uniformly, quickly adding 500ul of concentrated sulfuric acid, shaking, quickly moving to a water bath with the temperature of 80 ℃, preserving heat for 10min, cooling for 3min in the ice bath, and measuring the absorbance at 487 nm.
2.2. (fatty acid coupled amino acids)
And (3) after the reaction is finished, carrying out chromatographic purification, adding the reaction solution into a Shephadex G10 chromatographic column, eluting with normal saline, washing out the reaction product, collecting the eluent step by step, detecting by an indantrione method, and combining the 1 st elution peak to obtain the reaction product.
Detection by an indantrione method:
taking 200ul of a sample in the collecting tube, placing the sample in a test tube with a plug, taking deionized water as a blank, adding 300ul of 2% ninhydrin solution and 200ul of sodium acetate buffer (pH6), shaking and uniformly mixing, placing the mixture in a water bath at 90 ℃ for heating for 15min, cooling for 3min in the ice bath, adding 300ul of deionized water, uniformly mixing, and measuring the absorbance at 568 nm.
2.3. (fatty acid coupled ascorbic acid)
And (3) after the reaction is finished, carrying out chromatographic purification, adding the reaction solution into a Shephadex G10 chromatographic column, eluting with physiological saline, washing out the reaction product, collecting the eluent step by step, detecting the absorption value of the eluent by using an ultraviolet spectrophotometry (243nm), and combining the 1 st elution peak to obtain the reaction product.
2.4. (fatty acid coupled nucleotides)
And (3) after the reaction is finished, carrying out chromatographic purification, adding the reaction solution into a Shephadex G10 chromatographic column, eluting with physiological saline, washing out the reaction product, collecting the eluent step by step, detecting the absorption value of the eluent by using an ultraviolet spectrophotometry (260nm), and combining the 1 st elution peak to obtain the reaction product.
2.5. (fatty acid-PEG coupling)
And (3) after the reaction is finished, carrying out chromatographic purification, adding the reaction solution into a Shephadex G10 chromatographic column, eluting with physiological saline, washing out the reaction product, collecting the eluent step by step, detecting by using a barium chloride-iodine solution method, and combining the 1 st elution peak to obtain the reaction product.
A barium chloride-iodine solution detection method (refer to the method improvement of 3202 in the fourth general rule of the Chinese pharmacopoeia (2020 version)):
taking 100ul of a sample in the collecting tube, placing the sample in a test tube with a plug, taking deionized water as a blank, adding 300ul of deionized water, 100ul of 5% barium chloride solution and 50ul of 0.1mol/L iodine solution, shaking and uniformly mixing, placing at room temperature for incubation for 15min, and measuring the absorbance at 535 nm.
2.6. (steroid complexes)
And (3) after the reaction is finished, carrying out chromatographic purification, adding the reaction solution into a Shephadex G10 chromatographic column (phi 26mm multiplied by 50cm), eluting with normal saline at the flow rate of 50ml/h, collecting the eluent step by step, detecting by a formaldehyde sulfate color development method, and combining the 1 st elution peak to obtain the reaction product.
A formaldehyde sulfate color development method:
taking 100ul of a sample in the collecting tube, placing the sample in a test tube with a plug, taking deionized water as a blank, adding 500ul of concentrated sulfuric acid and 20ul of formaldehyde, shaking and mixing uniformly, placing the test tube at room temperature for incubation for 5min, adding 500ul of deionized water, shaking uniformly, and measuring the absorbance at 365 nm.
Specifically, the invention provides a preparation method of a compound (consisting of an action part and a macromolecular water-soluble part/binding part), which takes fatty acid as a carbon chain donor, and grafts the fatty acid to serum albumin under the action of a catalyst to form the compound, wherein the molar ratio of the fatty acid to the serum albumin (wherein, the total of 585 amino acids of the human serum albumin, the total of 607 amino acids of the bovine serum albumin, and the molecular weight is 66 kDa) is 20:1-1:1, the molar ratio of the catalyst to the fatty acid is 0.5:1-10:1, and the catalyst can be one or more of EDC, DCC, NHS, DMAP, HoBt and derivatives and analogues thereof. Wherein the carbon chain of the fatty acid is the active moiety and the serum albumin is the water soluble moiety and the binding moiety.
The reaction formula is as follows:
Figure BDA0003628692260001101
r respectively represents the carbon chains of fatty acids selected from fumaric acid, octanoic acid, undecanoic acid, hexadecenoic acid, oleic acid, linoleic acid, linolenic acid, eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA) and triacontenoic acid, respectively react with serum albumin,
preferably, the molar ratio of fatty acid to albumin (total 585 amino acids for human serum albumin and 607 amino acids for bovine serum albumin, all based on 66 kDa) is from 20:1 to 1:1, the molar ratio of catalyst to fatty acid is from 0.5:1 to 10:1, preferably from 1:1 to 10: 1; more preferably, the fatty acid to albumin molar ratio is 10:1, the molar ratio of the catalyst to the fatty acid is 1: 1; the catalyst can be one or more of EDC, DCC, NHS, DMAP, HoBt and derivatives and analogues thereof, and the catalyst is preferably selected from 1-ethyl- (3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysulfosuccinimide (sulfo-NHS), wherein the molar ratio of the 1-ethyl- (3-dimethylaminopropyl) carbodiimide (EDC) to the N-hydroxysulfosuccinimide (sulfo-NHS) is 0.1:1-10:1, and is preferably 1: 1. Further, in the compound obtained by reacting the above-mentioned fatty acid with serum albumin, it was found that the total binding efficiency of fatty acid to amino acid was 0.10 to 15%. Wherein one molecule of protein is combined with 7-24 molecules of linolenic acid, preferably 8 molecules of linolenic acid. Wherein one molecule of serum albumin is combined with 6-24 molecules of docosahexaenoic acid, preferably 10 molecules of docosahexaenoic acid in the compound obtained by reacting docosahexaenoic acid with serum albumin. Wherein one molecule of serum protein binds 1-24 molecules of oleic acid, preferably 1 molecule of oleic acid. Wherein one molecule of protein binds to the amino group of amino acids of about 6-24 eicosapentaenoic acid, preferably 17 EPA (eicosapentaenoic acid) molecules in a form deprived of one molecule of water to 1 protein molecule. Wherein one molecule of serum albumin is bonded with about 11-16 molecules of linoleic acid, preferably 13 molecular bonds of linoleic acid are bonded with 1 molecule of amino acid of protein in a form of removing one molecule of water, wherein all are bonded with free amino groups of lysine in a form of amido bonds, and the total degree of amino acid substitution is more than 1.9%. Wherein one serum albumin is bonded to about 6-15 DHA molecules, preferably 9 DHA molecules, in a dehydrated form to the amino group of the amino acid of 1 protein molecule.
In addition, the invention also provides a preparation method of the compound consisting of (action part + macromolecule water-soluble part/binding part), which takes unsaturated fatty acid as a carbon chain donor, and reacts the fatty acid with hyaluronic acid under the action of a catalyst to form the compound (wherein, the preferable preparation process comprises the steps of firstly adding the catalyst to react the fatty acid with the hyaluronic acid to obtain an intermediate product, then adding sodium hydroxide to adjust the pH value to be neutral, and continuously reacting to obtain the compound), wherein, the molar ratio of the carboxylic acid group of the fatty acid to the hydroxyl group of the hyaluronic acid is 4 n: 1-1: 1(n is the repetition number of a single molecule of hyaluronic acid), the molar ratio of the catalyst to the fatty acid is 0.5:1-10:1, preferably 1:1-10:1, the catalyst can be one or more of EDC, DCC, NHS, DMAP, HoBt and derivatives and analogues thereof, and the catalyst is preferably carbodiimide and succinimide, and the molar ratio of the carbodiimide to the succinimide is 0.1:1-10: 1. Wherein the carbon chain of the fatty acid is the active moiety and the hyaluronic acid is the water soluble moiety and the binding moiety.
The present invention also provides a method for preparing a complex having a (active moiety + target binding moiety/water-soluble moiety), comprising reacting a fatty acid, which is one or more of Oleic Acid (OA), Linoleic Acid (LA), linolenic acid (ALA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA), with a polypeptide, such as SBP1(ACE2 derived peptide; binding SARS-CoV-2 spike protein receptor domain, having a sequence of IEEQAKTFLDKFNHEAEDLFYQS (modified: Ser-23 ═ C-terminal amide), using an unsaturated fatty acid as a carbon chain donor, wherein the molar ratio of the carboxylic acid group of the fatty acid to the amino group of the polypeptide is 8:1 to 1:4, preferably 2: the molar ratio of the catalyst to the fatty acid is 0.5:1-10:1, the catalyst can be one or more of EDC, DCC, NHS, DMAP, HoBt and derivatives and analogues thereof, the catalyst is preferably carbodiimide and succinimide, and the molar ratio of the carbodiimide to the succinimide is 0.1:1-10:1, and is preferably 1:1-1: 10.
The invention also provides a preparation method of a compound having a structure of (action part + targeting binding part/water-soluble part), which takes unsaturated fatty acid as a carbon chain donor, and reacts the fatty acid with CD14 under the action of a catalyst to form the compound, wherein the fatty acid is one or more of oleic acid, linoleic acid, linolenic acid, eicosapentaenoic acid (EPA) and docosahexaenoic acid, the molar ratio of the carboxylic acid group of the fatty acid to the CD14 is 17:1-1:1, preferably 17:1, and the molar ratio of the catalyst to the fatty acid is 0.5; 1-10:1, the catalyst can be one or more of EDC, DCC, NHS, DMAP, HoBt and derivatives and analogues thereof, the catalyst is preferably carbodiimide and succinimide, and the molar ratio of the carbodiimide to the succinimide is 0.1:1-10:1, and is preferably 1:1-1: 10.
The invention also provides a method for preparing a complex composed of (a medium-long chain saturated carbon chain acting part + a macromolecular water-soluble part/binding part), wherein the medium-long chain saturated fatty acid is used as a carbon chain donor, and the fatty acid is reacted with the hyaluronic acid under the action of a catalyst to form the complex, wherein the fatty acid is one or more than two saturated fatty acids with the carbon number of 5-20, preferably one or more than two saturated fatty acids in valeric acid, caproic acid, enanthic acid, caprylic acid, pelargonic acid, capric acid, undecanoic acid, dodecanoic acid, tridecanoic acid, tetradecanoic acid, pentadecanoic acid, hexadecanoic acid, heptadecanoic acid, octadecanoic acid, nonadecanoic acid and eicosanoic acid, the molar ratio of the carboxylic acid group of the fatty acid to the hyaluronic acid is 4n:1-1:1(n is the number of repetition of a single molecular unit of the hyaluronic acid, and n is an integer of 1-2000), the molar ratio of the catalyst to the fatty acid is 0.5:1-10:1, preferably 0.5:1-2: 1; the catalyst is preferably carbodiimide and succinimide in a molar ratio of 0.1:1 to 10: 1.
Further, the fatty acid-serum protein complex test, the fatty acid-hyaluronic acid complex, the fatty acid-SBP 1 complex, the fatty acid-CD 14 complex, the fatty acid-glucan complex, and the like of the present invention show that they have a bactericidal/bacteriostatic effect on any one of the following bacteria: escherichia coli, staphylococcus aureus, methicillin-resistant staphylococcus aureus, streptococcus pneumoniae, klebsiella pneumoniae and pseudomonas aeruginosa, wherein the sterilization rate is more than 99%; can have the effects of killing and inhibiting bacteria on any fungus selected from the following fungi groups: candida albicans, Aspergillus niger, Actinomyces viscosus, Chaetomium globosum, Aspergillus verrucosus and Microsporum canis, and the sterilization rate is more than 99 percent; capable of having a virucidal effect against any one of the fungi selected from the group of viruses: H7N9 influenza virus, H5N1 influenza virus, HIV virus, new corona virus, HPV virus and rabies virus, and the virus killing rate reaches over 99 percent.
In addition, the invention also provides a preparation method of the compound consisting of the (action part + small molecule water-soluble part/binding part), which takes fatty acid as a carbon chain donor and leads the fatty acid and monosaccharide such as glucose and sucrose to react under the action of a catalyst; or reacting fatty acid with nucleotide (such as adenosine monophosphate), amino acid, water-soluble vitamin, PEG400-COOH with low polymerization degree, and substance with carbon chain having cyclic structure such as taurocholic acid (sodium) (such as 4-octenedioic acid and taurocholic acid complex) to form complex (preferably, adding catalyst to react fatty acid with monosaccharide such as glucose to obtain complex, optionally adding sodium hydroxide to the obtained intermediate solution to adjust pH to neutral to obtain final complex, and preferably, further purifying the obtained reaction product mixed solution), wherein fatty acid is preferably octanoic acid, and carboxylic acid group of fatty acid and water-soluble small molecule such as glucose, sucrose, nucleotide (such as adenosine monophosphate), amino acid, water-soluble vitamin complex, etc, The low-polymerization-degree PEG400-COOH and the like have a molar ratio of 1:1-1:4, the molar ratio of the catalyst to the fatty acid is 0.5: 1-10:1, preferably 1:1-10:1, and the catalyst is carbodiimide and succinimide or carbodiimide and dimethylaminopyridine, and the molar ratio of the two is 0.1: 1-10:1, preferably 1: 1. The fatty acid-micromolecule water-soluble molecule compound obtained by the invention has a bacteriostatic rate of more than 99%. The bacteria are any one of escherichia coli, methicillin-resistant staphylococcus aureus, streptococcus pneumoniae, klebsiella pneumoniae and pseudomonas aeruginosa; can have the effects of killing and inhibiting bacteria on any fungus selected from the following fungi groups: candida albicans, Aspergillus niger, Actinomyces viscosus, Chaetomium globosum, Aspergillus verrucosus and Microsporum canis, and the sterilization rate is more than 99 percent; capable of having a virucidal effect against any one of the fungi selected from the group of viruses: H7N9 influenza virus, H5N1 influenza virus, HIV virus, new corona virus, HPV virus and rabies virus, and the virus killing rate reaches over 99 percent.
Wherein in a further preferred scheme, the invention provides a compound obtained by reacting fatty acid with amino acid, wherein the fatty acid is selected from one or more than two of caprylic acid, linoleic acid, linolenic acid, eicosapentaenoic acid and docosahexaenoic acid; the amino acid is one or more than two of serine, threonine and lysine.
The present invention also provides a method for preparing a complex having a (water-soluble moiety + functional moiety non-covalently coupled) structure, which comprises using an unsaturated fatty acid, or a fatty acid ester, or lecithin as a carbon chain donor, and mixing the same with a protein, a polysaccharide, or an amino acid (for example, a liposome obtained by complexing a carboxylated lecithin, β -sitosterol, glycocholic acid sulfuric acid, pentacosanoic acid, or ethanol), a liposome obtained by complexing a fatty acid ethyl ester liposome (a liposome obtained by complexing a surfactant, aminated lecithin, cholesterol, or fatty acid ethyl ester), or a complex having a (water-soluble moiety + functional moiety non-covalently coupled) structure, wherein the fatty acid is a medium-chain caproic acid (ethyl caproate), heptanoic acid, octanoic acid, nonanoic acid, decanoic acid, stearic acid, oleic acid, linoleic acid, linolenic acid, eicosapentanoic acid, Docosahexaenoic acid, etc.). Experiments of the invention show that the liposome can be used for killing and inhibiting any one of the following bacteria: escherichia coli, staphylococcus aureus, methicillin-resistant staphylococcus aureus, streptococcus pneumoniae, klebsiella pneumoniae and pseudomonas aeruginosa, and the bacteriostasis rate is more than 99%. (ii) a Can also be used for killing and inhibiting any fungus selected from the following fungi groups: candida albicans, Aspergillus niger, Actinomyces viscosus, Chaetomium globosum, Aspergillus wart and Microsporum canis, and the bacteriostasis rate is more than 99 percent. The liposomes can also be used to have a virucidal effect against any one of the viruses selected from the following group of viruses: H7N9 influenza virus, H5N1 influenza virus, HIV virus, neocoronavirus, HPV virus, and rabies virus; wherein, the virus killing rate of the ethyl oleate liposome and the linoleic acid liposome can reach more than 99 percent.
The present invention also provides a process for the preparation of a complex (mixture) having an (amino/carboxyl + water-soluble + functional moiety non-covalently coupled) moiety, wherein preferably the mixture may be a lipid emulsion obtained by mixing a surfactant with a fatty acid ester having a carbon chain, such as a nanoliposome emulsion (particle size of 500nm-800nm) obtained by liposome emulsions of ethyl oleate, aminated lecithin, β -sitosterol and vitamin E palmitate; can be a preparation (particle size of 500-800 nm) obtained by compounding unsaturated fatty acid and carboxylated lecithin liposome, such as nanoliposome emulsion (particle size of 500-800 nm) obtained by mixing surfactant, linoleic acid, beta-sitosterol and carboxylated lecithin.
The experiment of the invention proves that the nano liposome emulsion can be used for having the effects of sterilization and bacteriostasis on any one of the following bacteria: escherichia coli, staphylococcus aureus, methicillin-resistant staphylococcus aureus, streptococcus pneumoniae, klebsiella pneumoniae and pseudomonas aeruginosa, and the bacteriostasis rate is more than 99%. Can also be used for killing and inhibiting any fungus selected from the following fungi groups: candida albicans, Aspergillus niger, Actinomyces viscosus, Chaetomium globosum, Aspergillus verrucosus and Microsporum canis, and the bacteriostasis rate is more than 99%. The above nanoliposome emulsion can also be used to have a virucidal effect against any one virus selected from the following group of viruses: H7N9 influenza virus, H5N1 influenza virus, HIV virus, neocoronavirus, HPV virus, and rabies virus; wherein, the virus killing rate of the ethyl oleate liposome and the linoleic acid liposome can reach more than 99 percent.
Furthermore, the compound can be prepared into injections, nasal sprays, dry powder inhalants, oral preparations, external preparations for skin, disinfectants and the like.
Oral formulations can be liquid (e.g., syrups, solutions, or suspensions) or solid (e.g., granules, tablets, or capsules), among others. The oral formulation may be coupled to a targeting ligand to cross the endothelial barrier. Some fatty acid derivative formulations may be spray dried, for example, with a disaccharide to form a fatty acid derivative powder. Solid compositions can be prepared in conventional manner using pharmaceutically acceptable excipients such as binders (e.g., pregelatinized corn starch, polyvinylpyrrolidone or hydroxypropylmethylcellulose); fillers (e.g., lactose, mannitol, microcrystalline cellulose, or dibasic calcium phosphate); lubricants (e.g., magnesium stearate, talc, or silicon dioxide); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulfate). Tablets may be coated by methods well known in the art, for example with sugar, film or enteric coating. Methods of making such dosage forms are known or apparent to those skilled in the art. The fatty acid emulsion can be taken orally, externally used or injected, the composition of auxiliary materials can be properly adjusted according to different administration modes during preparation, and other proper preparation methods are adopted according to different properties of the auxiliary materials.
Furthermore, for skin external preparations or disinfectant preparations, the compound can be directly dissolved in a solvent, and added with auxiliary materials to prepare a preparation for killing and preventing viruses, bacteria and fungi.
Further, the nasal spray and dry powder inhalation require the addition of a mucopromoting adsorbent;
the mucosa-promoting adsorbent comprises one or more of Hyaluronic Acid (HA), polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), polyethylene glycol (PEG), Carbomer (CP), sodium carboxymethylcellulose (CMC-Na), Methylcellulose (MC), hydroxyethyl cellulose (HEC), hydroxypropyl cellulose (HPC), and hydroxypropyl methylcellulose (HPMC).
Furthermore, the dry powder inhalant is prepared by adding the fatty acid compound solution into the mucopromoting adsorbent and then carrying out spray drying or freeze drying to obtain compound micro powder;
wherein, the freeze-drying method for preparing the dry powder inhalant needs to be added with a freeze-drying protective agent;
the freeze-drying protective agent comprises one or more of glycerol, mannitol, sorbitol, inositol, mercaptan, proline, tryptophan, sodium glutamate, alanine, glycine, lysine hydrochloride, sarcosine, L-tyrosine, phenylalanine, arginine, polyethylene glycol, polyvinylpyrrolidone, gelatin, glucose, alpha-D-mannopyranose, sucrose, lactose, trehalose, cellobiose, mannose, maltose, inositol, inulin, dextran, maltodextrin, maltopolysaccharose, sucrose octasulfate, heparin, 2-hydroxypropyl-beta cyclodextrin, Tween 80, Brij, Pluronic and sodium dodecyl sulfonate.
Further, the viruses include viruses with envelope and non-envelope viruses, such as coronavirus, human immunodeficiency virus (also known as AIDS virus), hepatitis B virus, hepatitis C virus, rabies virus, herpes virus, Ebola virus, hantaviruses, dengue virus, encephalitis B virus, Secat virus, influenza virus, hepatitis A virus, human papilloma virus, adenovirus, poliovirus, coxsackie virus;
the coronaviruses include HCoV-229E, HCoV-OC43, SARS-CoV, HCoV-NL63, HCoV-HKU1, MERS-CoV, and SARS-CoV-2.
Further, the bacteria include gram-positive bacteria and gram-negative bacteria, wherein
Gram-positive bacteria include Staphylococcus, Streptococcus, Bacillus, Clostridium, Listeria, Corynebacterium, including Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus agalactiae, enterococcus, Bacillus anthracis, Bacillus cereus, Clostridium botulinum, Clostridium perfringens, Clostridium difficile, Clostridium tetani, Listeria monocytogenes, Bacillus diphtheriae, and Bacillus tuberculosis.
The gram-negative bacteria include Escherichia coli, Pseudomonas aeruginosa, Bacillus proteus, Bacillus dysenteriae, Bacillus pneumoniae, Brucella, Haemophilus influenzae, Haemophilus parainfluenzae, Acinetobacter, Yersinia, Legionella pneumophila, Bordetella pertussis, Bordetella parapertussis, Neisseria, Shigella, Salmonella, Pasteurella, Vibrio cholerae, Parahemolytic bacillus, and Shigella shigella.
The bacteria include clinically common multidrug-resistant bacteria (MDRO), such as methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant and Shupprophyton (cefoperazone sodium and sulbactam sodium) enterococci (VRE), Extended Spectrum Beta Lactamase (ESBLs) producing bacteria of Enterobacteriaceae (such as Escherichia coli and Klebsiella pneumoniae), carbapenems-resistant bacteria of Enterobacteriaceae, multidrug-resistant Pseudomonas aeruginosa (MDR-PA), multidrug-resistant Acinetobacter baumannii (MDR-AB).
Further, the fungi include
Pathogenic fungi: histoplasma bacteria, coccidioidomycosis, blastomyces dermatitidis, chromomyces, mycetoma, sporothrix;
conditionally pathogenic fungi: fungi of the genera Candida, Cryptococcus, Aspergillus, Actinomycetes, Fusarium, and Nocardia, Serratia, Mucor and Aphanomyces.
Further said chlamydia include chlamydia trachomatis, chlamydia pneumoniae, chlamydia psittaci; the mycoplasma includes mycoplasma pneumoniae, ureaplasma urealyticum, mycoplasma hominis and mycoplasma genitalium.
Further, the mechanism of the complex acting on the microorganism is as follows:
(1) the binding part, the middle short chain/long chain action part and the macromolecular water-soluble part are coupled to form a compound I, the compound I can be retained on the surface of a respiratory tract mucous membrane or in blood circulation, virus, bacteria or fungi are inactivated for the first time, the virus, bacteria or fungi are prevented from diffusing in vivo, and the macromolecular compound I cannot enter normal tissues and only can enter inflammatory sites infected by the virus, bacteria or fungi to play a role;
(2) the binding part, the middle short chain/long chain action part, the water-soluble targeting polypeptide group and more than 2 small molecule water-soluble parts are coupled to form a compound II, and the compound II can penetrate through the blood vessel wall to enter into a tissue gap and interstitial fluid to play a role of a targeting microorganism;
(3) the binding part + the middle short chain/long chain action part + the water-soluble polypeptide targeting group/the small molecule water-soluble part + the water-soluble macromolecular polymer are coupled to form a compound III, the compound III can be retained on the surface of a respiratory tract mucous membrane or in blood circulation, and viruses, bacteria or fungi are inactivated for the first time so as to prevent the viruses, the bacteria or the fungi from diffusing in vivo; the macromolecular compound III can not enter normal tissues and can only enter inflammatory parts after virus, bacteria or fungus infection to play a role.
In some preferred embodiments of the invention, the complex may kill microorganisms including viruses, bacteria, fungi, chlamydia and mycoplasma; the virus includes enveloped virus such as coronavirus, influenza virus, AIDS virus, hepatitis B virus, hepatitis C virus, herpes virus, Secat virus, dengue virus, encephalitis B virus, Ebola virus, Hantaan virus, etc., and non-enveloped virus such as hepatitis A virus, human papilloma virus, poliovirus, coxsackievirus, etc.
The coronary disease preferably comprises HCoV-229E, HCoV-OC43, SARS-CoV, HCoV-NL63, HCoV-HKU1, MERS-CoV, and SARS-CoV-2.
In some preferred embodiments of the present invention, the pharmaceutical dosage form comprises a dry powder inhaler, a nasal spray, an injection, an oral dosage form, a skin external dosage form.
In some preferred embodiments of the present invention, the pharmaceutical dosage forms are dry powder inhalants, nasal sprays and injections, and skin external preparations against coronavirus (preferably SARS-CoV-2 virus) rabies virus, and influenza virus;
in some preferred embodiments of the present invention, the pharmaceutical dosage form for the HIV virus is injection, oral;
In some preferred embodiments of the present invention, the pharmaceutical dosage form for HPV virus is injection, oral preparation.
In some preferred embodiments of the present invention, the pharmaceutical dosage form is a nasal spray for upper respiratory tract infections.
In some preferred embodiments of the present invention, the pharmaceutical dosage form is a nasal spray for sinusitis.
The fatty acid and/or derivative thereof binds to the surface of the protein, polypeptide or polysaccharide in terms of the number of binding sites.
In some preferred embodiments of the present invention, the pharmaceutical excipient comprises a pharmaceutically acceptable excipient.
In some preferred embodiments of the present invention, the nasal spray adjuvant comprises: glucose, cyclodextrin, microcrystalline cellulose, sodium carboxymethylcellulose, sodium bisulfite, deoxycholic acid, thiourea, urea, hydroquinone, phenol, silica gel, graphite, protein, benzyl alcohol, phenethyl alcohol, benzalkonium chloride, tween 80 and other emulsifying agents, tocopherol, hydroxypropyl methyl methacrylate, gelatin, chitosan, alginate, Arabic gum, polylactic acid, polyglycolic acid and other high polymers, diluted hydrochloric acid, alcohol pure water and the like.
In some preferred embodiments of the invention, the dry powder inhaler excipients include an adhesion promoting film adsorbent, such as one or more of Hyaluronic Acid (HA), polyvinyl alcohol (PVA), polyvinyl pyrrolidone (PVP), polyethylene glycol (PEG), Carbomer (CP), sodium carboxymethylcellulose (CMC-Na), Methylcellulose (MC), Hydroxyethylcellulose (HEC), Hydroxypropylcellulose (HPC), Hydroxypropylmethylcellulose (HPMC);
Lyoprotectants such as one or more of glycerol, mannitol, sorbitol, inositol, thiol, proline, tryptophan, sodium glutamate, alanine, glycine, lysine hydrochloride, sarcosine, L-tyrosine, phenylalanine, arginine, polyethylene glycol, polyvinylpyrrolidone, gelatin, glucose, α -D-mannopyranose, sucrose, lactose, trehalose, cellobiose, mannose, maltose, inositol, inulin, dextran, maltodextrin, maltodextrins, sucrose octasulfate, heparin, 2-hydroxypropyl- β cyclodextrin, tween 80, brij, pluronic and sodium dodecyl sulfate.
The compound with the effects of preventing and treating virus, bacteria and fungal infection and the preparation thereof are applied to preventing or treating various virus, bacteria and fungal infectious diseases; the specific application mode comprises
Can be used before infection to prevent viral, bacterial and fungal infection;
can be used for killing virus, bacteria and fungi in vivo after infection;
the environment of the article can be sterilized to prevent the spread of viruses, bacteria and fungi.
In the present invention, it is understood that the use of the complex for the preparation of a medicament for the prevention and/or treatment of viral (enveloped and non-enveloped), bacterial and fungal infectious diseases is within the intended scope of the present invention.
The invention is further illustrated by the following specific examples, which, however, are to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. The methods used in the following examples are conventional methods unless otherwise specified; consumables and reagents used in the following examples were commercially available or synthesized by themselves unless otherwise specified.
The apparatus instrument list in the examples of the present invention is shown in the following Table 1-1, and the sources of the respective microorganisms are shown in the following tables 1-2, tables 1-3, and tables 1-4; in addition, in the present invention, the raw materials of each substance used for preparing the complex of the present invention are those which are conventionally commercially available to those skilled in the art, except that the preparation method thereof is specifically illustrated, and only the commercially available sources of some macromolecules or mesomolecules or oligomers in the examples of the present invention are listed below, which are specifically described in tables 1 to 5a and tables 1 to 5 b.
TABLE 1-1 names of respective instruments and equipments in examples
Serial number Name (R) Type number
1 Stirrer IKA RH basic1
2 Freeze dryer Telstar LYOQUEST-85
3 Fourier infrared spectrometer Nicolet iS 5
4 Transmission electron microscope Tecnai G2 Spirit BioTWIN
5 Scanning electron microscope Hitachi TM3030
6 Energy spectrometer Oxford AZtecOne
7 Inverted laboratory microscope Leica DM IL LED
8 Inverted fluorescence microscope Leica DM IL LED
9 CO 2 Culture box Thermo Heracell VIOS 160i
10 Microbial cultivation case Thermo Heratherm IMH100 SS
11 Multifunctional enzyme mark instrument TECAN Spark
12 FreezingCentrifugal machine Thermo Fresco 17
13 Vacuum freezing dryer Thermo Savant DNA120
14 Nanoliter liquid phase system Thermo Easy-nLC1200
15 High resolution mass spectrometer Thermo Q Exactive
Tables 1-2 examples for various viral sources
Figure BDA0003628692260001211
Tables 1-3 examples each bacterial source
Figure BDA0003628692260001212
Tables 1-4 examples the sources of the various fungi
Figure BDA0003628692260001221
Tables 1-5a examples the sources of each macromolecular, intermediate or oligomeric molecule
Figure BDA0003628692260001231
Sources of macromolecular, intermediate-molecular or oligomeric molecules in each of the examples of tables 1-5b
Figure BDA0003628692260001241
Example 1 preparation and characterization of human serum Albumin/bovine serum Albumin grafted fatty acid Complex (active moiety + macromolecular Water-soluble moiety/binding moiety)
Reaction scheme example formula 1-1 is as follows:
Figure BDA0003628692260001242
the molar ratio of fatty acid to albumin in this example was 10: 1, the fatty acid is selected from fumaric acid, caprylic acid, undecanoic acid, hexadecenoic acid, oleic acid, linoleic acid, linolenic acid, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), and triacontenoic acid reacts with serum albumin respectively. The molar ratio of the catalyst to the fatty acid is 1: 1, 1-ethyl- (3-dimethylaminopropyl) carbonyl diimine (EDC) and N-hydroxysulfosuccinimide (sulfo-NHS) are selected as catalysts. The reaction process is as follows:
precisely weighing 0.36mmol of fatty acid, 0.36mmol of EDC and 0.36mmol of sulfo-NHS; acid addition to catalytically activate the carboxyl group to form an activated fatty acid; activating with stirring in ice bath for 15 min. 0.0375mmol (2.5 g by mass) of bovine serum albumin is precisely weighed and dissolved in 5ml of PBS (phosphate buffer solution), NaOH solution is added to adjust the pH to be neutral, and the mixture is stirred uniformly to obtain serum albumin solution. And adding the obtained serum albumin solution into the stirred activated fatty acid, and continuously stirring and reacting overnight under ice bath to obtain a fatty acid-serum albumin compound solution with the concentration of 72 mM. Precipitating the solution by using glacial acetone, washing unreacted fatty acid in the precipitate by using ethanol, dialyzing the precipitate until the precipitate is completely dissolved, and simultaneously removing small molecular impurities (water with the molecular weight cutoff of 500-1000 is used for dialysis by using a dialysis bag with the molecular weight cutoff of 500-1000, and the molecular weight of small molecular compounds is less than 500 and can be removed every 4 h), then performing gradient low-temperature drying in a freezing vacuum dryer (pre-freezing at the temperature of-80 ℃ for 24h and vacuumizing for 12h, -vacuumizing at the temperature of-20 ℃ for 12h, and continuously vacuumizing at the temperature of 4 ℃ for more than 24h until the product is completely dried) to obtain a compound of the fatty acid and serum albumin, and using the compound obtained by vacuum drying for performance evaluation of later acting on microorganisms. Serum albumin compounds of fumaric acid, caprylic acid, undecanoic acid, hexadecenoic acid, oleic acid, linoleic acid, linolenic acid, EPA, DHA and triacontenoic acid were prepared according to the above methods, and the yields were 68.2% of fumaric acid, 68.7% of caprylic acid, 68.9% of undecanoic acid, 69.3% of hexadecenoic acid, 68.4% of oleic acid, 68.2% of linoleic acid, 69.0% of linolenic acid, 68.7% of EPA, 68.6% of DHA and 67.8% of triacontenoic acid, respectively. The yields here are all mass ratios of the end product relative to the total mass of the starting reactants.
Wherein the linolenic acid-serum albumin has Fourier infrared spectrum shown in figure 1, and the content of serum albumin grafted linolenic acid (ALA-HSA) is 1245cm compared with serum albumin -1 、1042cm -1 New absorption peaks appear at wavenumbers, and are classified as v of gamma ═ C-H (bending vibration out of unsaturated carbon-hydrogen bond plane) and amide C-N Absorption peaks, as evidenced by group recognition of amide bonds; after grafting the olefin was 1710cm -1 Absorption peaks at wave numbers are shifted to a low band due to the conjugation effect and are all derived from grafted small-molecule linolenic acid (ALA), so that the fact that ALA is successfully grafted to a serum albumin molecular chain can be preliminarily judged.
As shown in figure 2, the results of protein electrophoresis of fumaric acid-serum albumin complex, linolenic acid-serum albumin complex, eicosapentaenoic acid-serum albumin complex and docosahexaenoic acid-serum albumin complex show that the molecular weight of the product is between 6 and 7.5 ten thousand Da, which indicates that the serum albumin molecules themselves are not polymerized and the molecular weight change is not obvious because of the connection of small molecules.
The linolenic acid-serum albumin and DHA-serum albumin are analyzed by LC-MS method at the modified sites of serum albumin molecule, which are shown in FIG. 3A and FIG. 3B, and FIG. 4A and FIG. 4B, from the results of the graphs, the sequences of bovine serum albumin (607 total amino acids of bovine serum albumin, wherein 60 lysine, 17 glycine, 48 alanine, 38 valine, 65 leucine, 15 isoleucine, 30 phenylalanine, 3 tryptophan, 21 tyrosine, 40 aspartic acid, 14 asparagine, 59 glutamic acid, 20 glutamine, 5 methionine, 32 serine, 34 threonine, 35 cysteine, 28 histidine, 17 proline and 26 arginine) are aligned, and linolenic acid-serum albumin binding 8 molecules with a total fatty acid binding amino acid efficiency of 1.32% can be obtained, wherein, the 1 molecule is combined by threonine, the substitution degree of the threonine is 2.94 percent, the 1 molecule is combined by phenylalanine, the substitution degree of the phenylalanine is 3.33 percent, the 1 molecule is combined by proline, the substitution degree of the proline is 3.57 percent, the 5 molecule is combined by lysine, and the substitution degree of the fatty acid in the lysine is 8.33 percent;
1 molecule of serum albumin is combined with 10 molecules of docosahexaenoic acid, the total efficiency of fatty acid combination with amino acid is 1.65%, wherein, 1 molecule is combined with glutamic acid, the substitution degree of the glutamic acid is 1.69%, 1 molecule is combined with tyrosine, the substitution degree of the tyrosine is 4.76%, 2 molecule is combined with leucine, the substitution degree of the leucine is 3.08%, 2 molecule is cysteine, the substitution degree of the cysteine is 5.71%, 4 molecule is combined with lysine, and the substitution degree of the lysine is 6.67%.
In this example, serum albumin is both the water soluble and binding moieties, with the fatty acid carbon chain being the active moiety.
Example 2 bovine serum albumin grafted fatty acid composition preparation and characterization (active moiety + macromolecular Water soluble moiety/binding moiety)
Precisely weighing 0.36mmol of oleic acid; EDC 0.36 mmol; adding 0.36mmol of sulfo-NHS into acid to catalyze and activate carboxyl to obtain activated oleic acid; activating with stirring in ice bath for 10 min. 0.018mmol (1.2 g by mass) of bovine serum albumin is precisely weighed and dissolved in 5ml of PBS solution, NaOH solution is added to adjust the pH to be neutral, and the mixture is stirred uniformly to obtain serum albumin solution. Adding the serum albumin solution into the stirred activated oleic acid, and continuously stirring and reacting overnight under ice bath to obtain oleic acid-albumin primary solution with the oleic acid concentration of 72 mM. Precipitating the solution with glacial acetone, removing fatty acid which does not participate in the reaction by using ethanol, dialyzing to remove small molecular impurities (dialyzing by using a dialysis bag with the molecular weight cutoff of 500-plus-1000, changing water every 4h, wherein the molecular weight of small molecular compounds is less than 500 and can be removed), and then performing gradient low-temperature drying in a freeze vacuum dryer (the step of pre-freezing at the temperature of-80 ℃ for 24h and performing vacuum for 12h, vacuumizing at the temperature of-20 ℃ for 12h, and continuously vacuumizing at the temperature of 4 ℃ for more than 24h until the product is completely dried), wherein the yield is 68%; the composite obtained by this vacuum drying was used for the evaluation of the performance of the subsequent action on microorganisms.
The molecular structure of bovine serum albumin (bovine serum albumin with 607 amino acids, including 60 lysine, 17 glycine, 48 alanine, 38 valine, 65 leucine, 15 isoleucine, 30 phenylalanine, 3 tryptophan, 21 tyrosine, 40 aspartic acid, 14 asparagine, 59 glutamic acid, 20 glutamine, 5 methionine, 32 serine, 34 threonine, 35 cysteine, 28 proline, 17 histidine and 26 arginine) is analyzed by the LC-MS method, and it is found that amidation reaction without a part of water occurs at histidine position, and the mass spectrum and amino acid sequence alignment chart are respectively shown in FIG. 5 and FIG. 6, which can be concluded: the serum albumin coupled oleic acid is formed by coupling 1 oleic acid molecule with 1 serum albumin molecule, and the total substitution degree of fatty acid is 0.16%, wherein histidine is combined with 1 molecule, and the substitution degree of histidine is 5.88%.
In this example, serum albumin is both the water soluble and binding moieties, and the oleic acid carbon chain is the active moiety.
Example 3 preparation and characterization of human serum Albumin grafted fatty acid Complex (action moiety + macromolecular Water-soluble moiety/binding moiety)
Accurately weighing 0.036mmol of eicosapentaenoic acid (EPA); 0.036mmol of catalyst; adding acid to catalyze and activate carboxyl to obtain activated EPA; activating with stirring in ice bath for 20 min. 0.036mmol (about 2.4g by mass) of bovine serum albumin is precisely weighed and dissolved in 24ml of PBS solution, NaOH solution is added to adjust the pH to be neutral, and the mixture is stirred uniformly to obtain serum albumin solution. And adding the serum albumin solution into the activated EPA under stirring, and continuously stirring for reacting overnight under ice bath to obtain an EPA-serum albumin primary solution with the EPA concentration of 1.5 mM. Precipitating the solution with glacial acetone, removing fatty acid which does not participate in the reaction by using ethanol, dialyzing to remove small molecular impurities (dialyzing by using a dialysis bag with the molecular weight cutoff of 500 plus 1000, changing water every 4h, wherein the molecular weight of small molecular compounds is less than 500 and can be removed), and then performing gradient low-temperature drying in a freeze vacuum drier (the low temperature of 80 ℃ is pre-frozen for 24h and vacuum is performed for 12h, the vacuum is performed for 20h at the temperature of-20 ℃, the vacuum is continuously performed for more than 24h at the temperature of 4 ℃ until the product is completely dried), wherein the yield is 79%; the composite obtained by this vacuum drying was used for the evaluation of the performance of the subsequent action on microorganisms.
Fourier infrared spectrum of the sample is shown in FIG. 7, in which the serum albumin grafted eicosapentaenoic acid (EPA-HSA) was 1044cm in comparison with the human serum albumin -1 A strong new absorption peak appears at wave number, which is classified as gamma ═ C-H (bending vibration out of unsaturated carbon-hydrogen bond plane), and comes from grafted small molecular eicosapentaenoic acid (EPA), and the grafted olefin is 1708cm -1 The absorption peak at the wave number is shifted to a low band due to the conjugation to become a main characteristic peak v of amide C＝O Therefore, it can be preliminarily judged that EPA is successfully grafted to the molecular chain of serum albumin.
In this example, serum albumin is both the water soluble and binding moieties, with the eicosapentaenoic acid carbon chain being the active moiety.
Example 4 bovine serum albumin grafted fatty acid composition preparation and characterization (active moiety + macromolecular Water-soluble moiety/binding moiety)
Accurately weighing 0.036mmol of eicosapentaenoic acid (EPA); catalyst 0.036 mmol; adding acid to catalyze and activate carboxyl to obtain activated EPA; activating under stirring in ice bath for 15 min. 0.018mmol (about 1.2g by mass) of serum albumin is precisely weighed, dissolved in 10ml of PBS solution, added with NaOH solution to adjust the pH to be neutral, and stirred uniformly to obtain the serum albumin solution. And adding the serum albumin solution into the activated EPA in stirring, and continuously stirring for reacting overnight in ice bath to obtain an EPA-serum albumin primary solution with the EPA concentration of 36 mM. Precipitating the solution with glacial acetone, removing fatty acid which does not participate in the reaction by using ethanol, dialyzing to remove small molecular impurities (dialyzing by using a dialysis bag with the molecular weight cutoff of 500-1000, changing water every 4h, wherein the molecular weight of small molecular compounds is less than 500 and can be removed), and then performing gradient low-temperature drying in a freezing vacuum drier (the low temperature of 80 ℃ is pre-frozen for 24h and is vacuumized for 12h, the vacuum of-20 ℃ is pumped for 15h, the vacuum of 4 ℃ is continuously pumped for more than 24h until the product is completely dried), wherein the yield is 79%; the composite obtained by this vacuum drying was used for the evaluation of the performance of the subsequent action on microorganisms.
The setting of each time can be adjusted according to the amount of the preparation solution.
The structural change of molecules of bovine serum albumin (total 607 amino acids of bovine serum albumin, wherein 60 lysine, 17 glycine, 48 alanine, 38 valine, 65 leucine, 15 isoleucine, 30 phenylalanine, 3 tryptophan, 21 tyrosine, 40 aspartic acid, 14 asparagine, 59 glutamic acid, 20 glutamine, 5 methionine, 32 serine, 34 threonine, 35 cysteine, 28 proline, 17 histidine and 26 arginine) is analyzed by an LC-MS method, and a mass spectrum diagram and an amino acid sequence alignment diagram are respectively shown as a figure 8 and a figure 9. It can be concluded that: 17 EPA molecules are bonded to the amino group of the amino acid of 1 serum protein molecule in a mode of removing one molecule of water, and the total efficiency of fatty acid binding to the amino acid is 2.8%, wherein glutamic acid is bound to 2 molecules, the substitution degree of the glutamic acid is 3.39%, histidine 2 is bound to 11.76%, cysteine 2 is bound to 5.71%, and leucine, proline, aspartic acid, asparagine, valine and glutamine are respectively bound to one molecule, the substitution degrees are respectively 1.54%, 3.57%, 2.5%, 7.14%, 2.63% and 5%, lysine 5 is bound to 8.33%.
In this example, serum albumin is both the water soluble and binding moieties, with the eicosapentaenoic acid carbon chain being the active moiety.
Example 5 bovine serum albumin grafted fatty acid composition preparation and characterization (active moiety + macromolecular Water soluble moiety/binding moiety)
Precisely weighing 0.036mmol of linoleic acid; 0.036mmol of catalyst; adding acid to activate carboxyl; stirring and activating for 10-30min under ice bath to obtain activated linoleic acid. Accurately weighing 0.036mmol (about 2.4g by mass) of serum albumin, dissolving in 5ml of PBS solution, adding NaOH solution to adjust pH to neutrality, and stirring to obtain serum albumin solution. And adding the serum albumin solution into the stirred activated linoleic acid, and continuously stirring and reacting overnight under ice bath to obtain a linoleic acid-serum albumin primary solution with the linoleic acid concentration of 72 mM. Precipitating the solution with glacial acetone, removing fatty acid which does not participate in the reaction by using ethanol, dialyzing to remove small molecular impurities (dialyzing by using a dialysis bag with the molecular weight cutoff of 500-1000, changing water every 4h, wherein the molecular weight of small molecular compounds is less than 500 and can be removed), and then performing gradient low-temperature drying in a freeze vacuum drier (the low temperature of 80 ℃ is pre-frozen for 24h and vacuum is performed for 12h, the vacuum is performed for 12h at the temperature of-20 ℃, the vacuum is continuously performed for more than 24h at the temperature of 4 ℃ until the product is completely dried), wherein the yield is 79%; the composite obtained by this vacuum drying was used for the evaluation of the performance of the subsequent action on microorganisms.
The molecular weight of bovine serum albumin (607 total amino acids of bovine serum albumin, wherein 60 lysine, 17 glycine, 48 alanine, 38 valine, 65 leucine, 15 isoleucine, 30 phenylalanine, 3 tryptophan, 21 tyrosine, 40 aspartic acid, 14 asparagine, 59 glutamic acid, 20 glutamine, 5 methionine, 32 serine, 34 threonine, 35 cysteine, 28 proline, 17 histidine and 26 arginine) molecules is analyzed by the LC-MS method, and the conclusion can be obtained: 12 linoleic acid bonds were bonded to the amino acids of 1 serum albumin molecule in a one-molecule water-free manner, with a total degree of substitution of 1.98%, where lysine bound 11 molecules, lysine was 18.33%, aspartic acid bound 1 molecule, and aspartic acid was 2.5%. The mass spectrogram and the amino acid sequence alignment chart are respectively shown in FIG. 10 and FIG. 11.
In this example, serum albumin is both the water soluble and binding moieties, and the linoleic acid carbon chain is the active moiety.
Example 6 preparation and characterization of human serum Albumin/bovine serum Albumin grafted fatty acid Complex (active moiety + macromolecular Water-soluble moiety/binding moiety)
Accurately weighing 0.36mmol of docosahexaenoic acid (DHA); 0.36mmol of catalyst; adding acid to catalyze and activate carboxyl to obtain activated DHA; stirring and activating for 10-30min under ice bath. Precisely weighing 0.036mmol (about 2.4g by mass) of serum albumin, dissolving in 5ml of physiological saline, adding NaOH solution to adjust pH to neutrality, and stirring to obtain serum albumin solution. The serum albumin solution was added to the activated DHA while stirring, and the reaction was continued overnight with stirring in ice bath, whereby a DHA-albumin solution having a concentration of 72mM (the concentration here refers to the molar concentration of the complex DHA-albumin in the reaction mixture solution, and the same meanings apply in the following examples) was obtained. Precipitating the solution with glacial acetone, removing fatty acid which does not participate in the reaction by using ethanol, dialyzing to remove small molecular impurities (dialyzing by using a dialysis bag with the molecular weight cutoff of 500-1000, changing water every 4h, wherein the molecular weight of small molecular compounds is less than 500 and can be removed), and then performing gradient low-temperature drying in a freezing vacuum drier (the low temperature of 80 ℃ is pre-frozen for 24h and vacuumized for 12h, the low temperature of 20 ℃ is vacuumized for 12h, the high temperature of 4 ℃ is continuously vacuumized for more than 24h until the products are completely dried), wherein the yield is 79%; the composite obtained by this vacuum drying was used for the evaluation of the performance of the subsequent action on microorganisms.
Fourier spectrum shown in FIG. 12, the serum albumin grafted docosahexaenoic acid is 1044cm- 1 Strong new absorption peak appears at wavenumber, which is classified as gamma ═ C-H (bending vibration out of unsaturated carbon-hydrogen bond plane), comes from grafted small molecule docosahexaenoic acid (DHA), and the grafted olefin is 1708cm -1 The absorption peak at the wave number shifts to a low band due to conjugation because of v of carboxyl in docosahexaenoic acid C＝O V becomes amide bond after being combined with serum albumin C＝O Therefore, the successful grafting of DHA to the molecular chain of serum albumin can be preliminarily judged.
Further, the structural change, mass spectrum and amino acid sequence alignment chart of bovine serum albumin (total 607 amino acids of bovine serum albumin, wherein 60 lysine, 17 glycine, 48 alanine, 38 valine, 65 leucine, 15 isoleucine, 30 phenylalanine, 3 tryptophan, 21 tyrosine, 40 aspartic acid, 14 asparagine, 59 glutamic acid, 20 glutamine, 5 methionine, 32 serine, 34 threonine, 35 cysteine, 28 proline, 17 histidine and 26 arginine) molecules are respectively shown in fig. 13 and fig. 14 by LC-MS method. It can be concluded that: 9 DHA molecules were bonded to the amino groups of the amino acids of 1 serum albumin molecule with one molecule of water removed, and the total degree of fatty acid substitution was 1.48%, where phenylalanine was bound to 1 molecule, phenylalanine was 3.33%, glutamic acid was bound to 2 molecules, glutamic acid was 3.39%, cysteine was bound to 2 molecules, cysteine was 5.71%, lysine was bound to 4 molecules, and lysine was 6.67%.
In this example, serum albumin is both the water soluble and binding moieties, with the docosahexaenoic acid carbon chain being the active moiety.
Example 7 preparation of unsaturated fatty acid-conjugated hyaluronic acid Complex (active moiety + macromolecular Water-soluble moiety/binding moiety)
4 alcoholic hydroxyl groups on a single molecular unit of the hyaluronic acid can be subjected to esterification reaction with carboxyl groups of fatty acid molecules.
According to the molar ratio of fatty acid carboxyl to hyaluronic acid hydroxyl of 4 n: 1-1: 1, the molar ratio of the catalyst to the fatty acid carboxyl is 10: 1-1: the catalyst can be one or more of EDC, DCC, NHS, DMAP, HoBt and derivative analogues thereof.
In the embodiment, 0.001mmol of linoleic acid is precisely weighed, 0.001mmol of EDC and 0.001mmol of DMAP are added as catalysts, and an acid solution is added to be stirred and activated for 10min to obtain the activated linoleic acid. Precisely weighing 0.0025mmol (20 mg by mass in an example of molecular weight 200k Da) of hyaluronic acid, dissolving in 5ml of physiological saline, adding NaOH solution to adjust pH to neutrality, and stirring uniformly to obtain hyaluronic acid solution. Adding the hyaluronic acid solution into the stirred activated linoleic acid, and continuously stirring and reacting for 12 hours at room temperature to obtain the hyaluronic acid solution with the concentration of 72 And (3) a linoleic acid-hyaluronic acid solution in a mM concentration, wherein after the reaction is finished, the linoleic acid-hyaluronic acid reaction solution is dialyzed by a dialysis bag with the molecular weight cutoff of 500-1000 so as to purify the compound obtained by the reaction, water is replaced every 4 hours (the molecular weight of the catalyst and the unreacted fatty acid is less than 500 and can be removed), and the compound is dialyzed for 24 hours. The yield of the obtained product was calculated in the same manner as in examples 1 and 2 to be 75%. The Fourier IR spectrum of linoleic acid-hyaluronic acid is shown in FIG. 15, and 1735cm of the complex appears -1 The absorption peak at the wave number is attributed to a vC ═ O absorption peak, so that the successful grafting of linoleic acid to the molecular chain of the hyaluronic acid can be preliminarily judged.
In this embodiment, hyaluronic acid is both a water-soluble portion and a binding portion, and linoleic acid carbon chain is an active portion.
Example 8 preparation of polyunsaturated fatty acid-conjugated hyaluronic acid Complex (active moiety + macromolecular Water-soluble moiety/binding moiety)
Accurately weighing 0.36mmol of docosahexaenoic acid (DHA), adding 0.36mmol of EDC and 0.36mmol of DMAP, adding acid solution, stirring and activating for 10min to obtain activated DHA. Precisely weighing 0.045mmol (2.5 mg by mass in an example of a molecular weight of 50k Da) of hyaluronic acid, dissolving the hyaluronic acid in 5ml of physiological saline, adding a NaOH solution to adjust the pH to be neutral, and uniformly stirring to obtain a hyaluronic acid solution. Adding the hyaluronic acid solution into the activated DHA which is stirred, continuously stirring the mixture at room temperature for reacting for 8-24 hours to obtain a DHA-hyaluronic acid solution with the concentration of 72mM, and purifying the compound obtained by the reaction by adopting the same purification operation steps as in example 7 to remove unreacted fatty acid and the catalyst. The yield efficiency of the product was calculated in the same manner as in examples 1 and 2 and was 70%. The Fourier infrared spectrum of docosahexaenoic acid-hyaluronic acid is shown in FIG. 16, and 1412cm is caused by ring tension of six-membered ring of hyaluronic acid -1 V of (C) C＝C Frequency increase, Compound 1649cm -1 And 1568cm -1 Absorption peaks appear at the wave number, so that v C＝O Absorption peak (1649 cm) -1 ) Shifted to the lower band (characteristic v in general esters) C＝O The absorption peak is 1750-1735cm -1 ) Thus can be preliminaryJudging that the docosahexaenoic acid is successfully grafted to the hyaluronic acid molecular chain.
Eicosapentaenoic acid (EPA) -hyaluronic acid complex was prepared using the same method for subsequent experiments.
In this example, hyaluronic acid is both a water-soluble moiety and a binding moiety, with the carbon chains of eicosapentaenoic acid and docosahexaenoic acid being the active moieties.
Example 9 preparation of unsaturated fatty acid-polypeptide Complex (acting moiety + targeting binding moiety/Water soluble moiety)
The fatty acid is bonded to the molecular structure of the polypeptide via an amide bond (free carboxyl group of the fatty acid and free amino group of the polypeptide) and an ester bond (free carboxyl group of the fatty acid and free hydroxyl group of the polypeptide).
Taking SBP1(ACE2 derivative peptide; binds SARS-CoV-2 spike protein binding domain) as an example, the sequence is: IEEQAKTFLDKFNHEAEDLFYQS (modified: Ser-23 ═ C-terminal amide) containing 6 free amino groups; the carboxyl of the fatty acid reacts with the amino, the polypeptide is used as a carrier, the molar ratio of the fatty acid to the polypeptide is 8:1-1:4, the molar ratio of the catalyst to the fatty acid is 0.5: 1-10: 1, and the carbodiimide and the succinimide are used as catalysts, wherein the ratio of the two catalysts is 1:1-1: 10.
In this embodiment, Oleic Acid (OA), Linoleic Acid (LA), linolenic acid (ALA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA) were selected to react with SBP1, respectively.
Precisely weighing 0.36mmol of fatty acid; 0.36mmol of catalyst; adding acid to activate carboxyl; stirring and activating for 10min to obtain activated fatty acid. Accurately weighing polypeptide SBP10.72mmol, dissolving in 20ml physiological saline, adding NaOH solution to adjust pH, and stirring uniformly to obtain SBP1 solution. Adding the SBP1 solution into the stirred activated fatty acid, and continuing stirring for 1h in ice bath to obtain a 36mM fatty acid-SBP 1 solution, and purifying the compound obtained by the reaction by the same purification procedure as in example 7 to remove the unreacted fatty acid and the catalyst. Freezing at-80 deg.C, vacuum drying to obtain the final product, and evaluating the performance of the obtained compound on microorganism.
The prepared fatty acid-SBP 1 compound Fourier infrared spectrogram is shown in FIG. 17, and fatty acids (oleic acid (OA), Linoleic Acid (LA), linolenic acid (ALA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA)) are judged to be successfully grafted to an SBP1 molecular chain.
In this example, SBP1 is both a water soluble moiety and a binding moiety, with the unsaturated carbon chain of the fatty acid being the active moiety.
Example 10 preparation of unsaturated fatty acid-polypeptide Complex (acting moiety + targeting binding moiety/Water soluble moiety)
Taking the polypeptide SBP1 as a carrier, wherein the molar ratio of fatty acid to the polypeptide SBP1 is 7: 1-1: 2, the molar ratio of the catalyst to the fatty acid is 0.5: 1-10: 1, carbodiimide and succinimide are used as the catalyst, and the ratio of the two catalysts is 1: 1-1: 10.
in this example, 9-decatetraenoic acid was selected to react with SBP 1.
Precisely weighing 0.36mmol of fatty acid; 0.36mmol of catalyst; adding acid to catalytically activate carboxyl; stirring and activating for 10min to obtain activated fatty acid. SBP 10.36mmol is precisely weighed and dissolved in 20ml of normal saline, NaOH solution is added to regulate the pH value, and the mixture is stirred evenly to obtain SBP1 solution. The SBP1 solution was added to the stirred activated fatty acid, and the reaction was continued for 1 hour under ice-cooling to obtain a solution of 9-decatetraenoic acid-SBP 1 at a concentration of 18mM, and the compound obtained by the reaction was purified from the reaction solution by the same purification procedure as in example 7 to remove the unreacted fatty acid and the catalyst. Freezing at-80 deg.C, and vacuum drying.
Wherein, the Fourier infrared spectrum of the 9-decatetraenoic acid-SBP 1 is shown in figure 18, and the 9-decatetraenoic acid is preliminarily judged to be successfully grafted to the SBP1 molecular chain.
In this example, SBP1 is both a water soluble moiety and a binding moiety, with the unsaturated carbon chain of the fatty acid being the active moiety.
EXAMPLE 11 preparation of unsaturated fatty acid-CD 14 protein Complex (action moiety + targeting binding moiety/Water soluble moiety)
Fatty acids are bonded to the molecular structure of proteins through amide bonds (free carboxyl groups of fatty acids and free amino groups of proteins) and ester bonds (free carboxyl groups of fatty acids and free hydroxyl groups of proteins).
Taking CD14(34kDa) as an example, the sequence is:
TTPEPCELDDEDFRCVCNFSEPQPDWSEAFQCVSAVEVEIHAGGLNLEPFLKRVDADADPRQYADTVKALRVRRLTVGAAQVPAQLLVGALRVLAYSRLKELTLEDLKITGTMPPLPLEATGLALSSLRLRNVSWATGRSWLAELQQWLKPGLKVLSIAQAHSPAFSYEQVRAFPALTSLDLSDNPGLGERGLMAALCPHKFPAIQNLALRNTGMETPTGVCAALAAAGVQPHSLDLSHNSLRATVNPSAPRCMWSSALNSLNLSFAGLEQVPKGLPAKLRVLDLSCNRLNRAPQPDELPEVDNLTLDGN PFLVPG, containing 47 free amino groups.
In this embodiment, oleic acid, linoleic acid, linolenic acid, eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA) are selected to react with CD14, respectively. The molar ratio of carboxyl groups of the fatty acid to CD14 was 17:1 to 1:1, the carbodiimide and succinimide were used as catalysts in a ratio of 1:1 to 1:10, and the reaction methods were as in examples 1 and 2.
Precisely weighing 0.36mmol of fatty acid; 0.36mmol of catalyst; adding acid to activate carboxyl; stirring and activating for 30min to obtain activated fatty acid. Precisely weighing CD140.021mmol, dissolving in 100ml of physiological saline, adding NaOH solution to regulate pH, and stirring uniformly to obtain CD14 solution. The CD14 solution was added to the stirred activated fatty acid, and the stirring reaction was continued overnight in an ice bath to obtain a fatty acid-CD 14 solution having a concentration of 3.6mM, and the compound obtained by the reaction was purified from the reaction solution by the same purification procedure as in example 7 to remove the unreacted fatty acid and the catalyst. Freezing at-80 deg.C, and vacuum drying. The product yields were 87.3%, 87.6%, 86.8%, 89.0%, 88.7%, respectively.
In this example, CD14 is both the water soluble and binding moieties, with the unsaturated carbon chain of the fatty acid as the active moiety.
Example 12 preparation of saturated fatty acid-hyaluronic acid Complex (acting portion + macromolecular Water-soluble portion/binding portion)
In the embodiment, caproic acid, caprylic acid, pelargonic acid, dodecanoic acid, tetradecanoic acid, hexadecanoic acid, octadecanoic acid and eicosanoic acid are respectively reacted with hyaluronic acid to prepare a saturated fatty acid-hyaluronic acid compound, the molar ratio of carboxylic acid groups of fatty acids to the hyaluronic acid is 4n:1-1:1(n is the number of repetitions of a single molecular unit of the hyaluronic acid, and is an integer from 1 to 2000), the molar ratio of a catalyst to the fatty acids is 0.5:1-2:1, and the catalyst is carbodiimide and dimethylaminopyridine, and the molar ratio of the carbodiimide to the dimethylaminopyridine is 1:1-1: 10.
Specifically, the reaction is carried out according to the following reaction formula, for example, formula 12-1:
Figure BDA0003628692260001351
n is the number of repetition of the single molecular unit of hyaluronic acid, and n is an integer of 1-2000.
Wherein, R groups are saturated carbon chains of C5, C7, C9, C11, C13, C15, C17 and C19 respectively. The reaction process is as follows:
precisely weighing 0.36mmol of fatty acid, adding 0.4mmol of EDC and 0.4mmol of DMAP as catalysts, adding an acid solution, stirring and activating for 10min to obtain the activated fatty acid. Precisely weighing 0.00068mmol (taking the molecular weight of 500k Da as an example, the mass is 340mg) of hyaluronic acid, dissolving the hyaluronic acid in 5ml of physiological saline, adding NaOH solution to adjust the pH to be neutral, and uniformly stirring to obtain the hyaluronic acid solution. The hyaluronic acid solution was added to the activated fatty acid while stirring, and the reaction was continued at room temperature for 12 hours to obtain a fatty acid-hyaluronic acid solution having a concentration of 72mM, and the same purification procedure as in example 7 was applied to the reaction solution to purify the compound obtained by the reaction, and remove the unreacted fatty acid and the catalyst.
In the embodiment, hyaluronic acid is used as a water-soluble part and a binding part, and a fatty acid saturated carbon chain is used as an acting part.
Example 13 preparation of unsaturated fatty acid-hyaluronic acid Complex (acting portion + macromolecular Water-soluble portion/binding portion)
Glutaconic acid-hyaluronic acid, wherein the molar ratio of carboxylic acid groups of the fatty acid to the hyaluronic acid is 4 n: 1(n is the number of the repeated single molecular units of the hyaluronic acid, and n is an integer of 1-2000), wherein the molar ratio of the catalyst to the fatty acid is 0.5:1-2:1, and the catalyst is carbodiimide and dimethylaminopyridine, and the molar ratio of the catalyst to the fatty acid is 1:1-1: 10.
The reaction was carried out according to the following reaction scheme example formula 13-1:
Figure BDA0003628692260001361
wherein the R group is
Figure BDA0003628692260001362
Carbon chain of (2). The reaction process is as follows:
accurately weighing 0.36mmol of glutaconic acid, adding 0.4mmol of EDC and 0.4mmol of DMAP as catalysts, adding an acid solution, stirring and activating for 10min to obtain activated glutaconic acid. Precisely weighing 0.0014mmol (taking the molecular weight of 300k Da as an example, 420mg by mass), dissolving hyaluronic acid in 5ml of physiological saline, adding NaOH solution to adjust the pH to be neutral, and uniformly stirring to obtain hyaluronic acid solution. The hyaluronic acid solution was added to the active glutaconic acid while stirring, and the reaction was continued at room temperature for 12 hours to obtain a glutaconic acid-hyaluronic acid solution having a concentration of 72mM, and the same purification procedure as in example 7 was applied to the reaction solution to purify the compound obtained by the reaction, thereby removing the unreacted fatty acid and the catalyst.
In this embodiment, hyaluronic acid is both the water soluble portion and the binding portion, and the unsaturated carbon chain of glutaconic acid is the active portion.
Example 14 preparation of eight carbon saturated carbon chain-glucose Complex (Small molecule Water soluble portion + acting portion) and evaluation of Performance
This example uses octanoic acid to prepare a saturated fatty acid glucose complex, according to the following reaction example, formula 14-1:
Figure BDA0003628692260001371
the reaction process is as follows:
0.72mmol of octanoic acid, adding 0.72mmol of EDC and 0.72mmol of DMAP which are used as catalysts, and stirring and activating for 10min under ice bath to obtain activated octanoic acid; dissolving 1.44mmol of glucose in 10ml of deionized water, adding the solution into an activated octanoic acid solution, adjusting the pH value to 7.0-7.4 by NaOH, stirring the solution at room temperature for 12 hours to obtain a reaction product solution, carrying out chromatographic purification after the reaction is finished, adding the product solution into a Shephadex G10 chromatographic column (phi 26mm multiplied by 50cm), eluting the product solution by normal saline, carrying out flow rate of 50ml/h, collecting eluent step by step, detecting the eluent by a phenol-sulfuric acid method, and combining the 1 st elution peak to obtain a reaction product.
Thus obtaining the caprylic acid-glucose compound. The infrared spectrum of the prepared compound is shown in figure 19, and it can be seen from the figure that v is reserved after only hydroxyl of glucose is combined with n-caprylic acid c-o Peak 1000-1250cm -1 And the ester bond formed by the octanoic acid and the glucose has the absorption peak from 1740cm due to the conjugation with the hydroxyl of the glucose molecule -1 The characteristic peak of carboxyl at the position moves to 1660cm along a short wave band -1 。
The inhibition rate of bacteria (such as staphylococcus aureus) is tested, and the test process is as follows: LB agar is used for preparing the culture medium according to the commercial specification, and the pH value is 7.2-7.4. Inoculum preparation and inoculation: diluting the bacteria to 10 5 -10 6 CFU, 100ul bacteria are taken, 900ul bacteria are added and diluted into liquid medicines with different concentrations, the liquid medicines are incubated for 2 hours at 37 ℃, then the solution is diluted by 100 times, 100ul bacteria are taken and spread on a flat plate, the liquid medicines are cultured for 16 to 24 hours at 37 ℃, bacterial colonies are counted, the bacteriostasis rate is calculated, the bacteriostasis result is shown in figure 20, when the concentration is 72mM to 9mM, the bacteriostasis rate is more than 99 percent, the antibacterial property is achieved, the half bacteriostasis concentration is 4.5Mm, and when the concentration is 4.5mM to 0.14mM, the bacteriostasis rate is calculated<50% and has no bacteriostatic property
In this example, glucose is both a water-soluble part and a binding part, and a fatty acid saturated carbon chain is an active part, and the bacteriostatic results of the complex are summarized in table 2-1.
TABLE 2-1 Bactericidal and antibacterial Properties of eight carbon saturated carbon chain-glucose (9mM) (2h)
Testing microorganisms Sterilizing rate (%)
Escherichia coli >99
Methicillin-resistant staphylococcus aureus >99
Streptococcus pneumoniae >99
Klebsiella pneumoniae >99
Pseudomonas aeruginosa >99
Example 15 preparation of eight carbon saturated carbon chain-sucrose Complex (Small molecule Water soluble fraction + functional fraction) and Performance evaluation
In this example, a saturated fatty acid sucrose complex was prepared from octanoic acid and reacted according to the following reaction example formula 15-1.
Figure BDA0003628692260001391
The reaction process is as follows:
0.72mmol of octanoic acid, 0.72mmol of EDC and 0.72mmol of DMAP as catalysts are added, and stirring and activation are carried out for 10min under ice bath; 0.24mmol of sucrose is dissolved in 10ml of deionized water, added into the activated octanoic acid solution, the pH value is adjusted to be 7.0-7.4 by NaOH, the solution is stirred and reacted for 12 hours at room temperature, and the octanoic acid-sucrose compound is obtained after the reaction product solution is purified according to the same operation as the embodiment 14. Is made ofThe infrared spectrum of the prepared complex is shown in FIG. 21, the sucrose molecule is 1700-1500cm -1 The wave band has no absorption peak, while the n-octanoic acid is 1700cm -1 Has a strong absorption peak, and 1700-1500cm in the new compound generated by the reaction of the two -1 Two strong absorption peaks appear in the wave band, and the conclusion is that the peak shifts to a low wave band due to the conjugation effect of the formed ester bond and the hydroxyl of the sucrose molecule.
The inhibition rate of bacteria (such as staphylococcus aureus) is tested, and the test process is as follows: the preparation of the culture medium uses an LB agar culture medium, and the preparation is carried out according to a commercial specification, wherein the pH value is 7.2-7.4. Inoculum preparation and inoculation: diluting the bacteria to 10 5 -10 6 CFU, adding 100ul bacteria, diluting with 900ul bacteria to obtain different concentrations of medicinal liquid, incubating at 37 deg.C for 2 hr, diluting the solution 100 times, spreading 100ul bacteria on the plate, culturing at 37 deg.C for 16-24 hr, counting bacterial colonies, calculating antibacterial rate, wherein the antibacterial rate is greater than 99% when the concentration is 72mM-9mM, and has bactericidal effect, half antibacterial concentration is 4.5mM, and when the concentration is 4.5mM-0.14mM, the antibacterial rate is calculated<50%, has no bacteriostatic property.
In this example, sucrose is both a water-soluble part and a binding part, and a fatty acid saturated carbon chain is an active part, and specific bacteriostatic results are summarized in tables 2-2.
TABLE 2 Bactericidal and antibacterial Properties of 2 eight carbon saturated carbon chain-sucrose (9mM) (2h)
Testing microorganisms Sterilizing rate (%)
Escherichia coli >99
Methicillin-resistant staphylococcus aureus >99
Streptococcus pneumoniae >99
Klebsiella pneumoniae >99
Pseudomonas aeruginosa >99
Example 16 fatty acid-nucleotide Complex preparation (Small molecule Water soluble portion + acting portion) and Performance evaluation
The general formula of the reaction is shown in the specification, wherein octanoic acid, linolenic acid and docosapentaenoic acid are used as carbon chain donors and are respectively reacted with adenosine monophosphate, and R is respectively the carbon chain of octanoic acid, linolenic acid and docosapentaenoic acid.
The reaction was carried out according to the following reaction example formula 16-1.
Figure BDA0003628692260001401
The reaction process is as follows:
0.72mmol of fatty acid, 0.72mmol of EDC and 0.72mmol of DMAP as catalysts are added, and stirring and activation are carried out for 10min under ice bath; dissolving 0.72mmol of adenosine monophosphate in 10ml of deionized water, adding the solution into an activated octanoic acid solution, adjusting the pH value to 7.0-7.4 by NaOH, stirring and reacting for 12h at room temperature to obtain a fatty acid-adenosine complex solution, carrying out chromatographic purification on a reaction product solution (referred to as 'reaction solution') after the reaction is finished, adding the reaction solution into a Shephadex G10 chromatographic column (phi 26mm multiplied by 50cm), eluting by normal saline, carrying out flow rate of 50ml/h, collecting eluent step by step, detecting absorbance at the wavelength of 260nm by using an ultraviolet spectrophotometry, and combining the 1 st elution peak to obtain the reaction product.
In this examplePreparation of fatty acid-adenosine monophosphate Complex the infrared spectrum of the adenosine monophosphate complex is shown in FIG. 23, where the adenosine monophosphate molecule is at 3000-2700cm -1 The wave band has no absorption peak, and the content of the n-octanoic acid, the docosapentaenoic acid and the linolenic acid is 3000-2700cm -1 Has a strong absorption peak, and adenosine monophosphate molecules are positioned at 1700-1500cm -1 The wave band has no absorption peak, while the n-octanoic acid, docosapentaenoic acid and linolenic acid have a length of 1700cm -1 Has a strong absorption peak, and 1700-1500cm in the new compound generated by the reaction of the two -1 The band shows a strong absorption peak, and it is inferred that the peak shifts to the low band due to the conjugation effect between the formed ester bond and the hydroxyl group of the adenosine monophosphate molecule in the novel compound generated by the reaction between the formed ester bond and the hydroxyl group of the adenosine monophosphate molecule.
The fatty acid-adenosine monophosphate complex is subjected to bacterial (such as staphylococcus aureus) inhibition experiments, and the experimental process is as follows: the preparation of the culture medium uses an LB agar culture medium, and the pH value is 7.2-7.4 according to a commercial specification. Inoculum preparation and inoculation: diluting the bacteria to 10 5 -10 6 CFU, 100ul bacteria are taken, 900ul bacteria are added and diluted into liquid medicines with different concentrations, the liquid medicines are incubated for 2 hours at 37 ℃, then the solution is diluted by 100 times, 100ul bacteria are taken and spread on a flat plate, the liquid medicines are cultured for 16 to 24 hours at 37 ℃, bacterial colonies are counted, the bacteriostasis rate is calculated, the bacteriostasis result is shown in figure 24, when the concentration is 72mM to 9mM, the bacteriostasis rate is more than 99 percent, the antibacterial property is realized, the half bacteriostasis concentration is 4.5mM, and when the concentration is 4.5mM to 0.14mM, the bacteriostasis rate is calculated <50%, has no bacteriostatic property.
Adenosine monophosphate in this example is both a water soluble and binding moiety, with the fatty acid carbon chain being the active moiety. The bacteriostatic results are summarized in table 3.
TABLE 3 Bactericidal and antibacterial Properties of the fatty acid carbon chain-adenosine monophosphate complex (9mM) (2h)
Testing microorganisms Sterilizing rate (%)
Escherichia coli >99
Methicillin-resistant staphylococcus aureus >99
Streptococcus pneumoniae >99
Klebsiella pneumoniae >99
Pseudomonas aeruginosa >99
Example 17 eight carbon saturated carbon chain-Water soluble vitamin Complex preparation (Small molecule Water soluble fraction + Effect fraction) and Performance evaluation
This example was carried out in the following reaction example 17-1 by using octanoic acid to prepare a saturated fatty acid ascorbic acid complex.
Figure BDA0003628692260001421
The reaction process is as follows: 0.72mmol of octanoic acid, 0.72mmol of EDC and 0.72mmol of DMAP are added, and stirring and activation are carried out for 10min under ice bath; dissolving ascorbic acid 0.72mmol in 10ml deionized water, adding into activated octanoic acid solution, adjusting pH with NaOH to 7.0-7.4, stirring at room temperature for reaction for 12h, performing chromatographic purification on the reaction solution after the reaction is finished, adding the reaction solution into Shephadex G10 chromatographic column (phi 26mm × 50cm), eluting with normal saline, collecting eluate at flow rate of 50ml/h, and collecting eluate step by stepRemoving liquid, detecting the absorbance at the wavelength of 243nm by using an ultraviolet spectrophotometry, and combining the 1 st elution peak to obtain a reaction product, namely obtaining the caprylic acid-ascorbic acid compound. The infrared spectrum of the prepared octanoic acid-ascorbic acid compound is shown in figure 25, and it can be seen that no new functional group is generated after the reaction of octanoic acid and ascorbic acid, but v appears C＝O The shift of the characteristic peak to the lower band is presumably due to the intramolecular conjugation effect, and the lactonization of the carboxyl group of n-octanoic acid with the hydroxyl group of ascorbic acid.
The inhibition rate of bacteria (such as staphylococcus aureus) is tested, and the test process is as follows: the preparation of the culture medium uses an LB agar culture medium, and the preparation is carried out according to a commercial specification, wherein the pH value is 7.2-7.4. Inoculum preparation and inoculation: diluting the bacteria to 10 5 -10 6 CFU, 100ul bacteria are taken, 900ul bacteria are added and diluted into liquid medicines with different concentrations, the liquid medicines are incubated for 2 hours at 37 ℃, then the solution is diluted by 100 times, 100ul bacteria are taken and spread on a flat plate, the liquid medicines are cultured for 16 to 24 hours at 37 ℃, bacterial colonies are counted, the bacteriostasis rate is calculated, the bacteriostasis result is shown in figure 26, when the concentration is 72mM to 9mM, the bacteriostasis rate is more than 99 percent, the bactericidal performance is achieved, the half bacteriostasis concentration is 4.5Mm, and when the concentration is 4.5mM to 0.14mM, the bacteriostasis rate is calculated<50%, has no bacteriostatic property.
In this example, ascorbic acid is both the water soluble and binding moieties, with the fatty acid carbon chain being the active moiety. The specific bacteriostatic results are summarized in table 4.
TABLE 4 Bactericidal and antibacterial Properties of eight carbon saturated carbon chain-Water soluble vitamin Complex (9mM) (2h)
Testing microorganisms Sterilizing rate (%)
Escherichia coli >99.9
Methicillin-resistant staphylococcus aureus >99.9
Streptococcus pneumoniae >99.9
Klebsiella pneumoniae >99.9
Pseudomonas aeruginosa >99.9
Example 18 preparation of eight carbon saturated carbon chain-PEG-COOH Complex with Low polymerization degree (carboxyl group + Small molecule Water-soluble moiety + active moiety) and evaluation of Performance the saturated fatty acid-PEG 400-COOH complex prepared with octanoic acid was used in this example according to the following reaction example 18-1.
Figure BDA0003628692260001431
The reaction process is as follows: 0.72mmol of octanoic acid, 0.72mmol of EDC and 0.72mmol of DMAP are added, and stirring and activation are carried out for 10min under ice bath; 0.36mmol of PEG400-COOH is dissolved in 10ml of deionized water, added into the solution of the activated octanoic acid, the pH value is adjusted to 7.0-7.4 by NaOH, the mixture is stirred and reacted for 12h at room temperature, after the reaction is finished, the solution of the reaction product is chromatographically purified, the reaction solution is added into a Shephadex G10 chromatographic column (phi 26mm multiplied by 50cm), the elution is carried out by normal saline, the flow rate is 50ml/h, the eluent is collected step by step, the detection is carried out by a barium chloride-iodine solution method, the 1 st elution peak is combined to obtain the reaction product, and the octanoic acid-PEG 400-COOH compound is obtained. The IR spectrum of the prepared octanoic acid-PEG 400-COOH complex is shown in FIG. 27, in which it can be seen that no new functional group is generated after the reaction between octanoic acid and PEG400-COOH, but the absorption peak intensities at 1550cm-1 and 1020cm-1 are enhanced.
The inhibition rate of bacteria (staphylococcus aureus) is tested, and the test process is as follows: preparation of the Medium LB agar MediumTryptone(Tryptone)10g/L，Yeast extract(Yeast extract)5g/L, sodium chloride (NaCl)10g/L, 15 g-20 g agar powder/L) according to the commercial instructions, pH7.2-7.4. Inoculum preparation and inoculation: diluting the bacteria to 10 5 -10 6 CFU, 100ul of bacteria are taken, 900ul of the bacteria are added to be diluted into liquid medicines with different concentrations, the liquid medicines are incubated at 37 ℃ for 2 hours, then the solution is diluted by 100 times, 100ul of the bacteria are taken to be spread on a flat plate, the liquid medicines are cultured at 37 ℃ for 16-24 hours, bacterial colonies are counted, the bacteriostasis rate is calculated, the bacteriostasis result is shown in figure 28, when the concentration is 4.5mM-0.035mM, the bacteriostasis rate is greater than 99%, the bacteriostasis performance is achieved, when the concentration is 0.035mM-0.009mM, the bacteriostasis rate is 50-90%, the bacteriostasis performance is achieved, and the half bacteriostasis concentration is 0.009 mM.
In this embodiment, PEG400-COOH with low polymerization degree is not only a water-soluble part, but also a binding part, and a fatty acid carbon chain is an active part. The results of the bacteriostatic experiments are summarized in table 5.
TABLE 5 Bactericidal and antibacterial Properties (2h) of eight carbon saturated carbon chain-Low polymerization degree PEG400-COOH (0.035mM)
Testing microorganisms Sterilizing rate (%)
Escherichia coli >99
Methicillin-resistant staphylococcus aureus >99
Streptococcus pneumoniae >99
Klebsiella pneumoniae >99
Pseudomonas aeruginosa >99
Example 19 Liposome emulsion preparation and Performance evaluation (non-covalent coupling of amino/carboxyl + Water-soluble + functional moieties)
(1) Ethyl oleate and aminated lecithin preparations
1) 0.7g of aminated lecithin, 0.3g of beta-sitosterol, 0.2g of vitamin E palmitate and 10g of ethyl oleate, heating and stirring at 50 ℃ in a water bath for dissolving, adding a surfactant tween80, and uniformly dispersing;
2) slowly adding water into the mixture obtained in the step 2), uniformly stirring the mixture, homogenizing the mixture for 5min to obtain primary emulsion, performing ultrasonic treatment for 15min to obtain the lipid-loaded nano liposome emulsion, wherein a particle size graph observed under a transmission electron microscope of the lipid-loaded nano liposome emulsion is shown in figure 29, the particle size approximately keeps between 500nm and 800nm, and the appearance character of the lipid-loaded nano liposome emulsion is still stable after the lipid-loaded nano liposome emulsion is placed at normal temperature for 6 months, which indicates that the liposome emulsion has good stability.
In this example, the water-soluble liposome has an oil-in-water structure, the amino group on the phospholipid is a binding group, the carbon chains in the ethyl oleate and phospholipid molecules are acting groups, the phosphate group in the phospholipid molecule is a water-soluble group, and a complex (a nanoliposome encapsulating the ethyl oleate) is formed by hydrogen bonds and van der waals forces.
(2) Linoleic acid and carboxylated lecithin liposome preparation
1) 0.7g of carboxylated lecithin (purchased from Aladdin reagent company), 0.3g of beta-sitosterol and 2g of linoleic acid, heating and stirring the mixture in a water bath at the temperature of 50 ℃, adding a surfactant tween80, and dispersing the mixture evenly;
2) slowly adding the aqueous solution into the mixture obtained in the step 1), uniformly stirring, homogenizing for 5min to obtain primary emulsion, and performing ultrasonic treatment for 30min to obtain the lipid-loaded nano liposome emulsion.
FIG. 30 is a graph showing the particle size distribution observed by a transmission electron microscope, wherein the particle size distribution is maintained at approximately 500nm to 800nm, and the appearance is stable after 6 months of storage at room temperature, indicating that the liposome emulsion has good stability.
In this example, the water-soluble liposome is an oil-in-water structure, the carboxyl group on the phospholipid is a binding group, the carbon chains in the linoleic acid and phospholipid molecules are acting groups, and the phosphate group in the phospholipid molecule is a water-soluble group, and a complex (nanoliposome encapsulating linoleic acid) is formed through hydrogen bonds and van der waals force.
Verification of microbicidal effect of liposome
The virus inhibition experiment process comprises the following steps:
adding 260 mu L of high-pressure sterilized water into 36 holes on the periphery of the 96-hole plate, and sealing the edges to reduce errors caused by the evaporation of a culture medium in the edge holes; column 2 (cell control CC) was supplemented with DMEM complete medium 150. mu.L/well, column 3 (virus control VC) was supplemented with DMEM complete medium 100. mu.L/well, and the samples were assayed for liposomes having 2.25mM, 1.12mM, 0.56mM ethyl oleate (linoleic acid); a new coronavirus (B.1.526.2, purchased from Beijing Temple pharmaceutical biotechnology development company, No. 80062) is completely cultured in DMEM, the pseudovirus system carries Firefly luciferase (Firefly luciferase) reporter gene, the luminescent value of the pseudovirus can be detected by a fluorogenic substrate after infecting cells so as to determine the virus amount of infected cells, the pseudovirus takes Vesicular Stomatitis Virus (VSV) as a framework, new coronavirus Spike protein is embedded on the surface, the process that the euvirus is combined with a receptor and enters the cells can be simulated, a sample with neutralizing activity loses the capacity of infected cells after reacting with the pseudovirus, whether the sample has neutralizing activity can be judged by detecting the luminescent values of a control hole and a sample hole, and the effective action concentration of the sample, namely an EC50 value, can be calculated, the pseudovirus cannot autonomously replicate, only has single-round infection capacity, and has low biological safety level, the method is simple and convenient to operate and is a common means for evaluating the vaccine at present. ) Diluting to 1.3-2.3 × 10 4 Adding 50 mu L of TCID50/mL into each hole of the 3 rd to 11 th rows; the above 96-well plate was placed in a cell incubator (37 ℃, 5% CO) 2 ) Incubating for 1 h; to be incubated for 30mAfter in, digestion of Vero cells was started, and the cell concentration was diluted to 2X 10 5 Per mL; after incubation, 100. mu.L of cells were added to each well to give 2X 10 cells per well 4 A plurality of; put into a furnace at 37 ℃ and 5% CO 2 Culturing for 24h in a cell culture box; after the culture is finished, absorbing and removing 250 mu L of supernatant, adding 100 mu L of luciferase detection reagent, repeatedly blowing and beating after reacting for 2min in a dark place at room temperature, and transferring 100 mu L of liquid to a white board; reading luminescence value (RLU) using multifunctional imaging microplate reader
Calculating the neutralization inhibition rate according to the following formula E:
Figure BDA0003628692260001461
TABLE 6 Liposome (0.025mM) virucidal Performance (1h)
Figure BDA0003628692260001462
Experimental procedure for bacteriostasis
LB agar is used for preparing the culture medium according to the commercial specification, and the pH value is 7.2-7.4. Inoculum preparation and inoculation: diluting the bacteria to 10 5 -10 6 CFU, 100ul of bacteria were taken, 900ul of liquid medicine (the complex solution prepared by the present invention) was added, incubation was performed at 37 ℃ for 1-2 hours, then the solution was diluted 100 times, 100ul was taken and spread on a plate, incubation was performed at 37 ℃ for 16-24 hours, bacterial colonies were counted, and the bacteriostatic rate was calculated, and the results are summarized in tables 7 and 8 below.
TABLE 7 Bactericidal and antibacterial Properties of liposomes (0.025mM) (2h)
Figure BDA0003628692260001471
TABLE 8 fungicidal Performance of liposomes (0.025mM) (1h)
Figure BDA0003628692260001481
Example 20 serum Albumin graft fatty acid Complex preparation (active moiety + macromolecular Water-soluble moiety/binding moiety) and Performance evaluation
The linolenic acid-serum albumin compound freeze-dried powder is prepared according to the method of the embodiment example 1, namely the fatty acid-serum albumin compound freeze-dried powder for injection used for animal experiments is obtained, and the fatty acid-serum albumin compound freeze-dried powder is stored at 4 ℃ for standby. When in use, the composition is dissolved in normal saline, and filtered and sterilized by a 0.22um sterile filter membrane.
FIG. 31 is the injection solution of linolenic acid-serum albumin after freeze-drying and redissolved, FIG. 32 is the particle size distribution measured by Malvern particle sizer, in which it can be seen that the particle size 90% is distributed in the range of 200-300nm, the particle size dispersion coefficient is 0.136, the dispersibility is good, and FIG. 33 is the image under the transmission electron microscope.
In this example, serum albumin is both the water soluble and binding moieties, and the linolenic carbon chain is the active moiety.
Referring to the experimental procedure of the microorganism of example 19, the virucidal and bactericidal activity > 99% at a linolenic acid concentration of 0.035mM was obtained as shown in the results of tables 9, 10 and 11 below.
TABLE 9 virucidal Properties of linolenic acid-serum albumin complex (0.035mM) (1h)
Testing viruses Virucidal Rate (%)
H7N9 pseudovirus ＞99.9
H5N1 pseudovirus ＞99.9
HIV pseudovirus ＞99.9
Novel coronaviruses ＞99.9
TABLE 10 Bactericidal and antibacterial Properties of linolenic acid-serum albumin Complex (0.035mM) (2h)
Testing microorganisms Fungicidal ratio (%)
Escherichia coli >99
Staphylococcus aureus (Staphylococcus aureus) >99
Methicillin-resistant staphylococcus aureus >99
Streptococcus pneumoniae >99
Klebsiella pneumoniae >99
Pseudomonas aeruginosa >99
TABLE 11 fungicidal Properties (2h) of the linolenic acid-serum albumin complex (0.035mM)
Testing microorganisms Fungicidal ratio (%)
Candida albicans >99
Aspergillus niger >99
Actinomyces viscosus >99
Spherical shell >99
Aspergillus verrucosus >99
Microsporum canis >99
Example 21 evaluation of Properties of fatty acid-hyaluronic acid Complex lyophilized powder for injection (active portion + macromolecular Water-soluble portion/binding portion)
The linoleic acid-hyaluronic acid complex is prepared in example 7, and the docosahexaenoic acid-hyaluronic acid complex is prepared in example 8. The preparation process of the freeze-dried powder after reaction is as follows.
Dialyzing the reaction product in deionized water for 24h to remove small molecular impurities, and then performing gradient low-temperature drying in a freezing vacuum drier (pre-freezing at the temperature of-80 ℃ for 24h and vacuumizing for 12 h), vacuumizing at the temperature of-20 ℃ for 12h, and continuously vacuumizing at the temperature of 4 ℃ for more than 24h until the product is completely dried) to obtain the fatty acid-hyaluronic acid compound freeze-dried powder for injection for animal experiments, and storing at the temperature of 4 ℃ for later use. When in use, the composition is dissolved in normal saline, and is filtered and sterilized by a sterile filter membrane of 0.22 um.
FIG. 34 is a lyophilized powder injection of linoleic acid-hyaluronic acid, and FIG. 35 is a lyophilized powder injection of docosahexaenoic acid-hyaluronic acid;
taking linoleic acid-hyaluronic acid as an example, the lyophilized powder is re-dissolved in water, fig. 36 is an image under a transmission electron microscope, fig. 37 is a particle size distribution measured by a malvern particle size analyzer, wherein the particle size distribution can be seen at 100-400 nm, 80% of the particle size distribution is at 200-400nm, the average particle size is 360nm, and the particle size distribution coefficient is 0.231, which indicates that the dispersion performance is good.
In this embodiment, hyaluronic acid is both a water-soluble portion and a binding portion, and linoleic acid carbon chain is an active portion.
Referring to the experimental procedure of the microorganisms in example 19, the results shown in tables 12, 13 and 14 below were obtained, and the virucidal and bactericidal activity of the linolenic acid-hyaluronic acid complex was > 99% at a concentration of 0.035%.
TABLE 12 virucidal Properties (1h) of linoleic acid-hyaluronic acid Complex (0.035mM)
Testing viruses Virucidal Rate (%)
H7N9 pseudovirus ＞99.9
H5N1 pseudovirus ＞99.9
HIV pseudovirus ＞99.9
Novel coronaviruses ＞99.9
TABLE 13 Bactericidal and antibacterial Properties of linoleic acid-hyaluronic acid Complex (0.035mM) (2h)
Testing microorganisms Sterilizing rate (%)
Escherichia coli >99
Staphylococcus aureus >99
Methicillin-resistant staphylococcus aureus >99
Streptococcus pneumoniae >99
Klebsiella pneumoniae >99
Pseudomonas aeruginosa >99
TABLE 14 fungicidal Properties (2h) of linoleic acid-hyaluronic acid Complex (0.035mM)
Testing microorganisms Fungicidal ratio (%)
Candida albicans >99
Aspergillus niger >99
Actinomyces viscosus >99
Ball hair shell >99
Aspergillus verrucosus >99
Microsporophyte canis >99
Example 22 evaluation of the Performance of oral lyophilized powder formulation (active portion + Water-soluble portion)
The method comprises the following steps: take the octadecanoic acid-glutamic acid complex (reaction product of octadecanoic acid and glutamic acid) as an example
Uniformly grinding Arabic gum and cod liver oil, adding purified water, grinding to obtain primary emulsion, adding saccharin sodium water solution and octadecanoic acid-glutamic acid compound solution, wherein the mass ratio of oil, water and gel in the primary emulsion is 4: 2: 1, slowly adding the tragacanth mucilage and a proper amount of water, and uniformly stirring to obtain an emulsion containing 6mg/ml octadecanoic acid-glutamic acid compound.
Wherein octadecanoic acid is an active moiety, glutamic acid is a binding moiety, and glutamic acid, acacia and tragacanth are water-soluble moieties, forming a complex (emulsion) by hydrogen bonding or van der waals forces.
The method 2 comprises the following steps: take the case of a dodecanoic acid-aspartic acid complex (which is the reaction product of dodecanoic acid and aspartic acid)
188450 mg of poloxamer, 300mg of soybean lecithin and 10ml of purified water as a water phase; 200mg of dodecanoic acid-aspartic acid compound, 100mg of glyceryl stearate and 100mg of tricaprylin as oil phases, uniformly stirring, adding the water phases into the oil phases, continuously stirring for 20-30min, performing ultrasonic treatment for 30min to obtain an emulsion containing 2% (mass fraction) of the dodecanoic acid-aspartic acid compound, freezing the emulsion at-80 ℃, and performing vacuum drying at room temperature to obtain a liposome freeze-dried powder preparation of the dodecanoic acid-aspartic acid compound, wherein the figure is 38; FIG. 39 is a scanning electron microscope image thereof, and the particle size and the potential after redissolution in water are shown in FIGS. 40 and 41, which show that the average particle size is 110nm, the particle size distribution coefficient is 0.263, and the distribution property is good; the potential showed a potential distribution with good stability in which the absolute value of the average potential was 26.4mV (with good stability in which the absolute value of the potential was 20 or more).
Wherein, the carbon chain in the dodecanoic acid, the glyceryl stearate, the glyceryl tricaprylate and the soybean lecithin is an action part, the aspartic acid is a binding part, the aspartic acid, the soybean lecithin choline phosphate part, the gum arabic and the tragacanth are water-soluble parts, and a compound (liposome nano-emulsion) is formed by hydrogen bond or van der Waals force.
Referring to the experimental procedure for the microorganisms in example 19, the results in the following table are obtained, the virucidal and bactericidal power > 99% at a complex concentration of 0.05mM in the emulsion.
TABLE 15 Liposome (0.05mM) virucidal Properties (1h)
Figure BDA0003628692260001521
TABLE 16 Bactericidal and antibacterial Properties of liposomes (0.05mM) (2h)
Figure BDA0003628692260001531
TABLE 17 fungicidal Performance of liposomes (0.05mM) (1h)
Figure BDA0003628692260001532
Example 23 lecithin conjugate injection of fatty acids and derivatives (non-covalent conjugation of active moiety + Water soluble moiety + binding moiety)
(1) Liposome of pentacosane
0.7g of carboxylated lecithin (purchased from Aladdin reagent company), 0.3g of beta-sitosterol, 0.05g of glycocholic acid sulfate and 2g of pentacosanoic acid, 2ml of ethanol is added, the mixture is heated and stirred in a water bath at 50 ℃ and dissolved, the ethanol is removed by rotary evaporation, physiological saline and a surfactant tween80 are added, the mixture is stirred and dispersed uniformly, homogenized for 5min to obtain colostrum, and then the mixture is subjected to ultrasound treatment for 30min to obtain the nanometer liposome emulsion carrying the pentacosanoic acid.
The prepared pentacosanic acid liposome is a water-in-oil-in-water structure of a water-soluble liposome, glycocholic acid sulfate is an acting part and a combining part, wherein carboxyl and sulfonic group on glycocholic acid sulfonate are combining groups, and a cholestane skeleton is an acting group; the carbon chain in the pentacosanic acid and the phospholipid molecule is an acting group, the phosphate group in the phospholipid molecule is a water-soluble group, and a compound (the nanometer liposome for wrapping the pentacosanic acid) is formed through hydrogen bonds and Van der Waals force.
(2) Liposome of fatty acid ethyl ester
Water phase: tween-8020 mg for injection and 10ml of water;
oil phase: 0.7g of aminated lecithin, 0.3g of cholesterol and 0.72mmol of fatty acid ethyl ester.
Adding the water phase into the oil phase, stirring for 10-20min, and subjecting to ultrasonic treatment (320W).
The fatty acid may be medium-chain caproic acid (ethyl caproate), enanthic acid, caprylic acid, pelargonic acid, capric acid, stearic acid, oleic acid, linoleic acid, linolenic acid, eicosapentaenoic acid, docosahexaenoic acid, etc.
FIG. 42 is an image of eicosapentaenoic acid ethyl ester liposome under a transmission electron microscope, FIG. 43 is a particle size distribution measured by a Malvern particle sizer of the eicosapentaenoic acid ethyl ester liposome, in which it can be seen that 80% or more of the particle size distribution is around 100nm, a small portion is between 20-60nm and 140-200nm, and the dispersion coefficient is 0.162, indicating good dispersibility in water; FIG. 44 shows the potential distribution of eicosapentaenoic acid ethyl ester liposome measured by Malvern particle sizer, the potential average value was-25 mV, and the product stability was good. And after being placed for 6 months at normal temperature, the appearance character still keeps stable, which indicates that the liposome emulsion has good stability.
In this embodiment, the fatty acid ethyl ester liposome is a water-soluble liposome and a water-in-oil-in-water structure, wherein the fatty acid ethyl ester, cholesterol and fatty acyl carbon chains in phospholipid molecules are an active part, the aminated phospholipid is a binding part and a water-soluble part, amino groups in the molecules are binding groups, phosphate groups in the molecules are water-soluble groups, and a complex (fatty acid ethyl ester-encapsulated nanoliposome) is formed through hydrogen bonds and van der waals forces.
Referring to the experimental procedure of the microorganisms in example 19, the results shown in tables 18 and 19 below were obtained, and the virucidal and bactericidal activity of > 99% was obtained at an eicosapentaenoic acid concentration of 0.05% in the liposomes.
TABLE 18 virucidal Properties (1h) of liposomes of pentacosanic acid (0.05%)
Testing viruses Virucidal Rate (%)
H7N9 pseudovirus ＞99.9
H5N1 pseudovirus ＞99.9
HIV pseudovirus ＞99.9
New coronavirus ＞99.9
TABLE 19 Bactericidal and antibacterial Properties of Liposome of eicosapentaenoic acid (0.05%) (2h)
Testing microorganisms Fungicidal ratio (%)
Escherichia coli >99
Staphylococcus aureus (Staphylococcus aureus) >99
Methicillin-resistant staphylococcus aureus >99
Streptococcus pneumoniae >99
Klebsiella pneumoniae >99
Pseudomonas aeruginosa >99
Example 24 evaluation of unsaturated fatty acid-polypeptide Dry powder inhaler (action part + polypeptide targeting binding moiety/Water-soluble part) Performance
Preparation of unsaturated fatty acid-SBP 1 complex was carried out according to example 10.
The prepared compound freeze-dried powder can be used as a dry powder inhalant for animal experiments after being crushed.
For example, docosahexaenoic acid-SBP 1, the transmission electron microscope image thereof is shown in FIG. 45, and the particle size distribution measured by a Malvern particle sizer is shown in FIG. 46. FIG. 47 is a photograph of the products of grafting of the polypeptide SBP1 with different omega-3 fatty acids (ALA: linolenic acid, EPA: eicosapentaenoic acid, DHA: docosahexaenoic acid).
In this example, SBP1 is both a water soluble moiety and a targeting binding moiety, with the fatty acid carbon chain being the active moiety.
Referring to the experimental procedure of the microorganisms in example 19, the results shown in Table 20 below were obtained, and the virucidal and bactericidal activity was > 99% at a concentration of 0.035mM of omega-3 fatty acid-SBP 1.
TABLE 20 virucidal Performance of omega-3 fatty acid-SBP 1(0.035mM) (1h)
Figure BDA0003628692260001561
TABLE 21 antibacterial and fungicidal Properties of omega-3 fatty acid-SBP 1(0.035mM) (2h)
Figure BDA0003628692260001562
Example 25 evaluation of the Properties of lyophilized powder for injection (active moiety + targeting binding moiety/Water-soluble moiety) of fatty acid-CD 14
Fatty acid-CD 14 compound preparation referring to example 11, the prepared lyophilized powder can be used for fatty acid-CD 14 compound injection of animal experiments, and is stored at 4 ℃ for later use. When in use, the composition is dissolved in normal saline, and filtered and sterilized by a 0.22um sterile filter membrane.
FIGS. 48, 49, and 50 are TEM images of CD14 protein grafted dodecenoic acid, decatetraenoic acid, and eicosapentaenoic acid, respectively. As can be seen in the figure, the particle size of the lyophilized powder preparation which is reconstituted after freeze-drying is below 100nm, and the dispersibility is good.
In this example, CD14 is both a water soluble moiety and a targeting binding moiety, with the fatty acid carbon chain being the active moiety.
Referring to the experimental procedure of the microorganisms in example 19, the results shown in Table 22, Table 23 and Table 24 below were obtained, and the virucidal and bactericidal activity was > 99% at a fatty acid-CD 14 concentration of 0.035 mM.
TABLE 22 virucidal Performance of fatty acid-CD 14(0.035mM) (1h)
Figure BDA0003628692260001571
TABLE 23 Bactericidal and antibacterial Properties of fatty acid-CD 14(0.035mM) (2h)
Figure BDA0003628692260001572
TABLE 24 fungicidal Properties (2h) of fatty acid-CD 14(0.035mM)
Figure BDA0003628692260001581
Example 26 (eight carbon unsaturated carbon chain-threonine) Complex oral formulations (active moiety + Small molecule Water soluble moiety/binding moiety) Performance evaluation
Capsule
Taking starch and cyclodextrin, drying, crushing, sieving by a 120-mesh sieve, and mixing the starch and the cyclodextrin according to a mass ratio of 1: 1-1: 5, uniformly mixing, adding crushed and sieved eight-carbon unsaturated carbon chain-threonine powder, and uniformly grinding, wherein the mass ratio of the medicine to the auxiliary materials is 0.1: 2-1: 2, subpackaging into capsules.
In this example, the eight carbon unsaturated carbon chain can be considered to be the acting moiety and threonine can be both the water soluble moiety and the binding moiety, with the carboxyl group being considered to be the binding moiety.
Example 27 (Octadecane saturated carbon chain-serine) Complex oral formulations (active moiety + Small molecule Water soluble moiety/binding moiety)
Capsule
Taking starch, sucrose and cyclodextrin, drying, crushing, sieving by a 120-mesh sieve, and mixing the starch, the sucrose and the cyclodextrin according to a mass ratio of 1: 0.1: 1-1: 10: 10, mixing evenly, adding octadecane saturated carbon chain-threonine, grinding evenly, completely wrapping the medicine by the auxiliary materials, wherein the mass ratio of the medicine to the auxiliary materials is 0.1:2-1:2, and subpackaging into capsules.
In this example, the eighteen carbon saturated carbon chain can be considered as the active moiety and serine can be both the water soluble moiety and the binding moiety, with the carboxyl group being considered as the binding moiety.
EXAMPLE 28 preparation of carboxy-eight carbon saturated carbon chain-5' -adenosine monophosphate-four carbon unsaturated carbon chain-carboxy Complex (carboxy + four carbon unsaturated carbon chain + Small molecule Water soluble moiety + eight carbon saturated carbon chain + carboxy) oral formulation of the Complex
Tablet formulation
Preparation of carboxyl-eight carbon saturated carbon chain-5' -monophosphoadenosine-four carbon unsaturated carbon chain-carboxyl complex: weighing 1mmol of suberic acid and 1mmol of EDC, activating carboxyl for 10min under magnetic stirring, and adding 1mmol of DMAP (4-dimethylaminopyridine); weighing 1mmol of adenosine 5' -monophosphate monosodium, dissolving in 0.5ml of water, gradually adding 5' -monophosphate monosodium solution into suberic acid drop by drop, continuously stirring for reaction for 12h, adding activated (referring to an suberic acid activation method) 1mmol of butenedioic acid, continuously stirring for reaction for 12, precipitating with acetone, removing the solvent in vacuum, and removing the catalyst in a purification mode of column chromatography gradient elution to obtain a carboxyl-eight-carbon saturated carbon chain-5 ' -monophosphate adenosine-four-carbon unsaturated carbon chain-carboxyl compound; wherein the purification process is carried out by: and after the reaction is finished, carrying out chromatographic purification on the reaction product solution, adding the reaction solution into a Shephadex G10 chromatographic column (phi 26mm multiplied by 50cm), eluting with normal saline, carrying out flow rate of 50ml/h, collecting eluent step by step, detecting absorbance at the wavelength of 260nm by using an ultraviolet spectrophotometry, and combining the 1 st elution peak to obtain the reaction product, namely the carboxyl-eight-carbon saturated carbon chain-5' -monophosphonic adenosine-four-carbon unsaturated carbon chain-carboxyl compound.
Drying starch and cyclodextrin, pulverizing, sieving with 120 mesh sieve, mixing at a mass ratio of 1:1-1:5, and adding water to obtain 10% starch paste; adding 1-5g of the compound, 2.5g of hydroxypropyl methylcellulose and 8g of starch, mixing the materials by an equivalent progressive addition method, and sieving by a 80-mesh sieve to obtain uniform granules. Then adding the 10% starch paste into the particulate material, and continuously extruding and kneading by hands to fully mix the material and the starch slurry to prepare a soft material. And extruding and kneading the prepared soft material in a 16-mesh nylon sieve to obtain proper particles. The prepared granules were placed in an oven for drying. Sieving the dried granules again, weighing 1-3g of magnesium stearate, adding into the dried granules, mixing to obtain suitable granules, adding the granules into a tablet machine for tabletting to obtain the tablet of the carboxyl-eight-carbon saturated carbon chain-5' -adenosine monophosphate-four-carbon unsaturated carbon chain-carboxyl compound.
Wherein the eight-carbon saturated carbon chain and the four-carbon unsaturated carbon chain are both an active part and a binding part, wherein carboxyl is a binding group, and 5' -adenosine monophosphate is a water-soluble part.
The proportions in the above method can be continuously optimized and improved in the process.
Example 29 cytotoxicity validation (carboxy + saturated/unsaturated carbon chain + small molecule Water soluble moiety)
Preparation of amino acid complexes
Respectively weighing 1mmol of fatty acid (including n-octanoic acid, linoleic acid, linolenic acid, eicosapentaenoic acid and docosahexaenoic acid) and 1mmol of amino acid (including serine, threonine and lysine), respectively adding 1mmol of EDC into the fatty acid, activating carboxyl for 5min under magnetic stirring, adding 1mmol of NHS, stirring for 5min, dissolving the amino acid in 0.5ml of water, slowly dropwise adding an amino acid solution into the activated fatty acid, continuously stirring for reaction for 12h, carrying out chromatographic purification on a reaction product solution after the reaction is finished, adding the reaction solution into a Shephadex G10 chromatographic column (phi 26mm multiplied by 50cm), eluting with physiological saline at the flow rate of 50ml/h, collecting eluent step by step, detecting by an indetrione method, and combining the 1 st elution peak to obtain the amino acid compound for subsequent experiments.
Preparation of adenylic acid Complex
Weighing 1mmol of each of n-octanoic acid, 5 '-adenosine monophosphate monosodium salt, butenedioic acid, EDC and DMAP (4-dimethylaminopyridine), reacting according to the synthesis method in example 28, after the reaction is finished, carrying out chromatographic purification on a reaction product solution, adding the reaction solution into a Shephadex G10 chromatographic column (phi 26mm multiplied by 50cm), eluting with normal saline, carrying out flow rate of 50ml/h, collecting eluent step by step, detecting absorbance at a wavelength of 260nm by using an ultraviolet spectrophotometry, and combining the 1 st elution peak to obtain a reaction product, thus obtaining an octanoic acid-adenosine monophosphate-butenedioic acid complex (namely eight-carbon saturated carbon chain-5' -adenosine monophosphate-four-carbon unsaturated carbon chain-carboxyl) for subsequent experiments.
Preparation of N-methylglucamine complexes
Respectively weighing 1mmol of N-octanoic acid, N-nonanoic acid, N-methylglucamine, EDC and NHS, reacting according to an amino acid compound preparation method, after the reaction is finished, carrying out chromatographic purification on a reaction product solution, adding a reaction solution into a Shephadex G10 chromatographic column (phi 26mm multiplied by 50cm), eluting with normal saline at a flow rate of 50ml/h, collecting eluent step by step, detecting by a phenol-sulfuric acid method, combining the 1 st elution peak to obtain a reaction product, and obtaining the N-methylglucamine compound for subsequent experiments.
The prepared complex is used for cytotoxicity verification.
An eight carbon saturated carbon chain-threonine, wherein the eight carbon saturated carbon chain can be regarded as the acting moiety, threonine as the water soluble moiety, and the carboxyl group and amino group in the threonine molecule can be regarded as the binding moiety. The cytotoxicity results are shown in FIG. 51, which shows that the effect of the compound on cells from the concentration of 9mM to 0.035mM is less than 10%, and the safety is good;
an eight carbon saturated carbon chain-serine, wherein the eight carbon saturated carbon chain (octanoic acid carbon chain) can be regarded as an acting part, the serine can be regarded as a water-soluble part, and carboxyl and amino in a serine molecule can be regarded as binding parts. The cytotoxicity results are shown in FIG. 52, which shows that the effect of the compound on cells from the concentration of 9mM to 0.035mM is less than 10%, and the safety is good;
Octadecane monounsaturated carbon chain-serine, wherein, the octadecane monounsaturated carbon chain (oleic acid carbon chain) can be regarded as the acting part, the serine can be regarded as the water-soluble part, and the carboxyl and amino in the serine molecule can be regarded as the binding part. The cytotoxicity results are shown in figure 53, which shows that the effect of the compound on cells from the concentration of 9mM to 0.035mM is less than 10%, and the safety is good;
octadecane monounsaturated carbon chain-threonine, in which octadecane monounsaturated carbon chain (oleic acid carbon chain) can be regarded as the acting moiety, threonine as the water-soluble moiety, and carboxyl group and amino group in threonine molecule can be regarded as the binding moiety. The cytotoxicity results are shown in figure 54, which shows that the effect of the compound on cells from the concentration of 4.5mM to 0.035mM is less than 10 percent, and the safety is good;
the docosapolyunsaturated carbon chain-threonine, wherein the docosapolyunsaturated carbon chain (the carbon chain of docosahexaenoic acid) can be regarded as the acting moiety, threonine as the water-soluble moiety, and the carboxyl group and amino group in the threonine molecule can be regarded as the binding moiety. The cytotoxicity results are shown in figure 55, which shows that the influence of the compound on cells from the concentration of 4.5mM to 0.035mM is less than 10 percent, and the safety is good;
the docosapolyunsaturated carbon chain-serine, wherein the docosapolyunsaturated carbon chain (the carbon chain of docosahexaenoic acid) can be regarded as the active moiety, serine can be regarded as the water-soluble moiety, and carboxyl and amino groups in the serine molecule can be regarded as the binding moiety. The cytotoxicity results are shown in figure 56, which shows that the effect of the compound on cells from the concentration of 4.5mM to 0.035mM is less than 10%, and the safety is good;
Eighteen-carbon monounsaturated carbon chain-lysine, wherein the eighteen-carbon monounsaturated carbon chain (oleic acid carbon chain) can be used as an acting part, lysine can be used as a water-soluble part, and carboxyl and amino in lysine molecules can be used as binding parts. The cytotoxicity results are shown in figure 57, which shows that the effect of the compound on cells from the concentration of 4.5mM-0.035mM is less than 1%, and the safety is good;
the docosahexenoic acid chain is lysine, wherein the docosahexenoic acid chain (the carbon chain of docosahexaenoic acid) can be regarded as an acting part, lysine can be regarded as a water-soluble part, and carboxyl and amino in a lysine molecule can be regarded as binding parts. The cytotoxicity results are shown in figure 58, which shows that the effect of the compound on cells from the concentration of 4.5mM to 0.035mM is less than 10 percent, and the safety is good;
the octadecane polyunsaturated carbon chain-threonine, wherein the octadecane polyunsaturated carbon chain (oleic acid carbon chain) can be regarded as the acting moiety, threonine as the water-soluble moiety, and the carboxyl group and amino group in the threonine molecule can be regarded as the binding moiety. The cytotoxicity results are shown in figure 59, which shows that the effect of the compound on cells from the concentration of 4.5mM to 0.035mM is less than 10 percent, and the safety is good;
Eight carbon saturated carbon chain-5 '-adenosine monophosphate-four carbon unsaturated carbon chain-carboxyl, wherein the eight carbon saturated carbon chain and the four carbon unsaturated carbon chain can be regarded as an active part, the 5' -adenosine monophosphate serves as a water soluble part, and the carboxyl can be regarded as a binding part. The cytotoxicity results are shown in figure 60, which shows that the influence of the compound on cells from the concentration of 4.5mM to 0.035mM is less than 10 percent, and the safety is good;
N-octyl-N-methylglucamine, wherein an eight carbon saturated carbon chain can be considered as the active moiety, methylglucamine as the water soluble moiety and the hydroxyl group in the methylglucamine molecule can be considered as the binding moiety. The cytotoxicity results are shown in figure 61, which shows that the effect of the compound on cells from the concentration of 9mM to 0.035mM is less than 10 percent, and the safety is good;
N-nonyl-N-methylglucamine, wherein the nine carbon saturated carbon chain can be considered as the active moiety, methylglucamine as the water soluble moiety and the amino group in the methylglucamine molecule can be considered as the binding moiety. The cytotoxicity results are shown in FIG. 62, which shows that the effect of the compound on the cells (VERO E6 cells) from the concentration of 9mM to 0.035mM is less than 10%, and the safety is good.
EXAMPLE 30 verification of bacteriostatic Properties (carboxy + saturated/unsaturated carbon chain + Small molecule Water-soluble moiety)
In a bacteria inhibition experiment using the compound of example 29, a control solution (containing no fatty acid in the reaction solution, and the same other steps) was prepared according to the procedure of example 1 to exclude the influence of other components (as seen from the inhibition results, the inhibition effect of the control was almost 0, and the influence of other components in the composite product was excluded).
An eight carbon saturated carbon chain-threonine, wherein the eight carbon saturated carbon chain can be regarded as the acting moiety, threonine as the water soluble moiety, and the carboxyl group and amino group in the threonine molecule can be regarded as the binding moiety. The bacteriostatic results are shown in FIG. 63, in which the inhibitory rate of the control group is 0, the inhibitory rate of the experimental group is greater than 99% at a concentration of 0.035mM, and the bacteriostatic properties are exhibited, and the bacteriostatic rate is 50-99% at a concentration of 0.035mM-0.009mM, and the bacteriostatic properties are exhibited, and the half bacteriostatic concentration is 0.009 mM.
An eight carbon saturated carbon chain-serine, wherein the eight carbon saturated carbon chain can be regarded as an acting part, the serine can be regarded as a water-soluble part, and carboxyl and amino in a serine molecule can be regarded as binding parts. The bacteriostatic results are shown in fig. 64, in which it can be seen that the inhibitory rate of the control group is 0, the bacteriostatic rate of the experimental group is greater than 99% when the concentration is 0.035mM, and the bacteriostatic property is good, and the bacteriostatic rate of the experimental group is 50-99% when the concentration is 0.035mM-0.009mM, and the bacteriostatic property is good, and the half bacteriostatic concentration is 0.009 mM.
Octadecane monounsaturated carbon chain-serine, wherein, the octadecane monounsaturated carbon chain can be regarded as the acting part, the serine can be regarded as the water-soluble part, and the carboxyl and the amino in the serine molecule can be regarded as the binding part. The bacteriostatic results are shown in fig. 65, in which the inhibitory rate of the control group is 0, the bacteriostatic rate of the experimental group is greater than 99% when the concentration of the experimental group is 0.035mM, and the bacteriostatic performance is good, and the bacteriostatic rate of the experimental group is 50-99% when the concentration is 0.035mM-0.009mM, and the bacteriostatic performance is good, and the half bacteriostatic concentration is 0.009 mM.
Eighteen carbon monounsaturated carbon chain-threonine, in which the eighteen carbon unsaturated carbon chain can be regarded as the acting moiety, threonine as the water-soluble moiety, and the carboxyl group and amino group in the threonine molecule can be regarded as the binding moiety. The bacteriostatic results are shown in fig. 66, in which it can be seen that the inhibitory rate of the control group is 0, the bacteriostatic rate of the experimental group is greater than 99% when the concentration is 0.035mM, and the bacteriostatic property is good, and the bacteriostatic rate is 50-99% when the concentration is 0.035mM-0.009mM, and the bacteriostatic property is good, and the half bacteriostatic concentration is 0.009 mM.
A docosapolyunsaturated carbon chain-threonine, in which the docosapolyunsaturated carbon chain can be regarded as the active moiety, threonine as the water-soluble moiety, and the carboxyl and amino groups of the threonine molecule can be regarded as the binding moiety. The bacteriostatic results are shown in fig. 67, in which the inhibitory rate of the control group is 0, the bacteriostatic rate of the experimental group is greater than 99% when the concentration of the experimental group is 0.035mM, and the bacteriostatic performance is good, and the bacteriostatic rate of the experimental group is 50-99% when the concentration is between 0.035mM and 0.009mM, and the bacteriostatic performance is good, and the half bacteriostatic concentration is 0.009 mM.
The twenty-two carbon polyunsaturated carbon chain-serine, wherein, the twenty-two carbon polyunsaturated carbon chain can be regarded as the acting part, the serine is regarded as the water-soluble part, and the carboxyl and amino in the serine molecule can be regarded as the binding part. The bacteriostatic results are shown in fig. 68, in which it can be seen that the inhibitory rate of the control group is 0, the bacteriostatic rate of the experimental group is greater than 99% when the concentration is 0.035mM, and the bacteriostatic property is good, the bacteriostatic rate is 50-99% when the concentration is 0.035mM-0.009mM, and the bacteriostatic property is good, and the half bacteriostatic concentration is 0.009 mM.
Eighteen-carbon monounsaturated carbon chain-lysine, wherein the eighteen-carbon monounsaturated carbon chain can be regarded as an acting part, the lysine can be regarded as a water-soluble part, and carboxyl and amino in a lysine molecule can be regarded as binding parts. The bacteriostatic results are shown in FIG. 69, in which the inhibitory rate of the control group is 0, the inhibitory rate of the experimental group is greater than 99% at a concentration of 0.035mM, and the bacteriostatic properties are exhibited, and the bacteriostatic rate is 50-99% at a concentration of 0.035mM-0.009mM, and the bacteriostatic properties are exhibited, and the half bacteriostatic concentration is 0.009 mM.
A docosaur polyunsaturated carbon chain-lysine, wherein the docosaur polyunsaturated carbon chain can be regarded as an active moiety, lysine as a water-soluble moiety, and carboxyl and amino groups in the lysine molecule can be regarded as binding moieties. The bacteriostatic results are shown in FIG. 70, which shows that the control group has an inhibitory rate of 0, the experimental group has an inhibitory rate of more than 99% at a concentration of 0.035mM, and the control group has bactericidal activity, and has an inhibitory rate of 50-99% at a concentration of 0.035mM-0.009mM, and half of the control group has an inhibitory concentration of 0.009 mM.
Eighteen-carbon polyunsaturated carbon chain-threonine, wherein the eighteen-carbon polyunsaturated carbon chain can be regarded as an acting part, threonine as a water-soluble part, and carboxyl and amino groups in threonine molecules can be regarded as binding parts. The bacteriostatic results are shown in fig. 71, in which it can be seen that the inhibitory rate of the control group is 0, the bacteriostatic rate of the experimental group is greater than 99% when the concentration is 0.035mM, and the bacteriostatic property is good, and the bacteriostatic rate of the experimental group is 50-99% when the concentration is 0.035mM-0.009mM, and the bacteriostatic property is good, and the half bacteriostatic concentration is 0.009 mM.
Eight carbon saturated carbon chain-5 '-monophosphoadenosine-four carbon unsaturated carbon chain-carboxyl, wherein the eight carbon saturated carbon chain and the four carbon unsaturated carbon chain can be regarded as an active part, the 5' -monophosphoadenosine is used as a water soluble part, and the carboxyl can be regarded as a binding part. The bacteriostatic results are shown in FIG. 72, in which the inhibitory rate of the control group is 0, the inhibitory rate of the experimental group is greater than 99% at a concentration of 0.035mM, and the bacteriostatic properties are exhibited, and the bacteriostatic rate is 50-99% at a concentration of 0.035mM-0.009mM, and the bacteriostatic properties are exhibited, and the half bacteriostatic concentration is 0.009 mM.
N-octyl-N-methylglucamine, wherein an eight carbon saturated carbon chain can be considered as the active moiety, methylglucamine as the water soluble moiety and the hydroxyl group in the methylglucamine molecule can be considered as the binding moiety. The bacteriostatic results are shown in FIG. 73, in which the inhibitory rate of the control group is 0, the inhibitory rate of the experimental group is greater than 99% at a concentration of 0.009mM, and the bactericidal activity is exhibited, and the inhibitory rate of the experimental group is 50-99% at a concentration of 0.035mM-0.009mM, and the inhibitory rate is 0.003mM at half of the inhibitory concentration.
N-nonyl-N-methylglucamine, wherein the nine carbon saturated carbon chain is considered to be the active moiety, methylglucamine is the water soluble moiety and the hydroxyl group in the methylglucamine molecule is considered to be the binding moiety. The bacteriostatic results are shown in FIG. 74, in which the inhibitory rate of the control group is 0, the inhibitory rate of the experimental group is greater than 99% at a concentration of 0.009mM, and the bactericidal activity is exhibited, and the inhibitory rate of the experimental group is 50-99% at a concentration of 0.035mM-0.009mM, and the inhibitory rate is 0.003mM at half of the inhibitory concentration.
Further bacteriostatic experiments were carried out with the above compounds (using a concentration of 0.035mM) with reference to example 19, and the results are shown in Table 25, with bacteriostatic efficiency of > 99% for each compound.
TABLE 25
Figure BDA0003628692260001651
EXAMPLE 31 verification of virucidal Properties (carboxy + saturated/unsaturated carbon chain + Small molecule Water-soluble moiety)
The compound of example 29 (used at a concentration of 0.009mM) was subjected to virus neutralization test with reference to the test method of example 19, and the results are shown in Table 26, and the virus neutralization efficiency of each compound was > 99%.
Watch 26
Figure BDA0003628692260001661
Example 32 cytotoxicity of fatty acid-serum Albumin Complex (active moiety + macromolecular Water-soluble moiety/binding moiety)
Fatty acid-serum albumin complex cytotoxicity assay for VERO E6
In this example, toxicity of the linolenic acid-serum albumin (ALA-HSA), eicosapentaenoic acid-serum albumin (EPA-HSA) and docosahexaenoic acid-serum albumin (DHA-HSA) prepared in example 1 to Vero cells was examined.
In Vero cells, the cytotoxicity of fatty acid or its derivative complex was examined. VERO E6 cells (VERO E6: VERO African Green monkey Kidney cells, cat # iCell-c014, manufacturer iCell) at 5X 10 3 Inoculating cells/well into 96-well plate, sealing with sterile water or buffer solution at 37 deg.C and 5% CO for 4 weeks in each well with each well having a size of 100ul 2 Incubated in an incubator for 24h, treated with 72mM, 36mM, 18mM, 9mM, 4.5mM, 2.25mM, 1.12mM, 0.56mM, 0.28mM, 0.14mM, 0.07mM, 0.035mM fatty acid-serum albumin complex, each set of three duplicate wells, 10ul per well, blank controlThe culture medium with the same volume and the medicine 0 is a simple cell control, 10ul of CCK-8 dye solution is added into each hole after the medicine acts for 24h, the culture box is continuously incubated for 1-4h, the OD value is detected at the wavelength of 450nm, the cell survival rate is analyzed, and the safe and non-toxic range is determined when the concentration of the fatty acid is lower than 4.5mM as shown in figure 75 by tests.
In this example, serum albumin is both the water soluble and binding moieties, with the linolenic, eicosapentaenoic and docosahexaenoic acid carbon chains being the active moieties.
Example 33 cytotoxicity of the (active moiety + macromolecular Water-soluble moiety/binding moiety) Complex-fatty acid-serum Albumin Complex
Endothelial cytotoxicity assay for complexes of fatty acids or derivatives thereof
In this example, toxicity of the undecanoic acid-serum albumin (C11-HSA), hexadecenoic acid-serum albumin (C16-HSA), and triacontenoic acid-serum albumin (C30-HSA) prepared in example 1 to hepatocytes was examined.
The cytotoxicity of the fatty acid or derivative complex was examined in hepatocytes (LX-2 (human hepatic stellate cell), cat # CL-0560, manufacturer: Procell Punuisance). Cells were arranged at 5X 10 3 Inoculating cells/well into 96-well plate, sealing with sterile water or buffer solution at 37 deg.C and 5% CO for 4 weeks in each well with each well having a size of 100ul 2 Culturing in an incubator for 24h, respectively treating with 72mM, 7.2mM and 0.72mM fatty acid-serum albumin complex, each group comprises three multiple wells, each well is 10ul, a blank control group is culture medium with the same volume, a 0 drug adding group is simple cell control, after the drug acts for 24h, each well is added with 10ul CCK-8 dye solution, the incubator is continuously incubated for 1-4h, the OD value is detected at the wavelength of 450nm, the cell survival rate is analyzed, and tests show that the safe and nontoxic range is obtained when the fatty acid concentration is lower than 4.5mM, and the cytotoxicity is higher when the carbon chain is longer at high concentration as shown in figure 76.
In this example, serum albumin is both a water-soluble moiety and a binding moiety, with the carbon chains of undecanoic acid, hexadecenoic acid, and triacontenoic acid being the active moieties.
Example 34 cytotoxicity assays (binding moiety + acting moiety + water soluble moiety + binding moiety)
Synthesis of carboxyl-eight-carbon unsaturated carbon chain-taurocholic acid
Weighing 1mmol of 4-octenedioic acid and 1mmol of EDC, activating carboxyl for 10min under magnetic stirring, and adding 1mmol of DMAP (4-dimethylaminopyridine); weighing 1mmol of sodium taurocholate, dissolving the sodium taurocholate in 0.5ml of water, gradually and slowly adding the sodium taurocholate solution into activated 4-octenedioic acid, continuously stirring for reaction for 12h, carrying out chromatographic purification on the reaction mixture solution after the reaction is finished, adding the reaction solution into a Shephadex G10 chromatographic column (phi 26mm multiplied by 50cm), eluting with normal saline, carrying out flow rate of 50ml/h, collecting eluent step by step, detecting by a formaldehyde sulfate color development method, combining the 1 st elution peak to obtain a reaction product, and obtaining a 4-octenedioic acid-taurocholic acid compound (carboxyl-octacarbon unsaturated carbon chain-taurocholic acid) solution for subsequent experiments.
Toxicity assays on VERO E6 cells (VERO African Green monkey Kidney cells, cat # iCell-c014, manufacturer iCell):
preparing a carboxyl-eight-carbon unsaturated carbon chain-taurocholic acid solution. Cytotoxicity was examined in VERO E6 cells. The cells are inoculated into a 96-well plate cell culture plate according to 5 multiplied by 103 cells/well, each well is 100ul, the 96-well plate is sealed by sterile water or buffer solution for 4 weeks, the cells are cultured in a 5% CO2 incubator at 37 ℃ for 24h and are respectively treated by 72mM, 36mM, 18mM, 9mM, 4.5mM, 2.25mM, 1.12mM, 0.56mM, 0.28mM, 0.14mM, 0.07mM and 0.035mM of carboxyl-eight carbon unsaturated carbon chain-taurocholic acid, each group of three multiple wells is 10ul, a blank control group is culture medium with the same volume, 0 drug addition group is a pure cell control, 10ul of CCK-8 dye solution is added into each well after 24h of drug action, the incubator is continuously incubated for 1-4h, the OD value is detected at the wavelength of 450nm, the survival rate of the cells is analyzed, and the non-toxic effect is proved by the test as shown in figure 77, when the concentration of the fatty acid is less than or equal to 4.5mM, the safety range is the VERO E6 cells without obvious toxicity.
In this example, 4-octenedioic acid is the binding moiety, where the free carboxyl group is the binding group of the binding moiety, the eight-carbon unsaturated carbon chain and the cholestane ring structure in the taurocholic acid structure are the contributing moieties, and taurocholic acid is the water-soluble moiety.
Example 35 animal safety test (action part + macromolecular Water-soluble part/binding part)
(1) Animal safety experiments were conducted with the fatty acid-macromolecular water-soluble moiety complexes synthesized in examples 1 and 8 above
The drug is administered to the lung, the experimental group (ALA-HSA group, ALA-HA group, DHA-HSA group and DHA-HA group synthesized in examples 1 and 8) is injected into the tail vein of the mouse at a drug concentration of 562.5uM calculated by fatty acid, the drug is injected into the tail vein of the mouse at 1 ml/time and three times, blood is taken for detection, and the results are shown in a, b, c and d of fig. 78, which respectively show the detection results of liver function, liver/heart function, kidney/liver function and bone/liver and gall function, and the results show that the experimental group (ALA-HSA group, ALA-HA group, DHA-HSA group and DHA-HA group) and the control group have no difference, which indicates that the safety of the compound is good.
Mouse sources used in (2) to (5) below: c57BL/6 Normal grade, 4-6 weeks, female mouse.
(2) Animal safety experiments with the complexes synthesized in example 29
4.5mM of eight-carbon saturated carbon chain-threonine, eight-carbon saturated carbon chain-serine, eighteen-carbon monounsaturated carbon chain-serine and eighteen-carbon monounsaturated carbon chain-threonine are orally administrated (the mice drink water and eat in food, 5mg of medicine is eaten by each mouse every day, and the mice are fed for 5 days), the physiological state of the mice is observed, and the weight, the fur color, the food intake and the mental state are not obviously different from those of the control group of mice, so that the safety is good. Blood sampling tests are performed, as shown in a, b, c and d of fig. 79, which respectively show the results of tests on liver function, liver/heart function, kidney/liver function and bone/liver and gall bladder function, and the results show that the experimental group and the control group have no difference, which indicates that the compound has good safety.
(3) Animal safety experiments with the complexes synthesized in example 29
The physiological state of the mice is observed, and the weight, the fur color, the food intake and the mental state of the mice are not obviously different from those of the control group, so that the safety is good. Blood sampling tests are performed, as shown in a, b, c and d of fig. 80, which respectively show the results of the tests on liver function, liver/heart function, kidney/liver function and bone/liver and gall bladder function, and the results show that the experimental group and the control group have no difference, which indicates that the safety of the compound is good.
(4) Animal safety experiments with the complexes synthesized in example 29
The mice are orally administrated with octadecane polyunsaturated carbon chain-threonine, octane saturated carbon chain-5' -monophosphonic adenosine-tetracarbon unsaturated carbon chain-carboxyl, N-octyl-N-methylglucamine and N-nonyl-N-methylglucamine (drinking water, mixing with food for eating, each mouse takes 5mg of the medicine every day, and feeding is carried out for 5 days), the physiological state of the mice is observed, and the weight, the fur color, the food intake and the mental state of the mice are not obviously different from those of a control group, which indicates that the safety is good. Blood sampling tests are carried out, as shown in a, b, c and d of fig. 81, which respectively show the results of tests on liver function, liver/heart function, kidney/liver function and bone/liver and gall bladder function, and the results show that the experimental group and the control group have no difference, which indicates that the compound has good safety.
(5) Tail vein safety verification of ALA-SBP1, DHA-SBP1, ALA-CD14 and DHA-CD14 compound
The compound of ALA-SBP1, DHA-SBP1, ALA-CD14 and DHA-CD14 is administrated 100ul in tail vein at the concentration of 4.5mM, and is administrated 1 time per day for 5 days in total), the physiological state of the mice is observed, and the weight, the fur color, the food intake and the mental state are not obviously different from those of the mice in a control group, thereby indicating that the safety is good. Blood sampling tests are performed, as shown in a, b, c and d of fig. 82, which respectively show the results of the tests on liver function, liver/heart function, kidney/liver function and bone/liver and gall bladder function, and the results show that the experimental group and the control group have no difference, which indicates that the safety of the compound is good.
(6) Fresh blood (2.0mL) of healthy rats is collected in a centrifuge tube (the tube wall is coated with heparin in advance), and after centrifugation (4000rpm, 10min) at 4 ℃, the obtained precipitate is erythrocytes. The red blood cell pellet was diluted 10-fold with sterile PBS. 0.6mL of the red blood cell suspension was added with 0.4mL of the complex at different concentrations (dispersed with PBS) to give final concentrations of 0.56mM, 1.12mM, 2.25mM, 4.5mM, 9mM, 18mM, and gently pipetted uniformly and incubated at 37 ℃ for 6 h. Distilled water with the same volume is used as a positive control, and normal saline is used as a negative control group. After the incubation, each sample was centrifuged at 4000rpm for 10min, photographed, and 200. mu.L of the supernatant was transferred to a 96-well plate, and its absorbance at 560nm was measured with a microplate reader to calculate the hemolysis rate. As shown in fig. 83, the hemolysis rate increased slightly with increasing concentration, but all were within the normal range (5%). Wherein, the rat of hemolysis experiment described above: SD rats, normal grade 200-250 g, male.
EXAMPLE 36 detection of the antiviral Activity of the fatty acid serum albumin Complex against the novel coronavirus pseudovirus (active moiety + macromolecular Water-soluble moiety/binding moiety)
Antiviral activity assay Using linolenic acid-serum Albumin (ALA-HSA), eicosapentaenoic acid-serum Albumin (EPA-HSA), and docosahexaenoic acid-serum Albumin (DHA-HSA) synthesized in example 1 above
In this example, serum albumin is both a macromolecular water-soluble moiety and a binding moiety, with fatty acids as the active moiety.
Adding 260 mu L of high-pressure sterilized water into 36 holes on the periphery of the 96-hole plate, and sealing the edges to reduce errors caused by the evaporation of a culture medium in the edge holes; fatty acid-serum albumin at concentrations of 2.25mM, 1.12mM, 0.56mM, 0.28mM, 0.14mM, 0.07mM for sample detection, DMEM complete medium 150. mu.L/well (cell control CC) and DMEM complete medium 100. mu.L/well (virus control VC) in column 2; new coronaviruses [ b.1.526.2, purchased from beijing tiantan pharmaceutical biotechnology development company, No. with DMEM complete medium: 80062, the pseudovirus system carries Firefly luciferase (Firefly luciferase) reporter gene, and the luminescent value of the pseudovirus can be detected by a fluorogenic substrate after the pseudovirus infects cells, so as to determine the virus amount of the infected cells. The pseudovirus takes Vesicular Stomatitis Virus (VSV) as a framework, is embedded with a new coronavirus Spike protein on the surface, and can simulate a euvirus and a receptorIn the process of body combination and further entering cells, the sample with neutralizing activity loses the capability of infecting the cells after reacting with pseudoviruses, whether the sample has neutralizing activity can be judged by detecting the luminous values of the control hole and the sample hole, and the effective acting concentration of the sample, namely the EC50 value can be calculated. The pseudovirus can not be autonomously replicated, only has single-round infection capacity, low biological safety level and simple and convenient operation, and is a common means for evaluating the existing vaccines. Dilute to 1.3-2.3X 10 4 Adding 50 mu L of TCID50/mL to each hole of the 3 rd to 11 th rows; placing the 96-well plate in a cell incubator (37 ℃, 5% CO2) to incubate for 1 h; after incubation for 30min, digestion of Vero cells was started, diluting the cell concentration to 2X 10 5 Per mL; after the incubation was completed, 100. mu.L of cells were added to each well so that the cells per well became 2X 10 4 A plurality of; culturing in a 5% CO2 cell culture box at 37 deg.C for 24 hr; after the culture is finished, sucking and removing 250 mu L of supernatant, adding 100 mu L of luciferase detection reagent, repeatedly blowing and beating after reacting for 2min in a dark place at room temperature, and transferring 100 mu L of liquid to a white board; reading luminescence value (RLU) using multifunctional imaging microplate reader
The neutralization inhibition rate was calculated by the following inhibition rate formula E1:
Figure BDA0003628692260001711
meanwhile, a control solution (no fatty acid was added in the reaction of the solution, and the other steps were the same) was prepared according to the procedure of example 1 to exclude the influence of other components (as can be seen from the results of virus inhibition, the inhibitory effect of the control was almost 0, and the influence of other components in the complex product could be excluded).
The neutralization inhibition rate results are shown in FIG. 84, which shows that the neutralization inhibition rate of the control group is 0, the neutralization inhibition rate of the experimental group is 50% -60% when the concentration of ALA-HSA, EPA-HSA and DHA-HSA is 0.009mM, and the specific antibacterial and bactericidal results are summarized in Table 27.
TABLE 27 fatty acid-serum Albumin antibacterial Properties (2h)
Figure BDA0003628692260001721
Example 37 Effect of docosahexaenoic acid-serum Albumin Cyclodextrin encapsulation on rabies pseudoVirus (active moiety + Water-soluble macromolecule group/binding moiety)
Referring to the method of example 1, the docosahexaenoic acid-serum albumin complex is prepared, the prepared complex and the cyclodextrin are uniformly ground, and the mass ratio of the docosahexaenoic acid-serum albumin complex to the cyclodextrin is 1: 1-1: 10, the granules obtained by adopting a low-temperature drying or spray drying method, namely the oral preparation of the cyclodextrin inclusion compound, can also be filled into hard capsules. The auxiliary materials can also be other conventional auxiliary materials for oral preparations.
In this example, serum albumin and cyclodextrin are the water-soluble portion of the macromolecule, while serum albumin is also the binding portion, and docosahexaenoic acid is the active portion.
The antiviral activity of rabies pseudovirus CVS-11 (purchased from Beijing Temple pharmaceutical biotechnology development company, number 80052) was tested with the docosahexaenoic acid-serum albumin complex.
Meanwhile, a control solution (no hexacosenoic acid is added in the reaction of the solution, and other steps are the same) is prepared according to the same method steps to eliminate the influence of other components (as can be seen from the virus inhibition result, the virus inhibition effect of the control is almost 0, and the influence of other components in the compound product can be eliminated).
The method is the same as that of example 36, a multifunctional imaging microplate reader is used for reading a luminescence value (RLU), the result of the neutralization inhibition rate is shown in figure 85, the neutralization inhibition rate of the rabies pseudoviruses CVS-11 in the control group is 0, and the concentration of the rabies pseudoviruses CVS-11 half inhibition is shown in the experimental group to be 0.009 mM.
Example 38 detection of the antiviral Activity of the docosahexaenoic acid-SBP 1 Complex against New coronaviruses (action part + polypeptide targeting binding part/Water-soluble part)
In this example, SBP1 is both a polypeptide targeting binding moiety and a water soluble moiety, with docosahexaenoic acid as the active moiety.
The docosahexaenoic acid-SBP 1 complex prepared according to the method of example 10. The new coronaviruses [ B.1.526.2 ] were treated with the docosahexaenoic acid-SBP 1 complex, purchased from Beijing Temple pharmaceutical biotechnology development company, and numbered: 80062, the pseudovirus system carries Firefly luciferase (Firefly luciferase) reporter gene, and the luminescent value of the pseudovirus can be detected by a fluorescent substrate after infecting the cells to determine the virus amount of the infected cells. The pseudo virus takes Vesicular Stomatitis Virus (VSV) as a framework, a new corona virus Spike protein is embedded on the surface of the VSV, the process that a euvirus is combined with a receptor and then enters a cell can be simulated, a sample with neutralizing activity loses the capability of infecting the cell after the sample acts on the pseudo virus, whether the sample has the neutralizing activity can be judged by detecting the luminous values of a control hole and a sample hole, and the effective action concentration of the sample, namely the EC50 value can be calculated. The pseudovirus can not be autonomously replicated, only has single-round infection capacity, is low in biological safety level and simple and convenient to operate, and is a common means for evaluating the existing vaccines. Antiviral activity assay.
Meanwhile, a control solution (the reaction of the solution does not contain docosahexaenoic acid, and other steps are the same) is prepared to eliminate the influence of other components (as can be seen from the virus inhibition result, the inhibition effect of the control is almost 0, and the influence of other components in the compound product can be eliminated).
The method is the same as example 36, the luminescence value (RLU) is read by a multifunctional imaging microplate reader, the result of the neutralization inhibition rate is shown in FIG. 86, the neutralization inhibition rate of the control group is 0, the concentration of half inhibition (neutralization inhibition rate is 50%) of the experimental group is 0.009mM, and the specific antibacterial and bactericidal results are summarized in Table 28.
TABLE 28 Bactericidal and antibacterial Properties of the docosahexaenoic acid-SBP 1 Complex (2h)
Testing microorganisms Percent sterilization rate (%)
Escherichia coli >99
Staphylococcus aureus >99
Staphylococcus aureus (Staphylococcus aureus) >99
Streptococcus pneumoniae >99
Klebsiella pneumoniae >99
Pseudomonas aeruginosa >99
Example 39 Effect of caproic acid-hyaluronic acid Complex on HIV pseudovirus HIV18A-41 (short carbon chain active moiety + macromolecular Water soluble moiety/binding moiety)
In this example, hyaluronic acid is both a water-soluble portion and a binding portion of the macromolecule, and the short carbon chain portion of caproic acid serves as an active portion.
Test of antiviral Activity of HIV pseudoVirus HIV18A-41 with caproic acid-hyaluronic acid Complex prepared in example 12
Meanwhile, a control solution (caproic acid is not added in the solution reaction, and other steps are the same) is prepared to eliminate the influence of other components (as can be seen from the virus inhibition result, the inhibition effect of the control is almost 0, and the influence of other components in the compound product can be eliminated).
The method is the same as example 36, the luminescence value (RLU) is read by a multifunctional plate reader, the neutralization inhibition rate result is shown in FIG. 87, the neutralization inhibition rate of the control group is 0, the neutralization inhibition rate of the experimental group at the concentration of 1.12mM is 50% for HIV pseudovirus HIV18A-41, and the half inhibition concentration is 1.12 mM.
Example 40 Effect of N-nonanoic acid-hyaluronic acid Complex on influenza pseudovirus H7N9-Fluc (short carbon chain acting moiety + macromolecular Water-soluble moiety/binding moiety)
In this example, hyaluronic acid is both a water-soluble portion and a binding portion of the macromolecule, and the short carbon chain portion of pelargonic acid serves as an active portion.
The influenza pseudovirus H7N9-Fluc was tested for antiviral activity with the N-nonanoic acid-hyaluronic acid complex prepared in example 12.
Meanwhile, a control solution (solution reaction without adding n-nonanoic acid, and other steps are the same) was prepared to exclude the influence of other components.
As in example 36, the results of neutralization inhibition by luminescence value (RLU) reading using a microplate reader for multi-purpose imaging are shown in FIG. 88, in which neutralization inhibition is 0 in the control group, 54% in the test group at a concentration of 0.009mM, and 0.009mM in the half-inhibitory concentration, respectively, against H7N9-Fluc pseudovirus.
EXAMPLE 41 detection of the antiviral Activity of octadecanoic acid-serum albumin Complex against HIV pseudovirus (Long chain carbon chain acting moiety + macromolecular Water-soluble moiety/binding moiety)
In this embodiment, serum albumin is not only a water-soluble portion of macromolecule, but also a binding portion, and a long carbon chain portion of octadecanoic acid serves as an acting portion.
Reference example 1 preparation of octadecanoic acid-serum albumin complex antiviral activity test for HIV pseudovirus.
Meanwhile, a control solution (the solution reaction was carried out without adding octadecanoic acid, and the other steps were the same) was prepared to exclude the influence of other components.
The method is the same as example 36, the luminescence value (RLU) is read by a multifunctional microplate reader, the results of neutralization inhibition are shown in FIG. 89, the neutralization inhibition of the control group is 0, the neutralization inhibition of the test group at the concentration of 0.009mM is 52% for the HIV pseudovirus, and the concentration of half inhibition is 0.009 mM.
Example 42 Effect of hyaluronic acid coupled eicosanoic acid Complex on H7N9-Fluc pseudovirus (long carbon chain acting portion + macromolecular Water soluble portion/binding portion)
In this example, the hyaluronic acid macromolecule is both a water-soluble portion and a binding portion of the macromolecule, with the long carbon chain portion of eicosanoic acid serving as the active portion.
The eicosanoic acid-hyaluronic acid complex prepared in example 12 was tested for antiviral activity against H7N9-Fluc pseudovirus. Meanwhile, a control solution (the eicosanoic acid was not added in the solution reaction, and other steps were the same) was prepared to exclude the influence of other components.
The method is the same as example 36, the luminescence value (RLU) is read by a multifunctional microplate reader, the results of neutralization inhibition are shown in FIG. 90, the neutralization inhibition of the control group is 0, the neutralization inhibition of the experimental group is 53% at the concentration of 0.009mM, and the neutralization inhibition of the H7N9-Fluc pseudovirus is 0.009mM at the concentration of half inhibition.
EXAMPLE 43 Effect of octacosanoic acid-serum Albumin Complex on influenza pseudovirus H5N1-Fluc (Long chain carbon chain acting moiety + macromolecular Water-soluble moiety/binding moiety)
In this example, serum albumin is both a water-soluble portion of the macromolecule and a binding portion, with the long carbon chain portion of octacosanoic acid serving as the active portion.
The octacosanoic acid-serum albumin complex prepared by the method of reference example 1 was tested for antiviral activity against influenza pseudovirus H5N 1-Fluc.
A control solution (no octacosanoic acid was added to the solution reaction, and the other steps were the same) was also prepared to exclude the influence of other components.
The method was the same as example 36, and the light emission values (RLU) were read using a multi-functional microplate reader, and the results of the neutralization inhibition ratios are shown in FIG. 91, the neutralization inhibition ratio in the control group was 0, and the neutralization inhibition ratio in the test group at a concentration of 0.009mM against influenza pseudovirus H5N1-Fluc was 53%, and it was presumed that the half-inhibitory concentration was around 0.009 mM. The compound is used for bacteriostasis experiments, and specific results are summarized in table 29.
TABLE 29 Bactericidal and antibacterial Properties of octacosanoic acid-serum albumin complex (0.018mM) (2h)
Testing microorganisms Sterilizing rate (%)
Escherichia coli >99
Staphylococcus aureus >99
Methicillin-resistant staphylococcus aureus >99
Streptococcus pneumoniae >99
Klebsiella pneumoniae >99
Pseudomonas aeruginosa >99
EXAMPLE 44 Effect of serum Albumin coupling of omega-3 fatty acid Complex on HIV-derived lentiviruses (Long chain carbon chain acting moiety + macromolecular Water soluble moiety/binding moiety)
Effect of the linolenic acid-serum Albumin (ALA-HSA), eicosapentaenoic acid-serum Albumin (EPA-HSA), and docosahexaenoic acid-serum Albumin (DHA-HSA) tests prepared in example 1 on HIV-derived lentivirusesCells (LX-2 (human hepatic stellate cell), cat # CL-0560, Procell Punuisal) at 2X 10 4 Cells/well were plated in 96-well plates and incubated at 37 ℃ in a 5% CO2 incubator for 24 h. The virus was pretreated with a 2.25mM fatty acid serum albumin complex, incubated for 1h, added to cells in a 96-well plate, replaced with complete medium after 12-16h, and cultured for another 48h to observe cell fluorescence as shown in A, B, C and D of FIG. 92, which are the case of virus-infected cells in control group A (untreated group), ALA-HSA group B after 1 hour treatment, EPA-HSA group C after 1 hour treatment, and DHA-HSA group D after 1 hour treatment, respectively. As can be seen from the fluorescence of the treated cells, the fluorescence intensity of the experimental group is obviously weaker than that of the control group, and the average fluorescence intensity is analyzed by adopting the software of ImageJ to calculate the P value <0.01 (respectively, B: ALA-HSA group)<0.001, C: EPA-HSA group<0.001, D DHA-HSA group<0.001), the fatty acid serum albumin complex has obvious inhibiting effect on virus transfection.
EXAMPLE 45 antiviral action of the Complex, disruption of the viral envelope Structure (carboxy + short chain unsaturation)
The compounds in example 29 (eight carbon saturated carbon chain-threonine, eighteen carbon monounsaturated carbon chain-serine, twenty two carbon polyunsaturated carbon chain-threonine, twenty two carbon polyunsaturated carbon chain-lysine, eight carbon saturated carbon chain-5' -adenosine monophosphate-four carbon unsaturated carbon chain-carboxyl) were tested against the new crown pseudovirus [ b.1.526.2, purchased from beijing tiantan drug biotechnology development company, No.: 80062, the pseudovirus system carries Firefly luciferase (Firefly luciferase) reporter gene, and the luminescent value of the pseudovirus can be detected by a fluorogenic substrate after the pseudovirus infects cells, so as to determine the virus amount of the infected cells. The pseudo virus takes Vesicular Stomatitis Virus (VSV) as a framework, a new corona virus Spike protein is inlaid on the surface, the structural damage of the process that a euvirus is combined with a receptor and enters a cell can be simulated, the observation of a transmission electron microscope shows that the form of a control group of the new corona pseudo virus is shown as the form a of a figure 93, the form of the virus after 1 hour of treatment of each compound is shown as the form b-f of the figure 93, and the rupture of the membrane structure of the virus after 1 hour of treatment of each compound can be seen, so that the structural integrity is lost.
This example illustrates the destruction of the envelope structure of the enveloped virus by the fatty acid complex from the viewpoint of apparent structure, using the compounds eight carbon saturated carbon chain-threonine, eighteen carbon monounsaturated carbon chain-serine, twenty two carbon polyunsaturated carbon chain-threonine, 22 carbon polyunsaturated carbon chain-lysine, and eight carbon saturated carbon chain-5' -monophosphate adenosine-four carbon unsaturated carbon chain-carboxyl of example 29 as examples.
EXAMPLE 46 hydrophobic isolation of Complex to human papillomavirus (active portion + Small Water soluble portion/binding portion)
(1) Process of action of Compounds on non-enveloped viruses (FIG. 94 is a schematic diagram of Process of action)
The water-soluble compound improves the water solubility of the fatty acid, and the water-soluble compound can be general water-soluble compounds, such as polyethylene glycol, amino acid, polysaccharide and monosaccharide, and can also be antigen/polypeptide capable of recognizing regional sites of non-enveloped viruses; thus, the fatty acid loaded on the water-soluble compound molecule can easily find the non-enveloped virus and tightly surround the non-enveloped virus to form fat globules, and then the fat globules are discharged out of the body through a lymph system to play a role in preventing and treating non-enveloped virus infection.
(2) Simulating the HPV non-enveloped virus by using particles loaded with HPV L1 protein, and simulating the binding process of N-octanoyl-N-methylglucamine and the HPV non-enveloped virus in vitro
Preparation of HPV L1 protein-loaded microparticles: weighing 200mg of BSA and 10ug of HPV L1 recombinant protein, dissolving in 20ml of deionized water, stirring by a magnetic stirrer at a rotation speed of 500rpm, adjusting the pH value to 9 by 10% NaOH solution, adding 0.1% of sodium citrate (based on the mass fraction of the total protein), and stirring at a rotation speed of 500rpm overnight to complete hydration; after overnight, adding desolvation acetone, and adding the acetone with the same volume according to the rate of 1ml/min, wherein the solution is turbid subsequently; after addition of 200ul of 0.5mg/ml pyridine iodide (red fluorescence), 0.01ml of 4% paraformaldehyde ethanol solution was added for crosslinking, and the crosslinking was maintained at 500rpm at room temperature for 3 hours. And (3) centrifuging to remove protein which does not form particles after the crosslinking is finished, centrifuging for 30 min/time under the condition of 20000g twice to obtain particles loaded with HPV L1 protein, and uniformly dispersing the particles in 200ul deionized water.
Weighing 32.2mg of N-octanoyl-N-methylglucamine, 38.8mg of indocyanine green dye, 20mg of EDC and 100ul of deionized water, and incubating at 4 ℃ and shaking overnight to obtain the N-octanoyl-N-methylglucamine carrying a green luminescent group.
Sucking 20ul of HPV L1 protein-loaded particles, 20ul of N-octanoyl-N-methylglucamine carrying green luminescent groups, blowing, mixing uniformly, observing protein particles under a fluorescence microscope, as shown in A of figure 95, protein particles seen under white light, B of figure 95, red fluorescent protein particles carrying L1 protein prepared in a simulation way, C of figure 95, adding green fluorescent N-octanoyl-N-methylglucamine into a protein particle solution, mixing uniformly, and then, as can be seen, after mixing with the green luminescent group-carrying N-octanoyl-N-methylglucamine, a layer of green fluorescent light surrounds the protein particles, which shows that the N-octanoyl-N-methylglucamine can be attached and wrapped around the surface of the HPV L1 protein-loaded particles in a targeted way, the theoretical scenario in fig. 94 is verified. Specifically, as shown in fig. 94, a indicates that the surface of non-coated viruses such as HPV contain integrins and polysaccharides that can recognize cell surfaces by proteins L1 and L2, and are linked to each other, and enter the cell by endocytosis; b is the L1 protein which can be identified and connected with HPV by the drug-contained heparan fragment, the other end is a hydrophobic structure, a large number of drug molecules can form hydrophobic coating on the surface of HPV, and the virus is prevented from being connected with polysaccharide on the surface of cells to invade the cells.
EXAMPLE 47 inhibitory Effect of complexes on HPV pseudoviruses (Long chain acting moiety + binding group/Water soluble moiety)
Taking docosahexaenoic acid coupled serine as an example, sample dilution: and adjusting a multi-channel electric pipettor to 40 mu l of pipetting and 100 mu l of mixing procedure, gently and repeatedly blowing and sucking the liquid in the B4-B11 holes for 8 times to mix the liquid fully, then transferring 40 mu l of the liquid to the corresponding C4-C11 holes, gently and repeatedly blowing and sucking the liquid for 8 times, transferring the liquid to the D4-D11 holes, and so on, and finally sucking and discarding 40 mu l of the liquid from the G4-G11 holes.
HPV pseudovirus (HPV6-GFP) was diluted to 200TCID50 in DMEM complete medium and added in 120. mu.l/well in B3-G11 wells. The dilution plate was left at 4 ℃ for 1 hour. Sucking 100 mul of pseudovirus serum mixture (or culture medium) from each hole of the dilution plate, slowly adding the pseudovirus serum mixture (or culture medium) into the corresponding hole of the culture plate on which the cells are paved in advance, and lightly beating the periphery of the culture plate to mix evenly. The cell culture plate was incubated at 37 ℃ in a 5% CO2 incubator for 60-96 hours. The ELISPOT plate reader performs detection, and calculates the infection inhibition rate (%) as 1- (sample detection value-cell control value)/(virus control value-cell control value) × 100%.
The results of the neutralization inhibition are shown in FIG. 96, where it can be seen that the median neutralization concentration is 0.009 mM.
EXAMPLE 48 bacteriostatic action of the Complex, detachment of Staphylococcus aureus membrane (long carbon chain active moiety + macromolecular Water-soluble moiety/binding moiety) was observed under a transmission electron microscope
Method for grafting docosahexaenoic acid to serum albumin referring to example 1, the mother liquor concentration was 20mg/ml, it was diluted in a gradient of 18mM, 9mM, 4.5mM, 2.25mM, 1.12mM, 0.56mM, 0.28mM, 0.14mM, 0.07mM, 0.035mM, 0.018mM, 0.09mM, respectively, and methicillin sodium as a control (concentration of 18mM, 9mM, 4.5mM, 2.25mM, 1.12mM, 0.56mM, 0.28mM, 0.14mM, 0.07mM, 0.035mM, 0.018mM, 0.09mM, respectively), methicillin-resistant Staphylococcus aureus was diluted to 2X 10 mM 6 Taking 500ul of bacteria solution, taking 4.5ml of serum albumin fatty acid compound solution with different dilution concentrations, carrying out vortex mixing uniformly, continuously shaking and uniformly mixing for 2h at 37 ℃, calculating the bacteriostasis rate by adopting a plate counting method, wherein the bacteriostasis result is shown in a figure 97, and the graph shows that when the concentration of DHA-HSA is 18mM-0.14mM, the bacteriostasis rate is kept at 100%, and the half bacteriostasis concentration is 0.009 mM; the bacteriostasis test of the Escherichia coli is carried out in the same concentration gradient, the bacteriostasis result is shown in a graph 98, and the graph can show that when the concentration of DHA-HSA is 18mM-0.14mM, the bacteriostasis rate is kept at 100%, and the half bacteriostasis concentration is 0.009 mM. The bacterial structure was observed to change under scanning electron microscope, see A, B, C, D and E of FIG. 99, which are 10 min, 30 min, 1 hr and 2 hr of the action of Escherichia coli and drug respectively The appearance structure changes during the operation, and A, B, C, D and E in the graph 100 are respectively the appearance structure changes of the staphylococcus aureus and the medicament after 10 minutes, 30 minutes, 1 hour and 2 hours of action, so that the bacteria firstly change the structure of an external membrane, become unsmooth, have wrinkles and gradually lose the normal form along with the increase of the action time, and the bacteria can not be seen in the normal form after 2 hours of action and can be completely wrapped and damaged; the phenomenon of membrane detachment of staphylococcus aureus was observed under a transmission electron microscope, see fig. 101; where a is the staphylococcus aureus control and B, C, D is the bacterial change after 1 minute, 2 minutes and 5 minutes of treatment, respectively, the degree of membrane disruption increased with increasing duration of action, where detachment of the bacterial membrane was seen at 1 minute and partial disruption of the bacterial membrane resulted in bacterial cell disruption after 2 minutes.
This example illustrates the destruction of the structure of bacteria (e.g., Staphylococcus aureus) by a long-chain unsaturated fatty acid complex, docosahexaenoic acid serum albumin complex, from an apparent structural point of view.
Example 49
Taking docosahexaenoic acid-serum albumin labeled with DiI dye (red fluorescence) as an example, taking new coronavirus (less than or equal to 100nm), staphylococcus aureus (600-800nm), escherichia coli (2-3um), hepatic stellate cell (10-20um) as an acceptor, and taking fluorescence intensity as an inspection index, see fig. 102, it can be seen that smaller individuals can absorb the docosahexaenoic acid-serum albumin labeled with red fluorescence more rapidly, and more fluorescein is absorbed, indicating the compounds of this embodiment, namely: the fatty acid carbon chain is an action part, the serum albumin/hyaluronic acid/polypeptide/micromolecule water-soluble molecules and the like are water-soluble parts and also are components of the action part, and the speed of entering the microorganisms such as viruses, bacteria and the like is higher than that of larger cells.
Example 50 animal experiments-residence time of macromolecular drugs in lungs
The mice used below refer to transgenic mice: KI-hACE2 genotype C57BL/6 mouse, female mouse, 4-6 weeks, Experimental animals technologies, Inc. of Weitonglihua, Beijing.
(1) DiI dye-labeled docosahexaenoic acid-albumin (DHA-HSA prepared in example 1) was administered to the mice as a lung lavage, and lung tissues of the mice were removed after 1h, 4h, 8h, and 12h, as shown in A (after 1 h), B (after 4 h), C (after 8 h), D (after 12 h), and E (summary comparison) in FIG. 103, the complex exhibited a large retention capacity (8h also showed significant fluorescence). Thus, albumin complexes may be an ideal unknown factor for viral mutation to protect host cells from infection after inhalation.
(2) DiI dye-labeled eicosapentaenoic acid-hyaluronic acid complex was administered to mice in a lung perfused manner, and lung tissues of the mice were removed after 1h, 4h, 8h, and 12h, as shown in A (after 1 h), B (after 4 h), C (after 8 h), D (after 12 h), and E (summary contrast) of FIG. 104, the complex exhibited a large retention capacity (also significant fluorescence in 8 h). Thus, the hyaluronan complex may be an ideal unknown factor of viral mutation to protect host cells from infection after inhalation.
Example 51 animal experiments-pulmonary administration
New coronavirus B.1.526.2, purchased from Beijing Tiantan pharmaceutical Biotechnology development corporation, numbered: 80062, the pseudovirus system carries Firefly luciferase (Firefly luciferase) reporter gene, and the luminescent value of the pseudovirus can be detected by a fluorescent substrate after infecting the cells to determine the virus amount of the infected cells. The pseudovirus takes Vesicular Stomatitis Virus (VSV) as a framework, and a new coronavirus Spike protein is inlaid on the surface. The mice used below refer to transgenic mice: KI-hACE2 genotype C57BL/6 mouse, female mouse, 4-6 weeks, Experimental animals technologies, Inc. of Weitonglihua, Beijing.
(1) Validation of animal experiments was performed using a nasal spray of docosahexaenoic acid-serum albumin (prepared according to the method of example 1) as an example, and a new coronavirus carrying the LUCI gene was used instead of a true virus, and a C57 mouse overexpressing the hACE2 gene was used as an experimental animal. The administration mode of the lung perfusion is carried out, and the control group perfuses the new coronary pseudovirus of the lung by 50ul (the concentration is 7 multiplied by 10) 5 TCID 50 Ml), experimental groups were filled with lung neocoronaviruses (25ul, 1.4X 10) 6 TCID 50 Per ml) and twenty-two carbon sixA temporary mixture of a solution of alkenoic acid-serum albumin (25ul, 9M) was added to the lung immediately after mixing and 4h later, and after 3 days the lungs of the mice were taken for immunofluorescence of the lung tissue, see figure 105, where a is the non-drug treated positive control, B is the experimental, and C is the fluorescence of a and B as analyzed by ImageJ software. The fluorescence intensity of the experimental group was significantly weaker than that of the control group, and the P value was calculated to be 0.0164 by software analysis of ImageJ <0.05, indicating a significant difference.
(2) Validity verification of animal pulmonary drug administration experiments was performed by taking eicosapentaenoic acid-hyaluronic acid complex (prepared according to the method of example 8) as an example, and a new coronaviruse carrying the LUCI gene was used instead of a true virus, and a C57 mouse overexpressing the hACE2 gene was used as an experimental animal. The same (1), the control group was filled with 50ul of normal saline, the experimental group was filled with 50ul of eicosapentaenoic acid-hyaluronic acid (calculated as eicosapentaenoic acid, concentration is 9M), and after 4h, the experimental group was filled with more than 50ul of new coronaviruses (concentration is 1.4X 10X) 6 TCID 50 Per ml). The lungs of the mice were taken 3 days later for immunofluorescence of lung tissue, see figure 106, where a is the non-drug treated positive control group, B is the experimental group, and C is the fluorescence of a and B analyzed by ImageJ software. The fluorescence intensity of the experimental group was significantly weaker than that of the control group, and the P value was calculated to be 0.0017 by analysis using ImageJ software<0.01, indicating significant differences.
Example 52 animal experiments-oral administration
The mice used below refer to transgenic mice: KI-hACE2 genotype C57BL/6 mouse, female mouse, 4-6 weeks, Beijing Wintolite laboratory animals technologies, Inc.
The effectiveness of the animal oral administration experiments carried out with the compounds of example 29, eight carbon saturated carbon chain-threonine, eight carbon saturated carbon chain-serine, eighteen carbon monounsaturated carbon chain-threonine, twenty two carbon polyunsaturated carbon chain-serine, eighteen carbon monounsaturated carbon chain-lysine, twenty two carbon polyunsaturated carbon chain-lysine, eighteen carbon polyunsaturated carbon chain-threonine, eight carbon saturated carbon chain-5' -monophosphate adenosine tetraunsaturated carbon chain-carboxyl, N-octyl-N-methylglucamine, N-nonyl-N-methylglucamine, was verified by diluting the drug solution to a concentration of 4.5mM feeding the mice with an average of 5-10mg of drug (as the active portion) per day in a water and food mode Carbon chain mass), the pseudovirus (same as the virus of example 51 above) was perfused through the lungs 48h after continuous feeding (concentration 1.4 × 106TCID 50/ml). 2 days later, the lung of the mouse was taken for immunofluorescence of lung tissue, as shown in FIG. 107, wherein a, b, c, d, e, f, g, h, i, j, k, l and m are respectively a control group, an eight-carbon saturated carbon chain-threonine group, an eight-carbon saturated carbon chain-serine group, an eighteen-carbon monounsaturated carbon chain-threonine group, a twenty-two-carbon polyunsaturated carbon chain-serine group, an eighteen-carbon monounsaturated carbon chain-lysine group, a twenty-two-carbon polyunsaturated carbon chain-lysine group, an eighteen-carbon polyunsaturated carbon chain-threonine group, an eight-carbon saturated carbon chain-5' -monophosphonic acid adenosine-four-carbon unsaturated carbon chain-carboxyl group, an N-octyl-N-methylglucamine group, a, The fluorescence intensity of the experimental group was significantly weaker than that of the control group, the average fluorescence intensity was analyzed by the software of ImageJ, and the P value was calculated as <0.01 (in FIG. 108, the P values are b: <0.001, c: <0.001, d: <0.001, e: <0.001, f ═ 0.001, g: <0.001, h: <0.001, i: <0.001, j: <0.001, k: <0.001, l: <0.001, m: <0.001) in FIG. 108, indicating significant difference; where a in FIG. 108 is a control group and no P value.
Further selecting eight-carbon saturated carbon chain-threonine, eighteen-carbon monounsaturated carbon chain-serine, eighteen-carbon monounsaturated carbon chain-threonine, twenty-two-carbon polyunsaturated carbon chain-threonine and N-octyl-N-methylglucamine to carry out validity verification of animal experiments, and carrying out intragastric administration on mice according to the doses of 0mg/kg, 8.6mg/kg, 13mg/kg, 13.4mg/kg, 15mg/kg and 11.2mg/kg respectively, wherein the doses are 1 time per day, and pseudoviruses (the same as the viruses of the embodiment 51) are perfused through the lung after 2 days of administration (the concentration is 1.4 multiplied by 106TCID 50/ml). 2 days later, the lung of the mouse is taken for immunofluorescence of lung tissue, the result is shown in a graph 109, a, b, c, d, e, f and g in the graph are respectively a control group, an eight-carbon saturated carbon chain-threonine group, an eighteen-carbon monounsaturated carbon chain-serine group, an eighteen-carbon monounsaturated carbon chain-threonine group, a docosane polyunsaturated carbon chain-threonine group, an N-octyl-N-methylglucamine group and a fluorescence intensity comparative analysis result, compared with the control group, the fluorescence intensity of an experimental group is obviously weakened, the average fluorescence intensity is analyzed by adopting software of ImageJ, the P value is calculated to be less than 0.01, and the significant difference is shown.
EXAMPLE 53 Effect of heparin-oleic acid Complex on hepatitis B pseudovirus (Long chain carbon chain acting moiety + macromolecular Water soluble moiety/binding moiety)
In this example, heparin is both a macromolecular water-soluble moiety and a binding moiety, with the oleic acid long carbon chain moiety acting as the active moiety.
Reference example 7 heparin-oleic acid complex was prepared by substituting oleic acid for linoleic acid and heparin (weight average molecular weight 16200Da) for hyaluronic acid. Meanwhile, a control solution (no oleic acid was added to the solution reaction, and other steps were the same) was prepared to exclude the influence of other components. The obtained heparin-oleic acid complex has an infrared spectrum shown in figure 110, and the product retains 1561.50cm -1 The absorption peak comes from the v of oleic acid C＝C ，1645.71cm -1 The absorption peak is from ester bond generated after the esterification reaction of oleic acid and heparin, and the characteristic absorption peak of the ester bond shifts to a low wave band due to the conjugation effect of a side group (-OH/-CN).
The antiviral activity of hepatitis B pseudovirus (HBVpp) was tested with heparin-oleic acid complex. The hepatitis B pseudovirus is prepared by a method in the laboratory reference literature (Wang military science and the like, establishment of a primary hepatocyte model of tree shrew infected by the hepatitis B virus in vitro, biotechnological communication 2009,20 (06): 753-one 759):
hepatitis b pseudovirus (HBVpp) packaging: the HBsAgs plasmid, the lentivirus package and the shuttle plasmid are co-transfected into 293T cells, and 200 mu L of CaCl is added 2 Mixing the solution evenly to obtain DNA-CaCl 2 And (3) solution. The DNA is put into CaCl 2 Adding the solution into 200 mu L BBS solution, uniformly mixing, inoculating at room temperature for 10-20 min to obtain DNA-CaCl 2 -a BBS mixture; is provided withDNA-CaCl 2 The BBS mixture was added dropwise to the entire 6cm dish in 3% CO 2 Culturing in a 37 ℃ cell culture box, after 8 hours, replacing a complete culture medium containing 10mmol/L sodium butyrate for induction, after 8 hours of induction, replacing the solution, washing with PBS to remove residual sodium butyrate, and adding 4mL of fresh culture medium to continue culturing for 24-48 hours; collecting cell supernatant, centrifuging for 3-5 min (4 ℃) to remove cell crisp chips, filtering the supernatant by using a 0.45 mu m filter, and directly using the filtered cell supernatant for cell infection or freezing and storing at-80 ℃ for later use.
HBVpp infected HepG2 cells: adding 800 μ L cell supernatant containing pseudovirus (after adding 200 μ L200 g/L PEG8000 solution and mixing) into HepG2 cell hole before infection, setting 3 repeat holes for each sample, at 37 deg.C and 5% CO 2 Culturing for 15 hours in the cell culture box; sucking the cell supernatant containing HBVpp, washing the cell supernatant with PBS for 2-3 times, adding a DMEM culture medium to continue culturing, and changing the solution once every 2 days; and (3) observing and counting positive cells under a fluorescence microscope after the cells are infected with the pseudoviruses for 3-4 d, wherein the infection rate of the target cells is more than 80%.
HBVpp infection inhibition assay: adding heparin-glycerol compound into every 800 mu L of cell supernatant containing pseudoviruses, incubating for 1h at 4 ℃, adding 200 mu L of 200g/LPEG8000 solution, mixing uniformly, sucking 800 mu L of pseudovirus serum mixture (or culture medium) from each hole of a dilution plate, slowly adding into the corresponding hole of a culture plate in which cells are paved in advance, tapping the periphery of the culture plate, and mixing uniformly. The cell culture plate was placed at 37 ℃ in 5% CO 2 Is cultured in the incubator for 60 to 96 hours. Detecting by using an ELISPOT plate reading instrument, and calculating the neutralization inhibition rate according to the following formula:
neutralization inhibition ratio (%) of 1- (sample detection value-cell control value)/(virus control value-cell control value) × 100%
The results of the neutralization inhibition rates are shown in FIG. 111, the neutralization inhibition rate of the control group is 0, the neutralization inhibition rate of the experimental group at the concentration of 0.009mM on HBVpp is 53%, and the concentration of the half inhibition is presumed to be about 0.009 mM.
The compound was used for bacteriostatic experiments, and the specific results are summarized in table 30.
TABLE 30 Bactericidal and antibacterial Properties of oleic acid-hyaluronic acid complexes (2h)
Testing microorganisms Sterilizing rate (%)
Escherichia coli >99
Staphylococcus aureus >99
Methicillin-resistant staphylococcus aureus >99
Streptococcus pneumoniae >99
Klebsiella pneumoniae >99
Pseudomonas aeruginosa >99
Example 54 Effect of hyaluronic acid-DHA complexes on mouse sinusitis model
Weighing BALB/C mice, mixing distilled water and chloral hydrate according to a ratio of 10: 1, anesthetizing the mice by an abdominal cavity anesthesia mode according to 0.004mL/g after the chloral hydrate is fully dissolved, and obtaining the best anesthesia effect by the fact that the mice are weak in whole body, have nerves and are not closed in two eyes and have no response or little response to pain stimulation. The mouse head was disinfected with alcohol and the experiment was started after the alcohol was completely volatilized.
The cotton sliver is clamped by the axis of the microsurgical forceps, and is inserted into the right nostrils of the mice of the A group, the B group and the C group (5 mice in each group), the inserting depth of the forceps is not more than 1.5mm, and the cotton sliver is slowly inserted into the nasal cavity for 4 mm. Group A was applied to the tampon as a 10. mu.L drop in sterile saline. Groups B and C were dropped onto the tampon in an amount of 10. mu.L of a suspension containing 1.2X 109CFU of Staphylococcus aureus, and the remaining portion of the tampon exposed to the outside was inserted into the nostril.
Group A and group B mice were sacrificed by intraperitoneal injection of pentobarbital sodium (120mg/kg) at respiratory failure doses at 14 days. The skin of the head was removed. The mandible branch is cut off and the mandible is removed. A coronal incision was made 1mm behind the orbit and the nose was cut. Taking out the mucosa of the paranasal sinuses from an ice tray of a super clean bench, fixing the mucosa of the paranasal sinuses by using 4 percent paraformaldehyde solution, and preparing the mucosa of the paranasal sinuses into an HE slice by an HE staining method. Sinus mucosa changes were observed under a light scope. And nasal mucosa was taken for Myeloperoxidase (MPO) activity.
Group C the tampons were removed, and 2 drops of hyaluronic acid-DHA drug (concentration 4.5mM) (after adjusting the hyaluronic acid-DHA complex prepared in example 8 to the target concentration) were dropped into the right nasal cavity three times a day for 7 days as a total treatment period. Mice were sacrificed 7 days after treatment by intraperitoneal injection of pentobarbital sodium (120mg/kg) at the respiratory failure-causing dose. The skin of the head was removed. The mandible branch is cut off and the mandible is removed. A coronal incision was made 1mm posterior to the orbit and the nose was cut. Taking out the mucosa of the paranasal sinus from an ice tray of a super clean bench, fixing the mucosa of the paranasal sinus by using a 4% paraformaldehyde solution, and preparing the mucosa of the paranasal sinus into an HE slice by using an HE dyeing method. Sinus mucosa changes were observed under a light scope. And nasal mucosa was taken for Myeloperoxidase (MPO) activity.
As a result: histologically altered HE staining results are shown in A, B and C of fig. 112, in which the epithelial tissue structures of the nasal sinuses and nasal mucosa of group a are regular and regularly arranged, no obvious inflammatory cell infiltration is found in the submucosa, and a small amount of goblet cells are occasionally seen; the nasal sinuses and nasal mucosa in group B showed inflammatory manifestations, uneven distribution of mucosal epithelium, and infiltration of a large number of inflammatory cells. The epithelial tissues of nasal sinuses and nasal mucosa in the group C are the same as those in the group A, no obvious inflammation is shown, and a small amount of inflammatory cells infiltrate.
Myeloperoxidase activity (MPO) results are shown in A, B and group C of fig. 113, where MPO activity was significantly increased in mouse nasal mucosal tissue in group B compared to group a and significantly decreased in treated mouse nasal mucosal tissue in group C compared to group B in this figure 113.
Example 55 Tween-80-threonine Complex preparation and Performance evaluation (surfactants provide the carbon chain + binding moiety for the acting moiety)
The surfactant amino acid complex is prepared using Tween-80 and threonine, wherein Tween-80 serves as the active moiety, providing the carbon chain, and also serves as the water-soluble moiety, and threonine serves as the binding moiety.
The specific process is as follows:
tween-800.045 mmol (Cas: 9005-65-6, molecular weight 604.8Da) is precisely weighed, dissolved in 5ml of physiological saline, added with 0.36mmol of catalyst EDC and 0.36mmol of DMAP, and stirred for 10 min. Accurately weighing 0.36mmol of threonine, adding into Tween-80 solution at intervals of 4 times, wherein the addition interval is 30min each time, and continuously stirring at room temperature for reaction for 8-24h after the 4 th addition. After the reaction is finished, the reaction solution is dialyzed by a dialysis bag with the molecular weight cut-off of 500-1000 to purify the compound obtained by the reaction, water is replaced every 4h (the molecular weight of the catalyst and the unreacted threonine are both less than 500 and can be removed), and the dialysis is carried out for 24 h. The infrared spectrum of the Tween-80 threonine complex is shown in FIG. 114, and the product contains 1736.83cm -1 Characteristic absorption peak of ester bond (Tween 80 itself contains ester bond, reaction product of Tween 80 and threonine also contains ester bond), 1644.93cm -1 The absorption peak may be ν contained in tween 80 C＝C Slightly shifted to the lower band by 10cm compared with Tween 80 -1 。
Referring to the experimental procedure of the microorganisms in example 19, the results shown in tables 31 and 32 below were obtained, and the virucidal and bactericidal activity was > 99% at the complex concentration of 0.035 mM.
TABLE 31 virucidal Properties of Tween-80-threonine Complex (1h)
Testing viruses Virucidal Rate (%)
H7N9 pseudovirus ＞99.9
H5N1 pseudovirus ＞99.9
HIV pseudovirus ＞99.9
Novel coronaviruses ＞99.9
TABLE 32 Bactericidal and antibacterial Properties of Tween-80-threonine Complex (2h)
Testing microorganisms Sterilizing rate (%)
Escherichia coli >99
Staphylococcus aureus >99
Methicillin-resistant staphylococcus aureus >99
Streptococcus pneumoniae >99
Klebsiella pneumoniae >99
Pseudomonas aeruginosa >99
EXAMPLE 56 Cholesterol-PEG 400-fumaric acid Complex preparation and Performance evaluation (steroids provide the carbon chain + Water soluble moiety + binding moiety for the active moiety)
In the embodiment, cholesterol is selected as an action part to provide a carbon chain, PEG400 is a water-soluble part, and fumaric acid is a binding part, and the specific process is as follows:
PEG4001.5mmol, cholesteryl chloroformate 1mmol, triethylamine 1mmol were added, and the mixture was stirred at room temperature and reacted for 12 hours. 1mmol of fumaric acid, 1.2mmol of EDC and 1.2mmol of DMAP were added to the reaction mixture, and the reaction was continued for 12 hours while stirring at room temperature. After the reaction is finished, the reaction solution is dialyzed by a dialysis bag with the molecular weight cut-off of 500-1000 to purify the compound obtained by the reaction, water is replaced every 4h (the molecular weight of the catalyst and the unreacted fatty acid is less than 500 and can be removed), and the dialysis is carried out for 24 h. The infrared spectrogram of the product cholesterol-PEG 400-fumaric acid compound is shown in a figure 115, and compared with the peaks of fumaric acid and cholesterol, the product mainly increases a peak at 1715.11cm-1, retains 1642.52cm-1 (the original peak of the fumaric acid is 1646.62cm-1, esterification reaction occurs, and the peak is slightly shifted to a low band) of the fumaric acid due to the conjugation effect, and all the peaks of 1776.24cm-1, 1715.11cm-1 and 1642cm-1 are vC ═ O.
Referring to the experimental procedure of the microorganisms in example 19, the results shown in tables 33 and 34 below were obtained, and the virucidal and bactericidal activity was > 99% at the complex concentration of 0.035 mM.
TABLE 33 virucidal Properties of Cholesterol-PEG 400-fumaric acid Complex (1h)
Testing viruses Virucidal Rate (%)
H7N9 pseudovirus ＞99.9
H5N1 pseudovirus ＞99.9
HIV pseudovirus ＞99.9
New coronavirus ＞99.9
TABLE 34 Bactericidal and antibacterial Properties of Cholesterol-PEG 400-fumaric acid Complex (2h)
Testing microorganisms Sterilizing rate (%)
Escherichia coli >99
Staphylococcus aureus (Staphylococcus aureus) >99
Methicillin-resistant goldStaphylococcus aureus (CGS) >99
Streptococcus pneumoniae >99
Klebsiella pneumoniae >99
Pseudomonas aeruginosa >99
Example 57 preparation of Phosphatidylethanolamine-PEG 1000-suberic acid Complex and evaluation of Performance (Phosphatidylcholine provides the active moiety with the carbon chain + Water-soluble moiety + binding moiety)
In this example, phosphatidylethanolamine-PEG 1000 was used as the active moiety to provide a carbon chain, where PEG1000 is the water-soluble moiety and suberic acid is the binding moiety
Dissolving 0.36mmol of conjugate of phosphatidylethanolamine and PEG1000 in a molar ratio of 1:1 in 10ml of deionized water; 0.4mmol of suberic acid, 0.4mmol of EDC and 0.4mmol of DMAP, adding acid solution, stirring and activating for 10min under an ice bath; adding the activated suberic acid into phosphatidylethanolamine-PEG 1000 solution, adjusting pH value to 7.0-7.4 with NaOH, and stirring at room temperature for reaction for 12 hours. After the reaction is finished, the reaction solution is dialyzed by a dialysis bag with the molecular weight cut-off of 500-1000 to purify the compound obtained by the reaction, water is replaced every 4h (the molecular weight of the catalyst and the unreacted fatty acid is less than 500 and can be removed), and dialysis is carried out for 24 h. The infrared spectrum of the product phosphatidylethanolamine-PEG 1000-suberic acid compound is shown in figure 116, and the product is 1645.41cm -1 V of suberic acid C＝O Slightly shifted to the lower band, phospholipid v C＝O At 1718.58cm -1 The absorption peak at (a) is also shifted to the lower band.
Referring to the experimental procedure for the microorganisms in example 19, the results shown in tables 35 and 36 below were obtained, and the virucidal and bactericidal activity was > 99% at the complex concentration of 0.035 mM.
TABLE 35 virucidal Properties of phosphatidylethanolamine-PEG 1000-suberic acid complex (1h)
Testing viruses Virucidal Rate (%)
H7N9 pseudovirus ＞99.9
H5N1 pseudovirus ＞99.9
HIV pseudovirus ＞99.9
New coronavirus ＞99.9
TABLE 36 Bactericidal and antibacterial Properties of phosphatidylethanolamine-PEG 1000-suberic acid complex (2h)
Testing microorganisms Fungicidal ratio (%)
Escherichia coli >99
Staphylococcus aureus (Staphylococcus aureus) >99
Methicillin-resistant staphylococcus aureus >99
Streptococcus pneumoniae >99
Klebsiella pneumoniae >99
Pseudomonas aeruginosa >99
Example 58 preparation of alpha-tocopherol-hyaluronic acid complexes and evaluation of the Properties (fat-soluble vitamins provide the carbon chain + Water-soluble moiety/binding moiety for the active moiety)
In this example, alpha-tocopherol (vitamin E) was chosen as the active moiety, providing a carbon chain, and hyaluronic acid as the water-soluble/binding moiety.
The specific process is as follows:
precisely weighing hyaluronic acid 0.045mmol (2.5 mg by mass in an example of molecular weight 50k Da), dissolving in 5ml of physiological saline, adding catalyst EDC 0.045mmol and DMAP 0.045mmol, adding acid solution, stirring and activating for 10 min; adding 0.36mmol of alpha-tocopherol, stirring to disperse uniformly, adding NaOH solution to adjust the pH to be neutral, and continuously stirring and reacting for 8-24h at room temperature. After the reaction is finished, the reaction solution is dialyzed by a dialysis bag with the molecular weight cut-off of 500-1000 to purify the compound obtained by the reaction, water is replaced every 4h (the molecular weight of the catalyst and the unreacted fatty acid is less than 500 and can be removed), and the dialysis is carried out for 24 h. The infrared spectrum of the product alpha-tocopherol-hyaluronic acid complex is shown in FIG. 117, and the characteristic peak of the product is 1647.20cm -1 (ester bond between tocopherol and hyaluronic acid, which is shifted to a lower band due to benzene ring) and 1561.08cm -1 (Delta of hyaluronic acid) N-N )。
Referring to the experimental procedure of the microorganisms in example 19, the results shown in tables 37 and 38 below were obtained, and the virucidal and bactericidal activity was > 99% at the complex concentration of 0.035 mM.
TABLE 37 virucidal Properties of alpha-tocopherol-hyaluronic acid complexes (1h)
Testing viruses Virucidal Rate (%)
H7N9 pseudovirus ＞99.9
H5N1 pseudovirus ＞99.9
HIV pseudovirus ＞99.9
New coronavirus ＞99.9
TABLE 38 Bactericidal and antibacterial Properties of alpha-tocopherol-hyaluronic acid complexes (2h)
Testing microorganisms Sterilizing rate (%)
Escherichia coli >99
Staphylococcus aureus (Staphylococcus aureus) >99
Methicillin-resistant staphylococcus aureus >99
Streptococcus pneumoniae >99
Klebsiella pneumoniae >99
Pseudomonas aeruginosa >99
Example 59 preparation of sodium cholate-hyaluronic acid Complex and evaluation of Properties (steroid provides carbon chain + Water-soluble moiety/binding moiety for the acting moiety)
Sodium cholate was chosen as the active moiety in this example, providing a carbon chain with hyaluronic acid as the water soluble/binding moiety.
The specific process is as follows:
precisely weighing hyaluronic acid 0.045mmol (2.5 mg by mass in an example of molecular weight 50k Da), dissolving in 5ml of physiological saline, adding catalyst EDC 0.045mmol and DMAP 0.045mmol, adding acid solution, stirring and activating for 10 min; adding 0.36mmol of sodium cholate, stirring to disperse uniformly, adding NaOH solution to adjust the pH to be neutral, and continuously stirring at room temperature for reaction for 8-24 h. After the reaction is finished, the reaction solution is dialyzed by a dialysis bag with the molecular weight cut-off of 500-1000 to purify the reaction The compound obtained was dialyzed for 24 hours after every 4 hours by exchanging water (catalyst and unreacted fatty acid both having a molecular weight of less than 500 and being removable). The infrared spectrogram of the product sodium cholate-hyaluronic acid complex is shown in FIG. 118, and the product contains 1776.40cm -1 (esterification reaction product during the reaction, hyaluronic acid and sodium cholate v c＝o Characteristic peak of) 1669.15cm -1 V is N-H Absorption peak of (2).
Referring to the experimental procedure for the microorganisms in example 19, the results shown in tables 39 and 40 below were obtained, the virucidal and bactericidal activity being > 99% at a complex concentration of 0.035 mM.
TABLE 39 virucidal Properties of sodium cholate-hyaluronic acid complexes (1h)
Testing viruses Virucidal Rate (%)
H7N9 pseudovirus ＞99.9
H5N1 pseudovirus ＞99.9
HIV pseudovirus ＞99.9
Novel coronaviruses ＞99.9
TABLE 40 Bactericidal and antibacterial Properties of sodium cholate-hyaluronic acid Complex (2h)
Testing microorganisms Sterilizing rate (%)
Escherichia coli >99
Staphylococcus aureus (Staphylococcus aureus) >99
Methicillin-resistant staphylococcus aureus >99
Streptococcus pneumoniae >99
Klebsiella pneumoniae >99
Pseudomonas aeruginosa >99
Example 60 preparation of heparin-EPA-suberic acid-fumaric acid-threonine Complex and evaluation of Properties
In this example, heparin and threonine are the water-soluble moiety and the binding moiety, while the carbon chains of EPA, suberic acid and fumaric acid are the active moiety, and the free carboxyl groups of suberic acid and fumaric acid are also the binding moiety.
The specific process is as follows:
(1) heparin-EPA Complex (Complex A) preparation method referring to example 7
Compound A was synthesized by substituting hyaluronic acid with heparin (weight-average molecular weight 16200Da) and linoleic acid with EPA. After 12 hours of reaction, the reaction was carried out directly without dialysis.
(2) The compound A reacts with suberic acid to obtain heparin-EPA-suberic acid compound (compound B)
Accurately weighing 0.001mmol of suberic acid, adding 0.001mmol of EDC and 0.001mmol of DMAP, adding acid solution, stirring and activating for 10min to obtain activated suberic acid. Adding the activated suberic acid into the reaction solution in the step (1), adjusting the pH to be neutral by using NaOH solution, and continuously stirring and reacting for 12 hours at room temperature.
(3) The compound B reacts with fumaric acid to obtain a heparin-EPA-suberic acid-fumaric acid compound (compound C)
0.001mmol of fumaric acid is precisely weighed, 0.001mmol of EDC and 0.001mmol of DMAP are added as catalysts, and an acid solution is added to stir and activate for 10min to obtain activated fumaric acid. And (3) adding the activated fumaric acid into the reaction liquid in the step (2), adjusting the pH to be neutral by using a NaOH solution, and continuously stirring and reacting at room temperature for 12 hours.
(4) Reacting the compound C with threonine to obtain final product heparin-EPA-suberic acid-fumaric acid-threonine compound (compound D)
Accurately weighing 0.001mmol of threonine, adding 0.001mmol of EDC and 0.001mmol of DMAP as catalysts, adding acid solution, stirring and activating for 10min to obtain activated threonine. Adding the activated threonine into the reaction solution in the step (3), adjusting the pH to be neutral by using NaOH solution, and continuously stirring and reacting for 12h at room temperature. And (3) dialyzing the reaction solution by using a dialysis bag with the molecular weight cut-off of 500-1000 after the reaction is finished so as to purify the compound obtained by the reaction, changing water every 4h (the molecular weight of the catalyst and the unreacted fatty acid are both less than 500 and can be removed), and dialyzing for 24h to obtain a compound D solution.
The IR spectrum of the prepared composite A, B, C, D is shown in FIG. 119. After heparin is bonded with a compound having a carboxyl group, a strong absorption peak appears in the vicinity of a wavelength of 1650cm-1, and as an ester bond generated after bonding, since a molecule contains many unsaturated bonds and slightly affects the position of the ester bond, the position of the ester bond is shifted to a lower band than the position of a normal ester bond (v of a normal ester) C＝O Absorption peak is 1740cm -1 ). In addition, 1247-1250cm -1 Is present atA new strong absorption peak is the v of the ester as(C-O-C) I.e. the second characteristic peak of the ester.
The results in tables 41 and 42 below, obtained by performing virus inhibition and bacteriostasis experiments using the complex D according to the experimental method of microorganisms in example 19, show that the virucidal and bactericidal activity was > 99% at the complex concentration of 0.035 mM.
TABLE 41 virucidal Properties of Complex D (1h)
Testing viruses Virucidal Rate (%)
H7N9 pseudovirus ＞99.9
H5N1 pseudovirus ＞99.9
HIV pseudovirus ＞99.9
New coronavirus ＞99.9
TABLE 42 Bactericidal and antibacterial Properties of Compound D (2h)
Testing microorganisms Sterilizing rate (%)
Escherichia coli >99
Staphylococcus aureus (Staphylococcus aureus) >99
Methicillin-resistant staphylococcus aureus >99
Streptococcus pneumoniae >99
Klebsiella pneumoniae >99
Pseudomonas aeruginosa >99
The present invention has been described by way of example, but not by way of limitation, and reference to the description of the invention is made to other variations of the disclosed embodiments, as will be readily apparent to those skilled in the art, and such variations are intended to be within the scope of the invention as defined in the appended claims.

Claims (48)

1. A complex capable of preventing, deterring and/or treating a microbial infection, comprising an active moiety, a binding moiety and a water-soluble moiety,
the acting part is a fat-soluble saturated and/or unsaturated carbon chain with a branched chain, a cyclic structure and/or a straight chain structure, and the carbon chain is a molecule or a residue of the molecule; wherein the action part is a carbon chain or residue of a carbon chain with 3-100 carbon atoms formed by one or more than two substances selected from hydrophobic amino acid, fat-soluble vitamin, steroid lipid, phospholipid, sphingomyelin, glycolipid, surfactant, saturated and/or unsaturated aliphatic hydrocarbon and saturated and/or unsaturated aliphatic alcohol or oxo-aliphatic alcohol;
the water-soluble moiety is a water-soluble molecule containing one or two or more functional group groups selected from an amide group, a phosphoryloxy group, a carboxylic acid group, a phosphoric acid group, a sulfonic acid group, a sulfonyloxy group, a hydroxyl group, a quaternary ammonium group, a sulfide group, a disulfide group, an ether group, a mercapto group, an aldehyde group, an ester group, an amine group, an amino group, a urea group, and a guanidino group, or a residue of a molecule, and the water-soluble moiety may be the above one or two or more functional group groups linked to a carbon chain as an acting moiety;
The binding moiety is a molecule or residue of a molecule capable of binding to a microbial lipid membrane, a microbial surface protein, a microbial surface polysaccharide or cell wall component or capable of binding to a polysaccharide or protein or polypeptide in a microbe, which may be the same as the water-soluble moiety, i.e. a protein, polypeptide, amino acid, oligopeptide, oligosaccharide, mono-and/or polysaccharide molecule or residue thereof capable of binding to a microbial lipid membrane, surface domain;
wherein the number of any one of the acting portion, the water-soluble portion and the binding portion may be 1 or 2 or more.
2. The compound of claim 1, wherein the number of carbon atoms is from 3 to 48.
3. The composite of claim 1, said number of carbon atoms being from 3 to 26.
4. The complex according to any one of claims 1 to 3, wherein the water-soluble moiety is a residue of a water-soluble molecule or molecule containing one or two or more groups selected from a mercapto group, an amino group, a phosphoric acid group, a carboxylic acid group, a sulfonic acid group, a hydroxyl group, an amine group, a ureido group, a guanidino group, and a disulfide group;
the binding moiety has a group capable of binding to a microbial lipid membrane, a microbial surface protein, a microbial surface polysaccharide or a cell wall component or to a polysaccharide or protein or polypeptide in a microorganism, the group being derived from a water-soluble moiety or from two or more groups selected from mercapto groups, amino groups, phosphate groups, carboxylic acid groups, sulfonic acid groups, hydroxyl groups, amine groups, urea groups, guanidine groups and disulfide groups independently serving as the binding moiety or from one or two or more groups selected from mercapto groups, amino groups, phosphate groups, carboxylic acid groups, sulfonic acid groups, hydroxyl groups, amine groups, urea groups, guanidine groups and disulfide groups providing a carbon chain linkage to the carbon chain, so that the complex has one or two or more groups selected from mercapto groups, amino groups, phosphate groups, carboxylic acid groups, sulfonic acid groups, hydroxyl groups, amine groups, urea groups, guanidine groups and disulfide groups.
5. The complex of claim 4, wherein the binding moiety is selected from one or more of a di-or poly-fatty acid, an amino acid, a targeting protein, a targeting polypeptide, and a targeting polysaccharide.
6. The complex of any one of claims 1-4, which is a complex formed by the reaction of a surfactant and one or more selected from the group consisting of a di-or poly-fatty acid, an amino acid, a targeting protein, a targeting polypeptide, and a targeting polysaccharide.
7. The complex according to any one of claims 1 to 6, wherein the saturated and/or unsaturated fatty alcohol is an alcohol having a cyclic structure, a linear chain or a branched chain, of a saturated fat having 3 to 33 carbon atoms; and/or C3-33 unsaturated aliphatic straight-chain or branched-chain alcohol containing 1-3 hydroxyl groups and containing 1-5 double bonds and 1-5 triple bonds; the oxo-fatty alcohol is C8-31 alcohol ketone containing 1-3 double or triple bonds and 1-3 hydroxyl groups, and the ketone is monoketone or diketone.
8. The complex of any one of claims 1-5 and claim 7, the water-soluble moiety being a residue of a molecule or molecule containing one or more groups selected from thiol, amino, carboxylic, hydroxyl and disulfide groups; the molecule is selected from one or more than two water-soluble macromolecules or residues thereof in protein, polysaccharide, nucleic acid and artificially synthesized water-soluble high polymer;
And/or one or more than two medium molecules selected from polypeptide, oligopeptide, oligosaccharide, oligonucleotide and artificially synthesized water-soluble medium molecular weight polymer or residues thereof;
and/or, water-soluble small molecules selected from one or more than two of amino acid, monosaccharide, disaccharide, nucleotide, water-soluble vitamin and deoxynucleotide or residues thereof;
and/or a molecule or a residue of a molecule linked to the carbon chain as the acting moiety, said molecule or residue of a molecule comprising one or more groups selected from thiol, amino, carboxylic acid, hydroxyl, disulfide groups.
9. The complex according to claim 8, wherein the protein as the water-soluble macromolecule is one or more water-soluble macromolecules selected from the group consisting of serum albumin, immunoglobulin, water-soluble collagen, chaperonin, water-soluble glycoprotein, and CD 14; the polysaccharide as the macromolecule is one or more than two water-soluble macromolecules selected from dextran, hyaluronic acid, sialic acid, heparin sulfate, heparan sulfate, chondroitin sulfate, dermatan sulfate, keratan sulfate, acetyl water-soluble cellulose derivatives, beta-cyclodextrin and derivatives thereof and water-soluble chitosan derivatives; the water-soluble polymer as the macromolecule is one or more than two water-soluble macromolecules selected from polyethylene glycol, carboxylated or aminated polyethylene glycol, polyvinyl alcohol, carboxylated or quaternized polyvinyl alcohol, polyacrylic acid and ammonium polyacrylate;
The water-soluble polymer with medium molecular weight is selected from one or more than two of targeting polypeptide, oligopeptide, oligosaccharide, oligonucleotide and/or water-soluble polyamino acid;
the monosaccharide and/or disaccharide of the water-soluble micromolecule is one or more than two of glucose, fructose, rhamnose, sorbose, sucrose, maltose, lactose and trehalose; the nucleotide and/or deoxynucleotide as water-soluble small molecule is selected from adenylic acid, guanylic acid, uridylic acid, cytidylic acid, thymidylic acid, inosinic acid, deoxyadenylic acid, deoxyguanylic acid, deoxycytidylic acid, deoxythymidylic acid; amino acids such as one or more of serine, threonine, cysteine, asparagine, glutamine, tyrosine, lysine, arginine, histidine, aspartic acid, glutamic acid, citrulline, ornithine, taurine and aminobutyric acid; the vitamins as the water-soluble small molecules are selected from one or more of vitamin B1, pantothenic acid, vitamin B6 and vitamin C.
10. The complex of any one of claims 5-9, wherein the targeting polypeptide comprises any one of a protein or neutralizing antibody fragment that specifically targets microbial lipid membranes, bacterial and fungal cell walls, viral surface protein domains.
11. The complex of claim 9, wherein the water-soluble polyamino acid is selected from polyglutamic acid, polylysine and/or polyaspartic acid.
12. A complex according to any one of claims 1 to 11, wherein the binding moiety and the water-soluble moiety are the same, i.e. are proteins, polypeptides, oligopeptides, oligosaccharides, monosaccharides, disaccharides, amino acids, nucleotides, vitamins, water-soluble polymers, water-soluble polyamino acids and/or polysaccharide molecules or residues of such molecules, which molecules or residues of molecules comprise one or more groups selected from thiol, amino, carboxylic, hydroxyl, disulfide groups.
13. The complex according to any one of claims 1 to 12, which is a compound obtained by reacting a substance having a carbon chain having 3 to 100 carbon atoms as an active moiety with any one or two or more members selected from the group consisting of proteins, polypeptides, oligopeptides, oligosaccharides, amino acids, monosaccharides, disaccharides, nucleotides, vitamins, water-soluble polymers, water-soluble polyamines and/or polysaccharide molecules; or a compound obtained by reacting a substance having a carbon chain having 3 to 100 carbon atoms as an active moiety with any one or two or more members selected from the group consisting of proteins, polypeptides, oligopeptides, oligosaccharides, amino acids, monosaccharides, disaccharides, nucleotides, vitamins, water-soluble polymers, water-soluble polyamines and/or polysaccharide molecules, and a mixture of unreacted substances as an active moiety and/or unreacted molecules of the proteins, polypeptides, oligopeptides, oligosaccharides, amino acids, monosaccharides, disaccharides, oligonucleotides, vitamins, water-soluble polymers, water-soluble polyamines and/or polysaccharide molecules;
Wherein the substance having a carbon chain of 3 to 100 carbon atoms as an active moiety is selected from the group consisting of saturated and/or unsaturated aliphatic hydrocarbons, saturated and/or unsaturated aliphatic alcohols or oxoaliphatic alcohols, hydrophobic amino acids, fat-soluble vitamins, steroid lipids, phospholipids, sphingomyelin, glycolipids and/or surfactants.
14. The complex according to any one of claims 1 to 12, which is a mixture obtained by complexing a substance having a carbon chain with 3 to 100 carbon atoms as an acting moiety with a complex selected from any one or two of proteins, polypeptides, oligopeptides, oligosaccharides, amino acids, monosaccharides, disaccharides, nucleotides, vitamins, water-soluble polymers, water-soluble polyamines and polysaccharide molecules by a physicochemical action including a combination of hydrogen bonds or van der waals forces or both actions, or by direct physical mixing;
wherein the substance having a carbon chain of 3 to 100 carbon atoms as an active moiety is selected from the group consisting of saturated and/or unsaturated aliphatic hydrocarbons, saturated and/or unsaturated aliphatic alcohols or oxoaliphatic alcohols, hydrophobic amino acids, fat-soluble vitamins, steroid lipids, phospholipids, sphingomyelin, glycolipids and/or surfactants.
15. The complex according to claim 13, which is a compound obtained by reacting a substance having a carbon chain of 3 to 100 carbon atoms as an active moiety with at least one selected from the group consisting of a protein, a polypeptide, an oligopeptide and an amino acid; or a mixture of a compound obtained by reacting a substance having a carbon chain of 3 to 100 carbon atoms as an active moiety with at least one member selected from the group consisting of proteins, polypeptides, oligopeptides and amino acids, and an unreacted substance as an active moiety and/or an unreacted substance selected from the group consisting of at least one member selected from the group consisting of proteins, polypeptides, oligopeptides and amino acids.
16. The complex according to claim 13, which is a compound obtained by reacting a substance having a carbon chain of 3 to 100 carbon atoms as an active moiety with PEG and at least one selected from the group consisting of proteins, polypeptides, oligopeptides and amino acids; or a mixture of a substance having a carbon chain with 3 to 100 carbon atoms as an active moiety, a compound obtained by reacting PEG with at least one member selected from the group consisting of proteins, polypeptides, oligopeptides and amino acids, and an unreacted substance having a carbon chain with 3 to 100 carbon atoms as an active moiety, unreacted PEG and/or an unreacted at least one member selected from the group consisting of proteins, polypeptides, oligopeptides and amino acids.
17. The complex according to claim 13, which is a compound obtained by reacting a substance having a carbon chain of 3 to 100 carbon atoms as an active moiety with at least one selected from the group consisting of polysaccharides, monosaccharides, disaccharides and oligosaccharides; or a compound obtained by reacting a substance having a carbon chain having 3 to 100 carbon atoms as an active moiety with at least one member selected from the group consisting of polysaccharides, monosaccharides, disaccharides and oligosaccharides, and a mixture of the unreacted substance serving as the active moiety and/or the unreacted polysaccharides, monosaccharides, disaccharides and/or oligosaccharides.
18. The complex according to claim 13, which is a compound obtained by reacting a substance containing a carbon chain having 3 to 100 carbon atoms as an active moiety, PEG, and at least one selected from the group consisting of polysaccharide, monosaccharide, disaccharide, and oligosaccharide; or a compound obtained by reacting PEG with at least one selected from polysaccharides, monosaccharides, disaccharides and oligosaccharides, and a mixture of the unreacted acting portion substance, unreacted PEG and/or unreacted polysaccharides, monosaccharides, disaccharides and oligosaccharides.
19. The complex of claim 13, wherein the protein is selected from one or more of serum albumin, immunoglobulin, water-soluble collagen, chaperonin, water-soluble glycoprotein, and CD 14.
20. A complex as claimed in any one of claims 13 to 19, wherein the polysaccharide is selected from one or more of dextran and/or hyaluronic acid, sialic acid, heparin sulphate, heparan sulphate, chondroitin sulphate, dermatan sulphate, keratan sulphate, acetyl water-soluble cellulose derivatives, β -cyclodextrin and its derivatives and water-soluble chitosan derivatives.
21. The complex according to claim 13, which is a compound obtained by reacting a substance having a carbon chain having 3 to 100 carbon atoms as an active moiety, a linker and a thiol-group-containing protein; or a mixture of the compound obtained by the above reaction, the unreacted acting substance, the unreacted linker, and/or the unreacted thiol-group-containing protein; wherein the connecting matter is one or more than two of amino acid, succinic acid, butadiene acid, glutaconic acid, hexylamine diacid, carbamate, short peptide, N-hydroxyl crotonoimide, polyethylene glycol and derivatives of the compounds.
22. The compound according to claim 21, which is a compound obtained by reacting a substance having a carbon chain with 3 to 100 carbon atoms as an active moiety with N-hydroxybutylimide with a protein having a thiol group; or a mixture of the compound obtained by the above reaction, the unreacted substance as the acting portion, the unreacted N-hydroxybutylimide and/or the unreacted thiol-containing protein.
23. The complex according to claim 13, which is a compound obtained by reacting a substance containing a carbon chain having 3 to 100 carbon atoms as an active moiety, cystamine, and at least one selected from the group consisting of polysaccharides, monosaccharides, disaccharides, and oligosaccharides; or a compound obtained by reacting cystamine with at least one selected from the group consisting of polysaccharides, monosaccharides, disaccharides and oligosaccharides, and a mixture of unreacted substances acting as a part and unreacted polysaccharides or monosaccharides, disaccharides, oligosaccharides and/or unreacted cystamine.
24. The compound of any one of claims 13-23, wherein the resulting compound contains one or more of an amide group, an ester group, a thioether group, or an ether group as a linking moiety for the water-soluble moiety and the acting moiety.
25. The compound according to any one of claims 4 to 23, wherein the saturated and/or unsaturated aliphatic hydrocarbon, saturated and/or unsaturated aliphatic alcohol or oxoaliphatic alcohol has a carbon number of 3 to 50.
26. The compound of claim 25, wherein the number of carbon atoms is from 3 to 48.
27. The compound of claim 26, wherein the carbon atom is 3 to 26.
28. The complex of any one of claims 4-27, wherein the protein is human serum protein or bovine serum albumin, or CD 14; or the polysaccharide is dextran, heparin and/or hyaluronic acid.
29. The complex according to any one of claims 4 to 12, which is a compound obtained by reacting a surfactant having a carbon chain of 3 to 30 carbon atoms with at least one selected from the group consisting of a di-or poly-fatty acid, an amino acid, a targeting protein, a targeting polypeptide and a targeting polysaccharide; or a mixture of the compound obtained by the reaction, the unreacted surfactant and/or the unreacted dibasic or polybasic fatty acid, the amino acid, the targeting protein, the targeting polypeptide and/or the targeting polysaccharide.
30. The complex of any one of claims 1-29, wherein the surfactant is selected from one or more of fatty alcohol polyoxyethylene ether, fatty acid polyoxyethylene ester, alkyl glycoside, sucrose fatty acid ester, sorbitan polyoxyethylene fatty acid ester, mannosylerythritol ester, and N-fatty acyl-N-methylglucamine.
31. A complex according to any one of claims 1 to 30, wherein the microbial infection comprises an infection by any one or more of a virus, bacterium, fungus, chlamydia or mycoplasma.
32. A preparation for the prevention, prevention or treatment of a microbial infection made using a complex according to any one of claims 1 to 31.
33. The formulation of claim 32, which is a pharmaceutical formulation or an environmental kill formulation.
34. The formulation of claim 33, wherein the pharmaceutical formulation is one selected from the group consisting of an inhalant, a nasal spray, an injection, an oral formulation, and a skin external preparation.
35. Use of a complex according to any one of claims 1 to 31 for the preparation of a pharmaceutical preparation or an environmentally disinfectant micro-agent for the prevention, prevention and/or treatment of a microbial infection.
36. The use according to claim 35, wherein the microorganism is any one or more selected from viruses, bacteria, fungi, chlamydia or mycoplasma.
37. The use of claim 36, wherein the virus is an enveloped virus; and/or non-enveloped viruses.
38. The use according to claim 37, wherein the enveloped virus is one or more of coronavirus, influenza virus, aids virus, hepatitis b virus, hepatitis c virus, herpes virus, sakavirus, dengue virus, encephalitis b virus, ebola virus, rabies virus, and hantavirus; the non-enveloped virus is one or more than two of hepatitis A virus, human papilloma virus, poliovirus and coxsackievirus.
39. The use according to claim 36, wherein the virus is any one or more of coronavirus, aids virus, hepatitis b virus, hepatitis c virus, herpes virus, encephalitis b virus, rabies virus, human papilloma virus and ebola virus.
40. Use according to claim 36, wherein the bacterium is a gram-positive and/or gram-negative bacterium and the fungus is a pathogenic and/or conditionally pathogenic fungus; the chlamydia is chlamydia trachomatis, chlamydia pneumoniae and/or chlamydia psittaci; the mycoplasma includes mycoplasma pneumoniae, ureaplasma urealyticum, mycoplasma hominis, and/or mycoplasma genitalium.
41. The use according to claim 36, wherein the bacteria are selected from one or more of escherichia coli, staphylococcus aureus, methicillin-resistant staphylococcus aureus, streptococcus pneumoniae, klebsiella pneumoniae, and pseudomonas aeruginosa; the fungi is selected from one or more of Candida albicans, Aspergillus niger, Actinomyces viscosus, Chaetomium globosum, Aspergillus verrucosus and Microsporum canis.
42. The use according to claim 36, wherein the virus is selected from one or more of the group consisting of H7N9 influenza virus, H5N1 influenza virus, HIV virus, neocoronavirus, HPV virus, and rabies virus.
43. A method for preparing a complex as claimed in any one of claims 1 to 31, said complex being obtained by reacting a compound having a fat-soluble saturated and/or unsaturated carbon chain with a branched, cyclic and/or linear structure with a water-soluble molecule and, if desired, protein, polypeptide, amino acid, oligopeptide, oligosaccharide, monosaccharide and/or polysaccharide molecules capable of binding to a microbial lipid membrane, microbial surface domain or cell wall and, if desired, a linker molecule in the presence of a catalyst.
44. The method for producing a complex according to claim 44, wherein the complex is a product obtained by purifying a compound obtained by the reaction.
45. The method for preparing a complex as claimed in any one of claims 1 to 31, which is obtained by physically mixing a compound having a fat-soluble saturated and/or unsaturated carbon chain having a branched, cyclic and/or linear structure with a water-soluble molecule, and, if necessary, at least one molecule selected from the group consisting of proteins, polypeptides, amino acids, oligopeptides, oligosaccharides, monosaccharides, and polysaccharides capable of binding to a lipid membrane, a viral surface domain, or a cell wall of a microorganism.
46. The method for producing a complex as claimed in any one of claims 1 to 31, which is obtained by reacting a substance having a carbon chain with 3 to 100 carbon atoms as an active moiety with at least one substance selected from the group consisting of proteins, polypeptides, oligopeptides, oligosaccharides, amino acids, monosaccharides, disaccharides, nucleotides, vitamins, water-soluble polymers, water-soluble polyamines and polysaccharides in the presence of a catalyst.
47. The method according to claim 46, wherein the compound is a product obtained by purifying a compound obtained by the reaction.
48. The method for producing a complex as claimed in any one of claims 1 to 31, wherein the complex is obtained by physically mixing a substance having a carbon chain of 3 to 100 carbon atoms as an acting moiety with at least one member selected from the group consisting of proteins, polypeptides, oligopeptides, oligosaccharides, amino acids, monosaccharides, disaccharides, nucleotides, vitamins, water-soluble polymers, water-soluble polyamines and polysaccharide molecules in a physicochemical action or directly.
CN202210484471.9A 2022-05-06 2022-05-06 Compound for preventing, preventing or treating microbial infection and preparation and application thereof Pending CN114788872A (en)

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CN117017964A (en) * 2023-08-07 2023-11-10 华中农业大学 Application of arachidonic acid in preparation of rabies treatment drugs

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CN113647389A (en) * 2021-08-24 2021-11-16 太阳雨林（厦门）生物医药有限公司 Antibacterial and antiviral film agent with good use effect and preparation method and application thereof
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CN113647389A (en) * 2021-08-24 2021-11-16 太阳雨林（厦门）生物医药有限公司 Antibacterial and antiviral film agent with good use effect and preparation method and application thereof

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