CN112851756B - Phenolic acid polypeptide conjugate and preparation method and application thereof - Google Patents
Phenolic acid polypeptide conjugate and preparation method and application thereof Download PDFInfo
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Abstract
The invention provides a phenolic acid polypeptide conjugate and a preparation method and application thereof, wherein the phenolic acid polypeptide conjugate is formed by connecting phenolic acid and polypeptide through an amide bond, wherein the phenolic acid mainly comprises gallic acid, rosmarinic acid, ferulic acid, caffeic acid, protocatechuic acid, chlorogenic acid, sinapic acid and vanillic acid; the polypeptide amino acid sequence is KLVFFAED; the preparation method comprises the following steps: (1) synthesizing KLVFFAED polypeptide using a polypeptide solid phase synthesis method; (2) Under the protection of inert atmosphere, dissolving a phenolic acid solution, a catalyst and an alkaline reagent in DMF (dimethyl formamide) to couple with resin, and after reaction, cutting polypeptide from the resin by using a cutting reagent to obtain a crude product of a phenolic acid polypeptide conjugate; (3) Separating, purifying and freeze-drying to obtain the phenolic acid polypeptide conjugate. Compared with unmodified phenolic acid, the phenolic acid polypeptide conjugate can improve lipid solubility and stability, enables the phenolic acid polypeptide conjugate to be more easily taken by cells, can realize a targeting effect, reduces toxic and side effects on normal cells, and efficiently plays a role in drug treatment.
Description
Technical Field
The invention belongs to the technical field of medicines, and particularly relates to a phenolic acid polypeptide conjugate as well as a preparation method and application thereof.
Background
The phenolic acid compound refers to an organic acid containing a plurality of phenolic hydroxyl groups on the same benzene ring, and is widely present in plant seeds, roots, stems and leaves in nature. Because the molecule contains one or more phenolic hydroxyl groups and can react with the oxidized free radicals to generate semiquinone free radicals, the semiquinone free radicals can realize antioxidation, anti-free radical action, anti-inflammatory action, antibacterial and antiviral action, neuroprotective action and the like through different mechanisms. However, phenolic acid compounds have phenolic hydroxyl and carboxyl, and have short alkane chains in molecules and double bonds, so that the phenolic acid compounds have strong hydrophilicity, poor lipid solubility, difficulty in permeating a biolipid bilayer membrane, low bioavailability and limited application range.
Modifying the phenolic acid structure to increase lipid solubility, for example, linking lipid soluble molecules with phenolic acid compounds, is an effective way to expand the application range of phenolic acid compounds and improve the action effect. Conventional modification methods include the reaction of long chain fatty acids with the hydroxyl group of phenolic acids to form phenolic acid esters, or the reaction of long chain fatty alcohols with the carboxyl group of phenolic acids to form phenolic acid esters. However, phenolic acid contains carboxyl and hydroxyl in the molecule, so that intramolecular or intermolecular esterification of phenolic acid is easily formed under esterification reaction conditions, which results in a large amount of byproducts and a low yield. The formed phenolic acid ester has a hydrophilic head part and a hydrophobic tail part similar to those of the surfactant, can damage cell membranes through the action of the surfactant, has low safety and limits the application range of the phenolic acid ester. In addition, since the phenolic hydroxyl group is a main group exerting physiological activity in the phenolic acid compound, modification of the phenolic hydroxyl group may result in reduction or loss of physiological activity. Therefore, the method has important significance for more stably and efficiently modifying the phenolic acid on the basis of keeping the original physiologically active group.
Advanced Glycation end product Receptors (RAGE) are involved in the pathological processes of a variety of diseases, such as Alzheimer's disease, tumors, diabetes and the like, are widely distributed and are expressed on the surfaces of endothelial cells, mononuclear macrophages and neuronal cell nucleus microglia. RAGE belongs to a multi-ligand receptor and a β is one of the natural ligands. Under certain disease conditions such as Alzheimer's disease, the expression of RAGE in brain capillary endothelial cells of a patient is remarkably increased, and after peripheral A beta is combined with the RAGE, the A beta is promoted to cross a blood brain barrier and is aggregated in the brain to form a beta oligomer with neurotoxicity, so that a series of oxidative stress and inflammatory reactions are caused, and finally, nerve cell apoptosis is caused. The research finds that the binding domain of A beta and RAGE is mainly eight amino acids located at positions 16-23 of the sequence of A beta, and an octapeptide consisting of these eight amino acids has a good RAGE targeting effect.
The invention aims to couple a phenolic acid compound and RAGE targeting peptide to form a phenolic acid polypeptide conjugate through a solid-phase synthesis method, explore a new phenolic acid modification mode, improve the solubility and in-vivo distribution behavior of the phenolic acid compound, retain the activity, improve the stability and safety of the phenolic acid compound, enable the phenolic acid compound to be easily enriched at an action site and to be taken by cells, efficiently play the therapeutic action of a medicament and reduce the toxic action on normal cells.
Disclosure of Invention
The invention aims to provide a phenolic acid polypeptide conjugate and a preparation method and application thereof.
In order to achieve the purpose, the invention adopts the following technical scheme:
a phenolic acid polypeptide conjugate comprising a phenolic acid linked to a polypeptide by an amide bond, wherein the phenolic acid comprises predominantly gallic acid, rosmarinic acid, ferulic acid, caffeic acid, protocatechuic acid, chlorogenic acid, sinapic acid, and vanillic acid; the polypeptide amino acid sequence is KLVFFAED.
The structure of the phenolic acid polypeptide conjugate is specifically shown as the formula I:
wherein R is the structure of phenolic acid except carboxyl, and the carboxyl connected with R in the phenolic acid is connected with the amino of the polypeptide to form amido bond.
According to the phenolic acid polypeptide conjugate provided by the invention, phenolic acid is connected with polypeptide, and compared with a single phenolic acid compound, the solubility and stability of phenolic acid can be improved, so that the phenolic acid polypeptide conjugate can pass through cell membranes more easily. In specific diseases, the medicament can be highly enriched at the focus part by virtue of the targeting function of the polypeptide, so that the medicament can play the activity at the focus part without influencing the physiological functions of other normal tissues, the utilization rate of the medicament is increased, and the risk of damage to normal organs caused by systemic administration of the medicament is reduced.
The polypeptide sequence used in the invention is KLVFFAED, is obtained by screening a human endogenous substance A beta and a RAGE binding domain, and is a RAGE targeting polypeptide. The structure of the compound contains five continuous hydrophobic amino acid sequences Leu-Val-Phe-Phe-Ala (LVFFA), which causes the compound to have stronger hydrophobicity. Therefore, after the polypeptide is connected with the water-soluble phenolic acid, compared with the phenolic acid which is not modified by the polypeptide, the lipid solubility of the obtained phenolic acid polypeptide conjugate is improved, and the phenolic acid polypeptide conjugate can more easily penetrate a cell lipid bilayer membrane.
Preferably, the phenolic acid is gallic acid.
Exemplary such as: when the phenolic acid compound is gallic acid, the structure of the phenolic acid polypeptide conjugate is specifically shown in formula II:
in the invention, the phenolic acid is connected with the polypeptide through carboxyl, so that the antioxidant function of the phenolic acid is not influenced, the RAGE targeting function of the polypeptide is not influenced, and the toxic effect on cells due to the existence of the modified polypeptide is avoided. Preferably, the attachment site for the phenolic acid is selected as the free carboxyl group and the attachment site for the polypeptide is selected as the free amino group at the N-terminus. If the phenolic acid is linked to the polypeptide at other positions, the physiological activity of the phenolic acid is reduced or even lost.
The invention also provides a preparation method of the phenolic acid polypeptide conjugate, which at least comprises the following steps:
(1) Synthesizing a KLVFFAED polypeptide using a polypeptide solid phase synthesis method;
(2) After the synthesis of the polypeptide is finished, under the protection of inert atmosphere, phenolic acid solution, a catalyst and a basic reagent are dissolved in an organic solvent to be coupled with resin, so that phenolic carboxyl groups and polypeptide N-terminal amino groups are reacted to form amide bonds to be connected, and after the reaction is carried out for 8-24 hours, the polypeptide is cut from the resin by using a cutting reagent to obtain a crude product of the phenolic acid polypeptide conjugate;
(3) And separating and purifying the crude product of the phenolic acid polypeptide conjugate, and freeze-drying to obtain the phenolic acid polypeptide conjugate.
Preferably, the resin used in the solid phase synthesis method in steps (1) and (2) is 2-chlorotriphenylchloride resin.
Preferably, the organic solvent in step (2) is DMF.
Preferably, the catalyst in step (2) is selected from one or more of 4-Dimethylaminopyridine (DMAP), benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP), 1-hydroxybenzotriazole (HOBt), 1- (3-dimethylaminopropyl) -3-Ethylcarbodiimide (EDC), N, N-Diisopropylcarbodiimide (DIC), N-hydroxysuccinimide (NHS).
More preferably, the catalyst is benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP) or 4-Dimethylaminopyridine (DMAP).
Preferably, the molar ratio of the catalyst, phenolic acid and polypeptide in step (2) is (2-5); for example, 2.
Preferably, the alkaline reagent in step (2) is one or more selected from N, N-Diisopropylethylamine (DIEA), triethylamine (TEA) and pyridine (Py).
More preferably, the basic agent in step (2) is N, N-Diisopropylethylamine (DIEA).
Preferably, the molar ratio of the basic agent to the catalyst in step (2) is 1 to 3, and may be, for example, 1.
Preferably, the coupling reaction time of the phenolic acid and the polypeptide in the step (2) is 12h.
Preferably, the cleavage reagent in step (2) is TFA/TIA/water in a ratio of 2.5.
Preferably, the separation and purification steps in the step (3) are mainly washing, drying and HPLC purification.
More preferably, the washing manner in the separation and purification method in step (3) is washing with cold diethyl ether, and the purification manner is purification by reverse phase HPLC.
The application of the phenolic acid polypeptide conjugate provided by the invention comprises the following steps: (1) The application in treating Alzheimer's disease, cerebral apoplexy and amyloid cerebrovascular disease; (2) The application in preparing preparations for treating Alzheimer's disease, cerebral apoplexy and amyloid cerebrovascular disease; (3) The application in scavenging active oxygen substances and relieving oxidative stress; (4) The application in preparing the related disease targeted drugs of high expression advanced glycosylation end product receptors.
Compared with the prior art, the invention has the beneficial effects that:
(1) The hydrophobic polypeptide is used for modifying the hydrophilic phenolic acid compound, so that the lipophilicity of the medicine is improved, and the medicine is easier to be absorbed by cells;
(2) Phenolic acid is modified through a solid phase synthesis method, the reaction is efficient and mild, the generation of byproducts is reduced, and the yield is improved;
(3) The phenolic acid is condensed with the N-terminal amino group of the polypeptide to form an amido bond for connection, the active group of the phenolic acid is retained, the modified drug activity is not affected, and the drug stability is improved to a certain extent;
(4) After the polypeptide is modified, on one hand, the medicament can be selectively enriched at the focal site with high RAGE expression by virtue of the targeting effect of the polypeptide, and the cellular uptake is increased by virtue of the interaction of a ligand and a receptor, so that the treatment effect is efficiently exerted; on the other hand, the modified phenolic acid polypeptide conjugate has improved safety on cells, and reduces the toxic and side effects of the drug on normal cells.
Drawings
FIG. 1 is a chemical structure diagram of a phenolic acid polypeptide conjugate of examples 1-5 of the present invention.
FIG. 2 is a diagram showing the HPLC purification results of the phenolic acid polypeptide conjugate of examples 1-5 of the present invention.
FIG. 3 is a mass spectrum characterization of the conjugates of phenolic acid polypeptides of examples 1-5 of the present invention.
FIG. 4 is a fluorescence spectrum of the conjugate of phenolic acid polypeptide and the binding of phenolic acid and alizarin red in examples 1-5 of the present invention.
FIG. 5 is a fluorescence spectrum of stability observation of the conjugate of phenolic acid polypeptide and phenolic acid in pH 8.0 alkaline aqueous solution in examples 1-5 of the present invention.
FIG. 6 is a fluorescence spectrum of stability observation of the conjugate of phenolic acid polypeptide and phenolic acid in pH 8.5 alkaline aqueous solution in examples 1-5 of the present invention.
FIG. 7 is a graph of the safety evaluation of SH-SY5Y cells by the phenolic acid polypeptide conjugate and phenolic acid in examples 1-5 of the present invention.
FIG. 8 is a graph showing the safety evaluation of the conjugates of the phenolic acid polypeptide and the phenolic acid on BV-2 cells in examples 1-5.
Detailed Description
The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and should not be construed as limiting the invention.
Example 1
In this example, the gallic acid polypeptide conjugate is prepared by the following method, specifically including the following steps:
(1) The polypeptide solid phase synthesis method using FMOC strategy takes amino acid with amino terminal protected by FMOC as raw material, and uses a full-automatic microwave polypeptide synthesizer of Liberty Lite of CEM company to synthesize, and the operation method is carried out according to the instruction of the instrument.
The synthetic reagent is selected from:
(a) Carrier resin: 2-chlorotrityl chloride resin, degree of substitution: 0.5.
(b) Deprotection reagents: 20% piperidine in DMF.
(c) Coupling reagents were used for the condensation reaction: HOBT, acid-binding agent: DIEA.
(2) And sequentially connecting the required amino acids in the polypeptide sequence from right to left, after the reaction is finished and synthesizing the KLVFFAED polypeptide, dissolving gallic acid, pyBOP and DIEA in DMF, coupling with resin, and reacting for 12h. Wherein the molar ratio of the polypeptide to the gallic acid to the PyBOP to the DIEA is 1.5.
(3) Preparing a cutting reagent: 2.5% of TFA; TIA 2.5%; 95% of water, cracking the gallic acid polypeptide conjugate from the resin by using a cutting reagent, and volatilizing the cracking solution to obtain a crude product.
(4) The crude product was washed three times with cold ether, and the washed crude product was purified using an HPLC C18 semi-preparative column to collect the sample that flowed from the detector. The detection chromatographic conditions are as follows: detection wavelength: 214nm; mobile phase A: water (containing 0.1% trifluoroacetic acid), mobile phase B: acetonitrile (containing 0.1% trifluoroacetic acid) starting the gradient 30% B,70% A, ending the gradient 50% B,50% A, the gradient time 20min. Desalting and freeze-drying the product with the purity of more than 95% to obtain white powdery gallic acid polypeptide conjugate.
Example 2
In this example, the gallic acid polypeptide conjugate is prepared by the following method, specifically including the following steps:
(1) The polypeptide solid phase synthesis method using FMOC strategy takes amino acid with amino terminal protected by FMOC as raw material, and uses a full-automatic microwave polypeptide synthesizer of Liberty Lite of CEM company to synthesize, and the operation method is carried out according to the instruction of the instrument.
The synthetic reagent is selected from:
(a) Carrier resin: 2-chlorotrityl chloride resin, degree of substitution: 0.5.
(b) Deprotection reagents: 20% piperidine in DMF.
(c) Coupling reagents were used for the condensation reaction: HOBT, acid-binding agent: DIEA.
(2) And sequentially connecting the amino acids required in the polypeptide sequence from right to left, after the reaction is finished and synthesizing the KLVFFAED polypeptide, dissolving gallic acid, DMAP and DIEA in DMF, coupling with resin, and reacting for 12h. Wherein the molar ratio of the polypeptide, the gallic acid, the DMAP and the DIEA is 1.
(3) Preparing a cutting reagent: 2.5% of TFA; TIA 2.5%; 95% of water, cracking the gallic acid polypeptide conjugate from the resin by using a cutting reagent, and volatilizing the cracking solution to obtain a crude product.
(4) The crude product was washed three times with cold ether, and the washed crude product was purified using an HPLC C18 semi-preparative column to collect the sample that flowed from the detector. The detection chromatographic conditions are as follows: detection wavelength: 214nm; mobile phase A: water (containing 0.1% trifluoroacetic acid), mobile phase B: acetonitrile (containing 0.1% trifluoroacetic acid) starting the gradient 30% B,70% A, ending the gradient 50% B,50% A, the gradient time 20min. Desalting and freeze-drying the product with the purity of more than 95% to obtain white powdery gallic acid polypeptide conjugate.
Example 3
In this example, the gallic acid polypeptide conjugate is prepared by the following method, which specifically comprises the following steps:
(1) The polypeptide solid phase synthesis method using FMOC strategy takes amino acid with amino terminal protected by FMOC as raw material, and uses a full-automatic microwave polypeptide synthesizer of Liberty Lite of CEM company to synthesize, and the operation method is carried out according to the instruction of the instrument.
The synthetic reagent is selected from:
(a) Carrier resin: 2-chlorotrityl chloride resin, degree of substitution: 0.5.
(b) Deprotection reagents: 20% piperidine in DMF.
(c) Coupling reagents were used in the condensation reaction: HOBt, acid-binding agent: DIEA.
(2) And sequentially connecting the amino acids required in the polypeptide sequence from right to left, after the reaction is finished to synthesize the KLVFFAED polypeptide, dissolving gallic acid, pyBOP and TEA in DMF, coupling with resin, and reacting for 12h. Wherein the molar ratio of the polypeptide, the gallic acid, the PyBOP and the TEA is 1.5.
(3) Preparing a cutting reagent: 2.5% of TFA; TIA 2.5%; 95% of water, cracking the gallic acid polypeptide conjugate from the resin by using a cutting reagent, and volatilizing the cracking solution to obtain a crude product.
(4) The crude product was washed three times with cold ether, the washed crude product was purified using an HPLC C18 semi-preparative column, and the sample from the detector was collected. The detection chromatographic conditions are as follows: detection wavelength: 214nm; mobile phase A: water (containing 0.1% trifluoroacetic acid), mobile phase B: acetonitrile (containing 0.1% trifluoroacetic acid) starting the gradient 30% B,70% A, ending the gradient 50% B,50% A, the gradient time 20min. Desalting and freeze-drying the product with a purity of more than 95% to obtain a white powdery gallic acid polypeptide conjugate.
Example 4
In this example, the gallic acid polypeptide conjugate is prepared by the following method, specifically including the following steps:
(1) The polypeptide solid phase synthesis method using FMOC strategy takes amino acid with amino terminal protected by FMOC as raw material, and uses a full-automatic microwave polypeptide synthesizer of Liberty Lite of CEM company to synthesize, and the operation method is carried out according to the instruction of the instrument.
The synthetic reagent is selected from:
(a) Carrier resin: 2-chlorotrityl chloride resin, degree of substitution: 0.5.
(b) Deprotection reagents: 20% piperidine in DMF.
(c) Coupling reagents were used in the condensation reaction: HOBt, acid-binding agent: DIEA.
(2) And sequentially connecting the amino acids required in the polypeptide sequence from right to left, after the reaction is finished to synthesize the KLVFFAED polypeptide, dissolving gallic acid, DIC, HOBt and DIEA in DMF, coupling with resin, and reacting for 12h. Wherein the molar ratio of the polypeptide to the gallic acid to DIC to HOBt to DIEA is 1.
(3) Preparing a cutting reagent: 2.5% of TFA; TIA 2.5%; 95% of water, cracking the gallic acid polypeptide conjugate from the resin by using a cutting reagent, and volatilizing the cracking solution to obtain a crude product.
(4) The crude product was washed three times with cold ether, and the washed crude product was purified using an HPLC C18 semi-preparative column to collect the sample that flowed from the detector. The detection chromatographic conditions are as follows: detection wavelength: 214nm; mobile phase A: water (containing 0.1% trifluoroacetic acid), mobile phase B: acetonitrile (containing 0.1% trifluoroacetic acid) start the gradient 30% B,70% A, end the gradient 50% B,50% A, gradient time 20min. Desalting and freeze-drying the product with the purity of more than 95% to obtain white powdery gallic acid polypeptide conjugate.
Example 5
In this example, the gallic acid polypeptide conjugate is prepared by the following method, specifically including the following steps:
(1) The polypeptide solid phase synthesis method using FMOC strategy takes amino acid with amino terminal protected by FMOC as raw material, and uses a full-automatic microwave polypeptide synthesizer of Liberty Lite of CEM company to synthesize, and the operation method is carried out according to the instruction of the instrument.
The synthetic reagent is selected from:
(a) Carrier resin: 2-chlorotrityl chloride resin, degree of substitution: 0.5.
(b) Deprotection reagents: 20% piperidine in DMF.
(c) Coupling reagents were used in the condensation reaction: HOBt, acid-binding agent: DIEA.
(2) And sequentially connecting the needed amino acids in the polypeptide sequence from right to left, after the reaction is finished and synthesizing the KLVFFAED polypeptide, dissolving gallic acid, EDC, HOBt and TEA in DMF, coupling with resin, and reacting for 12h. Wherein the molar ratio of polypeptide, gallic acid, EDC, HOBt and TEA is 1.
(3) Preparing a cutting reagent: 2.5% of TFA; TIA 2.5%; 95% of water, cracking the gallic acid polypeptide conjugate from the resin by using a cutting reagent, and volatilizing the cracking solution to obtain a crude product.
(4) The crude product was washed three times with cold ether, and the washed crude product was purified using an HPLC C18 semi-preparative column to collect the sample that flowed from the detector. The detection chromatographic conditions are as follows: detection wavelength: 214nm; mobile phase A: water (containing 0.1% trifluoroacetic acid), mobile phase B: acetonitrile (containing 0.1% trifluoroacetic acid) start the gradient 30% B,70% A, end the gradient 50% B,50% A, gradient time 20min. Desalting and freeze-drying the product with a purity of more than 95% to obtain a white powdery gallic acid polypeptide conjugate.
TABLE 1
Table 1 shows the catalyst, alkaline reagent, ratio of each component and yield for synthesizing gallic acid polypeptide conjugate in examples 1-5. In examples 1-3, when PyBOP or DMAP was used as the catalyst, the product yield was high due to the high activity of the two catalysts. PyBOP is a catalyst with strong activity in phosphonium salt catalysts, DMAP is a novel acylation catalyst with strong nucleophilic action, and both the catalyst and the catalyst have high selectivity and catalytic activity, and the reaction is efficient and mild. In examples 4 to 5, the conventional carbodiimide-based catalysts DIC and EDC had low catalytic activities, and further required the addition of an acylation catalyst HOBt to inhibit the self-rearrangement to form urea as a by-product, and the reaction system was more complicated, and thus the yield was low in the actual reaction. In comparison with examples 1 and 3, the yield of DIEA, which is the basic reagent, is higher with the same other variables, probably because DIEA has a stronger electron-donating ability and a stronger basicity than TEA, and can promote the reaction more efficiently, and thus the product yield is higher.
TABLE 2
| Serial number | Retention time | Peak area | Peak height | |
| 1 | 6.454 | 210862 | 16342 | 2.491 |
| 2 | 7.033 | 8141295 | 755466 | 96.180 |
| 3 | 7.304 | 112519 | 7680 | 1.329 |
| Is totaled | 8464676 | 779488 | 100.000 |
The phenolic acid polypeptide conjugates prepared in examples 1-5 were characterized. Fig. 2 shows HPLC purification results, table 2 shows HPLC peak information, and fig. 3 shows mass spectrum characterization of the product. The result shows that the gallic acid polypeptide conjugate is successfully synthesized, and the purity meets the requirement.
Example 6
In this example, the lipophilicity of gallic acid and gallic acid polypeptide conjugate was evaluated by reverse thin layer chromatography as follows:
the stationary phase of the thin layer chromatography adopts a Merck RP-18F254s silica gel plate, the mobile phase is methanol/water, and the volume ratio is 90. Saturating silica gel plate with mobile phase for 30 min, loading gallic acid and gallic acid polypeptide conjugate in chromatography cylinder at a position 1cm away from the bottom of the plate, developing, observing under dark box type ultraviolet analyzer, selecting 254nm wavelength, and measuring specific shift value R f And R is calculated according to the following formula m The value: r m ＝log(1/R f -1)
The more lipophilic the compound, the more R m The smaller the absolute value. Calculating to obtain food deficiencyBronsted acid R m Is-1.276, gallic acid polypeptide conjugate R m Is-0.629, which shows that the lipid solubility of the drug is improved to about 2.03 times by polypeptide modification.
Example 7
In this example, the alizarin red fluorescence method was used to examine the phenolic hydroxyl activity of conjugate of gallic acid and gallic acid polypeptide, and the method was as follows:
3.61mg alizarin red powder is weighed and dissolved in 1mL pure water to obtain 10 mu mol/mL alizarin red mother liquor. Weighing 34 μ g (0.2 μmol) of gallic acid, dissolving in 100 μ L of purified water, weighing 224 μ g (0.2 μmol) of gallic acid polypeptide conjugate, dissolving in 100 μ L of purified water, adding 20 μ L of alizarin red mother liquor, reacting in dark for 10min, and scanning with multifunctional microplate reader.
As a result, alizarin red itself is almost non-fluorescent, and exhibits strong fluorescence when it is combined with a substance containing a catechol structure, as shown in fig. 4. Fluorescence spectra of gallic acid and gallic acid polypeptide conjugate after being combined with alizarin red are similar, the conjugate has a maximum emission wavelength at 620nm, and fluorescence values under the maximum emission wavelength are consistent, so that after the gallic acid and the polypeptide are synthesized to form the conjugate, a phenolic hydroxyl structure is still intact, the binding capacity is not influenced, and the original activity is retained.
Example 8
In this example, alizarin red fluorescence method was used to examine the stability of conjugate of gallic acid and gallic acid polypeptide in alkaline aqueous solution at pH 8.0, as follows:
3.61mg alizarin red powder is weighed and dissolved in 1mL pure water to obtain 10 mu mol/mL alizarin red mother liquor. Weighing 8.5 mu g (0.05 mu mol) of gallic acid, dissolving the gallic acid in 100 mu L of Tris-HCl solution with pH 8.0, weighing 56 mu g (0.05 mu mol) of gallic acid polypeptide conjugate, dissolving the gallic acid polypeptide conjugate in 100 mu L of Tris-HCl solution with pH 8.0, standing at room temperature for 24h, respectively adding 5 mu L of alizarin red mother liquor, reacting in a dark place for 10min, and performing fluorescence spectrum scanning on the two solutions by using a multifunctional microplate reader.
According to the analysis experiment result, after the phenolic acid substances are dissolved in water, the phenolic acid substances can easily react with oxygen, so that phenolic hydroxyl is oxidized into quinone. In alkaline solutions, this oxidation reaction is more rapid. In the alizarin red fluorescence spectrum, the oxidized quinoid structure is combined with alizarin red, and the fluorescence shows obvious red shift. As can be seen from FIG. 5, after standing in the buffer solution with pH 8.0 for 24h, the maximum emission wavelength of fluorescence of gallic acid combined with alizarin red is red-shifted from 620nm to 520nm, while the maximum emission wavelength of fluorescence of gallic acid polypeptide conjugate combined with alizarin red is almost not changed significantly, which proves that the stability of the drug is improved after the gallic acid is subjected to polypeptide coupling modification.
Example 9
In this example, alizarin red fluorescence method was used to examine the stability of conjugate of gallic acid and gallic acid polypeptide in alkaline aqueous solution at pH 8.5, as follows:
3.61mg alizarin red powder is weighed and dissolved in 1mL pure water to obtain 10 mu mol/mL alizarin red mother liquor. Weighing 8.5 mu g (0.05 mu mol) of gallic acid, dissolving the gallic acid in 100 mu L of Tris-HCl solution with pH 8.5, weighing 56 mu g (0.05 mu mol) of gallic acid polypeptide conjugate, dissolving the gallic acid polypeptide conjugate in 100 mu L of Tris-HCl solution with pH 8.5, standing at room temperature for 24h, respectively adding 5 mu L of alizarin red mother liquor, reacting in a dark place for 10min, and performing fluorescence spectrum scanning on the two solutions by using a multifunctional microplate reader.
The experimental result is shown in fig. 6, and it can be seen that after standing in the buffer solution of pH 8.5 for 24h, the maximum emission wavelength of the fluorescence of gallic acid and alizarin red is red-shifted from 620nm to 520nm, and the fluorescence value at 520nm is greater than that when standing in the buffer solution of pH 8.0 for 24h, indicating that the oxidation degree is higher. The gallic acid polypeptide conjugate and alizarin red combined fluorescence maximum emission wavelength hardly has obvious change, and the result proves that the stability of the medicine is improved after the gallic acid is subjected to polypeptide coupling modification.
Example 10
In this example, the SH-SY5Y cell safety of gallic acid polypeptide conjugate and gallic acid was examined by the following method:
SH-SY5Y cells are arranged at 2X 10 4 Inoculating each well into 96-well plate, culturing for 24 hr, discarding culture medium, adding 100uL gallic acid or gallic acid polypeptide conjugate solution prepared from serum-free culture medium at concentration of 0.01,0.1,1,5, 10, 20 μ M, culturing for 24 hr, discarding culture mediumAfter rinsing twice with PBS, 0.5% MTT solution prepared in 100uL serum-free medium was added, incubated at 37 ℃ for 4 hours, the supernatant was discarded, 100uL DMSO was added, and the mixture was shaken for 10min, and the absorbance was measured at 570nm using a microplate reader. The non-dosed cells served as 100% survival controls, and the non-dosed wells served as zero uptake calibrations.
The experimental results are shown in fig. 7, when the concentration range is 0.01-5 μ M, the survival rates of SH-SY5Y cells of both the gallic acid group and the gallic acid polypeptide conjugate group are above 90%. When the concentration reaches 5-20 μ M, the toxic effect on cells is increased along with the increase of the concentration of gallic acid, and the cell activity is only 76.87% at 20 μ M. The cell activity of the gallic acid polypeptide conjugate group is still over 90% under 20 mu M, which shows that the cytotoxicity is not increased by performing polypeptide modification on the gallic acid, and the drug safety is improved to a certain extent, and the phenomenon is more remarkable under the condition of higher drug concentration, namely the gallic acid polypeptide conjugate can also keep good cell safety under the condition of higher concentration.
Example 11
In this example, BV-2 cell safety of gallic acid polypeptide conjugate and gallic acid was examined as follows:
BV-2 cells were packed at 2X 10 4 Inoculating each well in a 96-well plate, culturing for 24h, discarding the culture medium, adding 100uL of gallic acid or gallic acid polypeptide conjugate solution prepared by serum-free culture medium, the concentration is 0.01,0.1,1,5, 10 and 20 μ M respectively, continuously culturing for 24h, discarding the culture medium, rinsing with PBS twice, adding 0.5 ml MTT solution prepared by 100uL of serum-free culture medium, incubating at 37 ℃ for 4h, discarding the supernatant, adding 100uL of DMSO, shaking for 10min, and measuring the absorption value at 570nm by using an enzyme-labeling instrument. The non-dosed cells served as 100% survival controls, and the non-dosed wells served as zero uptake calibrations.
The experimental results are shown in figure 8, when the concentration range is 0.01-5 mu M, the survival rate of BV-2 cells of the gallic acid group and the gallic acid polypeptide conjugate group is above 90%. When the concentration reaches 5-20 μ M, the toxic effect on cells is increased along with the increase of the concentration of gallic acid, and the cell activity is only 75.52% at 20 μ M. The cell activity of the gallic acid polypeptide conjugate group is still over 90% under 20 mu M, which shows that the cytotoxicity is not increased by performing polypeptide modification on the gallic acid, and the drug safety is improved to a certain extent, and the phenomenon is more remarkable under the condition of higher drug concentration, namely the gallic acid polypeptide conjugate can also keep good cell safety under the condition of higher concentration.
Example 12
In this example, the blood brain barrier permeability of gallic acid polypeptide conjugates was examined as follows:
hCMEC/D3 cells were plated at 1X 10 4 One well was inoculated into the upper chamber of Transwell to construct a blood brain barrier model. The cell resistance value was measured every day until it reached 180. Omega./cm 2 The above represents the success of the modeling. The upper chamber medium was discarded and 200. Mu.L of 20. Mu.M Abeta.was added 1-42 And (3) incubating the oligomer serum-free culture medium solution for 24 hours to simulate the pathological environment of Alzheimer's disease and stimulate the expression of RAGE of endothelial cells. After the incubation is finished, the culture medium in the upper chamber is replaced by 200 mu L of 50 mu g/mL gallic acid serum-free culture medium solution and 200 mu L of 50 mu g/mL gallic acid polypeptide conjugate serum-free culture medium solution, the culture medium in the lower chamber is replaced by 500 mu L of serum-free culture medium, and the incubation is continued for 4h. Taking the culture medium out of the chamber, and detecting the content of gallic acid or gallic acid polypeptide conjugate by HPLC injection.
Experimental results show that the blood brain barrier permeability of the gallic acid polypeptide conjugate is obviously higher than that of gallic acid, and the gallic acid polypeptide conjugate can be efficiently absorbed by cells through receptor-mediated transcytosis.
The protection of the present invention is not limited to the above embodiments. Variations and advantages that may occur to those skilled in the art are intended to be included within the invention without departing from the spirit and scope of the inventive concept, and the scope of the invention is to be protected by the following claims.
Claims (13)
1. A phenolic acid polypeptide conjugate, wherein the phenolic acid polypeptide conjugate is formed by connecting a phenolic acid and a polypeptide through an amide bond, wherein the phenolic acid is selected from the group consisting of gallic acid, rosmarinic acid, ferulic acid, caffeic acid, protocatechuic acid, chlorogenic acid, sinapic acid and vanillic acid; the amino acid sequence of the polypeptide is KLVFFAED.
2. The phenolic acid polypeptide conjugate of claim 1, wherein the phenolic acid polypeptide conjugate has the structure of formula i:
wherein R is a structure of phenolic acid except carboxyl, and the carboxyl connected with R in the phenolic acid is connected with the amino of the polypeptide to form amido bond.
3. The phenolic acid polypeptide conjugate of claim 2, wherein the structure of the phenolic acid polypeptide conjugate is according to formula ii:
4. a method of preparing a phenolic acid polypeptide conjugate of claims 1-3, comprising the steps of:
(1) Synthesizing a KLVFFAED polypeptide using a polypeptide solid phase synthesis method;
(2) After the synthesis of the polypeptide is finished, under the protection of inert atmosphere, phenolic acid solution, catalyst and alkaline reagent are dissolved in organic solvent to be coupled with resin, so that phenolic acid carboxyl and polypeptide N-terminal amino react to form amide bond connection, and after the reaction is carried out for 8-24h, the polypeptide is cut from the resin by using a cutting reagent to obtain a crude product of the phenolic acid polypeptide conjugate;
(3) Separating and purifying the crude product of the phenolic acid polypeptide conjugate, and freeze-drying to obtain the phenolic acid polypeptide conjugate; in the step (2), the catalyst is benzotriazole-1-yl-oxy tripyrrolidinyl phosphorus hexafluorophosphate or 4-dimethylaminopyridine.
5. The method for preparing a conjugate of phenolic acid polypeptide as claimed in claim 4, wherein the basic reagent in step (2) is one or more selected from N, N-diisopropylethylamine, triethylamine and pyridine.
6. The method for preparing a phenolic acid polypeptide conjugate according to claim 5, wherein the basic reagent in step (2) is N, N-diisopropylethylamine.
7. The method for preparing a phenolic acid polypeptide conjugate according to claim 4, wherein the molar ratio of the catalyst to the phenolic acid to the polypeptide in step (2) is 2-5.
8. The method for preparing a conjugate of a phenolic acid polypeptide as claimed in claim 7, wherein the molar ratio of the catalyst, the phenolic acid and the polypeptide in step (2) is 3.
9. The method for preparing a phenolic acid polypeptide conjugate according to claim 4, wherein the molar ratio of the basic agent to the catalyst in step (2) is 1-3.
10. The method for preparing a phenolic acid polypeptide conjugate according to claim 9, wherein the molar ratio of the basic agent to the catalyst in step (2) is 1.
11. The method for preparing a conjugate of phenolic acid polypeptide according to claim 4, wherein the separation and purification steps in step (3) are mainly washing, drying and HPLC purification.
12. The use of the phenolic acid polypeptide conjugate of claims 1-3 in the preparation of a medicament for the treatment of alzheimer's disease, stroke and amyloid cerebrovascular disease.
13. The use of claim 12, wherein the medicament is a formulation.
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| A conjugate of the lytic peptide Hecate and gallic acid: structure,activity against cervical cancer, and toxicity;Paulo R.S.Sanches 等;《Amino Acids》;20151231;第47卷;摘要，说明书第1434页，图1 * |
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