CN111499687A - Sphingosine-1-phosphate receptor 3 agonist, and preparation method and application thereof - Google Patents
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
- A61K47/542—Carboxylic acids, e.g. a fatty acid or an amino acid
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
- A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
- A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
- A61P9/14—Vasoprotectives; Antihaemorrhoidals; Drugs for varicose therapy; Capillary stabilisers
Abstract
The S1PR3 agonist comprises a polypeptide, a water-solubility enhancing part and a permeability enhancing part which are sequentially coupled at the N end of the polypeptide, wherein the polypeptide has an amino acid sequence shown in SEQ ID No.1, the water-solubility enhancing part and the permeability enhancing part are sequentially coupled at the N end of the polypeptide, the permeability enhancing part enables the sphingosine 1 phosphate receptor 3 agonist to have good cell membrane permeability and enhances the cell-entering capacity of the polypeptide, and the water-solubility enhancing part enables the sphingosine 1 phosphate receptor 3 agonist to have good water solubility and the solubility in ultra-pure water to reach 5mg/m L.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to a sphingosine-1-phosphate receptor 3 agonist, and a preparation method and application thereof.
Background
The Sphingosine-1-phosphate receptors (S1 PRs) are seven transmembrane G protein coupled receptors, and can start a cell transmembrane signal transduction effect after being combined with natural ligand Sphingosine-1-phosphate (S1P), thereby being widely involved in physiological processes such as blood vessel regeneration, cell proliferation, immune regulation and the like in a human body. The sphingosine-1-phosphate receptor 3(S1PR3) is a key member of the S1PRs superfamily, is widely expressed in endothelial cells, vascular smooth muscle cells and macrophages, and regulates angiogenesis and immune inflammatory response by activating mitogen-activated protein kinase (MAPKs) signal pathways.
Research (Sun X, Ma SF, Water MS, et a1.functional promoter variant to segment-associated with responsive therapy syndrome [ J ]. Am J Physiol L un Cell MolPhyol, 2013, 305 (7): L467-477. DOI: 10.1152/ajplung. 00010.2013.) found that mutation of the S1PR3 promoter leads to decreased expression of the S1PR3 protein in patients with sepsis-associated acute respiratory syndrome, while research (Hou J, Chen Q, Wu X, PRa1. S13 signaling bacteria expressing reduction sepsis ] suggests that activation of macrophage in MRJ, 024J, SR 11. PSI, SR JJ.7. and SR JJ.11. PSI, SR JK.11. PSI, SR JK.19, SR JK.7, SR JK.11, SR JK.PSR, SR JK.19, SR JK.K.K.K.K.K. 1, SR JK.K.K. 1. PSR.S.3, SR JK.K. PSR.K.K. 1, SR.K.K. PSR.K. 1, PSR.K.K.K. PSR.K. 1, PSR.K.K. PSR.K. K. PSR, PSR.K.K. No.1, PSR. K. 1, PSR.
On the basis, U.S. Pat. No. 4,2016/0333070A 1 discloses a polypeptide drug G { Nle } RPY-NH capable of specifically exciting S1PR32(GY 5 for short, having amide end capping at the C-terminal) and the amino acid sequence thereof is Gly-Nle-Arg-Pro-Tyr-NH2However, GY5 itself is difficult to enter the cell and thus fails to activate S1PR 3; therefore, the N end of the polypeptide sequence is further Myristoyl modified to enhance the cell-entering ability, and finally the high-activity S1PR3 agonist peptide Myristoyl-G { Nle } RPY-NH is obtained2(GPS-725.017 for short). Research shows that GPS-725.017 can significantly improve the survival rate of sepsis mice. However, GPS-725.017 is insoluble in water, which greatly limits its value in basic research and clinical transformation.
Disclosure of Invention
The invention aims to provide a sphingosine-1-phosphate receptor 3 agonist, and a preparation method and application thereof.
In order to achieve the above purpose, the technical solution of the present application is as follows:
a sphingosine-1-phosphate receptor 3 agonist comprises a polypeptide, wherein the amino acid sequence of the polypeptide is shown as SEQ ID No. 1; also includes a water-solubility enhancing moiety and a permeability enhancing moiety sequentially coupled to the N-terminus of the polypeptide.
The invention sequentially couples a water-solubility enhancing part and a permeability enhancing part at the N end of a polypeptide Gly-Nle-Arg-Pro-Tyr (abbreviated as G { Nle } RPY), wherein the permeability enhancing part ensures that the sphingosine-1-phosphate receptor 3 agonist has good cell membrane permeability and enhances the cell-entering capability of the polypeptide, and meanwhile, the water-solubility enhancing part is also arranged between the polypeptide and the permeability enhancing part, and the water-solubility enhancing part ensures that the sphingosine-1-phosphate receptor 3 agonist has excellent water solubility which can reach 5mg/m L in ultra-pure water.
In the aforementioned sphingosine-1-phosphate receptor 3 agonist, the water solubility-enhancing moiety comprises alanine-type PEG 11.
Wherein, the chemical formula of the propionic acid type PEG11 is as follows: NH (NH)2-(CH2CH2O)11-CH2CH2COOH。
In the aforementioned sphingosine-1-phosphate receptor 3 agonist, the permeability-enhancing moiety is a fatty acid, a steroid, a vitamin, a carbohydrate, an amino acid or a transporter peptide.
Preferably, in the sphingosine-1-phosphate receptor 3 agonist described above, the permeability-enhancing moiety is a fatty acid.
Further preferably, in the sphingosine-1-phosphate receptor 3 agonist described above, the permeability-enhancing moiety is stearic acid.
Preferably, the sphingosine-1-phosphate receptor 3 agonists of the invention have the formula:
the N-terminal amino group of the polypeptide G { Nle } RPY is subjected to dehydration condensation with the carboxyl group of propionic acid type PEG11, and the carboxyl group of stearic acid is subjected to dehydration condensation with the amino group of propionic acid type PEG11, so that the N-terminal amino group of the polypeptide G { Nle } RPY is sequentially coupled to the N-terminal of the polypeptide. The C-terminal carboxyl group of the polypeptide is amidated to ensure that the structure of the sphingosine-1-phosphate receptor 3 agonist remains stable.
The preparation method of the sphingosine-1-phosphate receptor 3 agonist comprises the following steps:
(1) synthesizing the polypeptide by adopting Fmoc solid phase synthesis method;
(2) dehydrating and condensing carboxyl of propionic acid type PEG11 and N-terminal amino of polypeptide to obtain an intermediate;
(3) dehydrating and condensing the carboxyl of stearic acid and the amino of the intermediate to obtain the sphingosine-1-phosphate receptor 3 agonist. Specifically, in the above method for preparing a sphingosine-1-phosphate receptor 3 agonist, the step (1) comprises the steps of:
(a) adding 2-chlorotrityl chloride resin into a solid phase synthesis reaction tube, adding Dichloromethane (DCM), and oscillating for 30 minutes to swell the resin;
(b) after dichloromethane in a solid phase synthesis reaction tube is removed, adding excessive Fmoc protected tyrosine, then adding N, N-Dimethylformamide (DMF) to fully dissolve, then adding excessive Diisopropylethylamine (DIEA), oscillating for 80-120min, finally sealing with methanol, and removing N, N-dimethylformamide;
(c) adding a deprotection solution into a solid-phase synthesis reaction tube, removing the reaction solution after full oscillation, adding the deprotection solution again, fully oscillating, then extracting the deprotection solution, taking a small amount of resin, and detecting whether the reaction is finished by adopting an indantrione method; after the reaction is finished, washing the resin by N, N-dimethylformamide and dichloromethane intermittently;
(d) adding excessive proline protected by Fmoc and O-benzotriazole-tetramethyluronium Hexafluorophosphate (HBTU) into a solid-phase synthesis reaction tube, adding a small amount of N, N-dimethylformamide for dissolving, immediately adding excessive diisopropylethylamine, and reacting for 20-40 min; taking a small amount of resin, and detecting whether the dehydration condensation reaction is finished by an indantrione method; after the reaction is finished, washing the resin by N, N-dimethylformamide and dichloromethane intermittently;
(e) and (d) repeating the steps (c) and (d), and adding excessive Fmoc-protected arginine, Fmoc-protected L-2-aminocaproic acid and Fmoc-protected glycine into a solid-phase synthesis reaction tube in sequence until all amino acids are dehydrated and condensed to obtain the polypeptide.
In the above method for preparing a sphingosine-1-phosphate receptor 3 agonist, the deprotection solution is DMF containing 20% piperidine.
In the above method for preparing a sphingosine-1-phosphate receptor 3 agonist, the step (2) comprises the steps of:
(f) repeating the step (c), and removing the Fmoc protecting group at the N end of the polypeptide;
(g) repeating step (d) and adding Fmoc-protected propionic acid type PEG11With the polypeptide G { Nle } RPY-NH2Dehydrating and condensing to obtain an intermediate.
In the above method for preparing a sphingosine-1-phosphate receptor 3 agonist, the step (3) comprises the steps of:
(h) repeating the step (c), and removing the Fmoc protecting group on the intermediate;
(i) repeating the step (d), and dehydrating and condensing the Fmoc-protected stearic acid and the intermediate;
(j) and adding a cutting agent into the solid phase synthesis reaction tube, cutting for 1.5-2.5h, and amidating the carboxyl of the C end of the polypeptide to obtain the sphingosine-1-phosphate receptor 3 agonist.
In the above method for preparing the sphingosine-1-phosphate receptor 3 agonist, the cleavage agent is composed of 95% TFA, 1% water, 2% ethanedithiol and 2% triisopropylsilane in percentage by volume.
The invention also provides the application of the sphingosine-1-phosphate receptor 3 agonist in the preparation of immunoregulation drugs, vascular protection drugs or antitumor drugs.
Compared with the prior art, the invention has the beneficial effects that:
the polypeptide G { Nle } RPY-NH of the invention2The N end of the polypeptide is sequentially coupled with a water-solubility enhancing part and a permeability enhancing part, wherein the permeability enhancing part enables the sphingosine-1-phosphate receptor 3 agonist to have good cell membrane permeability and enhances the cell-entering ability of the polypeptide; also, the present invention is directed to polypeptides and osmosesThe water-solubility enhancing parts are also arranged between the sexual enhancing parts, so that the sphingosine-1-phosphate receptor 3 agonist has excellent water solubility, and the solubility in ultra-pure water can reach 5mg/m L.
Drawings
FIG. 1 is a reversed phase high performance liquid chromatography detection profile of sphingosine-1-phosphate receptor 3 agonist G L D-001 according to the present invention;
wherein, sample Name represents sample Name, sample ID represents sample number, Time Processed represents processing Time (of sample), Month-Day-Year Processed represents processing Year-Month-Day (of sample); pump A: 0.065% trifluoracetic in 100% water (v/v) indicates a trifluoroacetic acid to water volume ratio of 0.065:100 in mobile phase A, Pump B: 0.05% trifluoracetic in 100% acetonitrile (v/v) represents that the volume ratio of trifluoroacetic acid to acetonitrile in the mobile phase B is 0.05:100, Total Flow represents Flow rate, and Wavelength represents detection Wavelength;
the mobile phase gradient elution procedure for chromatography was: the volume ratio of mobile phase a increased from 40% to 100% during elution time (time) from 0.01 to 25 min; the volume ratio (value) of mobile phase A is 95% during the elution time (time) of 25.01-32.00min, 40% during the elution time (time) of 32.01-40.00min, and the elution (stop) is stopped at the elution time (time) of 40.00 min;
column Performance represents Column information, Detecor a represents probe a, Equipment represents (operating) environment;
chromatogram for chromatography;
paek Table represents a liquid chromatogram Peak Table, Peak # represents a Peak serial number, ret. The same applies below;
FIG. 2 is a mass spectrometric profile of a sphingosine-1-phosphate receptor 3 agonist of the present invention;
wherein mass spectrum represents mass spectrum, positive intensity represents positive intensity, Acquired by Gary represents that the detection result is presented by Gary, Injection Volume represents Injection Volume, theoretical MW represents theoretical molecular weight, Observated MW represents actual molecular weight, Interface represents that ESI represents that the ion source is ESI, NebulilingGas Flow represents atomizing Gas Flow rate, CD L temp represents CD L temperature, block temp represents closing temperature, Interface bias represents atomizing voltage, Drying Gas Flow represents Drying Gas Flow rate, T.flow represents turbine Flow rate, and conc represents concentration;
FIG. 3 is a graph of the effect of sphingosine-1-phosphate receptor 3 agonists of the present invention on the expression levels of ERK and p-ERK in cells;
where ERK denotes extracellular regulated protein kinase, p-ERK denotes phosphorylated extracellular regulated protein kinase, blank denotes cells not treated with any sphingosine-1-phosphate receptor 3 agonist, GY5 denotes cells treated with the polypeptide GY5 (i.e., G { Nle } RPY), GPS-725.017 denotes cells treated with GPS-725.017, G L D-001 denotes cells treated with the sphingosine-1-phosphate receptor 3 agonist of the present invention G L D-001.
Detailed Description
The technical solution of the present invention will be further described in detail with reference to the accompanying drawings and the detailed description.
Example 1
The invention relates to a sphingosine-1-phosphate receptor 3 agonist G L D-001 which is consigned to Nanjing Kinshire Biotech, Inc. for synthesis, and the preparation method comprises the following steps:
(1) adopting Fmoc solid phase synthesis method to synthesize polypeptide G { Nle } RPY;
specifically, the method comprises the following steps:
(a) adding 2-chlorotrityl chloride resin into a solid phase synthesis reaction tube, adding Dichloromethane (DCM), and oscillating for 30 minutes to swell the resin;
(b) after dichloromethane in a solid phase synthesis reaction tube is removed, adding excessive Fmoc protected tyrosine, then adding N, N-Dimethylformamide (DMF) to fully dissolve, then adding excessive Diisopropylethylamine (DIEA), oscillating for 1h, finally sealing with methanol, and removing N, N-dimethylformamide;
(c) adding a deprotection solution (DMF containing 20% piperidine) into a solid-phase synthesis reaction tube, removing the reaction solution after full oscillation, adding the deprotection solution again, fully oscillating, then extracting the deprotection solution, taking a small amount of resin, and detecting whether the reaction is finished by adopting an indantrione method;
after the reaction is finished, washing the resin by N, N-dimethylformamide and dichloromethane intermittently;
(d) adding excessive proline and HBTU protected by Fmoc into a solid-phase synthesis reaction tube, adding a small amount of N, N-dimethylformamide for dissolving, immediately adding excessive diisopropylethylamine, and reacting for 30 min; taking a small amount of resin, and detecting whether the dehydration condensation reaction is finished by an indantrione method; after the reaction is finished, washing the resin by N, N-dimethylformamide and dichloromethane intermittently;
(e) repeating the steps (c) and (d), and sequentially adding excessive arginine protected by Fmoc, L-2-aminocaproic acid protected by Fmoc and glycine protected by Fmoc into a solid-phase synthesis reaction tube until all amino acids are dehydrated and condensed;
at this time, the polypeptide G { Nle } RPY is completely prepared, and the carboxyl group of the polypeptide G { Nle } RPY is bonded to the resin;
(2) dehydrating and condensing carboxyl of propionic acid type PEG11 and N-terminal amino of polypeptide to obtain an intermediate;
specifically, the method comprises the following steps:
(f) repeating the step (c), and removing the Fmoc protecting group at the N end of the polypeptide;
(g) repeating step (d) and adding Fmoc-protected propionic acid type PEG11Dehydrating and condensing with polypeptide GY5 to obtain an intermediate;
(3) dehydrating and condensing carboxyl of stearic acid and amino of the intermediate to obtain the sphingosine-1-phosphate receptor 3 agonist;
specifically, the method comprises the following steps:
(h) repeating the step (c), and removing the Fmoc protecting group on the intermediate;
(i) repeating the step (d), and dehydrating and condensing the Fmoc-protected stearic acid and the intermediate;
(j) adding a cutting agent into a solid phase synthesis reaction tube, cutting for 1.5-2.5h, amidating carboxyl of the C end of the polypeptide to obtain a target crude product, and purifying the target crude product by adopting reverse phase high performance liquid chromatography to finally obtain the sphingosine-1-phosphate receptor 3 agonist G L D-001 of the embodiment, wherein the chemical formula (shown in figure 1) is as follows:
wherein the cleavage agent consists of 95% TFA, 1% water, 2% ethanedithiol and 2% triisopropylsilane, in percent by volume.
Mass spectrometry (see FIG. 2) confirmed the successful synthesis of G L D-001, and HPLC analysis (see FIG. 1) indicated that the purity of G L D-001 in the final product was greater than 95%.
Further, the solubility of G L D-001 in each reagent was tested, and the test results are shown in Table 1.
TABLE 1
| Solvent(s) | Solubility in water | Solubility in water |
| Ultrapure water (ultra water) | Soluble in water | ≤5mg/mL |
| 0.1M PBS(pH 7.4) | Soluble in water | ≤5mg/mL |
| DMSO | Soluble in water | ≤10mg/mL |
As can be seen from Table 1, G L D-001 was sufficiently soluble in water and had a solubility as high as 5mg/m L.
Comparative example 1
Entrusted Nanjing King Shirui Biotechnology Ltd to synthesize the polypeptide G { Nle } RPY-NH2(GY 5) prepared in the same manner as in step (1) of example 1 by preparing G { Nle } RPY in a solid phase synthesis reaction tube, cleaving G { Nle } RPY with a cleavage reagent, and amidating the C-terminal carboxyl group to obtain GY 5.
Comparative example 2
The sphingosine-1-phosphate receptor 3 agonist GPS-725.017 was synthesized by Nanjing Kingsley Biotechnology Ltd, and its preparation method was referred to in the literature (Chenyue, Miyao, Sunyachi, Direction, Myristoyl-glycine modified S1PR3 specific agonist peptide transmembrane activation effect study. China Emergency medical journal 2017.26 (12): 1418-.
Using human umbilical vein endothelial cells HUVEC as an example, the effect of GY5, GPS-725.017, G L D-001 on the expression levels of intracellular ERK and p-ERK was tested.
Culturing HUVEC of human umbilical vein endothelial cell in DMEM medium containing 10% fetal calf serum and 1% double antibody, inoculating HUVEC growing in logarithmic phase into 12-well plate at inoculation density of 4 × 105Each well contains 500ul of pure DMEM medium, then GY5, GPS-725.017 and G L D-001 are added respectively for stimulation, the final concentration of GY5, GPS-725.017 and G L D-001 is 50uM, a blank control group is not stimulated by any substance, total protein in cells is extracted by RIPA lysate after 30min, and the expression levels of ERK and p-ERK in each group are detected by using a Western blot technique after the BCA kit protein is quantified, and the detection result is shown in figure 3.
As can be seen from FIG. 3, GY5 hardly promotes ERK phosphorylation due to poor permeability and poor cell-entering ability, while GPS-725.017 and G L D-001 can promote ERK phosphorylation, and the activity of G L D-001 is stronger than that of GPS-725.017, which shows that G L D-001 has not only higher water solubility, but also strong permeability and strong cell-entering ability after being modified by propionic acid type PEG11 and stearic acid.
Sequence listing
<110> Ningbo City first Hospital
<120> sphingosine-1-phosphate receptor 3 agonist, and preparation method and application thereof
<160>1
<170>SIPOSequenceListing 1.0
<210>2
<211>5
<212>PRT
<213> artificially synthesized sequence (Unknown)
<220>
<221>MOD_RES
<222>(2)..(2)
<223>Nle
<220>
<221>UNSURE
<222>(2)..(2)
<223>The 'Xaa' at location 2 stands for Gln, Arg, Pro, or Leu.
<400>2
Gly Xaa Arg Pro Tyr
1 5
Claims (10)
1. A sphingosine-1-phosphate receptor 3 agonist comprising a polypeptide having an amino acid sequence as shown in SEQ ID No. 1; the polypeptide is characterized by also comprising a water solubility enhancing part and a permeability enhancing part which are sequentially coupled at the N end of the polypeptide.
2. The sphingosine 1-phosphate receptor 3 agonist according to claim 1 wherein the water solubility enhancing moiety comprises alanine-type PEG 11.
3. The sphingosine 1-phosphate receptor 3 agonist according to claim 1 wherein the permeability enhancing moiety is a fatty acid, steroid, vitamin, carbohydrate, amino acid or transporter peptide.
4. The sphingosine 1-phosphate receptor 3 agonist according to claim 3 wherein the permeability enhancing moiety is stearic acid.
5. The sphingosine 1-phosphate receptor 3 agonist according to any of claims 1-4 having the formula:
6. the method of preparing a sphingosine 1-phosphate receptor 3 agonist according to any of claims 1-5 comprising the steps of:
(1) synthesizing the polypeptide by adopting Fmoc solid phase synthesis method;
(2) dehydrating and condensing carboxyl of propionic acid type PEG11 and N-terminal amino of polypeptide to obtain an intermediate;
(3) dehydrating and condensing the carboxyl of stearic acid and the amino of the intermediate to obtain the sphingosine-1-phosphate receptor 3 agonist.
7. The method of preparing a sphingosine-1-phosphate receptor 3 agonist according to claim 6, wherein step (1) comprises the steps of:
(a) feeding 2-chlorotrityl chloride resin into a solid phase synthesis reaction tube, adding dichloromethane, and oscillating for 30 minutes to swell the resin;
(b) after dichloromethane in a solid phase synthesis reaction tube is removed, adding excessive Fmoc protected tyrosine, then adding N, N-dimethylformamide for full dissolution, then adding excessive diisopropylethylamine, oscillating for 80-120min, finally sealing with methanol, and removing N, N-dimethylformamide;
(c) adding a deprotection solution into a solid-phase synthesis reaction tube, removing the reaction solution after full oscillation, adding the deprotection solution again, fully oscillating, then extracting the deprotection solution, taking a small amount of resin, and detecting whether the reaction is finished by adopting an indantrione method; after the reaction is finished, washing the resin by N, N-dimethylformamide and dichloromethane intermittently;
(d) adding excessive proline protected by Fmoc and O-benzotriazole-tetramethylurea hexafluorophosphate into a solid-phase synthesis reaction tube, adding a small amount of N, N-dimethylformamide for dissolving, immediately adding excessive diisopropylethylamine, and reacting for 20-40 min; taking a small amount of resin, and detecting whether the dehydration condensation reaction is finished by an indantrione method; after the reaction is finished, washing the resin by N, N-dimethylformamide and dichloromethane intermittently;
(e) and (d) repeating the steps (c) and (d), and adding excessive Fmoc-protected arginine, Fmoc-protected L-2-aminocaproic acid and Fmoc-protected glycine into a solid-phase synthesis reaction tube in sequence until all amino acids are dehydrated and condensed to obtain the polypeptide.
8. The method for preparing a sphingosine-1-phosphate receptor 3 agonist according to claim 7, wherein the step (2) comprises the steps of:
(f) repeating the step (c), and removing the Fmoc protecting group at the N end of the polypeptide;
(g) repeating step (d) and adding Fmoc-protected propionic acid type PEG11With the polypeptide G { Nle } RPY-NH2Dehydrating and condensing to obtain an intermediate.
9. The method for preparing a sphingosine-1-phosphate receptor 3 agonist according to claim 7, wherein the step (3) comprises the steps of:
(h) repeating the step (c), and removing the Fmoc protecting group on the intermediate;
(i) repeating the step (d), and dehydrating and condensing the Fmoc-protected stearic acid and the intermediate;
(j) and adding a cutting agent into the solid phase synthesis reaction tube, cutting for 1.5-2.5h, and amidating the carboxyl of the C end of the polypeptide to obtain the sphingosine-1-phosphate receptor 3 agonist.
10. Use of a sphingosine-1-phosphate receptor 3 agonist according to any of claims 1-5 for the preparation of an immunomodulatory, vasoprotective or antitumor drug.
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Citations (6)
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