CN1962691A - Thymus tetrapeptide active isomer and its preparation method and medicinal uses - Google Patents
Thymus tetrapeptide active isomer and its preparation method and medicinal uses Download PDFInfo
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Abstract
The invention discloses a thymic active isomer and preparing method for industrial manufacturing domain, which possesses amino sequence as X-Arg-Lys-Y-Val, wherein X is acetyl or hydrogen or random sequence with at least 70% isogenesis as the amino sequence; Y is Asp or Glu; Lys or Glu is substituted by corresponding beta-or delta-amino sequence. The invention also provides its medical utility, which can produce immune adjusting drug, tumour-resistance drug and oxidation resistance drug.
Description
Technical field
The present invention discloses a kind of thymus tetrapeptide active isomer and preparation method thereof, also discloses its application in preparation immunoregulation druge, antitumor drug and anti-oxidation medicine simultaneously, belongs to biological pharmacy technical field.
Background technology
Present biotechnology is separated from thymine, purifying several Zadaxin, the extrasin alpha that comprises the thymopoietin II that contains 49 amino-acid residues, constitutes by 28 amino-acid residues
1With thymulin etc.Native sequences thymus tripeptide (TP-3), tetrapeptide (TP-4), pentapeptide (TP-5) are the residue segments of thymopoietin II, and they still have the activity of thymopoietin bibliographical information.
Experiment showed, that Zadaxin can partially absorb at small intestine, and can improve immunologic function.But thymopeptide-5 is in vivo easily by various enzyme hydrolysis, as trypsinase, carboxypeptidase, aminopeptidase etc., so in the body transformation period have only 30 seconds.For healthy individual, peptide hormone plays in vivo and is eliminated rapidly after the regulating effect is very normal, but for the patient, when needing drug treatment, the transformation period is short unfavorable to treating.
Strengthen the stability of peptide hormone, the transformation period that prolongs it is to improve curative effect, this is that of peptide hormone generally needs the problem that solves, thus transform peptide hormone structure, to increase their in vivo stability, improve bioavailability be the problem to be solved in the present invention.
Summary of the invention
The present invention discloses a kind of thymus tetrapeptide active isomer and preparation method thereof, is applicable to suitability for industrialized production.
The invention also discloses the medicinal use of above-mentioned isomer, be used to prepare immunoregulation druge, antitumor drug and anti-oxidation medicine.
The thymus tetrapeptide active isomer of this disclosure of the Invention comprises one or multinomial following feature:
Compound has aminoacid sequence X-Arg-Lys-Y-Val, and X is ethanoyl or hydrogen, or arbitrary sequence therewith aminoacid sequence have 70% above homology at least; Y is Asp or Glu.
Thymus tetrapeptide active isomer, its structure are Lys among thymus tetrapeptide aminoacid sequence Arg-Lys-Asp-Val or the Arg-Lys-Glu-Val or Glu by its corresponding β-or aminoacid sequence of replacing of gamma-amino acid.
Thymus tetrapeptide active isomer aminoacid sequence table
| Thymus tetrapeptide active isomer | Aminoacid sequence |
| LW401 | Arg-Lys-β-Asp-Val |
| LW402 | Arg-β-Lys-Asp-Val |
| LW403 | Arg-Lys-γ-Glu-Val |
| LW404 | Arg-γ-Lys-Asp-Val |
| LW405 | Arg-β-Lys-β-Asp-Val |
| LW406 | Arg-β-Lys-γ-Glu-Val |
| LW407 | Arg-γ-Lys-β-Asp-Val |
| LW408 | Arg-γ-Lys-γ-Glu-Val |
The preparation method of isomer among the present invention: synthetic with the common Fmoc solid phase method of peptide synthesis.
With the amino acid derivative is raw material, is solvent with dimethyl formamide (DMF), and technological process comprises swelling-deprotection-coupling-deprotection coupling circulation-cutting.Said swelling be will connect peptide resin and DMF add in the column type reactor, the room temperature jolting is drained after connecing the peptide resin swelling, with draining after the DMF washing again; Said deprotection is to slough fluorenylmethyloxycarbonyl (Fmoc) protecting group that connects on the peptide resin, and method is that connecing after swelling adds in the peptide resin and take off Fmoc protecting group solution, and the room temperature jolting is drained the back and drained with the DMF washing again; Said coupling is to add a kind of amino acid derivative and condensing agent in column type reactor, adds DMF again, and the jolting of lucifuge room temperature was reacted 0.3-20 hour, drains the back and drains with DMF jolting washing again; Said deprotection coupling circulation is alternately to repeat deprotection and coupling process, the coupling multiplicity is by the chain length decision of small peptide, the amino acid derived raw material that each coupling is added is by the decision of forming of small peptide, and the multiplicity of deprotection is howed once than the coupling multiplicity; Said cutting is that deaminizating acid Side chain protective group reaches from connecing cleavage of peptide on the peptide resin; promptly add cutting reagent in the column type reactor after draining; in (25--20) ℃ vibration (1-5) hour, remove by filter and connect peptide resin, filtrate vacuumizes concentrated; concentrated solution adds ether/sherwood oil vibration; place (0--20) ℃ placement 0.2-2 hour, centrifugal, remove supernatant; precipitation vacuum-drying gets product.
In the above-mentioned technological process, connect peptide resin and can use Fmoc-NH-SAL resin, PAL resin, Wang resin, Fmoc-amide resins.Amino acid derivative can be Fmoc-Arg (pbf)-OH, Fmoc-Lys (Boc)-OH, Fmoc-Asp (otBu)-OH, Fmoc-β-Asp-otBu, Fmoc-Val-OH, Fmoc-Glu (otBu)-OH, Fmoc-γ-Glu-otBu, Fmoc-β-Lys (Boc)-OH, Fmoc-γ-Lys (Boc)-OH etc.The mol ratio that connects peptide resin and amino acid derivative is 1: (2-5).The condensing agent that the amino acid coupling is used can be benzotriazole-1-oxygen-three (dimethylamino) phosphorus hexafluorophosphate (BOP), 1-hydroxy benzo triazole (HOBT), N-methylmorpholine (NMM), in molar ratio 2: (1-3): (2-6) be mixed and made into; Amino acid derivative and condensing agent are 1 in molar ratio: (1--1.2).The cutting reagent main component is a trifluoroacetic acid.Take off the piperidines that Fmoc protecting group solution can be (15-25) %/DMF solution; Said with DMF jolting washing drain again need carry out repeatedly (5-8) inferior, each jolting (2-6) minute.
In order to obtain pure product, after the cutting technique process, also need carry out purge process.Said purifying is to adopt the high performance liquid chromatography chromatography purification, collects main peak.A pump solution is 0.1% trifluoroacetic acid aqueous solution, B pump solution is 0.1% trifluoroacetic acid, (60-80) % acetonitrile solution, carry out gradient elution---with overall flow rate (1-5) ml/ minute, in (10-60) minute, the A flow rate pump is changed into (50-20) % by (100--60) %, the B flow rate pump is changed into (50-80) % by (0-40) %, adopts the dual wavelength monitoring simultaneously, and wavelength is 214nm and 385nm.
Positively effect of the present invention is: the structure to TP-4 is transformed, designed the bioactive peptide isomer, on the basis of TP-4 original structure, with partial amino-acid in its aminoacid sequence with corresponding β-, gamma-amino acid replaces, bioactive peptide behind the gained reconstruction structure is keeping improving its stability greatly on the former activated basis, the transformation period in the extension body, improve its bioavailability, this to the exploitation of peptide medicament with significant.
By activity and the inside and outside stability of measuring them, their pharmacology, drug effect are studied.Experimental result shows that these isomer are keeping former activated basic upward stability to improve greatly.This invention provides theoretical foundation for the research and the new drug development of Zadaxin activity mechanism.
The present invention has made mensuration to the external activity of thymus tetrapeptide active isomer with vitro stability.
Human peripheral lymphocyte and the lymphocytic surface of porcine thymus T have sheep red blood cell (SRBC) (SRBC) acceptor, abbreviate the E acceptor as, and it makes the T cell form rosette with SRBC, and this is one of feature of mature T cells.Cell is handled through 45 ℃ of heating 1h or with pancreatin can slough the E acceptor.Zadaxin can promote to take off the synthetic again E acceptor of T cell of E acceptor, and therefore the E garland of the available E of taking off acceptor is tested the activity of measuring Zadaxin.
Thymopoietin can increase the T cell in various antigens or the former secretion that produces Interferon, rabbit (IFN), interleukin II (IL-2) and interleukin 3 multiple lymphokines such as (IL-3) as activation backs such as ConA of mitogenesis, increases the level of T cell surface lymphokine acceptor.Lymphocyte proliferation assay is the commonly used and leading indicator that lymphocyte function is examined in assessment, also is the effective means of examination pharmaceutical activity.
The mensuration of stability in the external blood plasma:
With sample in blood plasma 37 ℃ carry out enzymolysis, analyze hydrolysate with HPLC then, and compare with normal configuration, they are all longer than the normal configuration transformation period as a result.
The measuring result shows that the external immunocompetence of thymus tetrapeptide active isomer keeps or be higher than the level of thymopeptide-5 (TP-5).Thymus tetrapeptide active isomer stability strengthens, and the transformation period improves greatly.
The present invention has made general pharmacology to thymus tetrapeptide active isomer and has learned experiment, and the result shows animal nervous system, the cardiovascular system respiratory system of unifying is not all made significant difference.
The present invention has also made pharmacodynamic experiment to thymus tetrapeptide active isomer, shows to have significant immuno-potentiation, antitumor action and antioxygenation in the experimentation on animals.Its effect is significantly increased than thymopeptide-5 (TP-5's).
Test example 1
Cell proliferation experiment
1, the preparation of cell suspension
Kunming mouse is got spleen and is put into D-Hank ' liquid grinding release T cell, uses D-Hank ' liquid washing three times again, and being diluted to cell concn with the IMEM substratum at last is 2.5 * 10
7Individual/ml.
2, reaction system
Get cell 100 μ l, sample (50 μ g/ml) 50 μ l add in 96 orifice plates, and effect is 4 hours in the CO2gas incubator, add ConA50 μ l effect after 44 hours, add MTT20 μ l effect 4 hours in the system, and 2000 rev/mins centrifugal 5 minutes.Add DMSO jolting 20 minutes, microplate reader 570nm colorimetric is calculated SR.
Cell proliferation experiment result:
| Sample | TP5 | LW401 | LW402 | LW403 | LW404 | LW405 | LW406 | LW407 | LW408 |
| SR %) | 60.32 | 68.86 | 76.45 | 78.9 | 72.4 | 69.89 | 79.38 | 80.44 | 78.22 |
Conclusion: the influence of thymus gland active isomer LW401, LW402, LW403, LW404, LW405, LW406, LW407, LW408 on cell proliferation and TP-5 are quite or a little more than TP-5.
Test example 2
The mensuration of stability in the external blood plasma
For measuring the vitro stability of each isomer, we with sample in blood plasma 37 ℃ carry out enzymolysis, analyze hydrolysate with HPLC then, and compare with normal configuration, they are all longer than the normal configuration transformation period as a result.
Method: 37 ℃ of water-bath activation of Freshman blood plasma 30 minutes, add sample again, the final concentration that makes sample is 50 μ g/ml, 37 ℃ of water-bath hydrolysis, every interval sampling in 1 minute kind 400 μ l add acetic acid 400 μ l termination reactions.Sample is asked and is calculated the transformation period with the concentration that HPLC detects hydrolysate.
Measurement result:
| Sample | LW401 | LW402 | LW403 | LW404 | LW405 | LW406 | LW407 | LW408 | TP-5 |
| Transformation period (branch) | 3.3 | 3.2 | 5.4 | 5 | 5.8 | 6.3 | 6.2 | 7.6 | 1.2 |
Conclusion: the vitro stability of thymus gland active isomer LW401, LW402, LW403, LW404, LW405, LW406, LW407, LW408 is significantly better than TP-5.
Test example 3
Immuno-potentiation
1, to the influence of immunosuppressed mice mononuclear phagocyte system phagocytic function
Mouse is divided into group at random, 10 every group, male and female half and half.Be divided into control group, model group, positive drug group, administration group.Administration group 20 μ g/kg intravenous injection thymus gland bioactive peptides, control group and model group are pressed 10ml/kg intravenous injection equal-volume physiological saline, and the positive drug group is pressed 20 μ g/kg intravenous injection TP-5, once a day, continuous 6 days.In experiment the 6th day, each organized mouse tail vein injection india ink 0.1ml/10g, got blood 20 μ l from the eye socket venous plexus respectively in injection back 2 and 20min, was added to 2ml 0.1%Na
2CO
3Shake up in the solution, measure optical density(OD) at the 680nm place, calculate phagocytic index K and activate the phagocytic capacity α.Thymus gland peptide 20 μ g/kg intravenous injection can obviously enhancing endoxan inductive immunosuppressed mice mononuclear phagocyte system phagocytic function, effect obviously are better than contrast medicine TP-5 20 μ g/kg.
The thymus gland bioactive peptide is to the influence of immunosuppressed mice mononuclear phagocyte system phagocytic function (x ± s)
| Group | Dosage μ g/kg | Number of animals (only) | Phagocytic index K | Activate the phagocytic capacity α |
| Contrast | 10 | 0.029±0.016** | 5.24±1.01** | |
| Model | 10 | 0.016±0.008 | 3.12±0.45 | |
| TP-5 | 20 | 10 | 0.021±0.004* | 4.24±1.02* |
| LW401 | 20 | 10 | 0.038±0.019*** | 5.02±1.01*** |
| LW402 | 20 | 10 | 0.031±0.018** | 4.76±0.81** |
| LW403 | 20 | 10 | 0.028±0.015** | 4.52±0.75** |
| LW404 | 20 | 10 | 0.026±0.012* | 4.39±0.52* |
| LW405 | 20 | 10 | 0.030±0.016** | 4.63±0.51** |
| LW406 | 20 | 10 | 0.034±0.020** | 4.81±0.89** |
| LW407 | 20 | 10 | 0.027±0.013* | 4.52±0.67** |
| LW408 | 20 | 10 | 0.032±0.017** | 4.76±0.51** |
Compare * P＜0.05, * * P＜0.01, * * * P＜0.001 with model group
Conclusion: thymus gland active isomer LW401, LW402, LW403, LW404, LW405, LW406, LW407, LW408 20 μ g/kg intravenous injections can obviously enhancing endoxan inductive immunosuppressed mice mononuclear phagocyte system phagocytic function, effect obviously are better than contrast medicine TP-5 20 μ g/kg.
2, the influence that immunosuppressed mice serum hemolysin antibody is generated
Animal grouping administration is with experiment 1, administration the 1st day, every mouse carries out immunity with 5% chicken red blood cell 0.2ml abdominal injection, in experiment the 6th day, each is organized the mouse orbit venous plexus and gets blood 20 μ l, adding 1ml shakes up in the physiological saline, add 5% chicken red blood cell 0.5ml again, in ice bath, add 0.5ml complement (1: 10 fresh guinea pig serum), incubation 30min in 37 ℃ of waters bath with thermostatic control, termination reaction 3min in ice bath, the centrifugal 10min of 2000rpm gets supernatant 1ml, adds 3ml Dou Shi liquid, after room temperature leaves standstill 10min, at 540nm place photometry density value.The generation of thymus gland peptide 20 μ g/kg intravenous injection can obviously increasing endoxan inductive immunosuppressed mice serum hemolysin antibody, effect obviously are better than contrast TP-5 20 μ g/kg.
The influence that the thymus gland bioactive peptide generates immunosuppressed mice serum hemolysin antibody (x ± s)
| Group | Dosage (μ g/kg) | Number of animals (only) | Hemolytic antibody (OD) |
| Contrast | 10 | 0.032±0.011** | |
| Model | 10 | 0.018±0.010 | |
| TP-5 | 20 | 10 | 0.027±0.009* |
| LW401 | 20 | 10 | 0.039±0.018*** |
| LW402 | 20 | 10 | 0.033±0.007** |
| LW403 | 20 | 10 | 0.032±0.012** |
| LW404 | 20 | 10 | 0.031±0.008** |
| LW405 | 20 | 10 | 0.029±0.005* |
| LW406 | 20 | 10 | 0.035±0.011** |
| LW407 | 20 | 10 | 0.031±0.006** |
| LW408 | 20 | 10 | 0.032±0.007** |
Compare * P＜0.05, * * P＜0.01, * * * P＜0.001 with model group
Conclusion: the generation of thymus gland active isomer LW401, LW402, LW403, LW404, LW405, LW406, LW407, LW408 20 μ g/kg intravenous injections can obviously increasing endoxan inductive immunosuppressed mice serum hemolysin antibody, effect obviously are better than contrast TP-5 20 μ g/kg.
3, the interior influence of body to mice spleen lymphocytes proliferation
Mouse is divided into group at random, 10 every group, male and female half and half.Be divided into control group, positive drug group, administration group.Administration group 20 μ g/kg intravenous injection thymus gland bioactive peptides, control group is pressed 10ml/kg intravenous injection equal-volume physiological saline, and the positive drug group is pressed 20 μ g/kg intravenous injection TP-5, once a day, continuous 6 days.In experiment the 6th day, behind the last administration 1h, all mouse took off cervical vertebra and put to death.Get spleen and prepare splenocyte as stated above, the same method is cultivated the back and is measured.The 0 μ g/kg intravenous injection of thymus gland peptide 2 has remarkable promoter action to mice spleen lymphocytes proliferation, and effect obviously is better than contrast medicine TP-5 20 μ g/kg.
Body internal thymus immune-active peptides is to the influence of mice spleen lymphocytes proliferation (x ± s)
| Group | Dosage (μ g/kg) | Number of animals (only) | cpm/1×10 6Cell |
| Contrast | 0 | 10 | 4338±894 |
| TP-5 | 20 | 10 | 5125±1548** |
| LW401 | 20 | 10 | 7132±1056*** |
| LW402 | 20 | 10 | 6531±985*** |
| LW403 | 20 | 10 | 6357±1201*** |
| LW404 | 20 | 10 | 6036±1025*** |
| LW405 | 20 | 10 | 5986±1035*** |
| LW406 | 20 | 10 | 6612±1002*** |
| LW407 | 20 | 10 | 6056±1134*** |
| LW408 | 20 | 10 | 6214±1158*** |
Compare * P＜0.05, * * P＜0.01, * * * P＜0.001 with control group
Conclusion: thymus gland active isomer LW401, LW402, LW403, LW404, LW405, LW406, LW407, LW408 20 μ g/kg intravenous injections have remarkable promoter action to mice spleen lymphocytes proliferation, and effect obviously is better than contrast medicine TP-5 20 μ g/kg.
Test example 4
Antitumor action
1, to lotus S
180The influence of mice with tumor
50 mouse are divided into 5 groups at random, 10 every group, male and female half and half.Be divided into control group, positive drug group, administration group.Each organizes mouse under aseptic condition, with S
180Knurl strain (cultivating dilution in 1: 4 7) only is seeded in right side of mice oxter 0.2ml/.The administration group is by 20 μ g/kg intravenous injection thymus gland bioactive peptides, and control group is pressed 10ml/kg intravenous injection equal-volume physiological saline, positive drug group abdominal injection 20mg/kg endoxan, once a day, continuous 6 days, behind the last administration 1h, claim respectively to organize the mouse body weight, take off neck and put to death mouse.Getting knurl weighs.The 0 μ g/kg intravenous injection of thymus gland peptide 2 is to S
180It is 26～48% that the growth of transplantability mouse tumor has obvious suppression effect, inhibiting rate.
The thymus gland bioactive peptide is to lotus S
180The influence of mice with tumor (x ± s)
| Group | Dosage (μ g/kg) | Tumor weight (g) | Inhibiting rate (%) |
| Contrast | 0 | 2.41±0.31 | |
| Endoxan | 20000 | 0.74±0.29*** | 69.29 |
| LW401 | 20 | 1.43±0.31*** | 40.66 |
| LW402 | 20 | 1.66±0.32*** | 34.35 |
| LW403 | 20 | 1.79±0.39*** | 31.54 |
| LW404 | 20 | 1.95±0.46*** | 27.64 |
| LW405 | 20 | 1.71±0.33*** | 31.98 |
| LW406 | 20 | 1.63±0.30*** | 33.58 |
| LW407 | 20 | 1.82±0.45*** | 29.45 |
| LW408 | 20 | 1.68±0.35*** | 33.04 |
Compare * P＜0.05, * * P＜0.01, * * * P＜0.001 with model group
Conclusion: thymus gland active isomer LW401, LW402, LW403, LW404, LW405, LW406, LW407, LW408 20 μ g/kg intravenous injections are to S
180It is 28～41% that the growth of transplantability mouse tumor has obvious suppression effect, inhibiting rate.
2, to lotus H
22The influence of mice with tumor
Experimentation is the same, and the strain of inoculation knurl is H
22The knurl strain.The 0 μ g/kg intravenous injection of thymus gland peptide 2 is to H
22It is 29～49% that the growth of transplantability mouse tumor has obvious suppression effect, inhibiting rate.
The thymus gland bioactive peptide is to lotus H
22The influence of mice with tumor (x ± s)
| Group | Dosage (μ g/kg) | Tumor weight (g) | Inhibiting rate (%) |
| Contrast | 0 | 2.19±0.45 | |
| Endoxan | 20000 | 0.78±0.22*** | 64.38 |
| LW401 | 20 | 1.24±0.32*** | 43.38 |
| LW402 | 20 | 1.49±0.53*** | 33.12 |
| LW403 | 20 | 1.56±0.59*** | 31.08 |
| LW404 | 20 | 1.72±0.85** | 28.64 |
| LW405 | 20 | 1.79±0.87*** | 27.69 |
| LW406 | 20 | 1.45±0.53*** | 34.12 |
| LW407 | 20 | 1.52±0.61*** | 31.28 |
| LW408 | 20 | 1.67±0.71*** | 29.54 |
Compare * P＜0.05, * * P＜0.01, * * * P＜0.001 with model group
Conclusion: thymus gland active isomer LW401, LW402, LW403, LW404, LW405, LW406, LW407, LW408 20 μ g/kg intravenous injections to H22 transplantability mouse tumor growth obvious suppression effect, inhibiting rate are arranged is 28～43%.
The influence of 3 pairs of tumor-bearing mice serum interleukin IIs (IL-2)
Animal grouping administration is pressed literature method with 1
[2]Measure serum il-2 content.The 0 μ g/kg intravenous injection of thymus gland peptide 2 can obviously increase lotus S
180Mice with tumor serum interleukin II (IL-2) level, effect obviously are better than contrast medicine TP-5 20 μ g/kg.Show that the thymus gland bioactive peptide has the effect of the tumor-bearing mice of raising T cell growth factor.
The thymus gland bioactive peptide is to lotus S
180The influence of mice with tumor serum il-2 (x ± s)
| Group | Dosage (μ g/kg) | IL-2(μg/L) |
| Contrast | 1.74±0.65 | |
| TP-5 | 20 | 2.04±0.56 |
| LW401 | 20 | 3.02±0.64*** |
| LW402 | 20 | 2.85±0.72** |
| LW403 | 20 | 2.71±0.76** |
| LW404 | 20 | 2.64±0.61** |
| LW405 | 20 | 2.58±0.46** |
| LW406 | 20 | 2.89±0.78** |
| LW407 | 20 | 2.76±0.71** |
| LW408 | 20 | 2.71±0.67** |
Compare * P＜0.05**P＜0.01, * * * P＜0.001 with model group
Conclusion: thymus gland active isomer LW401, LW402, LW403, LW404, LW405, LW406, LW407, LW408 20 μ g/kg intravenous injections can obviously increase lotus S
180Mice with tumor serum interleukin II (IL-2) level, effect obviously are better than contrast medicine TP-5 20 μ g/kg.Show that the thymus gland bioactive peptide has the effect of the tumor-bearing mice of raising T cell growth factor.
Test example 5
Thymus gland bioactive peptide antioxygenation
1, to the influence of mouse lipid peroxidation product-mda (MDA)
60 mouse are divided into 6 groups at random, 10 every group, male and female half and half.Be divided into control group, model group, positive drug group, administration group.Administration group 20 μ g/kg intravenous injection thymus gland bioactive peptides, control group and model group are pressed 10ml/kg intravenous injection equal-volume physiological saline, and the positive drug group is pressed 20 μ g/kg intravenous injection TP-5, once a day, continuous 6 days.Behind the last administration 1h, model group and administration group are pressed 10ml/kg abdominal injection 0.1%CCl
4Soybean oil solution, normal group abdominal injection behind the fasting 16h, are got brain, liver and thymic tissue with volume soya-bean oil, press the Lowry method and measure protein content, press the TBA method and measure lipid peroxidation product-mda content.The 20 μ g/kg intravenous injections of thymus gland immune-active peptides are to CCl
4Cause that mouse brain, liver and thymus gland lipid peroxidation product-mda rising all has tangible reduction effect, effect obviously is better than contrast medicine TP-5 20 μ g/kg.
The thymus gland bioactive peptide is to the influence of mouse lipid peroxidation product-mda (MDA) (x ± s)
| Group | Dosage μ g/kg | Brain | MDA (nmol/mg) liver | Thymus gland |
| Contrast | 0 | 1.12±0.43* | 0.14±0.09*** | 0.10±0.04*** |
| Model | 0 | 1.51±0.22 | 0.34±0.09 | 0.31±0.07 |
| TP-5 | 20 | 1.03±0.30* | 0.28±0.08* | 0.25±0.08* |
| LW401 | 20 | 0.64±0.26*** | 0.20±0.05*** | 0.21±0.08*** |
| LW402 | 20 | 0.74±0.19*** | 0.23±0.02*** | 0.23±0.07** |
| LW403 | 20 | 0.8l±0.23** | 0.25±0.05*** | 0.25±0.11* |
| LW404 | 20 | 0.85±0.27** | 0.27±0.07*** | 0.28±0.12* |
| LW405 | 20 | 0.81±0.21*** | 0.23±0.03*** | 0.24±0.09** |
| LW406 | 20 | 0.76±0.21*** | 0.21±0.03*** | 0.21±0.09*** |
| LW407 | 20 | 0.77±0.22*** | 0.27±0.03** | 0.23±0.07*** |
| LW408 | 20 | 0.79±0.19*** | 0.22±0.04*** | 0.24±0.05*** |
Compare * P＜0.05, * * P＜0.01, * * * P＜0.001 with model group
Conclusion: thymus gland active isomer LW401, LW402, LW403, LW404, LW405, LW406, LW407, LW408 20 μ g/kg intravenous injections are to CCl
4Cause that mouse brain, liver and thymus gland lipid peroxidation product-mda rising all has tangible reduction effect, effect obviously is better than contrast medicine TP-5 20 μ g/kg.
2, the thymus gland bioactive peptide is to the active influence of mouse superoxide-dismutase (SOD)
Get experiment 10.1 murine liver tissue and serum, press kit method and measure the SOD activity.The 0 μ g/kg intravenous injection of thymus gland peptide 2 is to CCl
4The reduction that causes mouse lipid peroxidation hepatic tissue and activity of SOD in serum all has tangible enhancement, and effect obviously is better than contrast medicine TP-5 20 μ g/kg.
The thymus gland bioactive peptide is to the active influence of SOD in Mice (x ± s)
| Group | Dosage (μ g/kg) | Liver SOD (U/g) | SOD in serum (nU/ml) |
| Contrast | 0 | 29.26±6.87*** | 162.24±9.40*** |
| Model | 0 | 18.26±3.58 | 101.02±10.12 |
| TP-5 | 20 | 22.13±4.01* | 121.26±10.24* |
| LW401 | 20 | 26.84±5.01*** | 142.65±10.54*** |
| LW402 | 20 | 24.15±4.81** | 135.63±7.91** |
| LW403 | 20 | 23.85±4.12** | 132.32±7.01** |
| LW404 | 20 | 23.94±4.07** | 130.32±7.14** |
| LW405 | 20 | 24.15±4.24** | 1343.58±7.24** |
| LW406 | 20 | 24.98±4.86** | 134.58±7.97** |
| LW407 | 20 | 24.53±4.56** | 138.51±7.31** |
| LW408 | 20 | 24.22±4.17** | 134.57±7.12** |
Compare * P＜0.05, * * P＜0.01, * * * P＜0.001 with model group
Conclusion: thymus gland active isomer LW401, LW402, LW403, LW404, LW405, LW406, LW407, LW408 20 μ g/kg intravenous injections are to CCl
4The reduction that causes mouse lipid peroxidation hepatic tissue and activity of SOD in serum all has tangible enhancement, and effect obviously is better than contrast medicine TP-5 20 μ g/kg.
Test example 6
The rosette experiment
1, the preparation of T cell
Get fresh pig thymus gland and shred, 100 mesh sieves filter, and use the washing of D-Hank ' liquid, add lymphocyte separation medium in the filtrate, and 2000 left the heart 20 minutes, drew the T cell, and using D-Hank ' liquid to be diluted to cell concn is 2.5 * 10
6Individual/ml.
2, the preparation of sheep red blood cell (SRBC)
Get sheep blood 1ml, use D-Hank ' liquid washing three times, 1500 rev/mins centrifugal 3 minutes, obtain red corpuscle, it being diluted to cell concn is 2.5 * 10 again
7Individual/ml.
3, the generation of rosette
Get T cell 200 μ l and add sample (50 μ g/ml) 100 μ l, 30 ℃ are incubated 1 hour, add sheep red blood cell (SRBC) 200 μ l again, and 4 ℃ are spent the night.
Use the reaction solution smear, the microscopically counting.
| Sample | TP5 | LW401 | LW402 | LW403 | LW404 | LW405 | LW406 | LW407 | LW408 |
| Knot flower increment rate % | 19.76 | 28.56 | 23.98 | 21.45 | 29.7 | 19.51 | 20.2 | 22.39 | 25.12 |
Conclusion: the immunocompetence of thymus gland active isomer LW401, LW402, LW403, LW404, LW405, LW406, LW407, LW408 is equivalent to or a little more than TP-5.
Embodiment
The following example is intended to further describe for example the present invention, rather than limits the present invention by any way.Under the prerequisite that does not deviate from the spirit and principles in the present invention, any change or change that those of ordinary skills that the present invention did are realized easily all will fall within the claim scope that awaits the reply of the present invention.
Embodiment 1
The solid phase synthesis of LW401
LW401：Arg-Lys-β-Asp-Val
Raw material: Fmoc-Val-Wang resin, Fmoc-β-Asp-otBu, Fmoc-Arg (pbf)-OH, Fmoc-Lys (Boc)-OH, linked reaction condensing agent are benzene a pair of horses going side by side triazole-1-oxygen-three (dimethylamino) phosphorus hexafluorophosphate (BOP), 1-hydroxyl benzotriazole (HOBT), N-methylmorpholine (NMM).Deprotection agent is 20% piperidines/DMF solution.Cutting reagent is a trifluoroacetic acid.
Reaction: take by weighing Fmoc-Val-Wang resin room temperature swelling 30 minutes in DMF, with DMF washing three times.Add 20% piperidines/DMF solution, 1 hour deprotection of room temperature reaction.Wash three times with DMF again, Fmoc-β-Asp-otBu, room temperature reaction 3 hours, the back adds 20% piperidines/DMF solution, 1 hour deprotection of room temperature reaction with DMF washing three times.With DMF washing three times, add Fmoc-Lys (Boc)-OH again, room temperature reaction 3 hours, the back adds 20% piperidines/DMF solution, 1 hour deprotection of room temperature reaction with DMF washing three times.With DMF washing three times, add Fmoc-Arg (pbf)-OH more again, room temperature reaction 3 hours, the back adds 20% piperidines/DMF solution with DMF washing three times, and 1 hour deprotection of room temperature reaction is again with DMF washing three times.
Cutting: adding cutting reagent behind the resin drying of above-mentioned reaction is trifluoroacetic acid, and room temperature reaction 2 hours filters, and filtrate is precipitated in ether, and drying obtains thick product.
Purifying: crude product is soluble in water, use C
18Reversed-phase column carries out purifying, and elutriant is that A is the 0.1%TFA aqueous solution mutually, and B is acetonitrile mutually, and flow velocity is 20ml/min, and the detection wavelength is 214nm, collects main peak, and freeze-drying obtains pure product.
Embodiment 2
The solid phase synthesis of LW402
LW402：Arg-β-Lys-Asp-Val
Raw material: Fmoc-Val-Wang resin, Fmoc Asp otBu, Fmoc-Arg (pbf)-OH, Fmoc-β-Lys (Boc)-OH, linked reaction condensing agent are benzene a pair of horses going side by side triazole-1-oxygen-three (dimethylamino) phosphorus hexafluorophosphate (BOP), 1-hydroxyl benzotriazole (HOBT), N-methylmorpholine (NMM).Deprotection agent is 20% piperidines/DMF solution.Cutting reagent is a trifluoroacetic acid.
Reaction: take by weighing Fmoc-Val-Wang resin room temperature swelling 30 minutes in DMF, with DMF washing three times.Add 20% piperidines/DMF solution, 1 hour deprotection of room temperature reaction.Wash three times with DMF again, Fmoc-Asp-otBu, room temperature reaction 3 hours, the back adds 20% piperidines/DMF solution, 1 hour deprotection of room temperature reaction with DMF washing three times.With DMF washing three times, add Fmoc-β-Lys (Boc)-OH again, room temperature reaction 3 hours, the back adds 20% piperidines/DMF solution, 1 hour deprotection of room temperature reaction with DMF washing three times.With DMF washing three times, add Fmoc-Arg (pbf)-OH more again, room temperature reaction 3 hours, the back adds 20% piperidines/DMF solution with DMF washing three times, and 1 hour deprotection of room temperature reaction is again with DMF washing three times.
Cutting: adding cutting reagent behind the resin drying of above-mentioned reaction is trifluoroacetic acid, and room temperature reaction 2 hours filters, and filtrate is precipitated in ether, and drying obtains thick product.
Purifying: crude product is soluble in water, use C
18Reversed-phase column carries out purifying, and elutriant is that A is the 0.1%TFA aqueous solution mutually, and B is acetonitrile mutually, and flow velocity is 20ml/min, and the detection wavelength is 214nm, collects main peak, and freeze-drying obtains pure product.
Embodiment 3
The solid phase synthesis of LW403
LW403：Arg-Lys-γ-Glu-Val
Raw material: Fmoc-Val-Wang resin, Fmoc-Lys-(Boc)-OH, Fmoc-Arg (pbf)-OH, Fmoc-γ-Glu-otBu, linked reaction condensing agent are benzene a pair of horses going side by side triazole-1-oxygen-three (dimethylamino) phosphorus hexafluorophosphate (BOP), 1-hydroxyl benzotriazole (HOBT), N-methylmorpholine (NMM).Deprotection agent is 20% piperidines/DMF solution.Cutting reagent is a trifluoroacetic acid.
Reaction: take by weighing Fmoc-Val-Wang resin room temperature swelling 30 minutes in DMF, with DMF washing three times.Add 20% piperidines/DMF solution, 1 hour deprotection of room temperature reaction.With DMF washing three times, add Fmoc-γ-Glu-otBu again, room temperature reaction 3 hours, the back adds 20% piperidines/DMF solution, 1 hour deprotection of room temperature reaction with DMF washing three times.With DMF washing three times, add Fmoc-Lys (Boc)-OH again, room temperature reaction 3 hours, the back adds 20% piperidines/DMF solution, 1 hour deprotection of room temperature reaction with DMF washing three times.With DMF washing three times, add Fmoc-Arg (pbf)-OH more again, room temperature reaction 3 hours, the back adds 20% piperidines/DMF solution with DMF washing three times, and 1 hour deprotection of room temperature reaction is again with DMF washing three times.
Cutting: adding cutting reagent behind the resin drying of above-mentioned reaction is trifluoroacetic acid, and room temperature reaction 2 hours filters, and filtrate is precipitated in ether, and drying obtains thick product.
Purifying: crude product is soluble in water, use C
18Reversed-phase column carries out purifying, and elutriant is that A is the 0.1%TFA aqueous solution mutually, and B is acetonitrile mutually, and flow velocity is 20ml/min, and the detection wavelength is 214nm, collects main peak, and freeze-drying obtains pure product.
Embodiment 4
The solid phase synthesis of LW404
LW404：Arg-γ-Lys-Asp-Val
Raw material: Fmoc-Val-Wang resin, Fmoc-β-Lys-(Boc)-OH, Fmoc-Arg (pbf)-OH, Fmoc-Asp-otBu, linked reaction condensing agent are benzene a pair of horses going side by side triazole-1-oxygen-three (dimethylamino) phosphorus hexafluorophosphate (BOP), 1-hydroxyl benzotriazole (HOBT), N-methylmorpholine (NMM).Deprotection agent is 20% piperidines/DMF solution.Cutting reagent is a trifluoroacetic acid.
Reaction: take by weighing Fmoc-Val-Wang resin room temperature swelling 30 minutes in DMF, with DMF washing three times.Add 20% piperidines/DMF solution, 1 hour deprotection of room temperature reaction.With DMF washing three times, add Fmoc-Asp-otBu again, room temperature reaction 3 hours, the back adds 20% piperidines/DMF solution, 1 hour deprotection of room temperature reaction with DMF washing three times.With DMF washing three times, add Fmoc-γ-Lys (Boc)-OH again, room temperature reaction 3 hours, the back adds 20% piperidines/DMF solution, 1 hour deprotection of room temperature reaction with DMF washing three times.With DMF washing three times, add Fmoc-Arg (pbf)-OH more again, room temperature reaction 3 hours, the back adds 20% piperidines/DMF solution with DMF washing three times, and 1 hour deprotection of room temperature reaction is again with DMF washing three times.
Cutting: adding cutting reagent behind the resin drying of above-mentioned reaction is trifluoroacetic acid, and room temperature reaction 2 hours filters, and filtrate is precipitated in ether, and drying obtains thick product.
Purifying: crude product is soluble in water, use C
18Reversed-phase column carries out purifying, and elutriant is that A is the 0.1%TFA aqueous solution mutually, and B is acetonitrile mutually, and flow velocity is 20ml/min, and the detection wavelength is 214nm, collects main peak, and freeze-drying obtains pure product.
Embodiment 5
The solid phase synthesis of LW405
LW405：Arg-β-Lys-β-Asp-Val
Raw material: Fmoc-Val-Wang resin, Fmoc-β-Lys-(Boc)-OH, Fmoc-Arg (pbf)-OH, Fmoc-β-Asp-otBu, linked reaction condensing agent are benzene a pair of horses going side by side triazole-1-oxygen-three (dimethylamino) phosphorus hexafluorophosphate (BOP), 1-hydroxyl benzotriazole (HOBT), N-methylmorpholine (NMM).Deprotection agent is 20% piperidines/DMF solution.Cutting reagent is a trifluoroacetic acid.
Reaction: take by weighing Fmoc-Val-Wang resin room temperature swelling 30 minutes in DMF, with DMF washing three times.Add 20% piperidines/DMF solution, 1 hour deprotection of room temperature reaction.With DMF washing three times, add Fmoc-β-Asp-otBu again, room temperature reaction 3 hours, the back adds 20% piperidines/DMF solution, 1 hour deprotection of room temperature reaction with DMF washing three times.With DMF washing three times, add Fmoc-β-Lys (Boc)-OH again, room temperature reaction 3 hours, the back adds 20% piperidines/DMF solution, 1 hour deprotection of room temperature reaction with DMF washing three times.With DMF washing three times, add Fmoc-Arg (pbf)-OH more again, room temperature reaction 3 hours, the back adds 20% piperidines/DMF solution with DMF washing three times, and 1 hour deprotection of room temperature reaction is again with DMF washing three times.
Cutting: adding cutting reagent behind the resin drying of above-mentioned reaction is trifluoroacetic acid, and room temperature reaction 2 hours filters, and filtrate is precipitated in ether, and drying obtains thick product.
Purifying: crude product is soluble in water, use C
18Reversed-phase column carries out purifying, and elutriant is that A is the 0.1%TFA aqueous solution mutually, and B is acetonitrile mutually, and flow velocity is 20ml/min, and the detection wavelength is 214nm, collects main peak, and freeze-drying obtains pure product.
Embodiment 6
The solid phase synthesis of LW406
LW406：Arg-β-Lys-γ-Glu-Val
Raw material: Fmoc-Val-Wang resin, Fmoc-Glu-otBu, Fmoc-Arg (pbf)-OH, Fmoc-β-Lys (Boc)-OH, linked reaction condensing agent are benzene a pair of horses going side by side triazole-1-oxygen-three (dimethylamino) phosphorus hexafluorophosphate (BOP), 1-hydroxyl benzotriazole (HOBT), N-methylmorpholine (NMM).Deprotection agent is 20% piperidines/DMF solution.Cutting reagent is a trifluoroacetic acid.
Reaction: take by weighing Fmoc-Val-Wang resin room temperature swelling 30 minutes in DMF, with DMF washing three times.Add 20% piperidines/DMF solution, 1 hour deprotection of room temperature reaction.Wash three times with DMF again, Fmoc-Glu-otBu, room temperature reaction 3 hours, the back adds 20% piperidines/DMF solution, 1 hour deprotection of room temperature reaction with DMF washing three times.With DMF washing three times, add Fmoc-β-Lys (Boc)-OH again, room temperature reaction 3 hours, the back adds 20% piperidines/DMF solution, 1 hour deprotection of room temperature reaction with DMF washing three times.With DMF washing three times, add Fmoc-Arg (pbf)-OH more again, room temperature reaction 3 hours, the back adds 20% piperidines/DMF solution with DMF washing three times, and 1 hour deprotection of room temperature reaction is again with DMF washing three times.
Cutting: adding cutting reagent behind the resin drying of above-mentioned reaction is trifluoroacetic acid, and room temperature reaction 2 hours filters, and filtrate is precipitated in ether, and drying obtains thick product.
Purifying: crude product is soluble in water, use C
18Reversed-phase column carries out purifying, and elutriant is that A is the 0.1%TFA aqueous solution mutually, and B is acetonitrile mutually, and flow velocity is 20ml/min, and the detection wavelength is 214nm,
Collect main peak, freeze-drying obtains pure product.
Embodiment 7
The solid phase synthesis of LW407
LW407：Arg-γ-Lys-β-Asp-Val
Raw material: Fmoc-Val-Wang resin, Fmoc-β-Asp-otBu, Fmoc-Arg (pbf)-OH, Fmoc-γ-Lys (Boc)-OH, linked reaction condensing agent are benzene a pair of horses going side by side triazole-1-oxygen-three (dimethylamino) phosphorus hexafluorophosphate (BOP), 1-hydroxyl benzotriazole (HOBT), N-methylmorpholine (NMM).Deprotection agent is 20% piperidines/DMF solution.Cutting reagent is a trifluoroacetic acid.
Reaction: take by weighing Fmoc-Val-Wang resin room temperature swelling 30 minutes in DMF, with DMF washing three times.Add 20% piperidines/DMF solution, 1 hour deprotection of room temperature reaction.Wash three times with DMF again, Fmoc-β-Asp-otBu, room temperature reaction 3 hours, the back adds 20% piperidines/DMF solution, 1 hour deprotection of room temperature reaction with DMF washing three times.With DMF washing three times, add Fmoc-γ-Lys (Boc)-OH again, room temperature reaction 3 hours, the back adds 20% piperidines/DMF solution, 1 hour deprotection of room temperature reaction with DMF washing three times.With DMF washing three times, add Fmoc-Arg (pbf)-OH more again, room temperature reaction 3 hours, the back adds 20% piperidines/DMF solution with DMF washing three times, and 1 hour deprotection of room temperature reaction is again with DMF washing three times.
Cutting: adding cutting reagent behind the resin drying of above-mentioned reaction is trifluoroacetic acid, and room temperature reaction 2 hours filters, and filtrate is precipitated in ether, and drying obtains thick product.
Purifying: crude product is soluble in water, use C
18Reversed-phase column carries out purifying, and elutriant is that A is the 0.1%TFA aqueous solution mutually, and B is acetonitrile mutually, and flow velocity is 20ml/min, and the detection wavelength is 214nm,
Collect main peak, freeze-drying obtains pure product.
Embodiment 8
The solid phase synthesis of LW408
LW408：Arg-γ-Lys-γ-Glu-Val
Raw material: Fmoc-Val-Wang resin, Fmoc-γ-Glu-otBu, Fmoc-Arg (pbf)-OH, Fmoc-γ-Lys (Boc)-OH, linked reaction condensing agent are benzene a pair of horses going side by side triazole-1-oxygen-three (dimethylamino) phosphorus hexafluorophosphate (BOP), 1-hydroxyl benzotriazole (HOBT), N-methylmorpholine (NMM).Deprotection agent is 20% piperidines/DMF solution.Cutting reagent is a trifluoroacetic acid.
Reaction: take by weighing Fmoc-Val-Wang resin room temperature swelling 30 minutes in DMF, with DMF washing three times.Add 20% piperidines/DMF solution, 1 hour deprotection of room temperature reaction.Wash three times with DMF again, Fmoc-γ-Glu-otBu, room temperature reaction 3 hours, the back adds 20% piperidines/DMF solution, 1 hour deprotection of room temperature reaction with DMF washing three times.With DMF washing three times, add Fmoc-γ-Lys (Boc)-OH again, room temperature reaction 3 hours, the back adds 20% piperidines/DMF solution, 1 hour deprotection of room temperature reaction with DMF washing three times.With DMF washing three times, add Fmoc-Arg (pbf)-OH more again, room temperature reaction 3 hours, the back adds 20% piperidines/DMF solution with DMF washing three times, and 1 hour deprotection of room temperature reaction is again with DMF washing three times.
Cutting: adding cutting reagent behind the resin drying of above-mentioned reaction is trifluoroacetic acid, and room temperature reaction 2 hours filters, and filtrate is precipitated in ether, and drying obtains thick product.
Purifying: crude product is soluble in water, use C
18Reversed-phase column carries out purifying, and elutriant is that A is the 0.1%TFA aqueous solution mutually, and B is acetonitrile mutually, and flow velocity is 20ml/min, and the detection wavelength is 214nm,
Collect main peak, freeze-drying obtains pure product.
Embodiment 9
The method that takes by weighing embodiment 1,2,3,4,5,6,7,8 respectively obtains thymus gland active isomer LW401, LW402, LW403, LW404, LW405, LW406, LW407, each 100mg of LW408, be dissolved in 1000ml 0.9% sodium chloride solution, after mixing, be distributed into the injection liquid that 0.1mg/ml/ props up concentration and seal in medicine bottle, product is made in sterilization.
Embodiment 10
The method that takes by weighing embodiment 1,2,3,4,5,6,7,8 respectively obtains thymus gland active isomer LW401, LW402, LW403, LW404, LW405, LW406, LW407, each 100mg of LW408, be dissolved in the 1000ml water and make the aqueous solution, after mixing, being distributed into the injection liquid that 0.1mg/ml/ props up concentration seals in medicine bottle, sterilization, finished product is made in freeze-drying.
Embodiment 11
The method that takes by weighing embodiment 1,2,3,4,5,6,7,8 respectively obtains thymus gland active isomer LW401, LW402, LW403, LW404, LW405, LW406, LW407, each 100g of LW408, medical starch 0.5kg, make capsule by known capsule technology of preparing and equipment, every 10mg.Other project should meet requirement under Pharmacopoeia of People's Republic of China version capsule in 2000 item.
Embodiment 12
The method that takes by weighing embodiment 1,2,3,4,5,6,7,8 respectively obtains thymus gland active isomer LW401, LW402, LW403, LW404, LW405, LW406, LW407, each 100g of LW408, Microcrystalline Cellulose 560g, lactose hydrous 380g, Magnesium Stearate 200g, silicon oxide 30g, make tablet by known tabletting technology and equipment, every 10mg.Other project should meet requirement under Pharmacopoeia of People's Republic of China version tablet in 2000 item.
Claims (5)
1, a kind of thymus tetrapeptide active isomer comprises following feature:
Compound has aminoacid sequence X-Arg-Lys-Y-Val, and X is ethanoyl or hydrogen, or arbitrary sequence therewith aminoacid sequence have 70% above homology at least; Y is Asp or Glu;
2, the described thymus tetrapeptide active isomer of claim is characterized in that: structure is Lys among thymus tetrapeptide aminoacid sequence Arg-Lys-Asp-Val or the Arg-Lys-Glu-Val or Glu by its corresponding β-or aminoacid sequence of replacing of gamma-amino acid;
Thymus tetrapeptide active isomer aminoacid sequence table
3, the preparation method of thymus tetrapeptide active isomer may further comprise the steps:
With the amino acid derivative is raw material, is solvent with dimethyl formamide (DMF), will meet peptide resin and DMF and add in the column type reactor, and the room temperature jolting is drained after connecing the peptide resin swelling, with draining after the DMF washing again; Connecing after swelling adds in the peptide resin takes off Fmoc protecting group solution, and the room temperature jolting is drained the back and drained with the DMF washing again; Add a kind of amino acid derivative and condensing agent in column type reactor, add DMF again, the jolting of lucifuge room temperature was reacted 0.3-20 hour, drained the back and drained with DMF jolting washing again; Adopt and alternately repeat deprotection and coupling deprotection coupling circulation; Said cutting is that deaminizating acid Side chain protective group reaches from connecing cleavage of peptide on the peptide resin; promptly add cutting reagent in the column type reactor after draining; 25～-20 ℃ of vibrations 1～5 hour, remove by filter and connect peptide resin, filtrate vacuumizes concentrated; concentrated solution adds ether/sherwood oil vibration; place 0～-20 ℃ to place 0.2～2 hour, centrifugal, remove supernatant; precipitation vacuum-drying gets the present invention.
4, preparation method according to claim 3 is characterized in that:
Connect peptide resin and be selected from Fmoc-NH-SAL resin, PAL resin, Wang resin, Fmoc-amide resins;
Amino acid derivative is selected from Fmoc-Arg (pbf)-OH, Fmoc-Lys (Boc)-OH, Fmoc-Asp (otBu)-OH, Fmoc-β-Asp-otBu, Fmoc-Val-OH, Fmoc-Glu (otBu)-OH, Fmoc-γ-Glu-otBu, Fmoc-β-Lys (Boc)-OH, Fmoc-γ-Lys (Boc)-OH etc.; The mol ratio that connects peptide resin and amino acid derivative is 1: 2～5;
The condensing agent that the amino acid coupling is used is selected from benzotriazole-1-oxygen-three (dimethylamino) phosphorus hexafluorophosphate (BOP), 1-hydroxy benzo triazole (HOBT), N-methylmorpholine (NMM), and 2: 1 in molar ratio～3: 2～6 are mixed and made into; Amino acid derivative and condensing agent are 1: 1～1.2 in molar ratio;
The cutting reagent main component is a trifluoroacetic acid; Take off Fmoc protecting group solution and be piperidines/DMF solution of 15～25%.
5, the application of thymus tetrapeptide active isomer in preparation immunoregulation druge, antitumor drug and anti-oxidation medicine.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102477078A (en) * | 2010-11-23 | 2012-05-30 | 中国医学科学院药物研究所 | Preparation of thymic humoral factor (THF)-gamma2 structure modified peptide and application of pharmaceutical composition thereof |
| CN102558298A (en) * | 2011-12-23 | 2012-07-11 | 中国人民解放军第四军医大学 | Method for synthesizing tetrapeptide isomers by using solid phase peptide synthesis method and applications of tetrapeptide isomers |
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2006
- 2006-11-30 CN CN 200610131681 patent/CN1962691A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102477078A (en) * | 2010-11-23 | 2012-05-30 | 中国医学科学院药物研究所 | Preparation of thymic humoral factor (THF)-gamma2 structure modified peptide and application of pharmaceutical composition thereof |
| CN102477078B (en) * | 2010-11-23 | 2016-04-20 | 中国医学科学院药物研究所 | Thymus humoral factor (THF)-γ 2 changes the preparation of structure peptide and the purposes of pharmaceutical composition thereof |
| CN102558298A (en) * | 2011-12-23 | 2012-07-11 | 中国人民解放军第四军医大学 | Method for synthesizing tetrapeptide isomers by using solid phase peptide synthesis method and applications of tetrapeptide isomers |
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