CN102702318A - YIGSKYIGSK with target antithrombotic activity, and preparation and application thereof - Google Patents
YIGSKYIGSK with target antithrombotic activity, and preparation and application thereof Download PDFInfo
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- 238000002360 preparation method Methods 0.000 title claims abstract description 20
- 230000002785 anti-thrombosis Effects 0.000 title claims abstract description 17
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 11
- 229920001184 polypeptide Polymers 0.000 claims abstract description 9
- 102000004196 processed proteins & peptides Human genes 0.000 claims abstract description 9
- 239000003146 anticoagulant agent Substances 0.000 claims abstract description 6
- 238000000034 method Methods 0.000 claims description 9
- 239000007791 liquid phase Substances 0.000 claims description 3
- 230000015572 biosynthetic process Effects 0.000 claims description 2
- 238000003786 synthesis reaction Methods 0.000 claims description 2
- 239000003153 chemical reaction reagent Substances 0.000 claims 1
- 238000010171 animal model Methods 0.000 abstract description 2
- 229960004676 antithrombotic agent Drugs 0.000 abstract 1
- 230000021615 conjugation Effects 0.000 abstract 1
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 21
- SJRJJKPEHAURKC-UHFFFAOYSA-N N-Methylmorpholine Chemical compound CN1CCOCC1 SJRJJKPEHAURKC-UHFFFAOYSA-N 0.000 description 18
- 239000007864 aqueous solution Substances 0.000 description 17
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 description 16
- 230000006837 decompression Effects 0.000 description 16
- 239000000243 solution Substances 0.000 description 16
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 14
- 239000000706 filtrate Substances 0.000 description 14
- 238000006243 chemical reaction Methods 0.000 description 13
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 12
- 239000012141 concentrate Substances 0.000 description 12
- 241000700159 Rattus Species 0.000 description 11
- 238000002330 electrospray ionisation mass spectrometry Methods 0.000 description 11
- 238000003756 stirring Methods 0.000 description 11
- 150000001875 compounds Chemical class 0.000 description 10
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical class [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 9
- NPZTUJOABDZTLV-UHFFFAOYSA-N hydroxybenzotriazole Substances O=C1C=CC=C2NNN=C12 NPZTUJOABDZTLV-UHFFFAOYSA-N 0.000 description 8
- ASOKPJOREAFHNY-UHFFFAOYSA-N 1-Hydroxybenzotriazole Chemical compound C1=CC=C2N(O)N=NC2=C1 ASOKPJOREAFHNY-UHFFFAOYSA-N 0.000 description 7
- 238000001914 filtration Methods 0.000 description 7
- 239000000047 product Substances 0.000 description 7
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 6
- 208000007536 Thrombosis Diseases 0.000 description 6
- 229920006395 saturated elastomer Polymers 0.000 description 6
- 241001274216 Naso Species 0.000 description 5
- 238000004440 column chromatography Methods 0.000 description 5
- -1 methoxyl group Chemical group 0.000 description 5
- 239000011541 reaction mixture Substances 0.000 description 5
- 230000001105 regulatory effect Effects 0.000 description 5
- 210000001715 carotid artery Anatomy 0.000 description 4
- 239000003814 drug Substances 0.000 description 4
- 239000000843 powder Substances 0.000 description 4
- 150000003839 salts Chemical class 0.000 description 4
- 210000003462 vein Anatomy 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- ADFXKUOMJKEIND-UHFFFAOYSA-N 1,3-dicyclohexylurea Chemical compound C1CCCCC1NC(=O)NC1CCCCC1 ADFXKUOMJKEIND-UHFFFAOYSA-N 0.000 description 3
- 239000004698 Polyethylene Substances 0.000 description 3
- 239000002253 acid Substances 0.000 description 3
- 230000021164 cell adhesion Effects 0.000 description 3
- 229920000573 polyethylene Polymers 0.000 description 3
- 239000002994 raw material Substances 0.000 description 3
- FHOAKXBXYSJBGX-YFKPBYRVSA-N (2s)-3-hydroxy-2-[(2-methylpropan-2-yl)oxycarbonylamino]propanoic acid Chemical compound CC(C)(C)OC(=O)N[C@@H](CO)C(O)=O FHOAKXBXYSJBGX-YFKPBYRVSA-N 0.000 description 2
- 238000005160 1H NMR spectroscopy Methods 0.000 description 2
- 241000228143 Penicillium Species 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 125000000051 benzyloxy group Chemical group [H]C1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])O* 0.000 description 2
- 230000000903 blocking effect Effects 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 210000001772 blood platelet Anatomy 0.000 description 2
- 230000004087 circulation Effects 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 238000004821 distillation Methods 0.000 description 2
- 229920001971 elastomer Polymers 0.000 description 2
- 238000003810 ethyl acetate extraction Methods 0.000 description 2
- MVEAAGBEUOMFRX-UHFFFAOYSA-N ethyl acetate;hydrochloride Chemical compound Cl.CCOC(C)=O MVEAAGBEUOMFRX-UHFFFAOYSA-N 0.000 description 2
- 125000000956 methoxy group Chemical group [H]C([H])([H])O* 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 125000005931 tert-butyloxycarbonyl group Chemical group [H]C([H])([H])C(OC(*)=O)(C([H])([H])[H])C([H])([H])[H] 0.000 description 2
- DMBKPDOAQVGTST-GFCCVEGCSA-N (2r)-2-[(2-methylpropan-2-yl)oxycarbonylamino]-3-phenylmethoxypropanoic acid Chemical compound CC(C)(C)OC(=O)N[C@@H](C(O)=O)COCC1=CC=CC=C1 DMBKPDOAQVGTST-GFCCVEGCSA-N 0.000 description 1
- CNBUSIJNWNXLQQ-NSHDSACASA-N (2s)-3-(4-hydroxyphenyl)-2-[(2-methylpropan-2-yl)oxycarbonylamino]propanoic acid Chemical compound CC(C)(C)OC(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 CNBUSIJNWNXLQQ-NSHDSACASA-N 0.000 description 1
- WFIYPADYPQQLNN-UHFFFAOYSA-N 2-[2-(4-bromopyrazol-1-yl)ethyl]isoindole-1,3-dione Chemical compound C1=C(Br)C=NN1CCN1C(=O)C2=CC=CC=C2C1=O WFIYPADYPQQLNN-UHFFFAOYSA-N 0.000 description 1
- BSYNRYMUTXBXSQ-UHFFFAOYSA-N Aspirin Chemical compound CC(=O)OC1=CC=CC=C1C(O)=O BSYNRYMUTXBXSQ-UHFFFAOYSA-N 0.000 description 1
- 241000255925 Diptera Species 0.000 description 1
- JOYRKODLDBILNP-UHFFFAOYSA-N Ethyl urethane Chemical compound CCOC(N)=O JOYRKODLDBILNP-UHFFFAOYSA-N 0.000 description 1
- HTTJABKRGRZYRN-UHFFFAOYSA-N Heparin Chemical compound OC1C(NC(=O)C)C(O)OC(COS(O)(=O)=O)C1OC1C(OS(O)(=O)=O)C(O)C(OC2C(C(OS(O)(=O)=O)C(OC3C(C(O)C(O)C(O3)C(O)=O)OS(O)(=O)=O)C(CO)O2)NS(O)(=O)=O)C(C(O)=O)O1 HTTJABKRGRZYRN-UHFFFAOYSA-N 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 206010027476 Metastases Diseases 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 208000001132 Osteoporosis Diseases 0.000 description 1
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 1
- 230000003187 abdominal effect Effects 0.000 description 1
- 230000017531 blood circulation Effects 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 229910000071 diazene Inorganic materials 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 229960002897 heparin Drugs 0.000 description 1
- 229920000669 heparin Polymers 0.000 description 1
- 150000003949 imides Chemical class 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 210000004731 jugular vein Anatomy 0.000 description 1
- 235000020281 long black Nutrition 0.000 description 1
- 230000009401 metastasis Effects 0.000 description 1
- PSACHCMMPFMFAJ-UHFFFAOYSA-N nmm n-methylmorpholine Chemical compound CN1CCOCC1.CN1CCOCC1 PSACHCMMPFMFAJ-UHFFFAOYSA-N 0.000 description 1
- QWVGKYWNOKOFNN-UHFFFAOYSA-N o-cresol Chemical compound CC1=CC=CC=C1O QWVGKYWNOKOFNN-UHFFFAOYSA-N 0.000 description 1
- 239000000825 pharmaceutical preparation Substances 0.000 description 1
- 230000010118 platelet activation Effects 0.000 description 1
- CHKVPAROMQMJNQ-UHFFFAOYSA-M potassium bisulfate Chemical class [K+].OS([O-])(=O)=O CHKVPAROMQMJNQ-UHFFFAOYSA-M 0.000 description 1
- 238000011552 rat model Methods 0.000 description 1
- 238000004904 shortening Methods 0.000 description 1
- ITMCEJHCFYSIIV-UHFFFAOYSA-N triflic acid Chemical compound OS(=O)(=O)C(F)(F)F ITMCEJHCFYSIIV-UHFFFAOYSA-N 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
Landscapes
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention discloses a polypeptide YIGSKYIGSK with target antithrombotic activity, as well as a preparation method and application of the polypeptide YIGSKYIGSK as antithrombotic agent, which belongs to the biomedical field. According to the invention, the polypeptide with target antithrombotic activity is prepared through conjugation of YIGSK and YIGSK. Animal model tests prove that YIGSKYIGSK has better antithrombotic activity.
Description
The application is that the application number of on November 30th, 2006 application is 200610144234.9, and denomination of invention is divided an application for the patent of " polypeptide, its preparation method and application with target antithrombotic activity ".
Technical field
The present invention relates to have the polypeptide YIGSKYIGSK of target antithrombotic activity.The preparation method and it that the invention still further relates to it belong to biomedicine field as the application of pharmaceutical preparations having antithrombotic activity.
Background technology
Cell adhesion plays a crucial role in the evolution of cell adhesion property disease (metastasis of cancer, thrombosis, chemistry cause inflammation and osteoporosis).YIGSK can participate in the cell adhesion process.YIGSK optionally is attached on the activatory thrombocyte, and the compound that promptly contains the YIGSK sequence can make scleroproein combine to be obstructed with activated blood platelet to the enrichment of the position of platelet activation, thereby has the target antithrombotic effect.Under such prerequisite, put together YIGSK and YIGSK, just can further strengthen its target antithrombotic effect.
Summary of the invention
One of the object of the invention is to put together YIGSK and YIGSK, forms the polypeptide YIGSKYIGSK with target antithrombotic activity.
Two of the object of the invention provides a kind of method for preparing the YIGSKYIGSK of antithrombotic acitivity.This method comprises:
1. employing liquid-phase synthesis process is through progressively connecing the protection midbody of the synthetic YIGSK of peptide;
2. slough the N end protection base of the protection midbody of YIGSK;
3. slough the C end protection base of the protection midbody of YIGSK;
4. put together the protection midbody that obtains YIGSKYIGSK to the YIGSK protection midbody of the YIGSK of N end dissociative protection midbody and C end dissociative;
5. slough the protection base to the protection midbody of YIGSKYIGSK, obtain YIGSKYIGSK.
Wherein, described N end protection base is the blocking group that the N end of polypeptide is used always when protecting, and for example can be tertbutyloxycarbonyl (Boc); Described C end protection base is the blocking group that the C end of polypeptide is used always when protecting, and for example can be benzyloxy (OBzl), methoxyl group (OMe) etc.
Above-described liquid phase synthetic intermediate and the method for protecting are the conventional and technique known of this area.
Animal model test proves that YIGSKYIGSK of the present invention has stronger antithrombotic acitivity.
In order further to set forth the present invention, provide a series of embodiment below.These embodiment are illustrative fully, and they only are used for the present invention is specifically described, and are not to be understood that to be limitation of the present invention.
The explanation of the shortenings that is occurred among the present invention:
The THF THF
DCC dicyclohexyl imide
The DCU NSC 30023
The OBzl benzyloxy
The Boc tertbutyloxycarbonyl
The OMe methoxyl group
HOBt N-hydroxybenzotriazole
The NMM N-methylmorpholine
Embodiment
The preparation of embodiment 1 YIGSKYIGSK
1) preparation of Boc-Ser (Bzl)-Lys (Z)-OBzl
0.59g (2.0mmol) Boc-Ser (Bzl) is dissolved among the anhydrous THF of 6ml, and 0 ℃ adds 0.27g (2.0mmol) N-hydroxybenzotriazole (HOBt) and 0.45g (2.2mmol) dicyclohexyl carbonyl diimine (DCC) down.Stir the anhydrous THF solution that adds 1.084g (2.0mmol) TosLys (Z)-OBzl after 10 minutes.Reaction mixture is regulated pH 8-9 with N-methylmorpholine (NMM), and 0 ℃ was stirred 24 hours.Stopped reaction removes by filter the NSC 30023 (DCU) that is settled out.Filtrate decompression concentrates, and residue is dissolved in the ETHYLE ACETATE, and the solution that obtains is used saturated NaHCO successively
3The aqueous solution, 5%KHSO
4The aqueous solution and the saturated NaCl aqueous solution are washed.Ethyl acetate layer is used anhydrous Na SO
4Dry 3 hours.Filtering NaSO
4, filtrate decompression concentrates removes ETHYLE ACETATE.Residue uses column chromatography (CHCl
3: CH
3OH, 30: 1), obtain 1.216g (94%) target compound.ESI-MS(m/z)648[M+H]
+。
2) preparation of HClSer (Bzl)-Lys (Z)-OBzl
0.648g (1.0mmol) Boc-Ser (Bzl)-Lys (Z)-OBzl is dissolved in 10ml hydrogenchloride-ETHYLE ACETATE (6mol/l) solution; Stirring at room 2 hours; TLC detects raw material point and disappears, and reduces pressure and takes out ETHYLE ACETATE, adds a small amount of ether repeatedly and reduces pressure and bleed to remove the acid gas in the product.Add a small amount of ether at last product is ground to form pressed powder, directly be used for next step reaction.ESI-MS(m/z)548[M+H]
+。
3) preparation of Boc-Ile-Gly-OMe
With 0.498g (2.0mmol) Boc-IleH
2O is dissolved among the anhydrous THF of 5mL, and 0 ℃ adds 0.27g (2.0mmol) HOBt and 0.45g (2.18mmol) DCC down.Stir the anhydrous THF solution that adds 0.251g (2.0mmol) HClGly-OMe after 10 minutes.Reaction mixture is regulated pH 8-9 with NMM, and 0 ℃ was stirred 24 hours.Stopped reaction removes by filter the DCU that is settled out.Filtrate decompression concentrates, and residue is dissolved in the ETHYLE ACETATE, and the solution that obtains is used saturated NaHCO successively
3The aqueous solution, 5%KHSO
4The aqueous solution and the saturated NaCl aqueous solution are washed.Ethyl acetate layer is used anhydrous Na SO
4Dry 3 hours.Filtering NaSO
4, filtrate decompression concentrates removes ETHYLE ACETATE.Residue uses column chromatography (CHCl
3: CH
3OH, 60: 1).Obtain 0.567g (94%) target compound.ESI-MS(m/z)303[M+H]
+。
4) preparation of HClIle-Gly-OMe
0.567g (1.87mmol) Boc-Ile-Gly-OMe is dissolved in 15ml hydrogenchloride-ethyl acetate solution (6mol/l); Stirring at room 2 hours; TLC detects raw material point and disappears, and reduces pressure and takes out ETHYLE ACETATE, adds a small amount of ether repeatedly and reduces pressure and bleed to remove the acid gas in the product.Add a small amount of ether at last product is ground to form pressed powder, directly be used for next step reaction.ESI-MS(m/z)203[M+H]
+。
5) preparation of Boc-Tyr-Ile-Gly-OMe
0.525g (1.87mmol) Boc-Tyr is dissolved among the anhydrous THF of 5ml, and 0 ℃ adds 0.270g (2.0mmol) HOBt and 0.45g (2.18mmol) DCC down.Stir after 10 minutes, add the anhydrous THF solution of 0.377g (1.87mmol) HClIle-Gly-OMe.Reaction mixture is regulated pH 8-9 with NMM, and 0 ℃ was stirred 24 hours.Stopped reaction removes by filter the DCU that is settled out.Filtrate decompression concentrates, and residue is dissolved in the ETHYLE ACETATE, and the solution that obtains is used saturated NaHCO successively
3The aqueous solution, 5%KHSO
4The aqueous solution and the saturated NaCl aqueous solution are washed.Ethyl acetate layer is used anhydrous Na SO
4Dry 3 hours.Filtering NaSO
4, filtrate decompression concentrates removes ETHYLE ACETATE.Residue uses column chromatography (CHCl
3: CH
3OH, 60: 1).Obtain 0.808g (93%) target compound.ESI-MS(m/z)466[M+H]
+。
6) preparation of Boc-Tyr-Ile-Gly
0.808g (1.87mmol) Boc-Tyr-Ile-Gly-OMe is dissolved in the 10ml methyl alcohol, and 0 ℃ adds the NaOH aqueous solution (1.5mol/l) down, regulates pH value to 11, stirring reaction 0.5h, and the TLC detection reaction finishes.Add saturated KHSO4 and regulate pH value to 6～7, the salt that filtering is separated out, whole methyl alcohol are removed in the filtrate decompression distillation, and residuary water solution continues to use KHSO
4Regulate pH value to 2, with ethyl acetate extraction three times, ethyl acetate layer merges, with saturated NaCl come together give a baby a bath on the third day after its birth inferior, anhydrous Na SO
4Dry 3 hours, to filter, filtrate decompression concentrates and removes ETHYLE ACETATE, obtains 0.834g (99%) target compound.ESI-MS(m/z)450[M-H]
-。
7) preparation of Boc-Tyr-Ile-Gly-Ser (Bzl)-Lys (Z)-OBzl
0.857g (1.9mmol) Boc-Tyr-Ile-Gly is dissolved among the anhydrous THF of 5ml, and 0 ℃ adds 0.257g (1.9mmol) HOBt and 0.45g (2.18mmol) DCC down.Stir after 10 minutes, add the anhydrous THF solution of 1.039g (1.9mmol) HClSer (Bzl)-Lys (Z)-OBzl.Reaction mixture is regulated pH 8-9 with NMM, and 0 ℃ was stirred 24 hours.Stopped reaction removes by filter the DCU that is settled out.Filtrate decompression concentrates, and residue is dissolved in the ETHYLE ACETATE, and the solution that obtains is used saturated NaHCO successively
3The aqueous solution, 5%KHSO
4The aqueous solution and the saturated NaCl aqueous solution are washed.Ethyl acetate layer is used anhydrous Na SO
4Dry 3 hours.Filtering NaSO
4, filtrate decompression concentrates removes ETHYLE ACETATE.Residue uses column chromatography (CHCl
3: CH
3OH, 30: 1).Obtain 1.71g (92%) target compound.ESI-MS(m/z)981[M+H]
+。
8) preparation of HClTyr-Ile-Gly-Ser (Bzl)-Lys (Z)-OBzl
0.98g (1.0mmol) Boc-Tyr-Ile-Gly-Ser (Bzl)-Lys (Z)-OBzl is dissolved in 20ml hydrogenchloride-ethyl acetate solution (6mol/l); Stirring at room 2 hours; TLC detects raw material point and disappears; ETHYLE ACETATE is taken out in decompression, adds a small amount of ether repeatedly and reduces pressure and bleed to remove the acid gas in the product.Add a small amount of ether at last product is ground to form pressed powder, directly be used for next step reaction.ESI-MS(m/z)881[M+H]
+。
9) preparation of Boc-Tyr-Ile-Gly-Ser (Bzl)-Lys (Z)
0.784g (0.8mmol) Boc-Tyr-Ile-Gly-Ser (Bzl)-Lys (Z)-OBzl is dissolved in the 10ml methyl alcohol, and 0 ℃ adds the NaOH aqueous solution (1.5mol/) down, regulates pH value to 11, stirring reaction 0.5h, and the TLC detection reaction finishes.Add saturated KHSO
4Regulate pH value to 6～7, the salt that filtering is separated out, whole methyl alcohol are removed in the filtrate decompression distillation, and residuary water solution continues to use KHSO
4Regulate pH value to 2, with ethyl acetate extraction three times, ethyl acetate layer merges, with saturated NaCl come together give a baby a bath on the third day after its birth inferior, anhydrous Na SO
4Dry 3 hours, to filter, filtrate decompression concentrates and removes ETHYLE ACETATE, obtains 1.59g (96%) target compound.ESI-MS(m/z)889[M-H]
-。
The preparation of embodiment 2 YIGSKYIGSK
1) preparation of Boc-Tyr-Ile-Gly-Ser (Bzl)-Lys (Z)-Tyr-Ile-Gly-Ser (Bzl)-Lys (Z)-OBzl is dissolved in 0.89g (1.0mmol) Boc-Tyr-Ile-Gly-Ser (Bzl)-Lys (Z) among the anhydrous THF of 5ml, and 0 ℃ adds 0.135g (1.0mmol) HOBt and 0.23g (1.1mmol) DCC down.Stir after 10 minutes, add the anhydrous THF solution of 0.916g (1.0mmol) HClTyr-Ile-Gly-Ser (Bzl)-Lys (Z)-OBzl.Reaction mixture is regulated pH 8-9 with NMM, and 0 ℃ was stirred 24 hours.Stopped reaction removes by filter the DCU that is settled out.Filtrate decompression concentrates, and residue is dissolved in the ETHYLE ACETATE, and the solution that obtains is used saturated NaHCO successively
3The aqueous solution, 5%KHSO
4The aqueous solution and the saturated NaCl aqueous solution are washed.Ethyl acetate layer is used anhydrous Na SO
4Dry 3 hours.Filtering NaSO
4, filtrate decompression concentrates removes ETHYLE ACETATE.Residue uses column chromatography (CHCl
3: CH
3OH, 30: 1).Obtain 1.57g (90%) target compound.
1HNMR(BHSC-300，CDCl
3-d
3)δ＝7.88(d，J＝5.0Hz，12H)，7.19(m，25H)，6.95(d，J＝7.0Hz，4H)，6.68(d，J＝7.0Hz，4H)，5.34(s，6H)，5.0(s，2H)，4.92(q，J＝6.0Hz，2H)，4.86(q，J＝6.0Hz，2H)，4.63(s，4H)，4.52(t，J＝6.0Hz，3H)，4.42(q，J＝6.0Hz，1H)，4.09(d，J＝6.0Hz，4H)，3.87(d，J＝5.0Hz，4H)，3.05(d，J＝6.0Hz，4H)，2.96(q，J＝6.0Hz，4H)，2.5(m，2H)，1.7-1.9(q，J＝7.0Hz，4H)，1.55(m，4H)，1.4(s，9H)，0.8-1.3(m，J＝8.0Hz，20H)。ESI-MS(m/z)1753[M+H]
+。Ultimate analysis C
70H
88N
6O
21Theoretical value C 64.37, H, and 6.90, N 9.58. measured value C 64.49, H 6.81, and N 9.74.
2) preparation of YIGSKYIGSK
200mg (0.114mmol) Boc-Tyr-Ile-Gly-Ser (Bzl)-Lys (Z)-Tyr-Ile-Gly-Ser (Bzl)-Lys (Z)-OBzl is dissolved in the 4mL trifluoracetic acid; Add 1mL trifluoromethanesulfonic acid and 1mL methyl-phenoxide; 0 ℃ of following stirring reaction 1 hour, disposable adding 100mL ether was bled in decompression 5 minutes; The product pressed powder of separating out uses Sephadex G 10 to purify, and obtains 114mg (90%) target compound.
1HNMR(BHSC-300，CDCl
3-d
3)δ＝11.0(s，1H)，7.89(d，J＝5.0Hz，9H)，6.95(d，J＝7.0Hz，4H)，6.68(d，J＝7.0Hz，4H)，5.0(s，2H)，4.92(q，J＝6.0Hz，1H)，4.62(q，J＝6.0Hz，2H)，4.53(t，J＝6.0Hz，3H)，4.46(q，J＝6.0Hz，1H)，4.09(d，J＝6.0Hz，4H)，4.03(d，J＝7.0Hz，4H)，3.95(q，J＝6.0Hz，1H)，3.05(d，J＝6.0Hz，4H)，2.65(q，J＝6.0Hz，4H)，2.5(m，2H)，2.0(s，8H)，1.79(q，J＝7.0Hz，4H)，1.55(m，4H)，0.8-1.3(m，J＝8.0Hz，20H)。ESI-MS(m/z)1115[M+H]
+。Ultimate analysis C
52H
82N
12O
15Theoretical value C 56.00, H, and 7.41, N 15.07. measured value C 56.14.49, H 7.52, and N 15.20.
The antithrombotic acitivity test of Test Example 1 YIGSKYIGSK
1) rat operation and apparatus
(male, 220～230g) press 1200mgkg to the SD rat
-1Dosage abdominal injection urethane solution is anaesthetized.The anesthetized rat dorsal position is fixed, and separates RCCA, and in proximal part folder bulldog clamp, proximal part and distal end penetrate surgical thread respectively, and the surgical thread of distal end is clamped with mosquito forceps in fur, and preparation is in the distal end intubate.
1) intubate
Intubate is the polyethylene rubber tube that silylanization is crossed, and divides three sections, and the stage casing is a polyethylene rubber tube, long 60.0mm, internal diameter 3.5mm; Two ends are identical polyethylene tube, and pipe range 100mm, the end that internal diameter 1mm, external diameter 2mm should manage pull into point pipe (being used to insert rat carotid artery or vein), and external diameter is 1mm.Be respectively charged into the long black surgical thread of 6Gm in the clean penicillium mould bottle with the number of finishing, weigh; Take out silk thread then, put into the thicker intubate in stage casing of ready intubate according to numbering.
Open rat right side bulldog clamp, fill with heparin-saline solution (50IUkg in will managing through sharp pipe end with syringe
- 1), then the arterial end of intubate is inserted the rat right carotid, the heparin of calculated amount is slowly injected in the rat body.
2) administration
Medicine: saline water (3mlkg
-1), the physiological salt soln of Asprin (dosage is 30mg/kg), the physiological salt soln of YIGSKYIGSK (dosage is 10 μ mol/kg).
Folder closes rat right carotid artery folder, pulls up the syringe of intubate vein end, has the water-soluble medical fluid injector of medicine of calculated amount to insert the vein end of intubate suction, opens rat right carotid artery folder, and medicine is slowly pushed in the rat body.Folder closes right carotid artery folder, and the vein end in teahouse is inserted the rats with left jugular vein of separator well, opens bulldog clamp, makes blood begin circulation.And pick up counting simultaneously.Can produce thrombus because of blood circulation on the silk thread in this process in the extra heavy pipe of intubate central authorities.
3) thrombus is weighed
Timing is cut off venous incubation after beginning 15 minutes, stops circulation, carefully takes out silk thread with the ophthalmology tweezer, on filter paper, dips in drop of blood gently, puts into the penicillium mould bottle of weighing in advance, accurately weighs and record.Calculate the weight in wet base of thrombus.Each medicine repeats 11 administrations.The wet weight of thrombus
of each group of statistics is also done the t check.
4) result
Compound of the present invention all has good anti-thrombus activity.The result lists table 1 in.
The antithrombotic acitivity of table 1YIGSKYIGSK on rat model
N=11; A. compare P＜0.001 with saline water.
Claims (3)
1. the polypeptide YIGSKYIGSK that has target antithrombotic activity.
2. method for preparing the YIGSKYIGSK of claim 1 comprises:
(1) adopts liquid-phase synthesis process, through progressively connecing the protection midbody of the synthetic YIGSK of peptide;
(2) the N end of sloughing the protection midbody of YIGSK is protected base;
(3) the C end of sloughing the protection midbody of YIGSK is protected base;
(4) put together the protection midbody that obtains YIGSKYIGSK to the YIGSK protection midbody of the YIGSK of N end dissociative protection midbody and C end dissociative;
(5) slough the protection base to the protection midbody of YIGSKYIGSK, obtain YIGSKYIGSK.
3. the YIGSKYIGSK of claim 1 is in the purposes of preparation in the antithrombotic reagent.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2012101051478A CN102702318A (en) | 2006-11-30 | 2006-11-30 | YIGSKYIGSK with target antithrombotic activity, and preparation and application thereof |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
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|---|---|---|---|
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110577571A (en) * | 2018-06-08 | 2019-12-17 | 首都医科大学 | YIGSK modified heptacyclic aldehyde, synthesis, antithrombotic activity and application thereof |
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2006
- 2006-11-30 CN CN2012101051478A patent/CN102702318A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110577571A (en) * | 2018-06-08 | 2019-12-17 | 首都医科大学 | YIGSK modified heptacyclic aldehyde, synthesis, antithrombotic activity and application thereof |
| CN110577571B (en) * | 2018-06-08 | 2021-06-08 | 首都医科大学 | YIGSK modified heptacyclic aldehyde, synthesis, antithrombotic activity and application thereof |
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Application publication date: 20121003 |