CN109438555B - Preparation method of non-natural antioxidant peptide - Google Patents
Preparation method of non-natural antioxidant peptide Download PDFInfo
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- CN109438555B CN109438555B CN201811397810.XA CN201811397810A CN109438555B CN 109438555 B CN109438555 B CN 109438555B CN 201811397810 A CN201811397810 A CN 201811397810A CN 109438555 B CN109438555 B CN 109438555B
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- 101800000068 Antioxidant peptide Proteins 0.000 title claims abstract description 14
- 238000002360 preparation method Methods 0.000 title claims abstract description 6
- ZPGDWQNBZYOZTI-UHFFFAOYSA-N 1-(9h-fluoren-9-ylmethoxycarbonyl)pyrrolidine-2-carboxylic acid Chemical compound OC(=O)C1CCCN1C(=O)OCC1C2=CC=CC=C2C2=CC=CC=C21 ZPGDWQNBZYOZTI-UHFFFAOYSA-N 0.000 claims abstract description 85
- 125000003088 (fluoren-9-ylmethoxy)carbonyl group Chemical group 0.000 claims abstract description 72
- YEDUAINPPJYDJZ-UHFFFAOYSA-N 2-hydroxybenzothiazole Chemical compound C1=CC=C2SC(O)=NC2=C1 YEDUAINPPJYDJZ-UHFFFAOYSA-N 0.000 claims abstract description 46
- NPZTUJOABDZTLV-UHFFFAOYSA-N hydroxybenzotriazole Substances O=C1C=CC=C2NNN=C12 NPZTUJOABDZTLV-UHFFFAOYSA-N 0.000 claims abstract description 46
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 43
- 102000004196 processed proteins & peptides Human genes 0.000 claims abstract description 40
- 229920001184 polypeptide Polymers 0.000 claims abstract description 39
- NQRYJNQNLNOLGT-UHFFFAOYSA-N Piperidine Chemical compound C1CCNCC1 NQRYJNQNLNOLGT-UHFFFAOYSA-N 0.000 claims abstract description 36
- XOAAWQZATWQOTB-UHFFFAOYSA-N taurine Chemical compound NCCS(O)(=O)=O XOAAWQZATWQOTB-UHFFFAOYSA-N 0.000 claims abstract description 32
- 238000000034 method Methods 0.000 claims abstract description 19
- 230000002194 synthesizing effect Effects 0.000 claims abstract description 19
- LPNYRYFBWFDTMA-UHFFFAOYSA-N potassium tert-butoxide Chemical compound [K+].CC(C)(C)[O-] LPNYRYFBWFDTMA-UHFFFAOYSA-N 0.000 claims abstract description 18
- 238000010511 deprotection reaction Methods 0.000 claims abstract description 17
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- QUIXRGCMQOXUSV-SZMVWBNQSA-N Trp-Pro-Pro Chemical compound O=C([C@@H]1CCCN1C(=O)[C@H](CC=1C2=CC=CC=C2NC=1)N)N1CCC[C@H]1C(O)=O QUIXRGCMQOXUSV-SZMVWBNQSA-N 0.000 claims abstract description 6
- 241001339782 Scapharca broughtonii Species 0.000 claims abstract description 5
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-dimethylformamide Substances CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 claims description 80
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 60
- VFVBVEMVTNQIMZ-SFHVURJKSA-N (1s)-3-naphthalen-1-yloxy-1-phenylpropan-1-ol Chemical compound C1([C@H](CCOC=2C3=CC=CC=C3C=CC=2)O)=CC=CC=C1 VFVBVEMVTNQIMZ-SFHVURJKSA-N 0.000 claims description 53
- QCDWFXQBSFUVSP-UHFFFAOYSA-N 2-phenoxyethanol Chemical compound OCCOC1=CC=CC=C1 QCDWFXQBSFUVSP-UHFFFAOYSA-N 0.000 claims description 52
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- 239000003795 chemical substances by application Substances 0.000 claims description 45
- 230000003213 activating effect Effects 0.000 claims description 32
- 238000001914 filtration Methods 0.000 claims description 30
- 239000000047 product Substances 0.000 claims description 30
- 238000005406 washing Methods 0.000 claims description 30
- ONIBWKKTOPOVIA-BYPYZUCNSA-N L-Proline Chemical compound OC(=O)[C@@H]1CCCN1 ONIBWKKTOPOVIA-BYPYZUCNSA-N 0.000 claims description 25
- 239000007795 chemical reaction product Substances 0.000 claims description 20
- 238000002156 mixing Methods 0.000 claims description 18
- 238000004108 freeze drying Methods 0.000 claims description 15
- 239000002904 solvent Substances 0.000 claims description 15
- ADOHASQZJSJZBT-SANMLTNESA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-3-[1-[(2-methylpropan-2-yl)oxycarbonyl]indol-3-yl]propanoic acid Chemical compound C12=CC=CC=C2N(C(=O)OC(C)(C)C)C=C1C[C@@H](C(O)=O)NC(=O)OCC1C2=CC=CC=C2C2=CC=CC=C21 ADOHASQZJSJZBT-SANMLTNESA-N 0.000 claims description 12
- 239000000203 mixture Substances 0.000 claims description 6
- USIUVYZYUHIAEV-UHFFFAOYSA-N diphenyl ether Chemical compound C=1C=CC=CC=1OC1=CC=CC=C1 USIUVYZYUHIAEV-UHFFFAOYSA-N 0.000 claims 1
- 239000011347 resin Substances 0.000 abstract description 27
- 229920005989 resin Polymers 0.000 abstract description 27
- 239000003153 chemical reaction reagent Substances 0.000 abstract description 6
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- -1 Trp amino acid Chemical class 0.000 abstract description 2
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- 238000003786 synthesis reaction Methods 0.000 description 13
- DTQVDTLACAAQTR-UHFFFAOYSA-N trifluoroacetic acid Substances OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 13
- FDKXTQMXEQVLRF-ZHACJKMWSA-N (E)-dacarbazine Chemical compound CN(C)\N=N\c1[nH]cnc1C(N)=O FDKXTQMXEQVLRF-ZHACJKMWSA-N 0.000 description 12
- 210000004027 cell Anatomy 0.000 description 10
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 10
- 230000000694 effects Effects 0.000 description 8
- 239000011541 reaction mixture Substances 0.000 description 8
- RWSXRVCMGQZWBV-WDSKDSINSA-N glutathione Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@@H](CS)C(=O)NCC(O)=O RWSXRVCMGQZWBV-WDSKDSINSA-N 0.000 description 7
- 230000010355 oscillation Effects 0.000 description 7
- 239000003875 Wang resin Substances 0.000 description 6
- 230000009471 action Effects 0.000 description 6
- 230000004913 activation Effects 0.000 description 6
- 125000006239 protecting group Chemical group 0.000 description 6
- FEMOMIGRRWSMCU-UHFFFAOYSA-N ninhydrin Chemical compound C1=CC=C2C(=O)C(O)(O)C(=O)C2=C1 FEMOMIGRRWSMCU-UHFFFAOYSA-N 0.000 description 5
- 238000012360 testing method Methods 0.000 description 5
- 150000001413 amino acids Chemical class 0.000 description 4
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- BDNKZNFMNDZQMI-UHFFFAOYSA-N 1,3-diisopropylcarbodiimide Chemical compound CC(C)N=C=NC(C)C BDNKZNFMNDZQMI-UHFFFAOYSA-N 0.000 description 1
- ASOKPJOREAFHNY-UHFFFAOYSA-N 1-Hydroxybenzotriazole Chemical compound C1=CC=C2N(O)N=NC2=C1 ASOKPJOREAFHNY-UHFFFAOYSA-N 0.000 description 1
- 241000251468 Actinopterygii Species 0.000 description 1
- 241000238557 Decapoda Species 0.000 description 1
- MVORZMQFXBLMHM-QWRGUYRKSA-N Gly-His-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)CN)CC1=CN=CN1 MVORZMQFXBLMHM-QWRGUYRKSA-N 0.000 description 1
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 description 1
- 241000238413 Octopus Species 0.000 description 1
- 241000237502 Ostreidae Species 0.000 description 1
- ONIBWKKTOPOVIA-UHFFFAOYSA-N Proline Natural products OC(=O)C1CCCN1 ONIBWKKTOPOVIA-UHFFFAOYSA-N 0.000 description 1
- 241000238371 Sepiidae Species 0.000 description 1
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 description 1
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 description 1
- NERFNHBZJXXFGY-UHFFFAOYSA-N [4-[(4-methylphenyl)methoxy]phenyl]methanol Chemical compound C1=CC(C)=CC=C1COC1=CC=C(CO)C=C1 NERFNHBZJXXFGY-UHFFFAOYSA-N 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 210000005013 brain tissue Anatomy 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 230000003833 cell viability Effects 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 230000003013 cytotoxicity Effects 0.000 description 1
- 231100000135 cytotoxicity Toxicity 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 125000005519 fluorenylmethyloxycarbonyl group Chemical group 0.000 description 1
- 108010038983 glycyl-histidyl-lysine Proteins 0.000 description 1
- 206010073071 hepatocellular carcinoma Diseases 0.000 description 1
- 210000003292 kidney cell Anatomy 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 235000020636 oyster Nutrition 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 238000001243 protein synthesis Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 235000015170 shellfish Nutrition 0.000 description 1
- 238000010532 solid phase synthesis reaction Methods 0.000 description 1
- 229910052717 sulfur Inorganic materials 0.000 description 1
- 239000011593 sulfur Substances 0.000 description 1
- 125000005931 tert-butyloxycarbonyl group Chemical group [H]C([H])([H])C(OC(*)=O)(C([H])([H])[H])C([H])([H])[H] 0.000 description 1
- 230000014616 translation Effects 0.000 description 1
- 125000002221 trityl group Chemical group [H]C1=C([H])C([H])=C([H])C([H])=C1C([*])(C1=C(C(=C(C(=C1[H])[H])[H])[H])[H])C1=C([H])C([H])=C([H])C([H])=C1[H] 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/06—Dipeptides
- C07K5/06139—Dipeptides with the first amino acid being heterocyclic
- C07K5/06156—Dipeptides with the first amino acid being heterocyclic and Trp-amino acid; Derivatives thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P39/00—General protective or antinoxious agents
- A61P39/06—Free radical scavengers or antioxidants
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
- C07D403/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings
- C07D403/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings linked by a chain containing hetero atoms as chain links
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/06—Dipeptides
- C07K5/06139—Dipeptides with the first amino acid being heterocyclic
- C07K5/06165—Dipeptides with the first amino acid being heterocyclic and Pro-amino acid; Derivatives thereof
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/08—Tripeptides
- C07K5/0821—Tripeptides with the first amino acid being heterocyclic, e.g. His, Pro, Trp
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Abstract
The invention provides a preparation method of a non-natural antioxidant peptide, belonging to the field of biological medicine, and the synthesis method of the polypeptide comprises the following steps: carrying out resin grafting reaction on Fmoc-Pro (Boc) -OH, and then carrying out connection reaction on the Fmoc-Pro (Boc) -OH, Pro or Trp amino acid and taurine to obtain polypeptides with the structures of Tau-Trp-Pro, Trp-Tau-Pro, Tau-Trp-Pro, Trp-Pro-Tau-Pro, Trp-Tau-Pro-Pro and the like; in the reaction process, the Fmoc group removal adopts deprotection reagent combination as follows: piperidine, potassium tert-butoxide and HOBT. The taurine and the blood clam tripeptide Trp-Pro-Pro react to obtain the polypeptide with antioxidant activity, the amino group is completely deprotected in the process of synthesizing the polypeptide, and the purity of the synthesized polypeptide is high.
Description
Technical Field
The invention belongs to the field of biomedicine, and particularly relates to a preparation method of a non-natural antioxidant peptide.
Background
Taurine is a sulfur-containing non-protein amino acid, has wide physiological activities, and can regulate normal physiological activities of human body, such as promoting the development of infant brain tissue and intelligence, and enhancing the oxidation resistance of cells. The taurine is widely distributed, and marine organisms such as sea fish and shellfish such as cuttlefish, octopus, shrimp, oyster, conch and clam contain abundant taurine. Taurine exists in free form in the organism and is not involved in protein synthesis. Taurine can be reacted with other amino acids to form novel unnatural polypeptides by chemical synthesis, which can impart biological activity not possessed by natural protein peptides. The field has wide development prospect.
The tripeptide Trp-Pro-Pro from blood clam has remarkable antioxidant activity and anticancer activity. Through a chemical synthesis method, amino acids Trp, Pro and taurine are connected into tripeptide or tetrapeptide, which is beneficial to synthesizing the polypeptide with higher antioxidant or anticancer activity.
The prior Chinese patent with publication number CN 101230089A discloses a solid-phase synthesis method of glycyl histidyl lysine; mainly solves the technical problem that the artificial synthesis is difficult because the molecule contains 2 basic amino acids; however, the invention has the disadvantages of low amino group deprotection efficiency and low polypeptide synthesis purity.
Disclosure of Invention
The invention aims to provide a preparation method of non-natural antioxidant peptide, taurine reacts with blood clam tripeptide Trp-Pro-Pro to obtain polypeptide with enhanced antioxidant activity or anticancer activity, and an amino deprotection agent and a polypeptide synthesis promoter are utilized in the process of synthesizing the polypeptide to realize the purposes of complete amino deprotection and high purity of polypeptide synthesis; meanwhile, the defects of blindness and difficult separation in the process of preparing the active peptide only by enzymolysis of the protein are overcome, and the method has wide application prospect.
The technical scheme adopted by the invention for realizing the purpose is as follows:
By a chemical synthesis method, tripeptide Trp-Pro-Pro is reacted with taurine to obtain tripeptide or tetrapeptide with different structures, and the non-natural multifunctional peptide with enhanced antioxidant activity is obtained.
Based on the different order of attachment between the amino acids, the following newly synthesized tripeptides or tetrapeptides are obtained:
synthesizing tripeptide with a structure of Tau-Trp-Pro, comprising the following steps:
s1, mixing Fmoc-Pro (Boc) -OH and Wang Resin in proportion, adding activating agents of HOBT and DIC/HBTU, carrying out oscillation reaction for 2-3h at 42-44 ℃ by taking DMF as a solvent, and filtering a reaction product, washing the reaction product with DMF and ethanol to obtain Fmoc-Pro (Boc) -Wang Resin;
s2, removing Fmoc groups from Fmoc-Pro (Boc) -Wang Resin obtained in the step S1 to obtain H-Pro (Boc) -Wang Resin; then adding Fmoc-Trp (Trt) -OH, then adding (S) - (+) -3- (1-naphthoxy) -1-phenyl-1-propanol and ethylene glycol monophenyl ether, and reacting at 25-45 ℃ for 30-60min to obtain Fmoc-Trp (Trt) -Pro (Boc) -Wang Resin;
s3, removing Fmoc groups from Fmoc-Trp (Trt) -Pro (Boc) -Wang Resin obtained in the step S2 to obtain H-Trp (Trt) -Pro (Boc) -Wang Resin; then adding Fmoc-Tau-OH, then adding (S) - (+) -3- (1-naphthoxy) -1-phenyl-1-propanol and ethylene glycol monophenyl ether, and reacting for 30-60min at 25-45 ℃ to obtain Fmoc-Tau-Trp (Trt) -Pro (Boc) -Wang Resin;
S4, removing Fmoc groups from the product obtained in the step S3, adding TFA, oscillating to react for 0.5-2.5H, filtering, washing the product with DMF and ethanol respectively, and freeze-drying to obtain H-Tau-Trp-Pro-OH.
Wherein, the mole number of Fmoc-Pro (Boc) -OH in the step S1 is 2-3 times of that of the resin; the activating agent is HOBT and DIC or a mixture of HOBT and HBTU, and the mole number of the activating agent is 3-4 times that of the resin;
removing Fmoc groups, and combining by using deprotection agents: 10-25 vol% piperidine, 1-5 wt% potassium tert-butoxide and 0.1-3 wt% HOBT; the piperidine is easier to remove the amino protecting group under the activation of HOBT and the proper alkaline action of potassium tert-butoxide, and the removal efficiency is high;
the addition amounts of (S) - (+) -3- (1-naphthoxy) -1-phenyl-1-propanol and ethylene glycol monophenyl ether are 0.1-1.0% and 1-5% by mass of the reaction mixing system, respectively; the special existence of the (S) - (+) -3- (1-naphthoxy) -1-phenyl-1-propanol and the ethylene glycol monophenyl ether can activate and stretch polypeptide molecules, accelerate the connection reaction of the polypeptide molecules and taurine, inhibit racemization or by-product formation and improve the yield and purity of polypeptide synthesis.
Synthesizing tripeptide with a structure of Trp-Tau-Pro, comprising the following steps:
s1, mixing Fmoc-Pro (Boc) -OH and Wang Resin in proportion, adding activating agents of HOBT and DIC/HBTU, carrying out oscillation reaction for 2-3h at 42-44 ℃ by taking DMF as a solvent, and filtering a reaction product, washing the reaction product with DMF and ethanol to obtain Fmoc-Pro (Boc) -Wang Resin;
S2, removing Fmoc groups from Fmoc-Pro (Boc) -Wang Resin obtained in the step S1 to obtain H-Pro (Boc) -Wang Resin; then adding Fmoc-Tau-OH, then adding (S) - (+) -3- (1-naphthoxy) -1-phenyl-1-propanol and ethylene glycol monophenyl ether, and reacting for 30-60min at 25-45 ℃ to obtain Fmoc-Tau-Pro (Boc) -Wang Resin;
s3, removing Fmoc groups from Fmoc-Tau-Pro (Boc) -Wang Resin obtained in the step S2 to obtain H-Tau-Pro (Boc) -Wang Resin; then adding Fmoc-Trp (Boc) -OH, then adding (S) - (+) -3- (1-naphthoxy) -1-phenyl-1-propanol and ethylene glycol monophenyl ether, and reacting at 25-45 ℃ for 30-60min to obtain Fmoc-Trp (Boc) -Tau-Pro (Boc) -WangResin;
s4, removing Fmoc groups from the product obtained in the step S3, adding TFA, oscillating to react for 0.5-2.5H, filtering, washing the product with DMF and ethanol respectively, and freeze-drying to obtain H-Trp-Tau-Pro-OH.
Wherein, the mole number of Fmoc-Pro (Boc) -OH in the step S1 is 2-3 times of that of the resin; the activating agent is HOBT and DIC or a mixture of HOBT and HBTU, and the mole number of the activating agent is 3-4 times that of the resin;
removing Fmoc group, and combining with a deprotection agent as follows: 10-25 vol% piperidine, 1-5 wt% potassium tert-butoxide and 0.1-3 wt% HOBT;
the addition amounts of (S) - (+) -3- (1-naphthoxy) -1-phenyl-1-propanol and ethylene glycol monophenyl ether are 0.1-1.0% and 1-5% by mass of the reaction mixture system, respectively.
Synthesizing tetrapeptide with the structure of Tau-Trp-Pro-Pro, comprising the following steps:
s1, mixing Fmoc-Pro (Boc) -OH and Wang Resin in proportion, adding activating agents of HOBT and DIC/HBTU, carrying out oscillation reaction for 2-3h at 42-44 ℃ by taking DMF as a solvent, and filtering a reaction product, washing the reaction product with DMF and ethanol to obtain Fmoc-Pro (Boc) -Wang Resin;
s2, removing Fmoc groups from Fmoc-Pro (Boc) -Wang Resin obtained in the step S1 to obtain H-Pro (Boc) -Wang Resin; then adding Fmoc-Pro (Trt) -OH, then adding (S) - (+) -3- (1-naphthoxy) -1-phenyl-1-propanol and ethylene glycol monophenyl ether, and reacting for 30-60min at 25-45 ℃ to obtain Fmoc-Pro (Trt) -Pro (Boc) -Wang Resin;
s3, removing Fmoc groups from Fmoc-Pro (Trt) -Pro (Boc) -Wang Resin obtained in the step S2 to obtain H-Pro (Trt) -Pro (Boc) -Wang Resin; then adding Fmoc-Trp (Boc) -OH, then adding (S) - (+) -3- (1-naphthoxy) -1-phenyl-1-propanol and ethylene glycol monophenyl ether, and reacting at 25-45 ℃ for 30-60min to obtain Fmoc-Trp (Boc) -Pro (Trt) -Pro (Boc) -Wang Resin;
s4, removing Fmoc groups from Fmoc-Trp (Boc) -Pro (Trt) -Pro (Boc) -Wang Resin obtained in the step S3 to obtain H-Trp (Boc) -Pro (Trt) -Pro (Boc) -Wang Resin, adding Fmoc-Tau-OH, adding (S) - (+) -3- (1-naphthyloxy) -1-phenyl-1-propanol and ethylene glycol monophenyl ether, and reacting at 25-45 ℃ for 30-60min to obtain Fmoc-Tau-Trp (Boc) -Pro (Boc) -Wang Resin;
S5, removing Fmoc groups from the product obtained in the step S4, adding TFA, oscillating to react for 0.5-2.5H, filtering, washing the product with DMF and ethanol respectively, and freeze-drying to obtain H-Tau-Trp-Pro-Pro-OH.
Wherein, the mole number of Fmoc-Pro (Boc) -OH in the step S1 is 2-3 times of that of the resin; the activating agent is HOBT and DIC or a mixture of HOBT and HBTU, and the mole number of the activating agent is 3-4 times that of the resin;
removing Fmoc groups, and combining by using deprotection agents: 10-25 vol% piperidine, 1-5 wt% potassium tert-butoxide and 0.1-3 wt% HOBT;
the addition amounts of (S) - (+) -3- (1-naphthoxy) -1-phenyl-1-propanol and ethylene glycol monophenyl ether are 0.1-1.0% and 1-5% by mass of the reaction mixture system, respectively.
Synthesizing tetrapeptide with the structure of Trp-Tau-Pro-Pro, comprising the following steps:
s1, mixing Fmoc-Pro (Boc) -OH and Wang Resin in proportion, adding activating agents of HOBT and DIC/HBTU, carrying out oscillation reaction for 2-3h at 42-44 ℃ by taking DMF as a solvent, and filtering a reaction product, washing the reaction product with DMF and ethanol to obtain Fmoc-Pro (Boc) -Wang Resin;
s2, removing Fmoc groups from Fmoc-Pro (Boc) -Wang Resin obtained in the step S1 to obtain H-Pro (Boc) -Wang Resin; then adding Fmoc-Pro (Trt) -OH, then adding (S) - (+) -3- (1-naphthoxy) -1-phenyl-1-propanol and ethylene glycol monophenyl ether, and reacting for 30-60min at 25-45 ℃ to obtain Fmoc-Pro (Trt) -Pro (Boc) -Wang Resin;
S3, removing Fmoc groups from Fmoc-Pro (Trt) -Pro (Boc) -Wang Resin obtained in the step S2 to obtain H-Pro (Trt) -Pro (Boc) -Wang Resin; then adding Fmoc-Tau-OH, then adding (S) - (+) -3- (1-naphthoxy) -1-phenyl-1-propanol and ethylene glycol monophenyl ether, and reacting for 30-60min at 25-45 ℃ to obtain Fmoc-Tau-Pro (Trt) -Pro (Boc) -Wang Resin;
s4, removing Fmoc groups from Fmoc-Tau-Pro (Trt) -Pro (Boc) -Wang Resin obtained in the step S3 to obtain H-Tau-Pro (Trt) -Pro (Boc) -Wang Resin, adding Fmoc-Trp (Boc) -OH, adding (S) - (+) -3- (1-naphthyloxy) -1-phenyl-1-propanol and ethylene glycol monophenyl ether, and reacting at 25-45 ℃ for 30-60min to obtain Fmoc-Trp (Boc) -Tau-Pro (Trt) -Pro (Boc) -Wang Resin;
s5, removing Fmoc groups from the product obtained in the step S4, adding TFA, oscillating to react for 0.5-2.5H, filtering, washing the product with DMF and ethanol respectively, and freeze-drying to obtain H-Trp-Tau-Pro-Pro-OH.
Wherein, the mole number of Fmoc-Pro (Boc) -OH in the step S1 is 2-3 times of that of the resin; the activating agent is HOBT and DIC or a mixture of HOBT and HBTU, and the mole number of the activating agent is 3-4 times that of the resin;
removing Fmoc group, and combining with a deprotection agent as follows: 10-25 vol% piperidine, 1-5 wt% potassium tert-butoxide and 0.1-3 wt% HOBT;
the addition amounts of (S) - (+) -3- (1-naphthoxy) -1-phenyl-1-propanol and ethylene glycol monophenyl ether are 0.1-1.0% and 1-5% by mass of the reaction mixture system, respectively.
Synthesizing tetrapeptide with the structure of Trp-Pro-Tau-Pro, comprising the following steps:
s1, mixing Fmoc-Pro (Boc) -OH and Wang Resin in proportion, adding activating agents of HOBT and DIC/HBTU, carrying out oscillation reaction for 2-3h at 42-44 ℃ by taking DMF as a solvent, and filtering a reaction product, washing the reaction product with DMF and ethanol to obtain Fmoc-Pro (Boc) -Wang Resin;
s2, removing Fmoc groups from Fmoc-Pro (Boc) -Wang Resin obtained in the step S1 to obtain H-Pro (Boc) -Wang Resin; then adding Fmoc-Tau-OH, then adding (S) - (+) -3- (1-naphthoxy) -1-phenyl-1-propanol and ethylene glycol monophenyl ether, and reacting for 30-60min at 25-45 ℃ to obtain Fmoc-Tau-Pro (Boc) -Wang Resin;
s3, removing Fmoc groups from Fmoc-Tau-Pro (Boc) -Wang Resin obtained in the step S2 to obtain H-Tau-Pro (Boc) -Wang Resin; then adding Fmoc-Pro (Trt) -OH, then adding (S) - (+) -3- (1-naphthoxy) -1-phenyl-1-propanol and ethylene glycol monophenyl ether, and reacting at 25-45 ℃ for 30-60min to obtain Fmoc-Pro (Trt) -Tau-Pro (Boc) -WangResin;
s4, removing Fmoc groups from Fmoc-Pro (Trt) -Tau-Pro (Boc) -Wang Resin obtained in the step S3 to obtain H-Pro (Trt) -Tau-Pro (Boc) -Wang Resin, adding Fmoc-Trp (Boc) -OH, adding (S) - (+) -3- (1-naphthyloxy) -1-phenyl-1-propanol and ethylene glycol monophenyl ether, and reacting at 25-45 ℃ for 30-60min to obtain Fmoc-Trp (Boc) -Pro (Trt) -Tau-Pro (Boc) -Wang Resin;
S5, removing Fmoc groups from the product obtained in the step S4, adding TFA, carrying out oscillation reaction for 0.5-2.5H, filtering, washing the product with DMF and ethanol respectively, and freeze-drying to obtain H-Trp-Pro-Tau-Pro-OH.
Wherein, the mole number of Fmoc-Pro (Boc) -OH in the step S1 is 2-3 times of that of the resin; the activating agent is HOBT and DIC or a mixture of HOBT and HBTU, and the mole number of the activating agent is 3-4 times that of the resin;
removing Fmoc group, and combining with a deprotection agent as follows: 10-25 vol% piperidine, 1-5 wt% potassium tert-butoxide and 0.1-3 wt% HOBT;
the addition amounts of (S) - (+) -3- (1-naphthoxy) -1-phenyl-1-propanol and ethylene glycol monophenyl ether are 0.1-1.0% and 1-5% by mass of the reaction mixture system, respectively.
Some abbreviations commonly used in the present invention have the following meanings:
fmoc: fluorenylmethyloxycarbonyl;
wang Resin: resin queen;
boc: a tert-butoxycarbonyl group;
trt: a trityl group;
HOBT: 1-hydroxybenzotriazole;
DIC: n, N' -diisopropylcarbodiimide;
HBTU: o-benzotriazol-tetramethylurea hexafluorophosphate;
DMF: n, N-dimethylformamide;
TFA: trifluoroacetic acid;
trp: tryptophan;
pro: (ii) proline;
tau: taurine.
The invention has the beneficial effects that:
1) the taurine and the blood clam tripeptide Trp-Pro-Pro are reacted to obtain the polypeptide with the structure of Tau-Trp-Pro, Trp-Tau-Pro, Tau-Trp-Pro-Pro, Trp-Pro-Tau-Pro and Trp-Tau-Pro-Pro; by performing activity screening on the polypeptides, the polypeptides with enhanced antioxidant activity or anticancer activity can be obtained; in the process of synthesizing the polypeptide, an amino deprotection agent and a polypeptide synthesis promoter are utilized to achieve the purposes of complete amino deprotection and high polypeptide synthesis purity; meanwhile, the defects of blindness and difficult separation in the process of preparing the active peptide only by enzymolysis of protein are overcome, and the method has wide application prospect;
2) In the process of polypeptide synthesis, the special existence of (S) - (+) -3- (1-naphthoxy) -1-phenyl-1-propanol and ethylene glycol monophenyl ether is utilized to activate and spread polypeptide molecules, accelerate the connection reaction of the polypeptide molecules and taurine, inhibit racemization or by-product formation and improve the yield and purity of polypeptide synthesis.
The invention adopts the technical scheme to provide the model essay, makes up the defects of the prior art, and has reasonable design and convenient operation.
Drawings
FIG. 1 shows the polypeptide H-resistance to HepG-2 and HEK293 cells 2 O 2 Oxidative damage activity profile; a) HepG-2 cells; b) HEK293 cells.
Description of reference numerals: s1: Trp-Pro-Tau-Pro; s2: Trp-Tau-Pro-Pro; s3: Trp-Tau-Pro; s4: Tau-Trp-Pro; s5: Tau-Trp-Pro-Pro.
Detailed Description
The present invention is further described in detail with reference to the following examples:
example 1:
synthesizing tripeptide with a structure of Tau-Trp-Pro, comprising the following steps:
s1, mixing Fmoc-Pro (Boc) -OH and Wang Resin in proportion, adding activating agents of HOBT and DIC, taking DMF as a solvent, oscillating and reacting at 43 ℃ for 2.5h, and filtering and washing reaction products with DMF and ethanol to obtain Fmoc-Pro (Boc) -Wang Resin; wherein the mole number of Fmoc-Pro (Boc) -OH is 2.2 times of that of the resin; the mole number of the activating agent is 3.4 times of that of the resin;
S2, removing the Fmoc group from the Fmoc-Pro (Boc) -Wang Resin obtained in the step S1 to obtain H-Pro (Boc) -Wang Resin (the Resin is subjected to color testing by a ninhydrin color reagent, and the color is dark blue or blue purple, so that the Fmoc group is removed); then adding Fmoc-Trp (Trt) -OH, then adding (S) - (+) -3- (1-naphthoxy) -1-phenyl-1-propanol and ethylene glycol monophenyl ether, and reacting for 45min at 30 ℃ to obtain Fmoc-Trp (Trt) -Pro (Boc) -Wang Resin;
s3, removing Fmoc groups from Fmoc-Trp (Trt) -Pro (Boc) -Wang Resin obtained in the step S2 to obtain H-Trp (Trt) -Pro (Boc) -Wang Resin; then adding Fmoc-Tau-OH, then adding (S) - (+) -3- (1-naphthoxy) -1-phenyl-1-propanol and ethylene glycol monophenyl ether, and reacting for 45min at 30 ℃ to obtain Fmoc-Tau-Trp (Trt) -Pro (Boc) -WangResin;
s4, removing Fmoc groups from the product obtained in the step S3, adding TFA, oscillating to react for 2 hours, filtering, washing the product with DMF and ethanol respectively, and freeze-drying to obtain H-Tau-Trp-Pro-OH.
Wherein, removing Fmoc group, using deprotection agent combination as follows: 23 vol% piperidine, 4.2 wt% potassium tert-butoxide and 1.6 wt% HOBT; the piperidine is easier to remove the amino protecting group under the activation of HOBT and the proper alkaline action of potassium tert-butoxide, and the removal efficiency is high;
the addition amounts of (S) - (+) -3- (1-naphthoxy) -1-phenyl-1-propanol and ethylene glycol monophenyl ether are 0.56% and 3.8% by mass of the reaction mixing system, respectively; the special existence of the (S) - (+) -3- (1-naphthoxy) -1-phenyl-1-propanol and the ethylene glycol monophenyl ether can activate and stretch polypeptide molecules, accelerate the connection reaction of the polypeptide molecules and taurine, inhibit racemization or by-product formation and improve the yield and purity of polypeptide synthesis.
Example 2:
synthesizing tripeptide with a structure of Trp-Tau-Pro, comprising the following steps:
s1, mixing Fmoc-Pro (Boc) -OH and Wang Resin in proportion, adding activating agents of HOBT and HBTU, oscillating and reacting at 43 ℃ by taking DMF as a solvent for 2.5h, and filtering a reaction product, washing with DMF and ethanol to obtain Fmoc-Pro (Boc) -Wang Resin; wherein the mole number of Fmoc-Pro (Boc) -OH is 2.2 times of that of the resin; the mole number of the activating agent is 3.4 times of that of the resin;
s2, removing the Fmoc group from the Fmoc-Pro (Boc) -Wang Resin obtained in the step S1 to obtain H-Pro (Boc) -Wang Resin (the Resin is subjected to color testing by a ninhydrin color reagent, and the color is dark blue or blue purple, so that the Fmoc group is removed); then adding Fmoc-Tau-OH, then adding (S) - (+) -3- (1-naphthoxy) -1-phenyl-1-propanol and ethylene glycol monophenyl ether, and reacting for 45min at 30 ℃ to obtain Fmoc-Tau-Pro (Boc) -Wang Resin;
s3, removing Fmoc groups from Fmoc-Tau-Pro (Boc) -Wang Resin obtained in the step S2 to obtain H-Tau-Pro (Boc) -Wang Resin; then adding Fmoc-Trp (Boc) -OH, then adding (S) - (+) -3- (1-naphthoxy) -1-phenyl-1-propanol and ethylene glycol monophenyl ether, and reacting for 45min at 30 ℃ to obtain Fmoc-Trp (Boc) -Tau-Pro (Boc) -Wang Resin;
s4, removing Fmoc groups from the product obtained in the step S3, adding TFA, oscillating to react for 2 hours, filtering, washing the product with DMF and ethanol respectively, and freeze-drying to obtain H-Trp-Tau-Pro-OH.
Wherein, removing Fmoc group, using deprotection agent combination as follows: 23 vol% piperidine, 4.2 wt% potassium tert-butoxide and 1.6 wt% HOBT; the piperidine is easier to remove the amino protecting group under the activation of HOBT and the proper alkaline action of potassium tert-butoxide, and the removal efficiency is high;
the addition amounts of (S) - (+) -3- (1-naphthyloxy) -1-phenyl-1-propanol and ethylene glycol monophenyl ether were 0.56% and 3.8% by mass, respectively, of the reaction mixture system.
Example 3:
synthesizing tetrapeptide with the structure of Tau-Trp-Pro-Pro, comprising the following steps:
s1, mixing Fmoc-Pro (Boc) -OH and Wang Resin in proportion, adding activating agents of HOBT and HBTU, oscillating and reacting at 43 ℃ by taking DMF as a solvent for 2.5h, and filtering a reaction product, washing with DMF and ethanol to obtain Fmoc-Pro (Boc) -Wang Resin; wherein the mole number of Fmoc-Pro (Boc) -OH is 2.2 times of that of the resin; the mole number of the activating agent is 3.4 times of that of the resin;
s2, removing the Fmoc group from the Fmoc-Pro (Boc) -Wang Resin obtained in the step S1 to obtain H-Pro (Boc) -Wang Resin (the Resin is subjected to color testing by a ninhydrin color reagent, and the color is dark blue or blue purple, so that the Fmoc group is removed); then adding Fmoc-Pro (Trt) -OH, then adding (S) - (+) -3- (1-naphthoxy) -1-phenyl-1-propanol and ethylene glycol monophenyl ether, and reacting for 45min at 30 ℃ to obtain Fmoc-Pro (Trt) -Pro (Boc) -Wang Resin;
S3, removing Fmoc groups from Fmoc-Pro (Trt) -Pro (Boc) -Wang Resin obtained in the step S2 to obtain H-Pro (Trt) -Pro (Boc) -Wang Resin; then adding Fmoc-Trp (Boc) -OH, then adding (S) - (+) -3- (1-naphthoxy) -1-phenyl-1-propanol and ethylene glycol monophenyl ether, and reacting for 45min at 30 ℃ to obtain Fmoc-Trp (Boc) -Pro (Trt) -Pro (Boc) -Wang Resin;
s4, removing Fmoc groups from Fmoc-Trp (Boc) -Pro (Trt) -Pro (Boc) -Wang Resin obtained in the step S3 to obtain H-Trp (Boc) -Pro (Trt) -Pro (Boc) -Wang Resin, adding Fmoc-Tau-OH, adding (S) - (+) -3- (1-naphthyloxy) -1-phenyl-1-propanol and ethylene glycol monophenyl ether, and reacting at 30 ℃ for 45min to obtain Fmoc-Tau-Trp (Boc) -Pro (Boc) -Wang Resin;
s5, removing Fmoc groups from the product obtained in the step S4, adding TFA, oscillating to react for 2 hours, filtering, washing the product with DMF and ethanol respectively, and freeze-drying to obtain H-Tau-Trp-Pro-Pro-OH.
Wherein, removing Fmoc group, using deprotection agent combination as follows: 23 vol% piperidine, 4.2 wt% potassium tert-butoxide and 1.6 wt% HOBT; the piperidine is easier to remove the amino protecting group under the activation of HOBT and the proper alkaline action of potassium tert-butoxide, and the removal efficiency is high;
the addition amounts of (S) - (+) -3- (1-naphthyloxy) -1-phenyl-1-propanol and ethylene glycol monophenyl ether were 0.56% and 3.8% by mass, respectively, of the reaction mixture system.
Example 4:
synthesizing tetrapeptide with the structure of Trp-Tau-Pro-Pro, comprising the following steps:
s1, mixing Fmoc-Pro (Boc) -OH and Wang Resin in proportion, adding activating agents of HOBT and DIC, carrying out oscillation reaction at 43 ℃ for 2.5h by taking DMF as a solvent, and filtering and washing a reaction product with DMF and ethanol to obtain Fmoc-Pro (Boc) -Wang Resin; wherein the mole number of Fmoc-Pro (Boc) -OH is 2.2 times of that of the resin; the mole number of the activating agent is 3.4 times of that of the resin;
s2, removing the Fmoc group from the Fmoc-Pro (Boc) -Wang Resin obtained in the step S1 to obtain H-Pro (Boc) -Wang Resin (the Resin is subjected to color testing by a ninhydrin color reagent, and the color is dark blue or blue purple, so that the Fmoc group is removed); then adding Fmoc-Pro (Trt) -OH, then adding (S) - (+) -3- (1-naphthoxy) -1-phenyl-1-propanol and ethylene glycol monophenyl ether, and reacting for 45min at 30 ℃ to obtain Fmoc-Pro (Trt) -Pro (Boc) -WangResin;
s3, removing Fmoc groups from Fmoc-Pro (Trt) -Pro (Boc) -Wang Resin obtained in the step S2 to obtain H-Pro (Trt) -Pro (Boc) -Wang Resin; then adding Fmoc-Tau-OH, then adding (S) - (+) -3- (1-naphthoxy) -1-phenyl-1-propanol and ethylene glycol monophenyl ether, and reacting for 45min at 30 ℃ to obtain Fmoc-Tau-Pro (Trt) -Pro (Boc) -WangResin;
S4, removing Fmoc groups from Fmoc-Tau-Pro (Trt) -Pro (Boc) -Wang Resin obtained in the step S3 to obtain H-Tau-Pro (Trt) -Pro (Boc) -Wang Resin, adding Fmoc-Trp (Boc) -OH, adding (S) - (+) -3- (1-naphthyloxy) -1-phenyl-1-propanol and ethylene glycol monophenyl ether, and reacting at 30 ℃ for 45min to obtain Fmoc-Trp (Boc) -Tau-Pro (Trt) -Pro (Boc) -Wang Resin;
s5, removing Fmoc groups from the product obtained in the step S4, adding TFA, oscillating to react for 2 hours, filtering, washing the product with DMF and ethanol respectively, and freeze-drying to obtain H-Trp-Tau-Pro-Pro-OH.
Wherein, removing Fmoc group, using deprotection agent combination as follows: 23 vol% piperidine, 4.2 wt% potassium tert-butoxide and 1.6 wt% HOBT; piperidine is easy to remove amino protecting groups under the activation of HOBT and the proper alkaline action of potassium tert-butoxide, and has high removal efficiency;
the addition amounts of (S) - (+) -3- (1-naphthyloxy) -1-phenyl-1-propanol and ethylene glycol monophenyl ether were 0.56% and 3.8% by mass, respectively, of the reaction mixture system.
Example 5:
synthesizing tetrapeptide with the structure of Trp-Pro-Tau-Pro, comprising the following steps:
s1, mixing Fmoc-Pro (Boc) -OH and Wang Resin in proportion, adding activating agents of HOBT and HBTU, oscillating and reacting at 43 ℃ for 2.5h by taking DMF as a solvent, and filtering and washing a reaction product by DMF and ethanol to obtain Fmoc-Pro (Boc) -Wang Resin; wherein the mole number of Fmoc-Pro (Boc) -OH is 2.2 times of that of the resin; the mole number of the activating agent is 3.4 times of that of the resin;
S2, removing the Fmoc group from the Fmoc-Pro (Boc) -Wang Resin obtained in the step S1 to obtain H-Pro (Boc) -Wang Resin (the Resin is subjected to color testing by a ninhydrin color reagent, and the color is dark blue or blue purple, so that the Fmoc group is removed); then adding Fmoc-Tau-OH, then adding (S) - (+) -3- (1-naphthoxy) -1-phenyl-1-propanol and ethylene glycol monophenyl ether, and reacting for 45min at 30 ℃ to obtain Fmoc-Tau-Pro (Boc) -Wang Resin;
s3, removing Fmoc groups from Fmoc-Tau-Pro (Boc) -Wang Resin obtained in the step S2 to obtain H-Tau-Pro (Boc) -Wang Resin; then adding Fmoc-Pro (Trt) -OH, then adding (S) - (+) -3- (1-naphthoxy) -1-phenyl-1-propanol and ethylene glycol monophenyl ether, and reacting for 45min at 30 ℃ to obtain Fmoc-Pro (Trt) -Tau-Pro (Boc) -Wang Resin;
s4, removing Fmoc groups from Fmoc-Pro (Trt) -Tau-Pro (Boc) -Wang Resin obtained in the step S3 to obtain H-Pro (Trt) -Tau-Pro (Boc) -Wang Resin, adding Fmoc-Trp (Boc) -OH, adding (S) - (+) -3- (1-naphthyloxy) -1-phenyl-1-propanol and ethylene glycol monophenyl ether, and reacting at 30 ℃ for 45min to obtain Fmoc-Trp (Boc) -Pro (Trt) -Tau-Pro (Boc) -Wang Resin;
s5, removing Fmoc groups from the product obtained in the step S4, adding TFA, oscillating to react for 2 hours, filtering, washing the product with DMF and ethanol respectively, and freeze-drying to obtain H-Trp-Pro-Tau-Pro-OH.
Wherein, removing Fmoc group, using deprotection agent combination as follows: 23 vol% piperidine, 4.2 wt% potassium tert-butoxide and 1.6 wt% HOBT; the piperidine is easier to remove the amino protecting group under the activation of HOBT and the proper alkaline action of potassium tert-butoxide, and the removal efficiency is high;
the addition amounts of (S) - (+) -3- (1-naphthyloxy) -1-phenyl-1-propanol and ethylene glycol monophenyl ether were 0.56% and 3.8% by mass, respectively, of the reaction mixture system.
Example 6:
several polypeptides synthesized in examples 1-5 were tested for activity: detecting the antioxidant activity of cell level, and performing polypeptide cytotoxicity and H resistance by using human hepatoma cell HepG-2 and human embryonic kidney cell HEK293 2 O 2 Detecting and analyzing the oxidative damage capability; performing cell culture experiment with polypeptide concentration of 0.1, 0.2, 0.4, 0.6, 0.8, 1.0mg/mL, and adding H 2 O 2 Detecting polypeptide anti-H with reduced Glutathione (GSH) concentration of 0.1mg/mL as positive control to 1.0mM 2 O 2 The oxidative damage capacity, the cell viability of which is the cell activity, is shown in FIG. 1;
as can be seen from FIG. 1, the polypeptide synthesized by the present invention is resistant to H in HepG-2 and HEK293 cells 2 O 2 The oxidative damage capability has obvious enhancement effect, the activity of the polypeptides with different structures is respectively the best activity of Tau-Trp-Pro-Pro, and then Tau-Trp-Pro, Trp-Tau-Pro-Pro and Trp-Pro-Tau-Pro; meanwhile, under the culture of a positive control GSH (glutathione) of 0.1mg/mL, the survival rates of HepG-2 and HEK293 cells are respectively 62.1% and 55.8%, and compared with the polypeptide with the best activity synthesized by the invention, the survival rates of the HepG-2 and HEK293 cells are respectively 68.0% and 64.7%, which indicates that the polypeptide synthesized by the invention has stronger antioxidant activity.
Conventional techniques in the above embodiments are known to those skilled in the art, and therefore, will not be described in detail herein.
The above embodiments are merely illustrative, and not restrictive, of the present invention, and those skilled in the art can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, all equivalent technical solutions also belong to the scope of the present invention, and the protection scope of the present invention should be defined by the claims.
Claims (9)
1. A preparation method of non-natural antioxidant peptide is characterized by comprising the following steps: reacting taurine with tripeptide Trp-Pro-Pro extracted from blood clams to obtain polypeptide with antioxidant activity; the synthesized polypeptide is: Tau-Trp-Pro, Trp-Tau-Pro, Tau-Trp-Pro-Pro, Trp-Pro-Tau-Pro, Trp-Tau-Pro-Pro.
2. The method of claim 1, wherein the non-natural antioxidant peptide comprises: synthesizing tripeptide with a structure of Tau-Trp-Pro, comprising the following steps:
s1, mixing Fmoc-Pro (Boc) -OH and Wang Resin in proportion, adding activating agents of HOBT and DIC/HBTU, oscillating and reacting at 42-44 ℃ by taking DMF as a solvent for 2-3h, and filtering and washing reaction products with DMF and ethanol to obtain Fmoc-Pro (Boc) -Wang Resin;
S2, removing Fmoc groups from Fmoc-Pro (Boc) -Wang Resin obtained in the step S1 to obtain H-Pro (Boc) -Wang Resin; then adding Fmoc-Trp (Trt) -OH, then adding (S) - (+) -3- (1-naphthoxy) -1-phenyl-1-propanol and ethylene glycol monophenyl ether, and reacting at 25-45 ℃ for 30-60min to obtain Fmoc-Trp (Trt) -Pro (Boc) -Wang Resin;
s3, removing Fmoc groups from Fmoc-Trp (Trt) -Pro (Boc) -Wang Resin obtained in the step S2 to obtain H-Trp (Trt) -Pro (Boc) -Wang Resin; then adding Fmoc-Tau-OH, then adding (S) - (+) -3- (1-naphthoxy) -1-phenyl-1-propanol and ethylene glycol monophenyl ether, and reacting for 30-60min at 25-45 ℃ to obtain Fmoc-Tau-Trp (Trt) -Pro (Boc) -Wang Resin;
s4, removing Fmoc groups from the product obtained in the step S3, adding TFA, oscillating to react for 0.5-2.5h, filtering, washing the product with DMF and ethanol respectively, and freeze-drying to obtain Tau-Trp-Pro.
3. The method of claim 1, wherein the non-natural antioxidant peptide comprises: synthesizing tripeptide with a structure of Trp-Tau-Pro, comprising the following steps:
s1, mixing Fmoc-Pro (Boc) -OH and Wang Resin in proportion, adding activating agents of HOBT and DIC/HBTU, oscillating and reacting at 42-44 ℃ by taking DMF as a solvent for 2-3h, and filtering and washing reaction products with DMF and ethanol to obtain Fmoc-Pro (Boc) -Wang Resin;
S2, removing Fmoc groups from Fmoc-Pro (Boc) -Wang Resin obtained in the step S1 to obtain H-Pro (Boc) -Wang Resin; then adding Fmoc-Tau-OH, then adding (S) - (+) -3- (1-naphthoxy) -1-phenyl-1-propanol and ethylene glycol monophenyl ether, and reacting for 30-60min at 25-45 ℃ to obtain Fmoc-Tau-Pro (Boc) -Wang Resin;
s3, removing Fmoc groups from Fmoc-Tau-Pro (Boc) -Wang Resin obtained in the step S2 to obtain H-Tau-Pro (Boc) -Wang Resin; then adding Fmoc-Trp (Boc) -OH, then adding (S) - (+) -3- (1-naphthoxy) -1-phenyl-1-propanol and ethylene glycol monophenyl ether, and reacting at 25-45 ℃ for 30-60min to obtain Fmoc-Trp (Boc) -Tau-Pro (Boc) -Wang Resin;
s4, removing Fmoc groups from the product obtained in the step S3, adding TFA, oscillating to react for 0.5-2.5h, filtering, washing the product with DMF and ethanol respectively, and freeze-drying to obtain Trp-Tau-Pro.
4. The method of claim 1, wherein the non-natural antioxidant peptide comprises: synthesizing tetrapeptide with the structure of Tau-Trp-Pro-Pro, comprising the following steps:
s1, mixing Fmoc-Pro (Boc) -OH and Wang Resin in proportion, adding activating agents of HOBT and DIC/HBTU, oscillating and reacting at 42-44 ℃ by taking DMF as a solvent for 2-3h, and filtering and washing reaction products with DMF and ethanol to obtain Fmoc-Pro (Boc) -Wang Resin;
S2, removing Fmoc groups from Fmoc-Pro (Boc) -Wang Resin obtained in the step S1 to obtain H-Pro (Boc) -Wang Resin; then adding Fmoc-Pro (Trt) -OH, then adding (S) - (+) -3- (1-naphthoxy) -1-phenyl-1-propanol and ethylene glycol monophenyl ether, and reacting for 30-60min at 25-45 ℃ to obtain Fmoc-Pro (Trt) -Pro (Boc) -Wang Resin;
s3, removing Fmoc groups from Fmoc-Pro (Trt) -Pro (Boc) -Wang Resin obtained in the step S2 to obtain H-Pro (Trt) -Pro (Boc) -Wang Resin; then adding Fmoc-Trp (Boc) -OH, then adding (S) - (+) -3- (1-naphthoxy) -1-phenyl-1-propanol and ethylene glycol monophenyl ether, and reacting at 25-45 ℃ for 30-60min to obtain Fmoc-Trp (Boc) -Pro (Trt) -Pro (Boc) -Wang Resin;
s4, removing Fmoc groups from Fmoc-Trp (Boc) -Pro (Trt) -Pro (Boc) -Wang Resin obtained in the step S3 to obtain H-Trp (Boc) -Pro (Trt) -Pro (Boc) -Wang Resin, adding Fmoc-Tau-OH, adding (S) - (+) -3- (1-naphthyloxy) -1-phenyl-1-propanol and Boc monophenyl ether, and reacting at 25-45 ℃ for 30-60min to obtain Fmoc-Tau-Trp (Trt) -Pro (Boc) -Wang Resin;
s5, removing Fmoc group from the product obtained in the step S4, adding TFA, oscillating to react for 0.5-2.5h, filtering, washing the product with DMF and ethanol respectively, and freeze-drying to obtain Tau-Trp-Pro-Pro.
5. The method for preparing the non-natural antioxidant peptide according to claim 1, wherein: synthesizing tetrapeptide with the structure of Trp-Tau-Pro-Pro, comprising the following steps:
S1, mixing Fmoc-Pro (Boc) -OH and Wang Resin in proportion, adding activating agents of HOBT and DIC/HBTU, oscillating and reacting at 42-44 ℃ by taking DMF as a solvent for 2-3h, and filtering and washing reaction products with DMF and ethanol to obtain Fmoc-Pro (Boc) -Wang Resin;
s2, removing Fmoc groups from Fmoc-Pro (Boc) -Wang Resin obtained in the step S1 to obtain H-Pro (Boc) -Wang Resin; then adding Fmoc-Pro (Trt) -OH, then adding (S) - (+) -3- (1-naphthoxy) -1-phenyl-1-propanol and ethylene glycol monophenyl ether, and reacting for 30-60min at 25-45 ℃ to obtain Fmoc-Pro (Trt) -Pro (Boc) -Wang Resin;
s3, removing Fmoc groups from Fmoc-Pro (Trt) -Pro (Boc) -Wang Resin obtained in the step S2 to obtain H-Pro (Trt) -Pro (Boc) -Wang Resin; then adding Fmoc-Tau-OH, then adding (S) - (+) -3- (1-naphthoxy) -1-phenyl-1-propanol and ethylene glycol monophenyl ether, and reacting for 30-60min at 25-45 ℃ to obtain Fmoc-Tau-Pro (Trt) -Pro (Boc) -Wang Resin;
s4, removing Fmoc groups from Fmoc-Tau-Pro (Trt) -Pro (Boc) -Wang Resin obtained in the step S3 to obtain H-Tau-Pro (Trt) -Pro (Boc) -Wang Resin, adding Fmoc-Trp (Boc) -OH, adding (S) - (+) -3- (1-naphthyloxy) -1-phenyl-1-propanol and ethylene glycol monophenyl ether, and reacting at 25-45 ℃ for 30-60min to obtain Fmoc-Trp (Boc) -Tau-Pro (Trt) -Pro (Boc) -Wang Resin;
S5, removing Fmoc groups from the product obtained in the step S4, adding TFA, oscillating to react for 0.5-2.5h, filtering, washing the product with DMF and ethanol respectively, and freeze-drying to obtain Trp-Tau-Pro-Pro.
6. The method of claim 1, wherein the non-natural antioxidant peptide comprises: synthesizing tetrapeptide with the structure of Trp-Pro-Tau-Pro, comprising the following steps:
s1, mixing Fmoc-Pro (Boc) -OH and Wang Resin in proportion, adding activating agents of HOBT and DIC/HBTU, oscillating and reacting at 42-44 ℃ by taking DMF as a solvent for 2-3h, and filtering and washing reaction products with DMF and ethanol to obtain Fmoc-Pro (Boc) -Wang Resin;
s2, removing Fmoc groups from Fmoc-Pro (Boc) -Wang Resin obtained in the step S1 to obtain H-Pro (Boc) -Wang Resin; then adding Fmoc-Tau-OH, then adding (S) - (+) -3- (1-naphthoxy) -1-phenyl-1-propanol and ethylene glycol monophenyl ether, and reacting for 30-60min at 25-45 ℃ to obtain Fmoc-Tau-Pro (Boc) -Wang Resin;
s3, removing Fmoc groups from Fmoc-Tau-Pro (Boc) -Wang Resin obtained in the step S2 to obtain H-Tau-Pro (Boc) -Wang Resin; then adding Fmoc-Pro (Trt) -OH, then adding (S) - (+) -3- (1-naphthoxy) -1-phenyl-1-propanol and ethylene glycol monophenyl ether, and reacting at 25-45 ℃ for 30-60min to obtain Fmoc-Pro (Trt) -Tau-Pro (Boc) -Wang Resin;
S4, removing Fmoc groups from Fmoc-Pro (Trt) -Tau-Pro (Boc) -Wang Resin obtained in the step S3 to obtain H-Pro (Trt) -Tau-Pro (Boc) -Wang Resin, adding Fmoc-Trp (Boc) -OH, adding (S) - (+) -3- (1-naphthyloxy) -1-phenyl-1-propanol and ethylene glycol monophenyl ether, and reacting at 25-45 ℃ for 30-60min to obtain Fmoc-Trp (Boc) -Pro (Trt) -Tau-Pro (Boc) -Wang Resin;
s5, removing Fmoc groups from the product obtained in the step S4, adding TFA, oscillating to react for 0.5-2.5h, filtering, washing the product with DMF and ethanol respectively, and freeze-drying to obtain Trp-Pro-Tau-Pro.
7. The method for preparing the non-natural antioxidant peptide according to any one of claims 2 to 6, wherein: the mole number of Fmoc-Pro (Boc) -OH is 2-3 times that of Wang Resin; the activating agent is HOBT and DIC or a mixture of HOBT and HBTU, and the mole number of the activating agent is 3-4 times that of Wang Resin.
8. The method for preparing the non-natural antioxidant peptide according to any one of claims 2 to 6, wherein: the Fmoc group removal adopts a deprotection agent combination as follows: 10-25vol% of piperidine, 1-5wt% of potassium tert-butoxide and 0.1-3wt% of HOBT.
9. The method for preparing the non-natural antioxidant peptide according to any one of claims 2 to 6, wherein: the addition amounts of the (S) - (+) -3- (1-naphthoxy) -1-phenyl-1-propanol and the ethylene glycol monophenyl ether are respectively 0.1-1.0% and 1-5% of the mass percentage of the reaction mixing system.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5292962A (en) * | 1992-12-11 | 1994-03-08 | Eli Lilly And Company | Intermediates to 1-phenyl-3-naphthalenyloxy-propanamines |
| WO2016159773A1 (en) * | 2015-04-03 | 2016-10-06 | Tx Innovations B.V. | Organ preservation composition |
| CN106801081A (en) * | 2017-01-23 | 2017-06-06 | 嵊州市派特普科技开发有限公司 | A kind of method that activated protein is extracted from rice bran |
| CN108611391A (en) * | 2018-05-10 | 2018-10-02 | 浙江海洋大学 | A kind of method of modifying of collagen from black sea cucumbers from East China Sea oligopeptide and its application |
-
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- 2018-11-22 CN CN201811397810.XA patent/CN109438555B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5292962A (en) * | 1992-12-11 | 1994-03-08 | Eli Lilly And Company | Intermediates to 1-phenyl-3-naphthalenyloxy-propanamines |
| WO2016159773A1 (en) * | 2015-04-03 | 2016-10-06 | Tx Innovations B.V. | Organ preservation composition |
| CN106801081A (en) * | 2017-01-23 | 2017-06-06 | 嵊州市派特普科技开发有限公司 | A kind of method that activated protein is extracted from rice bran |
| CN108611391A (en) * | 2018-05-10 | 2018-10-02 | 浙江海洋大学 | A kind of method of modifying of collagen from black sea cucumbers from East China Sea oligopeptide and its application |
Non-Patent Citations (3)
| Title |
|---|
| Antioxidant and anticancer peptides from the protein hydrolysate of blood clam (Tegillarca granosa) muscle;Chang-Feng Chi等;《Journal of Functional Foods》;20150411;第15卷;301-313 * |
| 菲律宾蛤仔寡肽体外抗氧化、抗肿瘤活性研究;李荣等;《中国实验方剂学杂志》;20130630;第19卷(第11期);238-241 * |
| 菲律宾蛤仔提取物及生物活性的研究进展;徐律等;《浙江海洋学院学报(自然科学版)》;20130731;第32卷(第4期);357-361 * |
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