CN103012563A - Solid-phase synthesis method of antibacterial peptide Iseganan - Google Patents
Solid-phase synthesis method of antibacterial peptide Iseganan Download PDFInfo
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- CN103012563A CN103012563A CN201210541093XA CN201210541093A CN103012563A CN 103012563 A CN103012563 A CN 103012563A CN 201210541093X A CN201210541093X A CN 201210541093XA CN 201210541093 A CN201210541093 A CN 201210541093A CN 103012563 A CN103012563 A CN 103012563A
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- 238000000034 method Methods 0.000 title claims abstract description 23
- 238000010532 solid phase synthesis reaction Methods 0.000 title claims abstract description 10
- 239000003910 polypeptide antibiotic agent Substances 0.000 title abstract description 7
- 108010078256 antimicrobial peptide IB-367 Proteins 0.000 title abstract description 4
- GUCYBPFJNGVFEB-XELKFLSISA-N iseganan Chemical compound C([C@H]1C(=O)N[C@H]2CSSC[C@H](NC(=O)[C@H](CC=3C=CC=CC=3)NC(=O)[C@H](CCCNC(N)=N)NC(=O)CNC(=O)[C@H](CCCNC(N)=N)NC2=O)C(=O)N[C@H](C(=O)N[C@@H](CSSC[C@@H](C(N1)=O)NC(=O)[C@@H](NC(=O)CNC(=O)CNC(=O)[C@@H](N)CCCNC(N)=N)CC(C)C)C(=O)N[C@@H](C(C)C)C(=O)NCC(=O)N[C@@H](CCCNC(N)=N)C(N)=O)C(C)C)C1=CC=C(O)C=C1 GUCYBPFJNGVFEB-XELKFLSISA-N 0.000 title abstract description 4
- 229950000488 iseganan Drugs 0.000 title abstract description 4
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 56
- 229920005989 resin Polymers 0.000 claims abstract description 47
- 239000011347 resin Substances 0.000 claims abstract description 47
- 125000006239 protecting group Chemical group 0.000 claims abstract description 34
- 150000001413 amino acids Chemical class 0.000 claims abstract description 23
- 229920003180 amino resin Polymers 0.000 claims abstract description 22
- 230000008878 coupling Effects 0.000 claims abstract description 15
- 238000010168 coupling process Methods 0.000 claims abstract description 15
- 238000005859 coupling reaction Methods 0.000 claims abstract description 15
- ZCYVEMRRCGMTRW-UHFFFAOYSA-N 7553-56-2 Chemical compound [I] ZCYVEMRRCGMTRW-UHFFFAOYSA-N 0.000 claims abstract description 13
- 229910052740 iodine Inorganic materials 0.000 claims abstract description 13
- 239000011630 iodine Substances 0.000 claims abstract description 13
- 239000002253 acid Substances 0.000 claims abstract description 6
- 238000005336 cracking Methods 0.000 claims abstract description 4
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-dimethylformamide Substances CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 claims description 69
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 66
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 claims description 29
- 125000003088 (fluoren-9-ylmethoxy)carbonyl group Chemical group 0.000 claims description 28
- 239000007790 solid phase Substances 0.000 claims description 22
- 150000003053 piperidines Chemical group 0.000 claims description 21
- BWGNESOTFCXPMA-UHFFFAOYSA-N Dihydrogen disulfide Chemical compound SS BWGNESOTFCXPMA-UHFFFAOYSA-N 0.000 claims description 19
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical group CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 15
- NPZTUJOABDZTLV-UHFFFAOYSA-N hydroxybenzotriazole Substances O=C1C=CC=C2NNN=C12 NPZTUJOABDZTLV-UHFFFAOYSA-N 0.000 claims description 14
- HNICLNKVURBTKV-NDEPHWFRSA-N (2s)-5-[[amino-[(2,2,4,6,7-pentamethyl-3h-1-benzofuran-5-yl)sulfonylamino]methylidene]amino]-2-(9h-fluoren-9-ylmethoxycarbonylamino)pentanoic acid Chemical compound C12=CC=CC=C2C2=CC=CC=C2C1COC(=O)N[C@H](C(O)=O)CCCN=C(N)NS(=O)(=O)C1=C(C)C(C)=C2OC(C)(C)CC2=C1C HNICLNKVURBTKV-NDEPHWFRSA-N 0.000 claims description 12
- 230000003647 oxidation Effects 0.000 claims description 10
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- TZCYLJGNWDVJRA-UHFFFAOYSA-N 6-chloro-1-hydroxybenzotriazole Chemical compound C1=C(Cl)C=C2N(O)N=NC2=C1 TZCYLJGNWDVJRA-UHFFFAOYSA-N 0.000 claims description 8
- QWVGKYWNOKOFNN-UHFFFAOYSA-N o-cresol Chemical compound CC1=CC=CC=C1O QWVGKYWNOKOFNN-UHFFFAOYSA-N 0.000 claims description 8
- HNKJADCVZUBCPG-UHFFFAOYSA-N thioanisole Chemical compound CSC1=CC=CC=C1 HNKJADCVZUBCPG-UHFFFAOYSA-N 0.000 claims description 8
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- -1 Fmoc amino acid Chemical class 0.000 claims description 6
- 239000007821 HATU Substances 0.000 claims description 6
- FZTIWOBQQYPTCJ-UHFFFAOYSA-N 4-[4-(4-carboxyphenyl)phenyl]benzoic acid Chemical compound C1=CC(C(=O)O)=CC=C1C1=CC=C(C=2C=CC(=CC=2)C(O)=O)C=C1 FZTIWOBQQYPTCJ-UHFFFAOYSA-N 0.000 claims description 4
- 239000007787 solid Substances 0.000 claims description 4
- 238000010189 synthetic method Methods 0.000 claims description 3
- CVFXPOKENLGCID-KRWDZBQOSA-N (2s)-5-[[amino-[(2,2,4,6,7-pentamethyl-3h-1-benzofuran-5-yl)sulfonylamino]methylidene]amino]-2-[(2-methylpropan-2-yl)oxycarbonylamino]pentanoic acid Chemical compound CC1=C(S(=O)(=O)NC(N)=NCCC[C@H](NC(=O)OC(C)(C)C)C(O)=O)C(C)=C2CC(C)(C)OC2=C1C CVFXPOKENLGCID-KRWDZBQOSA-N 0.000 claims description 2
- 230000035484 reaction time Effects 0.000 claims description 2
- 230000035945 sensitivity Effects 0.000 claims description 2
- 150000001408 amides Chemical class 0.000 claims 2
- JDDWRLPTKIOUOF-UHFFFAOYSA-N 9h-fluoren-9-ylmethyl n-[[4-[2-[bis(4-methylphenyl)methylamino]-2-oxoethoxy]phenyl]-(2,4-dimethoxyphenyl)methyl]carbamate Chemical compound COC1=CC(OC)=CC=C1C(C=1C=CC(OCC(=O)NC(C=2C=CC(C)=CC=2)C=2C=CC(C)=CC=2)=CC=1)NC(=O)OCC1C2=CC=CC=C2C2=CC=CC=C21 JDDWRLPTKIOUOF-UHFFFAOYSA-N 0.000 claims 1
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- JGFZNNIVVJXRND-UHFFFAOYSA-N N,N-Diisopropylethylamine (DIPEA) Chemical compound CCN(C(C)C)C(C)C JGFZNNIVVJXRND-UHFFFAOYSA-N 0.000 description 11
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- DVBUCBXGDWWXNY-SFHVURJKSA-N (2s)-5-(diaminomethylideneamino)-2-(9h-fluoren-9-ylmethoxycarbonylamino)pentanoic acid Chemical compound C1=CC=C2C(COC(=O)N[C@@H](CCCN=C(N)N)C(O)=O)C3=CC=CC=C3C2=C1 DVBUCBXGDWWXNY-SFHVURJKSA-N 0.000 description 6
- WFDIJRYMOXRFFG-UHFFFAOYSA-N Acetic anhydride Chemical compound CC(=O)OC(C)=O WFDIJRYMOXRFFG-UHFFFAOYSA-N 0.000 description 6
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- UGNIYGNGCNXHTR-SFHVURJKSA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-3-methylbutanoic acid Chemical compound C1=CC=C2C(COC(=O)N[C@@H](C(C)C)C(O)=O)C3=CC=CC=C3C2=C1 UGNIYGNGCNXHTR-SFHVURJKSA-N 0.000 description 4
- ODKSFYDXXFIFQN-BYPYZUCNSA-N L-arginine Chemical compound OC(=O)[C@@H](N)CCCN=C(N)N ODKSFYDXXFIFQN-BYPYZUCNSA-N 0.000 description 4
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- LOBUWFUSGOYXQX-DHUJRADRSA-N (2r)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-3-[(4-methoxyphenyl)-diphenylmethyl]sulfanylpropanoic acid Chemical compound C1=CC(OC)=CC=C1C(C=1C=CC=CC=1)(C=1C=CC=CC=1)SC[C@@H](C(O)=O)NC(=O)OCC1C2=CC=CC=C2C2=CC=CC=C21 LOBUWFUSGOYXQX-DHUJRADRSA-N 0.000 description 3
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- CSMYOORPUGPKAP-IBGZPJMESA-N (2r)-3-(acetamidomethylsulfanyl)-2-(9h-fluoren-9-ylmethoxycarbonylamino)propanoic acid Chemical compound C1=CC=C2C(COC(=O)N[C@@H](CSCNC(=O)C)C(O)=O)C3=CC=CC=C3C2=C1 CSMYOORPUGPKAP-IBGZPJMESA-N 0.000 description 2
- JAUKCFULLJFBFN-VWLOTQADSA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-3-[4-[(2-methylpropan-2-yl)oxy]phenyl]propanoic acid Chemical compound C1=CC(OC(C)(C)C)=CC=C1C[C@@H](C(O)=O)NC(=O)OCC1C2=CC=CC=C2C2=CC=CC=C21 JAUKCFULLJFBFN-VWLOTQADSA-N 0.000 description 2
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- CBPJQFCAFFNICX-IBGZPJMESA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-4-methylpentanoic acid Chemical compound C1=CC=C2C(COC(=O)N[C@@H](CC(C)C)C(O)=O)C3=CC=CC=C3C2=C1 CBPJQFCAFFNICX-IBGZPJMESA-N 0.000 description 2
- QOSSAOTZNIDXMA-UHFFFAOYSA-N Dicylcohexylcarbodiimide Chemical compound C1CCCCC1N=C=NC1CCCCC1 QOSSAOTZNIDXMA-UHFFFAOYSA-N 0.000 description 2
- 241000233866 Fungi Species 0.000 description 2
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- QRUDEWIWKLJBPS-UHFFFAOYSA-N benzotriazole Chemical compound C1=CC=C2N[N][N]C2=C1 QRUDEWIWKLJBPS-UHFFFAOYSA-N 0.000 description 1
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- ZGYICYBLPGRURT-UHFFFAOYSA-N tri(propan-2-yl)silicon Chemical compound CC(C)[Si](C(C)C)C(C)C ZGYICYBLPGRURT-UHFFFAOYSA-N 0.000 description 1
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Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
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- Peptides Or Proteins (AREA)
Abstract
The invention relates to a solid-phase synthesis method of an antibacterial peptide Iseganan containing two pairs of disulfide bonds, which comprises the following steps: (1) according to a solid-phase synthesis method, sequentially coupling an amino resin used as a raw material with amino acids to synthesize a protected 17 peptide resin; (2) removing weak acid sensitive protecting groups, and then oxidizing to generate a first pair of disulfide bonds; (3) using iodine to remove Acm protecting groups, and cyclizing to generate a second pair of disulfide bonds; and (4) finally cracking from the resin, removing the protecting groups, and purifying to obtain the Iseganan antibacterial peptide. The route provided by the invention has the advantages of short operation steps, simple post treatment, fewer byproducts, high yield and low pollution, and is suitable for large-scale industrial production.
Description
Technical field
The present invention relates to the polypeptide drugs synthesis technical field, be specifically related to a kind of solid phase synthesis process that contains many antibacterial peptide Yi Segenan to disulfide linkage.
Background technology
Yi Segenan (Iseganan, the aminoacid sequence formula is as follows) be pig neutrophil leucocyte peptide-1(Protegrin-1, PG-1) analogue, can combine with the outer composition of the born of the same parents of bacterium such as lipopolysaccharides or adipose membrane acid etc., cause the bacterium expansion, break, by destroying the cytolemma killing bacteria.It all has restraining effect to Gram-positive, negative bacteria and fungi, yeast etc.MIC to gram positive organism is 0.13 ~ 0.16 μ g/mL, is 0.063 ~ 8 μ g/mL for the MIC of the relevant gram-negative bacteria of oral mucositis.IntraBiotics company is developing the pharmaceutical formulation of Yi Segenan, and the III clinical trial phase of 323 patients participation shows that Yi Segenan can prevent oral mucositis.Accept Yi Segenan treatment patient's stomatitis, oral cavity pain, have sore throat and the symptom such as dysphagia significantly alleviates.
The Yi Segenan aminoacid sequence
Yi Segenan is anti-microbial activity 17 peptides that contain 4 Cys residues, and whole molecule has β-hair clip type (conformation of β-hairpin).This compounds Main Function is in the cytolemma of target cell, and being gathered in the film surface and then importing into by the attraction on electronegative phosphatide head on the film affect membrane integrity or the formation passage makes membranolysis in the film.Active result shows that it has very strong restraining effect to multiple Gram-positive, negative bacteria, fungi, yeast, eucaryon protozoon, and very little to the HRBC hemolytic action.
It is synthetic that existing a large amount of document records contains many cyclic peptide to disulfide linkage.Its synthetic route can this be summarised as: the main chain sequence of first assembled peptide, then remove the Side chain protective group on the halfcystine, and make sulfydryl free, finally by suitable oxidizing condition, such as air, H
2O
2, I
2, Hg
2+Salt, Fe
3+Salt, DMSO etc., oxidation generates disulfide linkage.It should be noted, must control the concentration (generally below 1mmol/L) of free peptide when in solution, being oxidized to ring, otherwise can have the side reaction of intermolecular bridging.The dilution principle of intramolecular reaction that Here it is.In some cases, the dioxide giving that utilizes the false dilution effect (Pseudo-dilution effect, PDE) of solid phase carrier to carry out the solid phase mode can obtain than the better effect of liquid phase mode.
Patent WO03/062266 has reported the Yi Segenan synthetic method that solid phase is combined with liquid phase.Two fragments of solid phase synthesis (1-8,9-17) in the method form two pairs of disulfide linkage by deprotection base and oxidation after two fragments of liquid phase coupling and generate Yi Segenan.Yi Segenan is one and contains 17 amino acid whose polypeptide, is not difficult to the difficult amino acid of coupling in the structure, uses fragment condensation can increase reactions steps, increases intractability, reduces the combined coefficient of product.And the sulfydryl of 4 halfcystines all is with the Acm protection in the method, uses the methanol solution deprotection base of iodine and generates two pairs of disulfide linkage, makes the method not possess selectivity and forms (Cys
5-14, Cys
7-12) two pairs of disulfide linkage, be difficult to the Yi Segenan of synthetic correct structure.
Patent CN102336813A has reported the solid phase synthesis linear peptides, and then in liquid phase the method for Cheng Huan.The method is the synthesizing linear peptide in solid phase, forms first couple of disulfide linkage (Cys with hydrogen peroxide oxidation after removing the sulfhydryl protected basic Trt on 5 and 14 the halfcystine on the excision solid phase during full guard linear peptides
5-14); In the methanol solution of iodine, remove again the sulfhydryl protected basic Acm on 7 and 12 the halfcystine, and form second couple of disulfide linkage (Cys
7-12).This method on the excision solid phase during full guard linear peptides used high density TFA also can remove simultaneously part Acm protecting group, can't guarantee the correct pairing of disulfide linkage.Become ring to need high dilution to prevent the formation of intermolecular disulfide linkage in the liquid phase, therefore increase the difficulty of aftertreatment, reduce production efficiency, can't satisfy the large-scale industrial production requirement.
The pharmaceutical application of wanting in view of the Yi Sege south heavy is worth, and is necessary to provide more effective Yi Segenan synthetic schemes, can overcome the defective of above method and be fit to plant-scale production.
Summary of the invention
Goal of the invention: technical problem to be solved by this invention provides a kind of new Yi Segenan preparation method, and the method can overcome existing preparation method's deficiency and defective, and suitable suitability for industrialized production.
The invention scheme: technical scheme of the present invention may further comprise the steps:
(1) according to the method for solid phase synthesis; aminoresin is after sloughing Fmoc under the effect of Fmoc deprotecting regent; under the coupling system effect, in reaction solvent, connect successively Fmoc amino acid by the peptide order; 17 peptide resins of synthetic protection; the 5th identical with protecting group on the 14th halfcystine; the 7th identical with protecting group on the 12nd halfcystine, one group of protecting group X in two groups of protecting groups
1Be Xan, Tmob or Mmt, another group protecting group X
2Be Acm;
(2) remove protecting group X on the halfcystine
1, oxidation generates first pair of disulfide linkage;
(3) deprotection base X
2And cyclisation generates second pair of disulfide linkage;
(4) remove N end protecting group, cracking and remove Side chain protective group, De Yisegenan antibacterial peptide behind the purifying from the resin.
In the step (1); take Fmoc aminoresin as solid phase carrier, according to the solid state chemistry synthetic method, aminoresin is after sloughing Fmoc under the effect of Fmoc deprotecting regent; under the coupling system effect, in reaction solvent, connect successively Fmoc amino acid by the peptide order, 17 peptide resins of synthetic protection.Wherein last amino acid can use Fmoc-Arg (Pbf)-OH or Boc-Arg (Pbf)-OH.17 peptide resins of protection are PG-Arg (Pbf)-Gly-Gly-Leu-Cys (X
1)-Tyr (tBu)-Cys (X
2)-Arg (Pbf)-Gly-Arg (Pbf)-Phe-Cys (X
2)-Val-Cys (X
1)-Val-Gly-Arg (Pbf)-aminoresin, or PG-Arg (Pbf)-Gly-Gly-Leu-Cys (X
2)-Tyr (tBu)-Cys (X
1)-Arg (Pbf)-Gly-Arg (Pbf)-Phe-Cys (X
1)-Val-Cys (X
2)-Val-Gly-Arg (Pbf)-aminoresin.Wherein PG is Fmoc or Boc.
In the step (1), reaction solvent is DCM, any one among DMF or the NMP or more than one combination; Coupling system is any one or the combination of a plurality of and DIC among HOBt, HOAt, HOOBt or the Cl-HOBt, or is any one or a plurality of combinations among any one or two and HBTU, HATU, PyBOP or the PyAOP among any one or a plurality of and DPIEA or the NMM among HOBt, HOAt, HOOBt or the Cl-HOBt.The Fmoc deprotecting regent is piperidines, and the deprotection reaction temperature is 10-30 ℃, and the reaction times is 5-60min, and the volume ratio of Fmoc deprotecting regent and reaction solvent is 1:(1-5).
Preferably; take Rink Amide resin as raw material; take the amino acid of Fmoc protection as monomer; the DMF solution of the piperidines with 20% is for taking off Fmoc reagent; take HOBt as anti-racemization reagent, with DIC, HBTU or HATU are wherein a kind of for connecing the peptide condensing agent; coupling amino acid synthesizes 17 peptide resins of protection successively, and wherein protecting group Xan, Tmob or Mmt are the weak acid sensitivities.At first, remove Fmoc protecting group twice on the aminoresin with the DMF solution of 20% piperidines, the time is respectively 2-5min and 10-30min, alternately washs 3-10 time with DMF and DCM after removing Fmoc.By 1:(2-5): (2-5): (2-5): mol ratio (4-10) takes by weighing respectively aminoresin; the Fmoc protected amino acid; HOBt; HBTU and DIPEA; first with the Fmoc protected amino acid; HOBt; HBTU and DIPEA are dissolved among the DMF; pre-activation; then add the aminoresin after the swelling; room temperature concussion reaction 0.5 ~ 3h connects the Fmoc protected amino acid; connect next amino acid and remove twice of Fmoc protecting group with the DMF solution of piperidines before; time is respectively 2-5min and 10-30min; alternately wash 3-10 time with DMF and DCM after connecting a upper amino acid at every turn and removing Fmoc, and drain.
Deprotection base X in the step (2)
1With system be TFA:TIS:DCM=(1-5 by volume): (5-10): (85-94); Oxidation system is DMSO by volume: reaction solvent=1:(1-3), by volume.
Oxidation removal protecting group X in the step (3)
2And the system that forms second pair of disulfide linkage is: the DMF solution of iodine, the DMF:MeOH=(1-8 of iodine): 1 solution, the DCM solution of iodine or the DCM:MeOH=(1-8 of iodine): 1 solution, wherein the mol ratio of iodine and cyclisation peptide is (5-20): 1.
The cutting condition is in the step (4): with TFA: thioanisole: TIS: methyl-phenoxide: water=(96-80): (1-5): (1-5): (1-5): (1-5), by volume.
Contain the Yi Segenan antibacterial peptide of two pairs of disulfide linkage by route of the present invention preparation, operation steps is brief, and aftertreatment is simple, and by product is few, and productive rate is high, pollutes little, suitable large-scale industrial production.
Yi Segenan preparation method of the present invention sees that (wherein, the protecting group on the 5th and the 14th Cys is X to Fig. 7
2, the protecting group X on the 7th and the 12nd 's the Cys
1, X
1=Xan, Tmob or Mmt, X
2=Acm).
What deserves to be explained is that the Cys protecting group of step (2) and step (3) can be exchanged, is X such as the protecting group on the 7th and the 12nd Cys
2, the protecting group X on the 5th and the 14th 's the Cys
1In this case, then remove first the 5th and the 14th protecting group oxidation and form first pair of disulfide linkage, and then remove protecting group on the 7th and the 12nd Cys, form second pair of disulfide linkage.
Description of drawings
Fig. 1: Yi Segenan linear peptides crude product HPLC analyzes collection of illustrative plates;
Fig. 2: Yi Segenan linear peptides crude product MS collection of illustrative plates;
Fig. 3: Yi Segenan Cys
5-14One cyclic peptide HPLC analyzes collection of illustrative plates;
Fig. 4: Yi Segenan Cys
5-14One cyclic peptide MS collection of illustrative plates;
Fig. 5: Yi Segenan HPLC analyzes collection of illustrative plates;
Fig. 6: Yi Segenan MS collection of illustrative plates;
Fig. 7: Yi Segenan preparation method.
Embodiment
The present invention is including but not limited to following examples.
The reagent name abbreviation
DMF:N, dinethylformamide
The NMP:N-methyl-2-pyrrolidone
DCC: dicyclohexylcarbodiimide
TFA: trifluoroacetic acid
DCM: methylene dichloride
TIS: tri isopropyl silane
DIPEA:N, the N-diisopropylethylamine
The HOBt:1-hydroxybenzotriazole
HOAt:N-hydroxyl-7-azepine benzotriazole
Cl-HOBt:6-chloro-1-hydroxy benzo triazole
HBTU: benzotriazole-N, N, N', N'-tetramethyl-urea hexafluorophosphate
HATU:2-(7-azo benzotriazole)-N, N, N', N'-tetramethyl-urea phosphofluoric acid ester
PyBOP: phosphofluoric acid benzotriazole-1-base-oxygen base tripyrrole alkyl phosphorus
PyAOP:(3H-1,2,3-triazolo [4,5-b] pyridine-3-oxygen base) three-1-Bi coughs up Wan Ji Phosphonium hexafluorophosphate
Embodiment 1
Preparation and the analysis of Fmoc-Arg (Pbf)-Gly-Gly-Leu-Cys (Acm)-Tyr (tBu)-Cys (Mmt)-Arg (Pbf)-Gly-Arg-Phe-Cys (Mmt)-Val-Cys (Acm)-Val-Gly-Arg (Pbf)-aminoresin
(1) preparation H-Arg (Pbf)-aminoresin
Take by weighing the 2g substitution value and be the Rink Amide resin of 0.6mmol/g in solid phase reactor, add 10mL DCM swelling 20min, add subsequently the DMF solution of 10mL 20% piperidines, reaction 5min, the DMF washing once adds the DMF solution of the piperidines of 10mL20%, reaction 15min.Drain.In solid phase reactor, add Fmoc-Arg (Pbf)-OH (3.6mmol), HOBt (4.3mmol), DIC (4.3mmol), DMF(15mL), 20 ℃ of reaction 2h.The resin washing is drained, namely get Fmoc-Arg (Pbf)-resin.Add closed reagent 10mL(acetic anhydride (mmol) in resin: DIPEA(mmol): DMF=1:1:8), reaction 1h seals remaining amino, washs repeatedly with DCM, MeOH and DMF respectively, drains.Add 20%PIP/DMF solution reaction 20min and remove the Fmoc protecting group, wash repeatedly with DCM, MeOH and DMF respectively, drain, get H-Fmoc-Arg (Pbf)-resin.
(2) preparation
Fmoc-Arg (Pbf)-Gly-Gly-Leu-Cys (Acm)-Tyr (tBu)-Cys (Mmt)-Arg (Pbf)-Gly-Arg-Phe-Cys (Mmt)-Val-Cys (Acm)-Val-Gly-Arg (Pbf)-aminoresin
The H-Arg (Pbf) that embodiment 1 step (1) is made-resin adds amino acid Fmoc-Gly-OH(1mmol in solid phase reactor), HOBt(1.2mmol), DPIEA(3.6mmol), HBTU(1.2mmol) and DMF(10mL) 20 ℃ of reaction 2h.The coupling completeness can use the Kaiser test to detect.By known in this field, alternatively, coupling reagent can be selected from any one or a plurality of combinations among any one or two and HBTU, HATU, PyBOP or the PyAOP among any one or a plurality of and DPIEA or the NMM among HOBt, HOAt, HOOBt or the Cl-HOBt.
After detection is passed through, add the DMF solution of 10mL 20% piperidines, reaction 5min, DMF washs once, adds the DMF solution of the piperidines of 10mL 20%, reaction 15min.Drain.
With this remaining amino acid of coupling successively, namely get protection Yi Segenan linear peptides resin 4.05g.
It is as shown in the table for the amino acid consumption:
| Sequence | The amino acid title | Consumption |
| 1 | Fmoc-Arg(Pbf)-OH | 2.33g |
| 2 | Fmoc-Gly-OH | 1.07g |
| 3 | Fmoc-Val-OH | 1.22g |
| 4 | Fmoc-Cys(Acm)-OH | 1.49g |
| 5 | Fmoc-Val-OH | 1.22g |
| 6 | Fmoc-Cys(Mmt)-OH | 2.21g |
| 7 | Fmoc-Phe-OH | 1.39g |
| 8 | Fmoc-Arg(Pbf)-OH | 2.33g |
| 9 | Fmoc-Gly-OH | 1.07g |
| 10 | Fmoc-Arg(Pbf)-OH | 2.33g |
| 11 | Fmoc-Cys(Mmt)-OH | 2.21g |
| 12 | Fmoc-Tyr(tBu)-OH | 1.65g |
| 13 | moc-Cys(Acm)-OH | 1.49g |
| 14 | Fmoc-Leu-OH | 1.27g |
| 15 | Fmoc-Gly-OH | 1.07g |
| 16 | Fmoc-Gly-OH | 1.07g |
| 17 | Fmoc-Arg(Pbf)-OH | 2.33g |
(3) analysis of the linear thick peptide of Yi Segenan
Take by weighing Yi Segenan linear peptides resin that 0.5g embodiment 1 step (2) makes in solid phase reactor, add 5mL DCM swelling 10min, drain.Add subsequently 20%PIP/DMF solution reaction 20min and remove the Fmoc protecting group, wash repeatedly with DCM, MeOH and DMF respectively, drain.Configuration lysate 5mL, its each component volume ratio is TFA: thioanisole: TIS: methyl-phenoxide: water=90:2.5:2.5:2.5:2.5.The ice bath cooling, to the Yi Segenan linear peptides resin that wherein adds the side chain full guard, 15 ℃ of concussion reaction 3h; The filtering resin slowly drops to gained filtrate in the 50mL ice ether, precipitation, and centrifugal rear collection white precipitate, vacuum-drying gets 0.256g Yi Segenan linear peptides crude product.Product is analyzed (Fig. 1) and MS affirmation (Fig. 2) through HPLC.
Embodiment 2
Take by weighing the linear peptides resin that obtains in 3g embodiment 1 step (2), in solid phase reactor, add the DCM:DMSO=2:1(volume ratio of 30mL1%TFA) solution, at 25 ℃ of lower concussion reaction 4h.Drain.Respectively wash 3 times with DMF, anhydrous methanol, drain.Get Cys
5-14One cyclic peptide resin:
(2) Yi Segenan Cys
5-14The analysis of the thick peptide of one ring
Take by weighing the peptide resin that 0.5g embodiment 2 steps (1) obtain, in solid phase reactor, add 5mL DCM swelling 10min, drain.The DMF solution that adds 10mL 20% piperidines, reaction 5min, DMF washs once, adds the DMF solution of the piperidines of 10mL 20%, reaction 15min.Drain.With DMF washing 2 times, anhydrous methanol washing 3 times is drained respectively.Configuration lysate 5mL, its each component volume ratio is TFA: thioanisole: TIS: methyl-phenoxide: water=90:2.5:2.5:2.5:2.5.Behind 25 ℃ of lower concussion reaction 3h, reaction solution is injected ether, precipitation, centrifugal rear collection white precipitate, it is dry to put into vacuum drier, gets 0.233g.Obtain Yi Segenan Cys
5-14One cyclic peptide crude product.Product is analyzed (Fig. 3) and MS affirmation (Fig. 4) through HPLC.
Preparation and the analysis of embodiment 3 Yi Segenan
Take by weighing the peptide resin that obtains in 2g embodiment 2 steps (1), in solid phase reactor, add 10mL DCM swelling 10min, drain.Add 10mL DMF.Add in the peptide resin after being dissolved in 655.2mg iodine among the 10mL MeOH, mixed solution behind the concussion reaction 4h, respectively washs 3 times with DMF, anhydrous methanol at ambient temperature, drains.Get Cys
9-12, Cys
5-14Two cyclic peptide resin 1.78g.
(2) preparation of the thick peptide of Yi Segenan
The peptide resin that obtains in embodiment 3 steps (1) is added the DMF solution of 10mL 20% piperidines, reaction 5min, DMF wash once, adds the DMF solution of the piperidines of 10mL 20%, reacts 15min.Drain.With DMF washing 2 times, anhydrous methanol washing 3 times is drained respectively.Configuration lysate 20mL, its each component volume ratio is TFA: thioanisole: TIS: methyl-phenoxide: water=90:2.5:2.5:2.5:2.5.Behind 25 ℃ of lower concussion reaction 3h, reaction solution is injected ether, precipitation, centrifugal rear collection white precipitate, it is dry to put into vacuum drier.Get 0.89g Yi Segenan peptide crude product.
(3) Yi Segenan purifying crude
Concrete grammar is: device: C18 preparative column (20 * 250mm)
Elutriant A:0.1%(v/v) TFA/H
2O
Elutriant B: acetonitrile
Flow velocity: 12mL/min
Ultraviolet detection wavelength: 215nm
Operation: the aqueous solution of the Yi Sege South 2nd Ring Road crude product that (1) embodiment 3 steps (2) make is with 0.45 μ m filtering with microporous membrane;
(2) filtrate direct injection;
(3) 19-34%(v/v) acetonitrile-water eluent gradient wash-out;
(4) collect the purpose peptide;
(5) concentrated, lyophilize;
(6) De Yisegenan white solid 0.68g.
Product is analyzed (Fig. 5) and MS affirmation (Fig. 6) through HPLC.
Embodiment 4
Preparation and the analysis of Fmoc-Arg (Pbf)-Gly-Gly-Leu-Cys (Tmob)-Tyr (tBu)-Cys (Acm)-Arg (Pbf)-Gly-Arg-Phe-Cys (Acm)-Val-Cys (Tmob)-Val-Gly-Arg (Pbf)-aminoresin
(1) preparation H-Arg (Pbf)-aminoresin
Take by weighing the 2g substitution value and be the Rink Amide resin of 0.6mmol/g in solid phase reactor, add 10mL DCM swelling 20min, add subsequently the DMF solution of 10mL 20% piperidines, reaction 5min, the DMF washing once adds the DMF solution of the piperidines of 10mL20%, reaction 15min.Drain.In solid phase reactor, add Fmoc-Arg (Pbf)-OH (3.6mmol), HOBt (4.3mmol), DIC (4.3mmol), DMF(15mL), 20 ℃ of reaction 2h.The resin washing is drained, namely get Fmoc-Arg (Pbf)-resin.Add closed reagent 10mL(acetic anhydride (mmol) in resin: DIPEA(mmol): DMF=1:1:8), reaction 1h seals remaining amino, washs repeatedly with DCM, MeOH and DMF respectively, drains.Add 20%PIP/DMF solution reaction 20min and remove the Fmoc protecting group, wash repeatedly with DCM, MeOH and DMF respectively, drain, get H-Fmoc-Arg (Pbf)-resin.
(2) preparation
Fmoc-Arg (Pbf)-Gly-Gly-Leu-Cys (Tmob)-Tyr (tBu)-Cys (Acm)-Arg (Pbf)-Gly-Arg-Phe-Cys (Acm)-Val-Cys (Tmob)-Val-Gly-Arg (Pbf)-aminoresin
The H-Arg (Pbf) that step (1) is made-resin adds amino acid Fmoc-Gly-OH(1mmol in solid phase reactor), HOBt(1.2mmol), DPIEA(3.6mmol), HBTU(1.2mmol) and DMF(10mL) 20 ℃ of reaction 2h.The coupling completeness can use the Kaiser test to detect.By known in this field, alternatively, coupling reagent can be selected from any one or a plurality of combinations among any one or two and HBTU, HATU, PyBOP or the PyAOP among any one or a plurality of and DPIEA or the NMM among HOBt, HOAt, HOOBt or the Cl-HOBt.
After detection is passed through, add the DMF solution of 10mL 20% piperidines, reaction 5min, DMF washs once, adds the DMF solution of the piperidines of 10mL 20%, reaction 15min.Drain.
With this remaining amino acid of coupling successively, namely get protection Yi Segenan linear peptides resin 3.80g.
It is as shown in the table for the amino acid consumption:
| Sequence | The amino acid title | Consumption |
[0099]?
| 1 | Fmoc-Arg(Pbf)-OH | 2.33g |
| 2 | Fmoc-Gly-OH | 1.07g |
| 3 | Fmoc-Val-OH | 1.22g |
| 4 | Fmoc-Cys(Tmob)-OH | 1.49g |
| 5 | Fmoc-Val-OH | 1.22g |
| 6 | Fmoc-Cys(Acm)-OH | 2.21g |
| 7 | Fmoc-Phe-OH | 1.39g |
| 8 | Fmoc-Arg(Pbf)-OH | 2.33g |
| 9 | Fmoc-Gly-OH | 1.07g |
| 10 | Fmoc-Arg(Pbf)-OH | 2.33g |
| 11 | Fmoc-Cys(Mmt)-OH | 2.21g |
| 12 | Fmoc-Tyr(tBu)-OH | 1.65g |
| 13 | Fmoc-Cys(Tmob)-OH | 1.49g |
| 14 | Fmoc-Leu-OH | 1.27g |
| 15 | Fmoc-Gly-OH | 1.07g |
| 16 | Fmoc-Gly-OH | 1.07g |
| 17 | Fmoc-Arg(Pbf)-OH | 2.33g |
(3) analysis of the linear thick peptide of Yi Segenan
Take by weighing Yi Segenan linear peptides resin that 0.5g embodiment 4 steps (2) make in solid phase reactor, add 5mL DCM swelling 10min, drain.Add subsequently 20%PIP/DMF solution reaction 20min and remove the Fmoc protecting group, wash repeatedly with DCM, MeOH and DMF respectively, drain.Configuration lysate 5mL in round-bottomed flask, its each component volume ratio is TFA: thioanisole: TIS: methyl-phenoxide: water=90:2.5:2.5:2.5:2.5.The ice bath cooling, to the Yi Segenan linear peptides resin that wherein adds the side chain full guard, 15 ℃ of concussion reaction 3h; The filtering resin slowly drops to gained filtrate in the 50mL ice ether, precipitation, and centrifugal rear collection white precipitate, vacuum-drying gets 0.277g Yi Segenan linear peptides crude product.Product is analyzed and the MS affirmation through HPLC.
Embodiment 5
Take by weighing the linear peptides resin that obtains in 3g embodiment 4 steps (2), in solid phase reactor, add the DCM:DMSO=2:1(volume ratio of 30mL1%TFA) solution, at 25 ℃ of lower concussion reaction 4h.Drain.Respectively wash 3 times with DMF, anhydrous methanol, drain.Get Cys
9-12One cyclic peptide resin:
(2) Yi Segenan Cys
9-12The analysis of the thick peptide of one ring
Take by weighing the peptide resin that 0.5g embodiment 5 steps (1) obtain, in solid phase reactor, add 5mL DCM swelling 10min, drain.The DMF solution that adds 10mL 20% piperidines, reaction 5min, DMF washs once, adds the DMF solution of the piperidines of 10mL 20%, reaction 15min.Drain.With DMF washing 2 times, anhydrous methanol washing 3 times is drained respectively.Configuration lysate 5mL, its each component volume ratio is TFA: thioanisole: TIS: methyl-phenoxide: water=90:2.5:2.5:2.5:2.5.The linear peptides resin is added in the above-mentioned mixed solution, behind 25 ℃ of lower concussion reaction 3h, reaction solution is injected ether, precipitation, centrifugal rear collection white precipitate, vacuum-drying.Obtain Yi Segenan Cys
9-12One cyclic peptide crude product 0.245g.Product is analyzed and the MS affirmation through HPLC.
Preparation and the analysis of embodiment 6 Yi Segenan
Take by weighing the peptide resin that obtains in 2g embodiment 5 steps (1), in solid phase reactor, add 10mL DCM swelling 10min, drain.Add 10mL DMF.Add in the peptide resin after being dissolved in 655.2mg iodine among the 10mL MeOH, mixed solution behind the concussion reaction 4h, respectively washs 3 times with DMF, anhydrous methanol at ambient temperature, drains.Get Cys
9-12, Cys
5-14Two cyclic peptide resin 1.82g.
(2) preparation of the thick peptide of Yi Segenan
The peptide resin that obtains in embodiment 6 steps (1) is added the DMF solution of 10mL 20% piperidines, reaction 5min, DMF wash once, adds the DMF solution of the piperidines of 10mL 20%, reacts 15min.Drain.With DMF washing 2 times, anhydrous methanol washing 3 times is drained respectively.Configuration lysate 30mL, its each component volume ratio is TFA: thioanisole: TIS: methyl-phenoxide: water=90:2.5:2.5:2.5:2.5.The linear peptides resin is added in the above-mentioned mixed solution, and every 1g linear peptides resin uses the volume of mixed solution to be 10mL.Behind 25 ℃ of lower concussion reaction 3h, reaction solution is injected ether, precipitation, centrifugal rear collection white precipitate, it is dry to put into vacuum drier.Get 0.82g Yi Segenan peptide crude product.
(3) Yi Segenan purifying crude
Concrete grammar is: device: C18 preparative column (20 * 250mm)
Elutriant A:0.1%(v/v) TFA/H
2O
Elutriant B: acetonitrile
Flow velocity: 12mL/min
Ultraviolet detection wavelength: 215nm
Operation: the aqueous solution of the Yi Sege South 2nd Ring Road crude product that (1) makes embodiment 6 steps (2) is with 0.45 μ m filtering with microporous membrane;
(2) filtrate direct injection;
(3) 19-34%(v/v) acetonitrile-water eluent gradient wash-out;
(4) collect the purpose peptide;
(5) concentrated, lyophilize;
(6) De Yisegenan white solid 0.63g.
Claims (8)
1. the solid phase synthesis process of Yi Zhong Yi Segenan may further comprise the steps:
(1) according to the method for solid phase synthesis; aminoresin is after sloughing Fmoc under the effect of Fmoc deprotecting regent; under the coupling system effect, in reaction solvent, connect successively Fmoc amino acid by the peptide order; synthetic 17 peptide aminoresin; wherein the 5th identical with protecting group on the 14th halfcystine; the 7th identical with protecting group on the 12nd halfcystine, one group of protecting group X in two groups of protecting groups
1Be the Xan to the weak acid sensitivity, Tmob or Mmt, another group protecting group X
2Be Acm;
(2) deprotection base X
1, oxidation generates first pair of disulfide linkage;
(3) deprotection base X
2And cyclisation generates second pair of disulfide linkage;
(4) remove N end protecting group, cracking and remove Side chain protective group, De Yisegenan behind the purifying from the aminoresin.
2. method according to claim 1 is characterized in that, used aminoresin is the RinkAmide resin in the step (1), Rink Amide AM resin, Rink Amide mbha resin, Rink Amide PEGA resin, PAL resin.
3. method according to claim 1 is characterized in that, 17 peptide resins of protection are PG-Arg (Pbf)-Gly-Gly-Leu-Cys (X in the step (1)
1)-Tyr (tBu)-Cys (X
2)-Arg (Pbf)-Gly-Arg (Pbf)-Phe-Cys (X
2)-Val-Cys (X
1)-Val-Gly-Arg (Pbf)-aminoresin, or PG-Arg (Pbf)-Gly-Gly-Leu-Cys (X
2)-Tyr (tBu)-Cys (X
1)-Arg (Pbf)-Gly-Arg (Pbf)-Phe-Cys (X
1)-Val-Cys (X
2)-Val-Gly-Arg (Pbf)-aminoresin, wherein PG is Fmoc or Boc.
4. method according to claim 1; it is characterized in that; in the step (1) take Fmoc aminoresin as solid phase carrier; according to the solid state chemistry synthetic method; hold N to hold successively condensation reaction to connect the amino acid of Fmoc protection from C; last amino acid uses Fmoc-Arg (Pbf)-OH or Boc-Arg (Pbf)-OH, obtains linearity 17 peptide resins of protection.
5. method according to claim 1 is characterized in that, in the step (1), reaction solvent is DCM, any one among DMF or the NMP or more than one combination; Coupling system is any one or the combination of a plurality of and DIC among HOBt, HOAt, HOOBt or the Cl-HOBt, or is any one or a plurality of combinations among any one or two and HBTU, HATU, PyBOP or the PyAOP among any one or a plurality of and DPIEA or the NMM among HOBt, HOAt, HOOBt or the Cl-HOBt; The Fmoc deprotecting regent is piperidines, and the deprotection reaction temperature is 10-30 ℃, and the reaction times is 5-60min, and the volume ratio of Fmoc deprotecting regent and reaction solvent is 1:(1-5).
6. method according to claim 1 is characterized in that, deprotection base X in the step (2)
1The system that adopts is TFA:TIS:DCM=(1-5 by volume): (5-10): (94-85); Oxidation system is DMSO by volume: reaction solvent=1:(1-3).
7. method according to claim 1 is characterized in that, oxidation removal X described in the step (3)
2Protecting group and the system that forms disulfide linkage are following wherein a kind of: the DMF solution of (1) iodine; (2) the DMF/MeOH solution of iodine; (3) the DCM solution of iodine; (4) the DCM/MeOH solution of iodine.Wherein the mol ratio of iodine and cyclisation peptide is (5-20): 1.
8. method according to claim 1, it is characterized in that cracking condition is by volume in the step (4): TFA: thioanisole: TIS: methyl-phenoxide: water=(80-96): (1-5): (1-5): (1-5): (1-5).
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| CN105504012A (en) * | 2014-09-30 | 2016-04-20 | 深圳翰宇药业股份有限公司 | Preparation method of polypeptide |
| CN106496315A (en) * | 2016-10-24 | 2017-03-15 | 合肥国肽生物科技有限公司 | A kind of efficient ziconotide synthesis preparation method |
| CN106554391A (en) * | 2015-09-29 | 2017-04-05 | 深圳翰宇药业股份有限公司 | A kind of synthetic method of Marine biologic peptide Xen2174 |
| WO2017114240A1 (en) * | 2015-12-31 | 2017-07-06 | 深圳翰宇药业股份有限公司 | Method for synthesizing etelcalcetide |
| CN111100188A (en) * | 2018-10-25 | 2020-05-05 | 成都医学院 | Method for preparing spider antibacterial peptide |
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| CN105504012A (en) * | 2014-09-30 | 2016-04-20 | 深圳翰宇药业股份有限公司 | Preparation method of polypeptide |
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| US10647742B2 (en) | 2015-12-31 | 2020-05-12 | Hybio Pharmaceutical Co., Ltd. | Method for synthesizing etelcalcetide |
| CN106496315A (en) * | 2016-10-24 | 2017-03-15 | 合肥国肽生物科技有限公司 | A kind of efficient ziconotide synthesis preparation method |
| CN111100188A (en) * | 2018-10-25 | 2020-05-05 | 成都医学院 | Method for preparing spider antibacterial peptide |
| CN112500454A (en) * | 2020-12-13 | 2021-03-16 | 江苏新瑞药业有限公司 | Preparation method of Iseganan |
| CN112500453A (en) * | 2020-12-13 | 2021-03-16 | 江苏新瑞药业有限公司 | Solid-phase synthesis method of fragment of antibacterial peptide Iseganan |
| CN114044817A (en) * | 2021-10-12 | 2022-02-15 | 杭州固拓生物科技有限公司 | Preparation method of beta-defensin 2 antibacterial peptide |
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