CN115368437A - Method for solid-phase synthesis of cyclic polypeptide - Google Patents
Method for solid-phase synthesis of cyclic polypeptide Download PDFInfo
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- CN115368437A CN115368437A CN202210708912.9A CN202210708912A CN115368437A CN 115368437 A CN115368437 A CN 115368437A CN 202210708912 A CN202210708912 A CN 202210708912A CN 115368437 A CN115368437 A CN 115368437A
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- polypeptide
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- amino acid
- polypeptide resin
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- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 153
- 229920001184 polypeptide Polymers 0.000 title claims abstract description 145
- 102000004196 processed proteins & peptides Human genes 0.000 title claims abstract description 144
- 238000000034 method Methods 0.000 title claims abstract description 30
- 238000010532 solid phase synthesis reaction Methods 0.000 title claims abstract description 7
- 125000004122 cyclic group Chemical group 0.000 title claims description 18
- 239000011347 resin Substances 0.000 claims abstract description 86
- 229920005989 resin Polymers 0.000 claims abstract description 86
- 150000001413 amino acids Chemical class 0.000 claims abstract description 48
- 238000006243 chemical reaction Methods 0.000 claims abstract description 23
- JRZJOMJEPLMPRA-UHFFFAOYSA-N olefin Natural products CCCCCCCC=C JRZJOMJEPLMPRA-UHFFFAOYSA-N 0.000 claims abstract description 4
- 150000001336 alkenes Chemical class 0.000 claims abstract description 3
- 238000000354 decomposition reaction Methods 0.000 claims abstract description 3
- 125000006239 protecting group Chemical group 0.000 claims description 24
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 claims description 21
- 239000003054 catalyst Substances 0.000 claims description 21
- JGFZNNIVVJXRND-UHFFFAOYSA-N N,N-Diisopropylethylamine (DIPEA) Chemical compound CCN(C(C)C)C(C)C JGFZNNIVVJXRND-UHFFFAOYSA-N 0.000 claims description 13
- 239000003638 chemical reducing agent Substances 0.000 claims description 10
- NQRYJNQNLNOLGT-UHFFFAOYSA-N Piperidine Chemical compound C1CCNCC1 NQRYJNQNLNOLGT-UHFFFAOYSA-N 0.000 claims description 8
- 239000003795 chemical substances by application Substances 0.000 claims description 8
- 125000003088 (fluoren-9-ylmethoxy)carbonyl group Chemical group 0.000 claims description 7
- 238000006722 reduction reaction Methods 0.000 claims description 7
- VHYFNPMBLIVWCW-UHFFFAOYSA-N 4-Dimethylaminopyridine Chemical compound CN(C)C1=CC=NC=C1 VHYFNPMBLIVWCW-UHFFFAOYSA-N 0.000 claims description 6
- WFDIJRYMOXRFFG-UHFFFAOYSA-N Acetic anhydride Chemical compound CC(=O)OC(C)=O WFDIJRYMOXRFFG-UHFFFAOYSA-N 0.000 claims description 6
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 claims description 6
- 239000002253 acid Substances 0.000 claims description 6
- 230000009471 action Effects 0.000 claims description 6
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 claims description 6
- QNAYBMKLOCPYGJ-UWTATZPHSA-N D-alanine Chemical compound C[C@@H](N)C(O)=O QNAYBMKLOCPYGJ-UWTATZPHSA-N 0.000 claims description 5
- QNAYBMKLOCPYGJ-UHFFFAOYSA-N D-alpha-Ala Natural products CC([NH3+])C([O-])=O QNAYBMKLOCPYGJ-UHFFFAOYSA-N 0.000 claims description 5
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 claims description 5
- 125000003277 amino group Chemical group 0.000 claims description 5
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims description 5
- 125000002777 acetyl group Chemical group [H]C([H])([H])C(*)=O 0.000 claims description 4
- 239000007822 coupling agent Substances 0.000 claims description 4
- 229910052736 halogen Inorganic materials 0.000 claims description 4
- 150000002367 halogens Chemical class 0.000 claims description 4
- 150000007530 organic bases Chemical class 0.000 claims description 4
- GQHTUMJGOHRCHB-UHFFFAOYSA-N 2,3,4,6,7,8,9,10-octahydropyrimido[1,2-a]azepine Chemical compound C1CCCCN2CCCN=C21 GQHTUMJGOHRCHB-UHFFFAOYSA-N 0.000 claims description 3
- 229960000549 4-dimethylaminophenol Drugs 0.000 claims description 3
- KPFBUSLHFFWMAI-HYRPPVSQSA-N [(8r,9s,10r,13s,14s,17r)-17-acetyl-6-formyl-3-methoxy-10,13-dimethyl-1,2,7,8,9,11,12,14,15,16-decahydrocyclopenta[a]phenanthren-17-yl] acetate Chemical compound C1C[C@@H]2[C@](CCC(OC)=C3)(C)C3=C(C=O)C[C@H]2[C@@H]2CC[C@](OC(C)=O)(C(C)=O)[C@]21C KPFBUSLHFFWMAI-HYRPPVSQSA-N 0.000 claims description 3
- 238000005865 alkene metathesis reaction Methods 0.000 claims description 3
- 150000001408 amides Chemical class 0.000 claims description 3
- 125000000539 amino acid group Chemical group 0.000 claims description 3
- 229910052799 carbon Inorganic materials 0.000 claims description 3
- 150000001875 compounds Chemical class 0.000 claims description 3
- 238000002156 mixing Methods 0.000 claims description 3
- OKDQKPLMQBXTNH-UHFFFAOYSA-N n,n-dimethyl-2h-pyridin-1-amine Chemical compound CN(C)N1CC=CC=C1 OKDQKPLMQBXTNH-UHFFFAOYSA-N 0.000 claims description 3
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 claims description 3
- 230000009467 reduction Effects 0.000 claims description 3
- 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 description 2
- WWZKQHOCKIZLMA-UHFFFAOYSA-N Caprylic acid Natural products CCCCCCCC(O)=O WWZKQHOCKIZLMA-UHFFFAOYSA-N 0.000 claims description 2
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 claims description 2
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 claims description 2
- NQPDZGIKBAWPEJ-UHFFFAOYSA-N Valeric acid Natural products CCCCC(O)=O NQPDZGIKBAWPEJ-UHFFFAOYSA-N 0.000 claims description 2
- 125000003172 aldehyde group Chemical group 0.000 claims description 2
- 125000003545 alkoxy group Chemical group 0.000 claims description 2
- 125000000217 alkyl group Chemical group 0.000 claims description 2
- 238000003379 elimination reaction Methods 0.000 claims description 2
- 238000005984 hydrogenation reaction Methods 0.000 claims description 2
- 150000002466 imines Chemical class 0.000 claims description 2
- 239000000203 mixture Substances 0.000 claims description 2
- FUZZWVXGSFPDMH-UHFFFAOYSA-N n-hexanoic acid Natural products CCCCCC(O)=O FUZZWVXGSFPDMH-UHFFFAOYSA-N 0.000 claims description 2
- 125000001624 naphthyl group Chemical group 0.000 claims description 2
- 229910052700 potassium Inorganic materials 0.000 claims description 2
- 239000011591 potassium Substances 0.000 claims description 2
- 235000017281 sodium acetate Nutrition 0.000 claims description 2
- 239000001632 sodium acetate Substances 0.000 claims description 2
- 239000012279 sodium borohydride Substances 0.000 claims description 2
- 229910000033 sodium borohydride Inorganic materials 0.000 claims description 2
- BEOOHQFXGBMRKU-UHFFFAOYSA-N sodium cyanoborohydride Chemical compound [Na+].[B-]C#N BEOOHQFXGBMRKU-UHFFFAOYSA-N 0.000 claims description 2
- 125000003107 substituted aryl group Chemical group 0.000 claims description 2
- GOJUJUVQIVIZAV-UHFFFAOYSA-N 2-amino-4,6-dichloropyrimidine-5-carbaldehyde Chemical group NC1=NC(Cl)=C(C=O)C(Cl)=N1 GOJUJUVQIVIZAV-UHFFFAOYSA-N 0.000 claims 1
- 238000003786 synthesis reaction Methods 0.000 abstract description 18
- 230000015572 biosynthetic process Effects 0.000 abstract description 17
- 125000001424 substituent group Chemical group 0.000 abstract description 13
- 239000007790 solid phase Substances 0.000 abstract description 6
- 238000007363 ring formation reaction Methods 0.000 abstract description 5
- 238000006467 substitution reaction Methods 0.000 abstract description 3
- -1 amino acid olefin Chemical class 0.000 abstract description 2
- 230000001737 promoting effect Effects 0.000 abstract description 2
- 238000000926 separation method Methods 0.000 abstract description 2
- 230000004888 barrier function Effects 0.000 abstract 1
- 239000011203 carbon fibre reinforced carbon Substances 0.000 abstract 1
- 230000009466 transformation Effects 0.000 abstract 1
- 229940024606 amino acid Drugs 0.000 description 25
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 22
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 15
- 239000002904 solvent Substances 0.000 description 15
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 12
- 239000003814 drug Substances 0.000 description 10
- 238000004895 liquid chromatography mass spectrometry Methods 0.000 description 10
- 229940079593 drug Drugs 0.000 description 9
- 239000000243 solution Substances 0.000 description 9
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 8
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 6
- 238000003776 cleavage reaction Methods 0.000 description 6
- 230000014759 maintenance of location Effects 0.000 description 6
- 230000007017 scission Effects 0.000 description 6
- 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 6
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 5
- 238000004458 analytical method Methods 0.000 description 5
- 239000007864 aqueous solution Substances 0.000 description 5
- 239000012634 fragment Substances 0.000 description 4
- 238000004128 high performance liquid chromatography Methods 0.000 description 4
- 108090000623 proteins and genes Proteins 0.000 description 4
- 102000004169 proteins and genes Human genes 0.000 description 4
- 102000001189 Cyclic Peptides Human genes 0.000 description 3
- 108010069514 Cyclic Peptides Proteins 0.000 description 3
- 239000007821 HATU Substances 0.000 description 3
- 229960003767 alanine Drugs 0.000 description 3
- 239000002173 cutting fluid Substances 0.000 description 3
- 201000010099 disease Diseases 0.000 description 3
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- OBMZMSLWNNWEJA-XNCRXQDQSA-N C1=CC=2C(C[C@@H]3NC(=O)[C@@H](NC(=O)[C@H](NC(=O)N(CC#CCN(CCCC[C@H](NC(=O)[C@@H](CC4=CC=CC=C4)NC3=O)C(=O)N)CC=C)NC(=O)[C@@H](N)C)CC3=CNC4=C3C=CC=C4)C)=CNC=2C=C1 Chemical compound C1=CC=2C(C[C@@H]3NC(=O)[C@@H](NC(=O)[C@H](NC(=O)N(CC#CCN(CCCC[C@H](NC(=O)[C@@H](CC4=CC=CC=C4)NC3=O)C(=O)N)CC=C)NC(=O)[C@@H](N)C)CC3=CNC4=C3C=CC=C4)C)=CNC=2C=C1 OBMZMSLWNNWEJA-XNCRXQDQSA-N 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical compound N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 description 2
- CMWYAOXYQATXSI-UHFFFAOYSA-N n,n-dimethylformamide;piperidine Chemical compound CN(C)C=O.C1CCNCC1 CMWYAOXYQATXSI-UHFFFAOYSA-N 0.000 description 2
- 230000004850 protein–protein interaction Effects 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 230000008685 targeting Effects 0.000 description 2
- LMDZBCPBFSXMTL-UHFFFAOYSA-N 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide Substances CCN=C=NCCCN(C)C LMDZBCPBFSXMTL-UHFFFAOYSA-N 0.000 description 1
- FPQQSJJWHUJYPU-UHFFFAOYSA-N 3-(dimethylamino)propyliminomethylidene-ethylazanium;chloride Chemical compound Cl.CCN=C=NCCCN(C)C FPQQSJJWHUJYPU-UHFFFAOYSA-N 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 208000000094 Chronic Pain Diseases 0.000 description 1
- 241000725303 Human immunodeficiency virus Species 0.000 description 1
- 102000004877 Insulin Human genes 0.000 description 1
- 108090001061 Insulin Proteins 0.000 description 1
- 108010052285 Membrane Proteins Proteins 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 208000001132 Osteoporosis Diseases 0.000 description 1
- 208000002193 Pain Diseases 0.000 description 1
- 125000002252 acyl group Chemical group 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 230000003197 catalytic effect Effects 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 230000008878 coupling Effects 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 206010012601 diabetes mellitus Diseases 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 125000000524 functional group Chemical group 0.000 description 1
- 238000007429 general method Methods 0.000 description 1
- NPZTUJOABDZTLV-UHFFFAOYSA-N hydroxybenzotriazole Substances O=C1C=CC=C2NNN=C12 NPZTUJOABDZTLV-UHFFFAOYSA-N 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 229940125396 insulin Drugs 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 231100000053 low toxicity Toxicity 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 201000006417 multiple sclerosis Diseases 0.000 description 1
- 230000000149 penetrating effect Effects 0.000 description 1
- 230000035515 penetration Effects 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 239000000523 sample Substances 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 238000001308 synthesis method Methods 0.000 description 1
- 238000010189 synthetic method Methods 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 230000009385 viral infection Effects 0.000 description 1
Classifications
-
- 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
- C07K5/10—Tetrapeptides
- C07K5/1002—Tetrapeptides with the first amino acid being neutral
- C07K5/1005—Tetrapeptides with the first amino acid being neutral and aliphatic
- C07K5/1008—Tetrapeptides with the first amino acid being neutral and aliphatic the side chain containing 0 or 1 carbon atoms, i.e. Gly, Ala
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/50—Cyclic peptides containing at least one abnormal peptide link
- C07K7/54—Cyclic peptides containing at least one abnormal peptide link with at least one abnormal peptide link in the ring
- C07K7/56—Cyclic peptides containing at least one abnormal peptide link with at least one abnormal peptide link in the ring the cyclisation not occurring through 2,4-diamino-butanoic acid
-
- 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
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention provides a novel method for promoting polypeptide solid-phase cyclization reaction by amido bond substitution, which reduces the polypeptide conformation transformation energy barrier by using an amido bond substitution means. The method promotes the double decomposition reaction of the unnatural amino acid olefin containing the terminal group olefin to be carried out under very mild solid phase conditions by introducing removable amido bond substituent groups (Hmb, hnb, hmnb and the like) in solid phase synthesis. Can efficiently synthesize stable polypeptide cyclized by carbon-carbon bonds, and synthesize polypeptide molecules with stable frameworks with higher synthesis efficiency. The method has the following advantages: 1) The amino acid is cheap and easy to obtain, and complex synthesis is not needed; 2) The length of a side chain is convenient to regulate and control, and the tolerance of the cyclization reaction to the size of a ring system is high; 3) Aiming at the characteristic of polypeptide reaction sequence dependence, an amido bond substituent group is introduced, so that the reaction has high sequence tolerance, special modified resin is not needed, the condition is mild, the product purity is high, and the separation is easy.
Description
Technical Field
The invention belongs to the field of polypeptide synthesis, and particularly relates to a method for solid-phase synthesis of cyclic polypeptide.
Background
Including intracellular protein-protein interactions (PPIs), it is currently difficult to target about 75% of disease-associated proteins with small molecules and biologies. The polypeptide molecule has the advantages of large binding area with a target, high selectivity and capability of selectively targeting a single subtype protein in different subtypes of the same family protein, so that the polypeptide molecule becomes one of potential choices for solving the problem of the proteins related to diseases which are difficult to target. Since 1922, the first polypeptide drug was approved by the insulin molecule, the pharmaceutical use of polypeptide molecules in disease has been for eighty years, and over eighty polypeptide drugs have been approved for the market in succession. The polypeptide medicine has wide treatment fields, including diabetes, cancer, osteoporosis, multiple sclerosis, AIDS virus infection, chronic pain and the like. The polypeptide drug has the advantages of high selectivity and low toxicity of macromolecular drugs and low production cost of micromolecular drugs. However, the polypeptide drug also has the defects that the macromolecular drug is easy to degrade and has poor capability of penetrating cell membranes, so that the polypeptide drug is widely applied to targeting extracellular targets at present.
In order to truly exert the advantages of PPI in polypeptide-targeted cells, develop an efficient polypeptide synthesis method and improve the membrane penetration of polypeptides, the application value of polypeptide molecules in the development of chemical and biological probes and drugs is greatly improved. The development of synthetic modification means of polypeptide is abundant so far, but is still limited by the diversity of polypeptide side chain functional groups and solid phase loading reaction to a great extent. The polypeptide cyclization method is difficult to carry out in a solid phase or overcome the limitation of polypeptide sequence diversity.
Disclosure of Invention
In order to improve the technical problem, the invention provides a method for solid-phase synthesis of cyclic polypeptide, which comprises the following steps:
G 0 is-NH-, -O-, -NH-peptide chain-, -O-peptide chain-, wherein the peptide chain has n 1 Amino acids; n is a radical of an alkyl radical 1 Is greater than or equal to1；
G 1 Is n 2 A plurality of amino acid residues; n is 2 Greater than or equal to 1;
G 2 is n 3 A plurality of amino acid residues; n is 3 Is greater than or equal to 1;
R 1 is-CH 2 -R 3 ，R 3 Is a substituted aryl group;
R 2 is H, -NH-R', or a peptide chain, wherein the peptide chain has n 4 Amino acids; n is 4 Is greater than or equal to 1; r' is an amino protecting group (e.g., acetyl);
x and Y are the same or different and are independently selected from- (CH) 2 ) m1 (CR 4 R 5 )(CH 2 ) m2 -、-(CH 2 ) m1 S(CH 2 ) m2 -、-(CH 2 ) m1 O(CH 2 ) m2 -；R 4 、R 5 Are identical or different and are independently selected from H, - (CH) 2 ) m3 CH 3 、-OCH 3 Or phenyl; m1, m2 and m3 are independently of one another selected from integers from 0 to 8, preferably from 0 to 5, for example 1, 2, 3, 4;
(A) Mixing the polypeptide resin (IV) with a catalyst, carrying out olefin double decomposition reaction on two double bonds in the polypeptide resin in the presence of the catalyst to obtain a cyclic polypeptide resin (III) with a ring containing a double bond, and then adding a reducing agent to carry out reduction reaction to obtain a cyclic polypeptide resin (II), wherein the polypeptide resin (IV) is a polypeptide chain connected to the resin;
(B) Removing the group R by subjecting the saturated cyclic polypeptide resin (II) to elimination reaction 1 (ii) a Then removing the resin to obtain the cyclic polypeptide (I).
According to the invention, R 3 Is optionally substituted by one, two or more OH, NO 2 Halogen, CN, C 1-6 Alkyl radical, C 1-6 Alkoxy, -SO-C 1-6 Alkyl, -SO 2 -C 1-6 Alkyl-substituted groups as follows: phenyl, naphthyl; is exemplified by
According to the invention, n 1 Is an integer from 1 to 100, such as an integer from 1 to 50, 1 to 20, 1 to 10; n is 4 Is an integer from 1 to 100, such as an integer from 1 to 50, from 1 to 20, from 1 to 10;
according to the invention, n 2 Is an integer from 1 to 6, such as 1, 2, 3, 4, 5; n is a radical of an alkyl radical 3 Is an integer from 1 to 6, such as 1, 2, 3, 4, 5;
according to an embodiment of the invention, the catalyst in step (A) may be an olefin metathesis catalyst, such as a Hovyda-Grubbs-II (HG-II) catalyst, a Grubbs primary catalyst, or a Grubbs secondary catalyst;
according to an embodiment of the present invention, in the step (a), the reduction reaction may be performed under the action of an organic base, such as at least one of piperidine, triethylamine, diisopropylethylamine, pyridine, N-dimethylaminopyridine, DBU;
according to an embodiment of the invention, in step (a), the reducing agent is a hydrogenation reduction catalyst, for example 2-NBSH. The equivalent weight of the reducing agent can be from 5 to 30, for example from 10 to 25, illustratively 20.
According to an embodiment of the invention, the radical R is removed in step (B) 1 The reaction of (a) may be carried out in the presence of an acid or in the presence of ultraviolet light. The reaction in the presence of an acid may be carried out in a reducing agent, which may be SnCl 2 HCl/MeOH solution (b); the reaction under ultraviolet light may use, for example, an ultraviolet lamp.
According to an embodiment of the present invention, the reaction of removing the resin in step (B) may be carried out in a cleavage agent such as TFA/TIPS/phenol/H 2 A mixed solution of O; the volume ratio is preferably 88.
According to the invention, the polypeptide resin (IV) is prepared by a method comprising the following steps:
optionally, (1) reacting the resin containing amino group with N-terminal protected amino acid, removing the N-terminal protecting group, and optionally repeating the above steps to obtain the product with N attached 1 Polypeptide resin 1,n of amino acids 1 Greater than or equal to 1;
(2) Reacting amino-containing resin or the polypeptide resin 1 in the step (1) with unnatural amino acid which is protected by the N terminal and contains double bonds, and then removing the protecting group of the N terminal to obtain polypeptide resin 2;
(3) Reacting the polypeptide resin 2 in the step (2) with N-terminal protected amino acid, then removing the N-terminal protecting group, and optionally repeating the steps to obtain the peptide with N 2 Polypeptide resin of amino acid 3,n 2 Greater than or equal to 1;
(4) Polypeptide resin 3 and compound R 3 -CHO mixing, wherein aldehyde group reacts with amino group in the polypeptide resin to obtain imine polypeptide resin, then reducing agent is added to obtain N end connected with R 3 -CH 2 -polypeptide resin 4;
(5) Reacting the polypeptide resin 4 obtained in the step (4) with N-terminal protected amino acid, then removing the N-terminal protecting group, and optionally repeating the steps to obtain the polypeptide resin with N connected 3 Polypeptide resin of amino acid 5,n 3 Greater than or equal to 1;
(6) Polypeptide resin 5 and C with double bond as terminal group 3-10 Reacting olefinic acid (such as 4-alkenyl pentanoic acid, 5-alkenyl hexanoic acid, etc.) to obtain polypeptide resin (IV);
or reacting the polypeptide resin 5 with the N-terminal protected unnatural amino acid containing the double bond to obtain a polypeptide resin (IV); if necessary, the N-terminal protecting group can be removed, and another protecting group (such as acetyl) can be introduced into the N-terminal;
or, a) reacting the polypeptide resin 5 with the unnatural amino acid which is protected at the N terminal and contains double bonds, then removing the protecting group at the N terminal to obtain the polypeptide resin 6, b) reacting the polypeptide resin 6 with the amino acid which is protected at the N terminal, then removing the protecting group at the N terminal, and optionally repeating the steps to obtain the unnatural amino acid which is connected with N 4 Polypeptide resin of amino acids 6', n 4 Greater than or equal to 1; c) Then, a protecting group (e.g., acetyl group) is introduced into the N-terminus of the polypeptide resin 6' to obtain a polypeptide resin (IV) with the N-terminus connected with the protecting group.
The amino acids are identical or different and are selected, independently of one another, from natural amino acids or synthetic unnatural amino acids. The natural amino acids are for example selected from: l-alanine, D-alanine;
according to an embodiment of the invention, R 3 Selected from optionally substituted by one, two or more OH, NO 2 Halogen, CN, -S (O) C 1-6 Alkyl radical, C 1-6 Alkyl radical, C 1-6 Alkoxy-substituted phenyl; r 3 For example, in the case of a,
R 3 -CHO is preferably:
according to an embodiment of the invention, the unnatural amino acid containing a double bond is selected from the group consisting of:
according to an embodiment of the present invention, the amino group-containing resin in step (1) may be Rink amide AM resin, rink amide MBHA resin after Fmoc removal.
According to an embodiment of the invention, the N-terminally protected amino acids in steps (1), (3), (5), (7) have an equivalent weight of 2-10, such as 3-8, exemplary 5;
according to an embodiment of the present invention, the reaction in steps (1), (3), (5), (7) may be performed under the action of an organic base, which may be selected from at least one of piperidine, triethylamine, diisopropylethylamine, pyridine, N-dimethylaminopyridine, DBU;
according to an embodiment of the present invention, the reaction in steps (1), (3), (5), (7) may be performed under the action of a condensing agent, which may be at least one of HATU, EDCI, DIC, HOBt, DCC;
according to an embodiment of the invention, the organic base in steps (1), (3), (5), (7) has an equivalent weight of 2 to 20, such as 3 to 15, exemplary 10;
according to an embodiment of the invention, the equivalent weight of the condensing agent in steps (1), (3), (5), (7) is 2 to 10, such as 3 to 8, exemplary 5;
according to an embodiment of the present invention, in the above step, the protecting group at the N-terminus may be an Fmoc protecting group.
According to an embodiment of the present invention, the reducing agent in step (4) may be at least one of sodium borohydride, sodium cyanoborohydride, sodium acetate borohydride, and potassium borohydride;
according to an embodiment of the present invention, the reaction of the polypeptide resin 4 with the N-terminal protected amino acid in the step (5) may be performed by the action of a coupling agent, which may be Oxyma; the equivalent weight of the coupling agent can be from 2 to 10, for example from 3 to 8, illustratively 5. The reaction may be carried out with a catalyst, which may be DMAP;
the invention also provides the cyclic polypeptide prepared by the method.
A cyclic polypeptide selected from the structures:
advantageous effects
The invention provides a novel method for promoting polypeptide solid-phase cyclization reaction by amido bond substitution. The method firstly introduces removable amido bond substituent groups (Hmb, hnb and Hmnb) in solid phase synthesis to promote double bond-containing unnatural amino acid (such as terminal group double bond-containing unnatural amino acid) olefin metathesis reaction to be carried out under very mild solid phase conditions. The method has the following advantages: 1) The amino acid is cheap and easy to obtain (100 RMB/g), and complex synthesis is not needed; 2) The length of a side chain is convenient to regulate, and the tolerance of cyclization reaction on the size of a ring system is high; 3) Aiming at the characteristic of polypeptide reaction sequence dependence, an amido bond substituent group is introduced, so that the reaction has high sequence tolerance, special modified resin is not needed, the condition is mild, the product purity is high, and the separation is easy.
Detailed Description
The technical solution of the present invention will be further described in detail with reference to specific embodiments. It is to be understood that the following examples are only illustrative and explanatory of the present invention and should not be construed as limiting the scope of the present invention. All the technologies realized based on the above-mentioned contents of the present invention are covered in the protection scope of the present invention.
Unless otherwise indicated, the raw materials and reagents used in the following examples are all commercially available products or can be prepared by known methods.
Obtaining Hmb, hnb and Hmnb:
hmb purchased from graduate pharmaceutical group;
hnb was obtained by a synthetic method reported in the literature (An Activated O → N Acyl Transfer Autoxiliary: effective Amide-Backbone subscription of Hindered "Difficult" Peptides, journal of Organic Chemistry,65 (18), 5460-5468;
1 H NMR(300MHz,CDCl 3 )δ12.12(s,1H),10.34(s,1H),7.64(t,J＝8.1Hz,1H),7.60–7.54(m,1H),7.31(d,J＝8.3Hz,1H).
hmnb was obtained by reference Synthesis (Robust Chemical Synthesis of Membrane Proteins through a General Method of Removable Back carbon modification. Journal of the American Chemical Society,138 (10), 3553-3561 2016
1 H NMR(300MHz,CDCl 3 )δ11.78(s,1H),9.81(s,1H),8.33(s,1H),6.59(s,1H),4.04(s,3H).
Unnatural amino acids and numbering used in the invention:
example 1 Synthesis of Cyclic peptide 1 with Hmnb as substituent group
1.1 Synthesis of Linear polypeptide 1d
Rink amide AM resin was first added to the polypeptide tube and swollen with DCM for 30min. The polypeptide was then treated with 25% piperidine in DMF for 25min and washed three times with DMF, DCM and DMF in that order to give Fmoc (9-fluorenylmethoxycarbonyl) cleaved resin H 2 N-Resin. Coupling the first amino acid fragment 5-fold equivalents of SX 5 5 times equivalent of HATU,10 times equivalent of DIPEA (N, N-diisopropylethylamine) was dissolved in DMF, added to a polypeptide synthesis tube and reacted for 2 hours, and the resulting resin was washed three times with DMF, DCM, and DMF in this order. The polypeptide was then treated with 25% piperidine DMF for 25min and washed three times with DMF, DCM and DMF in that order to give Fmoc-removed resin H 2 N-SX 5 -a Resin. Then, the freshly prepared DMF solution (5 equivalents of Fmoc-protected L-alanine, 5 equivalents of HATU,10 equivalents of DIPEA) was added thereto and reacted for 2 hours, and the resulting resin was washed three times with DMF, DCM and DMF in that order, and then 25% of DMF was addedTreatment of the polypeptide with piperidine DMF for 25min followed by three washes with DMF, DCM, DMF in that order provided polypeptide fragment 1a. Dissolving 2 times of equivalent of Hmnb in DMF, adding into 1a, reacting for 30min, and adding 5 times of equivalent of NaBH 4 Reducing the solution in DMF for 5min to obtain the polypeptide fragment 1b. 5 equivalents of Fmoc-protected L-alanine, 5 equivalents of DIC,5 equivalents of Oxyma, and a catalytic amount of DMAP were dissolved in DMF, and the mixture was added to a polypeptide tube containing the polypeptide 1b to react for 4 hours, and Fmoc was removed by the same method as above to obtain a polypeptide fragment 1c. And (2) adding Fmoc-protected L-alanine into the obtained 1c by adopting the same method, reacting, removing Fmoc to obtain a product connected with a fourth amino acid fragment, adding Fmoc-protected (S) -2-aminohex-5-olefine acid, and removing Fmoc to obtain a product connected with a fifth amino acid fragment. Finally, acetic anhydride is used: DIEA: DMF =1:1:8 to obtain polypeptide 1d. Polypeptide 1d + : calcd 750.3, found: 750.6; retention time (min): 21.618;
1.2 Synthesis of Cyclic peptide 1 from polypeptide 1d
A0.25 equivalent solution of Hovyda-Grubbs-II (HG-II) in DCM was added to the polypeptide tube containing polypeptide 1d for 3 hours and the reaction was repeated, followed by three washes with DCM, DMF, DMSO to give polypeptide fragment 1e, which was cleaved with a small amount of the resinoid 1e and confirmed to completion by LCMS analysis.
To a polypeptide tube containing 1e was added 20 equivalents of 2-NBSH followed by DCM and Triethylamine (TEA) separately and the reaction was repeated at 40 ℃ for 4 hours, followed by DCM, DMF/H 2 After three washes of O (1).
Adding 6M SnCl into polypeptide tube containing 1f 2 And 10mM HCl/MeOH in DMF and reacted at room temperature for two hours, followed by DMF, DMF/H 2 O (1). The resulting resinAfter drying in vacuo, the cleavage agent (TFA (trifluoroacetic acid)/TIPS (triisopropylsilane)/phenol/H was added 2 O,88/2/5, v/v/v/v) for 4-5 hours to obtain a cutting fluid containing the polypeptide 1. It was then concentrated and precipitated three times with frozen ether and the resulting product was analyzed by LCMS and purified by HPLC to give polypeptide 1.
Polypeptide 1M + H + : calcd 501.2, found: 501.4; retention time (min): 10.218;
preparation of 2-Nitrobenzenesulfonylhydrazide (2-NBSH) reference is made to: buszek K R, brown N.improved Method for the derivative Reduction of Multiple Bonds on Solid-Supported Substrates [ J ]. Journal of Organic Chemistry,2007,72 (8): 3125.
Example 2 Synthesis of Cyclic peptides 2R and 2S with Hnb as substituent
Polypeptide 2a was synthesized using a synthetic route similar to 1d with Hnb instead of Hmnb.
A0.25 equivalent solution of Hovyda-Grubbs-II (HG-II) in DCM catalyst was added to the polypeptide tube containing polypeptide 2a for 3 hours and the reaction was repeated, followed by three washes with DCM, DMF, DMSO, DCM. The resulting resin was dried in vacuo and 20 equivalents of 2-NBSH were added followed by DCM and Triethylamine (TEA) respectively and the reaction was repeated at 40 ℃ for 4 hours followed by DMF, DMF/H 2 O (1), THF, DMF were added after three washes and reacted under uv lamp (PLS-LAM 250) for 4 hours, followed by three washes with DMF, DCM. The resulting resin was dried in vacuo and the cleavage agent (TFA/TIPS/phenol/H) was added 2 O,88/2/5, v/v/v/v) for 2 hours to obtain a cutting fluid containing the polypeptides 2S and 2R. It was then concentrated and precipitated three times with frozen ether, and the resulting product was analyzed by LCMS and purified using HPLC to afford polypeptides 2S and 2R.
Polypeptide 2S: m + H + : calculated 497.3, found: 497.6; retention time (min): 16.280;
polypeptide 2R: m + H + : the calculated value is 497.3, and the calculated value,measured value: 497.6; retention time (min): 20.299;
example 3 Synthesis of polypeptide 3 with Hmnb as substituent group
A0.25 equivalent solution of Hovyda-Grubbs-II (HG-II) in DCM catalyst was added to the polypeptide tube containing polypeptide 3a for 3 hours and the reaction was repeated, followed by three washes with DCM, DMF, DMSO, DCM. The resulting resin was dried in vacuo and the cleavage agent (TFA/TIPS/phenol/H) was added 2 O,88/2/5, v/v/v/v) for 2 hours to obtain a cutting fluid containing the polypeptide 3. It was then concentrated and precipitated three times with frozen ether and the resulting product analyzed by LCMS and purified using HPLC to give polypeptide 3.
Polypeptide 3: m + H + : calcd 831.4, found: 832.4; retention time (min): 10.524;
example 4 Synthesis of polypeptide 4 with Hmnb as substituent group
A0.25 equivalent solution of Hovyda-Grubbs-II (HG-II) in DCM catalyst was added to the polypeptide tube containing polypeptide 4a for 3 hours and the reaction was repeated, followed by three washes with DCM, DMF, DMSO, DCM. The resulting resin was dried in vacuo and the cleavage agent (TFA/TIPS/phenol/H) was added 2 O,88/2/5, v/v/v/v) for 2 hours to obtain a cleavage solution containing the polypeptide 4. It was then concentrated and precipitated three times with frozen ether, and the resulting product was analyzed by LCMS and purified using HPLC to afford polypeptide 4.
Polypeptide 4: m +2H + : calculated 551.8, found: 551.9; retention time (min): 10.084;
example 5 Synthesis of polypeptide 5 with Hmnb as substituent group
LCMS analytical methods: solvent A:0.1% aqueous solution of tfa, solvent B:0.1% TFA in acetonitrile; gradient: the solvent B is 5 to 50 percent in 20 min; column temperature: 25 ℃; column type: c18,250 x 4.6mm x 5 μm. M +2H + : calculated values are: 692.9, found: 692.1.
example 6 Synthesis of polypeptide 6 with Hnb as substituent group
Polypeptide 6 was prepared using a similar method as in example 1.2.
LCMS analytical methods: solvent A:0.1% aqueous solution of tfa, solvent B:0.1% TFA in acetonitrile; gradient: the solvent B is 5 to 60 percent in 20 min; column temperature: 25 ℃; column type: c18,250 x 4.6mm x 5 μm.
M+H + : calculated values: 1406.7, found: 1407.0.
example 7 Synthesis of polypeptide 7 with Hnb as substituent
Polypeptide 7 was prepared using a similar method as in example 2.
LCMS analytical methods: solvent A:0.1% aqueous solution of tfa, solvent B:0.1% TFA in acetonitrile; gradient: the solvent B is 5 to 95 percent in 20 min; column temperature: 25 ℃; column type: c18,250 x 4.6mm x 5 μm.
M+H + : calculated values: 1420.7, found: 1421.7.
example 8 Synthesis of polypeptide 8 with Hnb as substituent group
Polypeptide 8 was prepared using a similar method as in example 2.
LCMS analytical methods: solvent A:0.1% aqueous solution of tfa, solvent B:0.1% TFA in acetonitrile; gradient: the solvent B is 5 to 95 percent in 20 min; column temperature: 25 ℃; column type: c18,250 x 4.6mm x 5 μm.
M+H + : calculated values are: 1272.7, found: 1272.9.
example 9 Synthesis of polypeptide 9 with Hnb as substituent group
Polypeptide 9 was prepared using a similar method as in example 2.
LCMS analytical methods: solvent A:0.1% aqueous solution of tfa, solvent B:0.1% TFA in acetonitrile; gradient: the solvent B is 5 to 95 percent in 20 min; column temperature: 25 ℃; column type: c18,250 x 4.6mm x 5 μm.
M+H + : calculated values: 1286.7, found: 1289.1.
the embodiments of the present invention have been described above. However, the present invention is not limited to the above embodiment. Any modification, equivalent replacement, or improvement made without departing from the spirit and principle of the present invention shall fall within the protection scope of the present invention.
Claims (9)
1. A method for solid phase synthesis of a cyclic polypeptide comprising the steps of:
G 0 is-NH-, -O-, -NH-peptide chain-, -O-peptide chain-, wherein the peptide chain has n 1 Amino acids; n is a radical of an alkyl radical 1 Greater than or equal to 1;
G 1 is n 2 Amino acid residues; n is a radical of an alkyl radical 2 Is greater than or equal to 1;
G 2 is n 3 Amino acid residues; n is a radical of an alkyl radical 3 Greater than or equal to 1;
R 1 is-CH 2 -R 3 ，R 3 Is a substituted aryl group;
R 2 is H, -NH-R', or a peptide chain, wherein the peptide chain has n 4 Amino acids; n is 4 Greater than or equal to 1; r' is an amino protecting group (e.g., acetyl);
x and Y are the same or different and are independently selected from- (CH) 2 ) m1 (CR 4 R 5 )(CH 2 ) m2 -、-(CH 2 ) m1 S(CH 2 ) m2 -、-(CH 2 ) m1 O(CH 2 ) m2 -；R 4 、R 5 Are identical or different and are independently selected from H, - (CH) 2 ) m3 CH 3 、-OCH 3 Or phenyl; m1, m2 and m3 are independently of one another selected from integers from 0 to 8, preferably from 0 to 5, for example 1, 2, 3, 4;
(A) Mixing polypeptide resin (IV) with a catalyst, carrying out olefin double decomposition reaction on two double bonds in the polypeptide resin in the presence of the catalyst to obtain cyclic polypeptide resin (III) with a double bond on the ring, and then adding a reducing agent to carry out reduction reaction to obtain cyclic polypeptide resin (II), wherein the polypeptide resin (IV) is a polypeptide chain connected to the resin;
(B) Removing the group R by subjecting the saturated cyclic polypeptide resin (II) to elimination reaction 1 (ii) a Then removing the resin to obtain the cyclic polypeptide (I);
preferably, R 3 Is optionally substituted by one, two or more OH, NO 2 Halogen, CN, C 1-6 Alkyl radical, C 1-6 Alkoxy, -SO-C 1-6 Alkyl, or-SO 2 -C 1-6 Alkyl-substituted groups as follows: phenyl, naphthyl; is exemplified by
2. The method of claim 1, wherein n is 1 Is an integer from 1 to 100; n is 2 Is an integer of 1 to 6; n is a radical of an alkyl radical 3 Is an integer of 1 to 6; n is a radical of an alkyl radical 4 Is an integer of 1 to 100.
3. The process according to claim 1 or 2, characterized in that the catalyst in step (a) is an olefin metathesis catalyst, such as Hovyda-Grubbs-II (HG-II) catalyst, grubbs primary catalyst or Grubbs secondary catalyst;
preferably, in step (a), the reduction reaction is performed under the action of an organic base, such as at least one of piperidine, triethylamine, diisopropylethylamine, pyridine, N-dimethylaminopyridine, DBU;
preferably, in step (a), the reducing agent is a hydrogenation reduction catalyst, for example 2-NBSH;
preferably, the group R is eliminated in step (B) 1 The reaction of (a) is carried out in the presence of an acid or in the presence of ultraviolet light;
preferably, the reaction of removing the resin in the step (B) may be performed in a cutting agent.
4. The method according to any one of claims 1 to 3, wherein the polypeptide resin (IV) is prepared by a method comprising:
optionally, (1) reacting the amino group-containing resin with an N-terminally protected amino acid, followed by removal of the N-terminally protecting group; optionally repeating the above steps to obtain a linkage n 1 Polypeptide resin 1,n of amino acids 1 Is greater than or equal to 1;
(2) Reacting amino-containing resin or the polypeptide resin 1 in the step (1) with unnatural amino acid which is protected by the N terminal and contains double bonds, and then removing the protecting group of the N terminal to obtain polypeptide resin 2;
(3) Reacting the polypeptide resin 2 in the step (2) with the N-terminal protected amino acid, then removing the N-terminal protecting group, and optionally repeating the steps to obtain the peptide with N attached 2 Polypeptide resin of amino acid 3,n 2 Is greater than or equal to 1;
(4) Polypeptide resin 3 and compound R 3 -CHO mixtures wherein aldehyde groups are bound to polypeptidesReacting amino in the resin to obtain imine polypeptide resin, adding a reducing agent to obtain a product with an N-terminal connected with an R 3 -CH 2 -polypeptide resin 4;
(5) Reacting the polypeptide resin 4 obtained in the step (4) with N-terminal protected amino acid, then removing the N-terminal protecting group, and optionally repeating the above steps to obtain the peptide with N 3 Polypeptide resin of amino acid 5,n 3 Is greater than or equal to 1;
(6) Polypeptide resin 5 and C with double bond as terminal group 3-10 Reacting olefinic acid (such as 4-alkenyl pentanoic acid, 5-alkenyl hexanoic acid, etc.) to obtain polypeptide resin (IV);
or reacting the polypeptide resin 5 with the N-terminal protected unnatural amino acid containing the double bond to obtain a polypeptide resin (IV); if necessary, the protecting group at the N terminal can be removed, and other protecting groups can be introduced into the N terminal;
or, a) reacting the polypeptide resin 5 with an unnatural amino acid protected at the N-terminus and containing a double bond, followed by removal of the N-terminal protecting group to give a polypeptide resin 6, b) reacting the polypeptide resin 6 with an N-terminal protected amino acid, followed by removal of the N-terminal protecting group, optionally repeating the above steps to give a compound having N attached thereto 4 Polypeptide resin of amino acids 6', n 4 Greater than or equal to 1; c) Introducing a protecting group (for example, reacting with acetic anhydride) into the N end of the polypeptide resin 6' to obtain a polypeptide resin (IV) with the N end connected with the protecting group;
the amino acids are the same or different and are independently selected from natural amino acids or synthetic unnatural amino acids; the natural amino acids are for example selected from: l-alanine, D-alanine.
5. The method of any one of claims 1-4, wherein R is 3 Selected from optionally substituted by one, two or more OH, NO 2 Halogen, CN, -S (O) C 1-6 Alkyl radical, C 1-6 Alkyl radical, C 1-6 Alkoxy-substituted phenyl; r 3 For example, the order of a,
R 3 -CHO is preferably:
6. the method of claim 4, wherein the double bond-containing unnatural amino acid is selected from the group consisting of:
7. the method according to claim 4, wherein the amino group-containing resin in step (1) is Rink amide AM resin, rink amide MBHA resin after Fmoc removal;
preferably, the reducing agent in the step (4) is at least one of sodium borohydride, sodium cyanoborohydride, sodium acetate borohydride and potassium borohydride;
preferably, the reaction of the polypeptide resin 4 and the N-terminal protected amino acid in the step (5) is carried out under the action of a coupling agent, wherein the coupling agent can be Oxyma; the reaction may be carried out over a catalyst, which may be DMAP.
8. A cyclic polypeptide produced by the method of any one of claims 1-7.
9. A cyclic polypeptide selected from the structures:
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Application publication date: 20221122 |