CN113621022A - Synthetic method of cyclic peptide - Google Patents
Synthetic method of cyclic peptide Download PDFInfo
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- CN113621022A CN113621022A CN202110810189.0A CN202110810189A CN113621022A CN 113621022 A CN113621022 A CN 113621022A CN 202110810189 A CN202110810189 A CN 202110810189A CN 113621022 A CN113621022 A CN 113621022A
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- 108010069514 Cyclic Peptides Proteins 0.000 title claims abstract description 17
- 102000001189 Cyclic Peptides Human genes 0.000 title claims abstract description 17
- 238000010189 synthetic method Methods 0.000 title abstract description 6
- 229920005989 resin Polymers 0.000 claims abstract description 37
- 239000011347 resin Substances 0.000 claims abstract description 37
- 238000010168 coupling process Methods 0.000 claims abstract description 34
- JDKLPDJLXHXHNV-MFVUMRCOSA-N (3s,6s,9r,12s,15s,23s)-15-[[(2s)-2-acetamidohexanoyl]amino]-9-benzyl-6-[3-(diaminomethylideneamino)propyl]-12-(1h-imidazol-5-ylmethyl)-3-(1h-indol-3-ylmethyl)-2,5,8,11,14,17-hexaoxo-1,4,7,10,13,18-hexazacyclotricosane-23-carboxamide Chemical compound C([C@@H]1C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CC=2C3=CC=CC=C3NC=2)C(=O)N[C@@H](CCCCNC(=O)C[C@@H](C(N[C@@H](CC=2NC=NC=2)C(=O)N1)=O)NC(=O)[C@@H](NC(C)=O)CCCC)C(N)=O)C1=CC=CC=C1 JDKLPDJLXHXHNV-MFVUMRCOSA-N 0.000 claims abstract description 33
- 238000005859 coupling reaction Methods 0.000 claims abstract description 33
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- OJBNDXHENJDCBA-QFIPXVFZSA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-6-(prop-2-enoxycarbonylamino)hexanoic acid Chemical compound C1=CC=C2C(COC(=O)N[C@@H](CCCCNC(=O)OCC=C)C(=O)O)C3=CC=CC=C3C2=C1 OJBNDXHENJDCBA-QFIPXVFZSA-N 0.000 claims abstract description 17
- 125000003088 (fluoren-9-ylmethoxy)carbonyl group Chemical group 0.000 claims abstract description 16
- 238000007363 ring formation reaction Methods 0.000 claims abstract description 13
- FBNFRRNBFASDKS-IBGZPJMESA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-4-oxo-4-prop-2-enoxybutanoic acid Chemical compound C1=CC=C2C(COC(=O)N[C@@H](CC(=O)OCC=C)C(=O)O)C3=CC=CC=C3C2=C1 FBNFRRNBFASDKS-IBGZPJMESA-N 0.000 claims abstract description 9
- SJVFAHZPLIXNDH-JOCHJYFZSA-N (2r)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-3-phenylpropanoic acid Chemical compound C([C@H](C(=O)O)NC(=O)OCC1C2=CC=CC=C2C2=CC=CC=C21)C1=CC=CC=C1 SJVFAHZPLIXNDH-JOCHJYFZSA-N 0.000 claims abstract description 7
- 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 abstract description 7
- 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 abstract description 7
- 102000004196 processed proteins & peptides Human genes 0.000 claims abstract description 7
- 239000007790 solid phase Substances 0.000 claims abstract description 7
- XXMYDXUIZKNHDT-QNGWXLTQSA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-3-(1-tritylimidazol-4-yl)propanoic acid Chemical compound C([C@@H](C(=O)O)NC(=O)OCC1C2=CC=CC=C2C2=CC=CC=C21)C(N=C1)=CN1C(C=1C=CC=CC=1)(C=1C=CC=CC=1)C1=CC=CC=C1 XXMYDXUIZKNHDT-QNGWXLTQSA-N 0.000 claims abstract description 6
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- 238000010532 solid phase synthesis reaction Methods 0.000 claims abstract description 6
- VCFCFPNRQDANPN-IBGZPJMESA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)hexanoic acid Chemical compound C1=CC=C2C(COC(=O)N[C@@H](CCCC)C(O)=O)C3=CC=CC=C3C2=C1 VCFCFPNRQDANPN-IBGZPJMESA-N 0.000 claims abstract description 5
- 230000021736 acetylation Effects 0.000 claims abstract description 4
- 238000006640 acetylation reaction Methods 0.000 claims abstract description 4
- 238000006467 substitution reaction Methods 0.000 claims description 16
- KDLHZDBZIXYQEI-UHFFFAOYSA-N Palladium Chemical compound [Pd] KDLHZDBZIXYQEI-UHFFFAOYSA-N 0.000 claims description 12
- 230000002194 synthesizing effect Effects 0.000 claims description 10
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 9
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 9
- FEMOMIGRRWSMCU-UHFFFAOYSA-N ninhydrin Chemical compound C1=CC=C2C(=O)C(O)(O)C(=O)C2=C1 FEMOMIGRRWSMCU-UHFFFAOYSA-N 0.000 claims description 8
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 6
- 229910052763 palladium Inorganic materials 0.000 claims description 6
- 239000012071 phase Substances 0.000 claims description 6
- PARWUHTVGZSQPD-UHFFFAOYSA-N phenylsilane Chemical compound [SiH3]C1=CC=CC=C1 PARWUHTVGZSQPD-UHFFFAOYSA-N 0.000 claims description 6
- 238000003776 cleavage reaction Methods 0.000 claims description 5
- JGFZNNIVVJXRND-UHFFFAOYSA-N N,N-Diisopropylethylamine (DIPEA) Chemical compound CCN(C(C)C)C(C)C JGFZNNIVVJXRND-UHFFFAOYSA-N 0.000 claims description 4
- 239000000243 solution Substances 0.000 claims description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 4
- FPIRBHDGWMWJEP-UHFFFAOYSA-N 1-hydroxy-7-azabenzotriazole Chemical compound C1=CN=C2N(O)N=NC2=C1 FPIRBHDGWMWJEP-UHFFFAOYSA-N 0.000 claims description 3
- 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 3
- 230000005526 G1 to G0 transition Effects 0.000 claims description 3
- 239000007821 HATU Substances 0.000 claims description 3
- 239000007864 aqueous solution Substances 0.000 claims description 3
- 238000004108 freeze drying Methods 0.000 claims description 3
- YTJSFYQNRXLOIC-UHFFFAOYSA-N octadecylsilane Chemical compound CCCCCCCCCCCCCCCCCC[SiH3] YTJSFYQNRXLOIC-UHFFFAOYSA-N 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
- -1 Rink Amide-AM Resin Chemical compound 0.000 claims description 2
- 238000003556 assay Methods 0.000 claims description 2
- 239000011248 coating agent Substances 0.000 claims description 2
- 238000000576 coating method Methods 0.000 claims description 2
- 230000009089 cytolysis Effects 0.000 claims description 2
- 238000004007 reversed phase HPLC Methods 0.000 claims description 2
- 239000011259 mixed solution Substances 0.000 claims 1
- 230000008569 process Effects 0.000 abstract description 8
- 150000003949 imides Chemical class 0.000 abstract description 6
- 238000005897 peptide coupling reaction Methods 0.000 abstract description 3
- 239000003814 drug Substances 0.000 abstract description 2
- 229940079593 drug Drugs 0.000 abstract description 2
- 101710201349 Metallothionein B Proteins 0.000 abstract 1
- 102100031347 Metallothionein-2 Human genes 0.000 abstract 1
- 101710094505 Metallothionein-2 Proteins 0.000 abstract 1
- 238000006243 chemical reaction Methods 0.000 description 21
- 230000015572 biosynthetic process Effects 0.000 description 17
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 15
- WFDIJRYMOXRFFG-UHFFFAOYSA-N Acetic anhydride Chemical compound CC(=O)OC(C)=O WFDIJRYMOXRFFG-UHFFFAOYSA-N 0.000 description 12
- 239000000047 product Substances 0.000 description 12
- 238000003786 synthesis reaction Methods 0.000 description 12
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 8
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 description 8
- 235000001014 amino acid Nutrition 0.000 description 8
- 238000005303 weighing Methods 0.000 description 8
- YDNMHDRXNOHCJH-UHFFFAOYSA-N 3-aminopyrrolidine-2,5-dione Chemical compound NC1CC(=O)NC1=O YDNMHDRXNOHCJH-UHFFFAOYSA-N 0.000 description 7
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 6
- 238000005406 washing Methods 0.000 description 6
- 238000002360 preparation method Methods 0.000 description 5
- NQRYJNQNLNOLGT-UHFFFAOYSA-N Piperidine Chemical compound C1CCNCC1 NQRYJNQNLNOLGT-UHFFFAOYSA-N 0.000 description 4
- 239000003875 Wang resin Substances 0.000 description 4
- 229940024606 amino acid Drugs 0.000 description 4
- 150000001413 amino acids Chemical class 0.000 description 4
- 230000005587 bubbling Effects 0.000 description 4
- 229910052757 nitrogen Inorganic materials 0.000 description 4
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 description 4
- 238000003746 solid phase reaction Methods 0.000 description 4
- 230000008961 swelling Effects 0.000 description 4
- 230000003213 activating effect Effects 0.000 description 3
- 239000000706 filtrate Substances 0.000 description 3
- 239000005457 ice water Substances 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 125000006239 protecting group Chemical group 0.000 description 3
- YDNMHDRXNOHCJH-REOHCLBHSA-N (3s)-3-aminopyrrolidine-2,5-dione Chemical class N[C@H]1CC(=O)NC1=O YDNMHDRXNOHCJH-REOHCLBHSA-N 0.000 description 2
- BDNKZNFMNDZQMI-UHFFFAOYSA-N 1,3-diisopropylcarbodiimide Chemical compound CC(C)N=C=NC(C)C BDNKZNFMNDZQMI-UHFFFAOYSA-N 0.000 description 2
- YEDUAINPPJYDJZ-UHFFFAOYSA-N 2-hydroxybenzothiazole Chemical compound C1=CC=C2SC(O)=NC2=C1 YEDUAINPPJYDJZ-UHFFFAOYSA-N 0.000 description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 2
- 230000004913 activation Effects 0.000 description 2
- 239000007822 coupling agent Substances 0.000 description 2
- 238000010511 deprotection reaction Methods 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- NPZTUJOABDZTLV-UHFFFAOYSA-N hydroxybenzotriazole Substances O=C1C=CC=C2NNN=C12 NPZTUJOABDZTLV-UHFFFAOYSA-N 0.000 description 2
- 230000002035 prolonged effect Effects 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- 238000007086 side reaction Methods 0.000 description 2
- DCXYFEDJOCDNAF-UHFFFAOYSA-N Asparagine Natural products OC(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-N 0.000 description 1
- DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical compound OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 0.000 description 1
- 238000005917 acylation reaction Methods 0.000 description 1
- 150000001408 amides Chemical class 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 229960001230 asparagine Drugs 0.000 description 1
- 235000009582 asparagine Nutrition 0.000 description 1
- 238000005815 base catalysis Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- WVZWEMOFSIEEMU-UHFFFAOYSA-N indene-1,2,3-trione Chemical compound C1=CC=C2C(=O)C(=O)C(=O)C2=C1 WVZWEMOFSIEEMU-UHFFFAOYSA-N 0.000 description 1
- 238000011031 large-scale manufacturing process Methods 0.000 description 1
- 125000003473 lipid group Chemical group 0.000 description 1
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- 108010080780 melanotan-II Proteins 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
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Images
Classifications
-
- 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
-
- 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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- 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 discloses a synthetic method of cyclic peptide, belongs to the technical field of polypeptide drug biochemistry, and aims to solve the problem that hetero-peaks of imide are formed by Asp in the conventional linear peptide coupling process. The method comprises selecting amino resin, preparing Fmoc-Lys (alloc) -amino resin, coupling Fmoc-Trp (Boc) -OH, Fmoc-Arg (pbf) -OH, Fmoc-D-Phe-OH and Fmoc-His (Trt) -OH in sequence according to Fmoc solid phase synthesis strategy; Fmoc-Asp (OAll) -OH; removing Alloc and OAll groups on Lys and Asp in one time; solid phase cyclization; removing the Fmoc group at the N end of Asp, continuing coupling Fmoc-Nle-OH, and then performing acetylation to complete coupling; cutting off the polypeptide from the resin to obtain MT-II crude peptide; and (5) purifying.
Description
Technical Field
The invention belongs to the technical field of polypeptide drug biochemistry, and particularly relates to a synthetic method of cyclic peptide.
Background
In Fmoc solid phase synthesis, the most common side reaction affecting Asp is the formation of asparagine, strong bases such as piperidine and DBU are selected as effective removal reagents for Fmoc in each cycle, N on the alpha-amido bond of Asp and the beta-carbonyl of the side chain form a ring, and the lipid protecting group of the side chain is simultaneously lost. Aspartimides are susceptible to base catalysis to stereoisomerism and to ring-opening reactions, resulting in the formation of various forms of by-products.
The amount of aspartimide formation is determined primarily by the time of exposure to the base and the strength of the base, and the use of DBU is more likely to result in the formation of aspartimides than piperidine. Some methods for overcoming the side reaction exist, for example, Hmb protection is introduced into an amino acid before Asp, but acylation reaction of an amino acid after Hmb protection amino acid coupling is difficult, and related raw materials are expensive, so that large-scale production is not facilitated.
This also occurs during the synthesis of MT-II. The peptide sequence of MT-II (Melanotan II, Chinese name: melanotan II) is as follows: Ac-Nle-cyclo [ Asp-His-D-Phe-Arg-Trp-Lys ] -NH2, molecular weight 1024.2, required to form a side chain amide cyclized structure, the structure of which is shown in figure 1.
The conventional synthesis method of MT-II is to synthesize Ac-Nle-Asp (OAll) -His (Trt) -D-Phe-Arg (Pbf) -Trp (Boc) -Lys (Alloc) -RinkResin, then remove OAll and Alloc protecting groups simultaneously, and then carry out solid phase side chain cyclization. The patent CN101195654B "a solid phase synthesis method of melanotan-II" is to synthesize linear peptide and cyclize on resin to obtain MT-II.
However, during the synthesis of linear peptides, when Asp is defmoc, a significant hetero-peak (13.533) appears after coupling Nle, which is the peak where Asp forms an imide, as shown in FIG. 2.
After the linear peptide resin is coupled, removing OAll and Alloc protecting groups by adopting palladium tetratriphenylphosphine/phenyl silane, and then adding a coupling agent for solid phase cyclization, wherein two peaks with the molecular weight of 1024 are formed in the crude peptide, wherein the content of a hetero-peak is about 25 percent (RT13.318), and the content of a target peak is about 38 percent (RT13.4.653). Proves that in the process of coupling linear peptide, the Asp has the phenomenon of aspartimide due to the use of strong base in the process of Fmoc removal.
Disclosure of Invention
The invention aims to provide a synthetic method of cyclic peptide, which aims to solve the problem that the quality of melanotan II is influenced by a hetero-peak of imide formed by Asp in the conventional linear peptide coupling process.
In order to solve the problems, the technical scheme of the invention is as follows:
a method for synthesizing cyclic peptide comprises the following steps:
step A, selecting an amino resin, and preparing Fmoc-Lys (alloc) -amino resin;
step B, coupling Fmoc-Trp (Boc) -OH, Fmoc-Arg (pbf) -OH, Fmoc-D-Phe-OH and Fmoc-His (Trt) -OH sequentially to the product obtained in the step A according to an Fmoc solid phase synthesis strategy; Fmoc-Asp (OAll) -OH;
c, removing Alloc and OAll groups on Lys and Asp of the product obtained in the step B by adopting palladium tetratriphenylphosphine and phenyl silane;
step D, performing solid phase cyclization on the product obtained in the step C on resin;
e, removing the Fmoc group at the N end of Asp from the product obtained in the step D, continuing coupling Fmoc-Nle-OH, and then performing acetylation to complete coupling;
step F, cleaving the polypeptide from the resin by cleavage reaction of the product obtained in step E to obtain crude peptide of MT-II (Melanotan II, Chinese name: melanotan II);
and G, purifying the MT-II prepared in the step F to obtain MT-II refined peptide.
Further, the amino Resin in the step A is Rinkamide Resin, Rinkamide-AM Resin, Rink Amide-MBHAresin; the amino acid substitution degree is 0.2-0.6 mmol/g.
Further, the amino Resin is Rinkamide-AM Resin; the degree of amino acid substitution was 0.4 mmol/g.
Further, the sequential coupling of Fmoc-Trp (Boc) -OH, Fmoc-Arg (pbf) -OH, Fmoc-D-Phe-OH, Fmoc-His (Trt) -OH as described in step B; during Fmoc-Asp (OAll) -OH, the completion of coupling was determined by ninhydrin assay; it is necessary to ensure that the N-terminal Fmoc group cannot be removed after coupling of Fmoc-Asp (OAll) -OH.
Further, the cyclization conditions in the step D are as follows: during coupling, the side chain coupling system of Lys and Asp is HATU/HOAt/DIPEA.
Further, the lysis solution of the cleavage reaction in step F is a mixture of TFA, EDT and water, and the volume ratio is: TFA: EDT (electro-thermal transfer coating): water 90: 8: 2.
and further, in the step F, purifying by adopting a reversed-phase high-pressure liquid chromatography, taking reversed-phase octadecylsilane chemically bonded silica as a stationary phase and taking 0.1% acetic acid aqueous solution and acetonitrile as a mobile phase, collecting target peak fractions, concentrating and freeze-drying to obtain the MT-II refined peptide.
The invention has the following beneficial effects:
(1) the invention aims at the situation that the aspartimide is formed in the process of synthesizing MT-II, and effectively avoids the formation of the aspartimide by changing the synthesis strategy. The strategy breaks through the conventional thinking that linear full-chain synthesis is carried out firstly and then cyclization is carried out. After Asp synthesis, cyclization is carried out and then the synthesis is continued. After linear peptide resin is coupled to Asp (OAll), removing Fmoc, adding palladium tetratriphenylphosphine and phenyl silane to remove OAll and Alloc protecting groups, adding a coupling agent to carry out solid phase cyclization, removing Fmoc protection on an Asp main chain, and continuing to couple other amino acids. This effectively solves the phenomenon of aspartimide from the source. Further solves the problem that hetero-peak of imide formed by Asp in the process of coupling linear peptide, and leads the finally prepared product-melanotan II to have higher purity.
(2) The whole processing course of the invention does not adopt high-price raw materials, and skillfully changes the synthesis sequence, thereby reducing the exposure times and time of Asp side chains in a strong alkaline environment, reducing the risk of forming aspartimide and greatly reducing the synthesis cost of the melanotan II.
Drawings
FIG. 1 is a structural diagram of MT-II (Melanotan II, Chinese name: melanotan II);
FIG. 2 is a chromatogram of the product after coupling Nle when Asp is Fmoc-removed in a conventional linear coupling method; wherein the distinct hetero-peak (RT 13.533min) is the Asp-forming imide peak;
FIG. 3 is a chromatogram of a crude peptide prepared by cyclization after linear coupling; wherein the content of the hetero-peak is about 25% (RT13.318 min), and the content of the target peak is about 38% (RT13.4.653 min);
FIG. 4 is a chromatogram of a crude MT-II peptide resin prepared in step E of example 4.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions of the embodiments of the present invention will be described clearly and completely with reference to the accompanying drawings of the embodiments of the present invention, and it is obvious that the described embodiments are some, but not all embodiments of the present invention. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention.
To demonstrate the feasibility and effectiveness of the synthetic method of the present invention, the steps were performed separately.
Example 1
A method of synthesis of a cyclic peptide, step a: amino resin was selected and Fmoc-Lys (alloc) -amino resin was prepared.
Preparation of Resin with substitution degree of 0.60mmolFmoc-Lys (alloc) -Rinkamide Resin is selected, and the specific process is as follows:
weighing 100g of Rinkamide Resin with the substitution degree of 1.0mmol/g into a solid phase reaction column, adding DMF, and carrying out nitrogen bubbling for swelling for 60 minutes;
weighing Fmoc-Lys (alloc) -OH45.3 g (100mmol) and HOBt16.2 g (120mmol), dissolving with DMF, adding 20.3ml of LDIC at 0 ℃, activating for 5 minutes, and adding into a reaction column;
after two hours of reaction, 70mL of acetic anhydride and 60mL of pyridine were added, mixed and blocked for 24 hours, washed three times with DCM, and the resin was drained after methanol shrinkage to give Fmoc-Lys (alloc) -Wang resin with a degree of substitution of 0.58 mmol/g.
Example 2
A method of synthesis of a cyclic peptide, step a: amino resin was selected and Fmoc-Lys (alloc) -amino resin was prepared.
Preparation of Resin with 0.40mmol of Fmoc-Lys (alloc) -Rinkamide-AM Resin selection substitution degree, which comprises the following steps:
weighing 100g of Rink Amide-AM Resin with the substitution degree of 1.0mmol/g into a solid phase reaction column, adding DMF, and carrying out bubbling swelling for 60 minutes by nitrogen;
weighing Fmoc-Lys (alloc) -OH31.7 g (70mmol) and HOBt11.3 g (84mmol), dissolving with DMF, adding 14.2mL of LDIC at 0 ℃, activating for 5 min, and adding into a reaction column;
after two hours of reaction, 70mL of acetic anhydride and 60mL of pyridine were added, mixed and blocked for 24 hours, washed three times with DCM, and the resin was drained after methanol shrinkage to give Fmoc-Lys (alloc) -Wang resin with a degree of substitution of 0.40 mmol/g.
Example 3
A method of synthesis of a cyclic peptide, step a: amino resin was selected and Fmoc-Lys (alloc) -amino resin was prepared.
The preparation process of the Resin with the selection degree of 0.20mmolFmoc-Lys (alloc) -Rinkamide-MBHA Resin is as follows:
weighing 100g of Rink Amide-AM Resin with the substitution degree of 1.0mmol/g into a solid phase reaction column, adding DMF, and carrying out bubbling swelling for 60 minutes by nitrogen;
weighing Fmoc-Lys (alloc) -OH15.9 g (35mmol) and HOBt5.7 g (42mmol), dissolving with DMF, adding 7.1mL of LDIC at 0 ℃, activating for 5 minutes, and adding into a reaction column;
after two hours of reaction, 70mL of acetic anhydride and 60mL of pyridine were added, mixed and blocked for 24 hours, washed three times with DCM, and the resin was drained after methanol shrinkage to give Fmoc-Lys (alloc) -Wang resin with a degree of substitution of 0.22 mmol/g.
The results of the measured substitution degrees of Fmoc-Lys (alloc) -Wang resins finally obtained in examples 1 to 3 were not out of tolerance with the amino acid substitution degree of each resin, and it was confirmed that the amino resin selected in the present invention and the amino acid substitution degree thereof were feasible to be set to 0.2 to 0.6 mmol/g.
Since the properties of Fmoc-Lys (alloc) -amino Resin finally prepared in 3 examples were similar, they were arbitrarily selected and used in the following example 4, and the Fmoc-Lys (alloc) -Rinkamide-AM Resin finally prepared in example 2 was selected and used for subsequent synthesis.
Example 4
A method for synthesizing cyclic peptide, the steps after step A are as follows:
step B, coupling Fmoc-Trp (Boc) -OH, Fmoc-Arg (pbf) -OH, Fmoc-D-Phe-OH and Fmoc-His (Trt) -OH sequentially to the product obtained in the step A according to an Fmoc solid phase synthesis strategy; Fmoc-Asp (OAll) -OH.
Specifically, the method comprises the following steps:
weighing 100g of Fmoc-Lys (alloc) -Rink Amide-AM Resin with the substitution degree of 0.40mmol/g prepared in example 2 into a solid phase reaction column, adding DMF, and carrying out bubbling swelling for 60 minutes by nitrogen; then deprotected with DBLK for 6min +8min, washed 6 times with DMF.
63.2g (120mmol) of Fmoc-Trp (Boc) -OH and 22.5g (144mmol) of HOBT were weighed and dissolved in DMF, 25mL (144mmol) of DIPCDI was added under ice-water bath to activate for 3min, and then the mixture was put into a reaction column and reacted at room temperature for 2h, and the reaction end point was detected by ninhydrin (the reaction was stopped when the resin was colorless and transparent; the reaction was prolonged for 1 h when the resin was colored).
After the reaction is finished, washing the resin with DMF for 3 times, adding DBLK to perform deprotection for 6min +8min, washing the resin with DMF for 6 times, and detecting the color of the resin by ninhydrin.
Repeating the above coupling operation, and coupling Fmoc-Arg (pbf) -OH, Fmoc-D-Phe-OH and Fmoc-His (Trt) -OH sequentially according to peptide sequence; Fmoc-Asp (OAll) -OH.
And step C, removing Alloc and OAll groups on Lys and Asp of the product obtained in the step B in one step by adopting palladium tetratriphenylphosphine and phenylsilane.
Specifically, the method comprises the following steps:
the peptide resin obtained in step B was swollen with DCM, 98.5ml phenylsilane (800mmol) was added, 5.6g tetratriphenylphosphine palladium (8mmol) was added, the reaction was carried out at room temperature for 60min, and the removal of Alloc and OAll was confirmed by ninhydrin detection.
And D, performing solid-phase cyclization on the product obtained in the step C on the resin.
Specifically, the method comprises the following steps:
preparation of Fmoc-cyclo [ Asp-His-D-Phe-Arg-Trp-Lys ] -Rink Amide-AM Resin was performed:
weighing 15.2g of HATU (40mmol) and 6.5g of HOAt (48mmol), dissolving with 200ml of DMMF, adding 13.9ml of LDIPEA (80mmol) under ice water bath for activation for 3 minutes, adding into a reaction column for reaction for 2 hours, and detecting by an indantrione method to judge the reaction end point.
Washing with DMF for 6 times, shrinking and drying with methanol to obtain Fmoc-cyclo [ Asp-His-D-Phe-Arg-Trp-Lys ] -Rink Amide-AM Resin.
And E, removing the Fmoc group at the N end of Asp from the product obtained in the step D, continuing coupling Fmoc-Nle-OH, and then performing acetylation to complete coupling.
Specifically, the method comprises the following steps:
preparation of Ac-Nle-cyclo [ Asp-His-D-Phe-Arg-Trp-Lys ] -Rinkamide-AM Resin:
and D, treating the Fmoc-cyclo [ Asp-His-D-Phe-Arg-Trp-Lys ] -Rink Amide-AM Resin in the step D with DBLK for 6+8min, removing the Fmoc group, and washing with DMF for 6 times.
42.4g (120mmol) of Fmoc-Nle-OH and 22.5g (144mmol) of HOBT are weighed and dissolved in DMF, 25mL (144mmol) of DIPCDI is added in an ice-water bath for activation for 3min, the mixture is added into a reaction column and reacted for 2h at room temperature, and the reaction end point is detected by ninhydrin (the reaction is stopped if the resin is colorless and transparent; the reaction is prolonged for 1 h if the resin is colored). After the reaction is finished, washing the resin with DMF for 3 times, adding DBLK to perform deprotection for 6min +8min, washing the resin with DMF for 6 times, and detecting the color of the resin by ninhydrin.
76.1ml (800mmol) of acetic anhydride and 66.1ml (800mmol) of pyridine are weighed out and mixed into the resin, the reaction is carried out for 2h, and the resin is colorless and transparent when detected by ninhydrin. The resin was washed 6 times with DMF and then the resin was shrunk with methanol and dried under vacuum to give 182.4g of MT-II peptide resin.
After Nle coupling, a small sample fragment is cleaved for analysis, and the chromatogram is shown in FIG. 4, comparing with the conventional linear peptide coupling method (shown in FIG. 3), it can be seen that:
FIG. 3 is a chromatogram of a crude peptide prepared by conventional linear post-coupling cyclization, wherein the content of the hetero-peak is about 25% (RT13.318 min) and the content of the desired peak is about 38% (RT13.4.653 min);
FIG. 4 is a chromatogram of a crude peptide prepared according to this example, showing a purity of the fragment of about 65%, which is an improvement of 27% over the original method.
Therefore, the method of the invention effectively solves the phenomenon of the aspartimide from the source. Further solves the problem that hetero-peak of imide formed by Asp in the process of coupling linear peptide, and leads the finally prepared product-melanotan II to have higher purity.
And F, cleaving the polypeptide from the resin by using a cleavage reaction of the product obtained in the step E to obtain the crude peptide of MT-II (Melanotan II, Chinese name: melanotan II).
182.4g of the peptide resin obtained in step E was added to a 3L three-necked flask, and TFA pre-cooled to 0 ℃ or below, EDT: H2O ═ 90: 8: 2(V: V)1.82L, reacting at room temperature for 2 hours, filtering the resin, and collecting the filtrate. The resin was washed with a small amount of TFA and the filtrates combined.
The filtrate was slowly added to 18.4L of ethyl acetate for precipitation, centrifuged, washed 5 times with ethyl acetate and dried under reduced pressure to give 39.7g of crude peptide in 96.9% yield.
And G, purifying the MT-II prepared in the step F to obtain MT-II refined peptide.
39.7g of the solid obtained in step F were directly prepared by purification in a high performance liquid phase.
And (3) taking reverse-phase octadecylsilane as a stationary phase and taking 0.1% acetic acid aqueous solution and acetonitrile as mobile phases, collecting target peak fractions, concentrating and freeze-drying to obtain a pure product 18.8g, wherein the purity is higher than 99.0%, and the yield is 45.9%.
Claims (7)
1. A method for synthesizing cyclic peptide is characterized by comprising the following steps:
step A, selecting an amino resin, and preparing Fmoc-Lys (alloc) -amino resin;
step B, coupling Fmoc-Trp (Boc) -OH, Fmoc-Arg (pbf) -OH, Fmoc-D-Phe-OH and Fmoc-His (Trt) -OH sequentially to the product obtained in the step A according to an Fmoc solid phase synthesis strategy; Fmoc-Asp (OAll) -OH;
c, removing Alloc and OAll groups on Lys and Asp of the product obtained in the step B by adopting palladium tetratriphenylphosphine and phenyl silane;
step D, performing solid phase cyclization on the product obtained in the step C on resin;
e, removing the Fmoc group at the N end of Asp from the product obtained in the step D, continuing coupling Fmoc-Nle-OH, and then performing acetylation to complete coupling;
step F, cleaving the polypeptide from the resin by cleavage reaction of the product obtained in step E to obtain crude peptide of MT-II (Melanotan II, Chinese name: melanotan II);
and G, purifying the MT-II prepared in the step F to obtain MT-II refined peptide.
2. A method of synthesizing a cyclic peptide according to claim 1, wherein: the amino Resin in the step A is Rink Amide Resin, Rink Amide-AM Resin, Rink Amide-MBHA Resin; the amino acid substitution degree is 0.2-0.6 mmol/g.
3. A method of synthesizing a cyclic peptide according to claim 2, wherein: the amino Resin is Rink Amide-AM Resin; the degree of amino acid substitution was 0.4 mmol/g.
4. A method of synthesizing a cyclic peptide according to claim 1, wherein: the sequential coupling of Fmoc-Trp (Boc) -OH, Fmoc-Arg (pbf) -OH, Fmoc-D-Phe-OH, Fmoc-His (Trt) -O H as described in step B; during Fmoc-Asp (OAll) -OH, the completion of coupling was determined by ninhydrin assay; it is necessary to ensure that the N-terminal Fmoc group cannot be removed after coupling of Fmoc-Asp (OAll) -OH.
5. A method of synthesizing a cyclic peptide according to claim 1, wherein: the cyclization conditions in the step D are as follows: during coupling, the side chain coupling system of Lys and Asp is HATU/HOAt/DIPEA.
6. A method of synthesizing a cyclic peptide according to claim 1, wherein: and F, preparing a mixed solution of TFA, EDT and water as a lysis solution of the cleavage reaction in the step F, wherein the volume ratio is as follows: TFA: EDT (electro-thermal transfer coating): water 90: 8: 2.
7. a method of synthesizing a cyclic peptide according to claim 1, wherein: and F, purifying by adopting a reversed-phase high-pressure liquid chromatography, collecting target peak fractions by adopting reversed-phase octadecylsilane as a stationary phase and adopting 0.1% acetic acid aqueous solution and acetonitrile as mobile phases, concentrating and freeze-drying to obtain the MT-II refined peptide.
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| CN101092451A (en) * | 2006-06-20 | 2007-12-26 | 中国人民解放军军事医学科学院毒物药物研究所 | Solid phase synthesis method for PT 141 and MT II |
| CN101195654A (en) * | 2006-12-08 | 2008-06-11 | 吉尔生化（上海）有限公司 | Solid phase synthesis technique for melanotan-II |
| CN102260327A (en) * | 2010-05-28 | 2011-11-30 | 吉尔生化（上海）有限公司 | Preparation method of Melanotan II |
| CN105037488A (en) * | 2015-08-25 | 2015-11-11 | 南京肽业生物科技有限公司 | Purification method of melanotan II |
| CN111808169A (en) * | 2020-04-29 | 2020-10-23 | 杭州固拓生物科技有限公司 | Solid-phase synthesis method of melanotan I |
| CN112724193A (en) * | 2021-02-05 | 2021-04-30 | 华南理工大学 | Solid-phase synthesis method and application of polypeptide-manganese-carbonyl compound-based CO release molecule |
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| CN101092451A (en) * | 2006-06-20 | 2007-12-26 | 中国人民解放军军事医学科学院毒物药物研究所 | Solid phase synthesis method for PT 141 and MT II |
| CN101195654A (en) * | 2006-12-08 | 2008-06-11 | 吉尔生化（上海）有限公司 | Solid phase synthesis technique for melanotan-II |
| CN102260327A (en) * | 2010-05-28 | 2011-11-30 | 吉尔生化（上海）有限公司 | Preparation method of Melanotan II |
| CN105037488A (en) * | 2015-08-25 | 2015-11-11 | 南京肽业生物科技有限公司 | Purification method of melanotan II |
| CN111808169A (en) * | 2020-04-29 | 2020-10-23 | 杭州固拓生物科技有限公司 | Solid-phase synthesis method of melanotan I |
| CN112724193A (en) * | 2021-02-05 | 2021-04-30 | 华南理工大学 | Solid-phase synthesis method and application of polypeptide-manganese-carbonyl compound-based CO release molecule |
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