CN113621022B - Synthesis method of cyclic peptide - Google Patents
Synthesis method of cyclic peptide Download PDFInfo
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- CN113621022B CN113621022B CN202110810189.0A CN202110810189A CN113621022B CN 113621022 B CN113621022 B CN 113621022B CN 202110810189 A CN202110810189 A CN 202110810189A CN 113621022 B CN113621022 B CN 113621022B
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- 102000001189 Cyclic Peptides Human genes 0.000 title claims abstract description 11
- 108010069514 Cyclic Peptides Proteins 0.000 title claims abstract description 11
- 238000001308 synthesis method Methods 0.000 title description 2
- 229920005989 resin Polymers 0.000 claims abstract description 37
- 239000011347 resin Substances 0.000 claims abstract description 37
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 32
- 238000010168 coupling process Methods 0.000 claims abstract description 30
- 238000005859 coupling reaction Methods 0.000 claims abstract description 28
- 230000008878 coupling Effects 0.000 claims abstract description 26
- 238000000034 method Methods 0.000 claims abstract description 26
- 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 25
- 101710201349 Metallothionein B Proteins 0.000 claims abstract description 20
- 102100031347 Metallothionein-2 Human genes 0.000 claims abstract description 20
- 101710094505 Metallothionein-2 Proteins 0.000 claims abstract description 20
- 125000003088 (fluoren-9-ylmethoxy)carbonyl group Chemical group 0.000 claims abstract description 18
- 229920003180 amino resin Polymers 0.000 claims abstract description 18
- 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 16
- 238000007363 ring formation reaction Methods 0.000 claims abstract description 12
- 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
- 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
- 229920001184 polypeptide Polymers 0.000 claims abstract description 6
- 102000004196 processed proteins & peptides Human genes 0.000 claims abstract description 6
- 238000010532 solid phase synthesis reaction Methods 0.000 claims abstract description 6
- 230000002194 synthesizing effect Effects 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
- 238000005520 cutting process Methods 0.000 claims abstract description 4
- 238000006243 chemical reaction Methods 0.000 claims description 20
- 238000006467 substitution reaction Methods 0.000 claims description 16
- KDLHZDBZIXYQEI-UHFFFAOYSA-N Palladium Chemical compound [Pd] KDLHZDBZIXYQEI-UHFFFAOYSA-N 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
- 238000001514 detection method Methods 0.000 claims description 8
- FEMOMIGRRWSMCU-UHFFFAOYSA-N ninhydrin Chemical compound C1=CC=C2C(=O)C(O)(O)C(=O)C2=C1 FEMOMIGRRWSMCU-UHFFFAOYSA-N 0.000 claims description 7
- 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
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 5
- 150000001413 amino acids Chemical class 0.000 claims description 5
- 229910052763 palladium Inorganic materials 0.000 claims description 5
- USFPINLPPFWTJW-UHFFFAOYSA-N tetraphenylphosphonium Chemical compound C1=CC=CC=C1[P+](C=1C=CC=CC=1)(C=1C=CC=CC=1)C1=CC=CC=C1 USFPINLPPFWTJW-UHFFFAOYSA-N 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
- -1 RINK AMIDE-AM Resin Chemical compound 0.000 claims description 4
- 238000005336 cracking Methods 0.000 claims description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 4
- 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
- 239000007864 aqueous solution Substances 0.000 claims description 3
- 238000003776 cleavage reaction Methods 0.000 claims description 3
- YTJSFYQNRXLOIC-UHFFFAOYSA-N octadecylsilane Chemical compound CCCCCCCCCCCCCCCCCC[SiH3] YTJSFYQNRXLOIC-UHFFFAOYSA-N 0.000 claims description 3
- FPIRBHDGWMWJEP-UHFFFAOYSA-N 1-hydroxy-7-azabenzotriazole Chemical compound C1=CN=C2N(O)N=NC2=C1 FPIRBHDGWMWJEP-UHFFFAOYSA-N 0.000 claims description 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 description 2
- 239000007821 HATU Substances 0.000 claims description 2
- 238000000746 purification Methods 0.000 claims description 2
- 238000004007 reversed phase HPLC Methods 0.000 claims description 2
- 239000007788 liquid Substances 0.000 claims 2
- 239000012141 concentrate Substances 0.000 claims 1
- 238000005897 peptide coupling reaction Methods 0.000 abstract description 4
- 239000003814 drug Substances 0.000 abstract description 2
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 15
- 239000000047 product Substances 0.000 description 15
- 230000015572 biosynthetic process Effects 0.000 description 14
- WFDIJRYMOXRFFG-UHFFFAOYSA-N Acetic anhydride Chemical compound CC(=O)OC(C)=O WFDIJRYMOXRFFG-UHFFFAOYSA-N 0.000 description 12
- DCXYFEDJOCDNAF-UHFFFAOYSA-N Asparagine Natural products OC(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-N 0.000 description 9
- DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical compound OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 0.000 description 9
- 229960001230 asparagine Drugs 0.000 description 9
- 235000009582 asparagine Nutrition 0.000 description 9
- 238000003786 synthesis reaction Methods 0.000 description 9
- 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
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 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
- 239000002585 base Substances 0.000 description 4
- 230000005587 bubbling Effects 0.000 description 4
- 150000003949 imides Chemical class 0.000 description 4
- 229910052757 nitrogen Inorganic materials 0.000 description 4
- 238000010647 peptide synthesis reaction Methods 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
- 239000000706 filtrate Substances 0.000 description 3
- 239000005457 ice water Substances 0.000 description 3
- 125000006239 protecting group Chemical group 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 238000005303 weighing Methods 0.000 description 3
- 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
- 239000007822 coupling agent Substances 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 125000005842 heteroatom Chemical group 0.000 description 2
- NPZTUJOABDZTLV-UHFFFAOYSA-N hydroxybenzotriazole Substances O=C1C=CC=C2NNN=C12 NPZTUJOABDZTLV-UHFFFAOYSA-N 0.000 description 2
- 239000000203 mixture Substances 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
- 238000005406 washing Methods 0.000 description 2
- 238000005917 acylation reaction Methods 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 150000001408 amides Chemical class 0.000 description 1
- 238000004458 analytical method Methods 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
- 238000005119 centrifugation Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 238000011031 large-scale manufacturing process Methods 0.000 description 1
- 125000003473 lipid group Chemical group 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- NFHFRUOZVGFOOS-UHFFFAOYSA-N palladium;triphenylphosphane Chemical compound [Pd].C1=CC=CC=C1P(C=1C=CC=CC=1)C1=CC=CC=C1.C1=CC=CC=C1P(C=1C=CC=CC=1)C1=CC=CC=C1.C1=CC=CC=C1P(C=1C=CC=CC=1)C1=CC=CC=C1.C1=CC=CC=C1P(C=1C=CC=CC=1)C1=CC=CC=C1 NFHFRUOZVGFOOS-UHFFFAOYSA-N 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 238000007142 ring opening reaction Methods 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 238000010189 synthetic method Methods 0.000 description 1
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
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 discloses a method for synthesizing cyclic peptide, which belongs to the technical field of polypeptide medicine biochemistry and aims to solve the problem that Asp forms an imide hetero peak in the traditional linear peptide coupling process. The method comprises the steps of selecting amino resin, preparing Fmoc-Lys (Alloc) -amino resin, and sequentially coupling Fmoc-Trp (Boc) -OH, fmoc-Arg (pbf) -OH, fmoc-D-Phe-OH and Fmoc-His (Trt) -OH according to an Fmoc solid-phase synthesis strategy; fmoc-Asp (OAll) -OH; disposable removing Alloc and OAll groups on Lys and Asp; solid phase cyclization; removing Fmoc group at the N end of Asp, continuing to couple 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 medicine biochemistry, and particularly relates to a method for synthesizing cyclic peptides.
Background
In Fmoc solid phase synthesis, the most common side reaction affecting Asp is formation of asparagine, and strong bases such as piperidine and DBU are selected as Fmoc effective removal reagents during each cycle, and N on the alpha-amide bond of Asp forms a ring with the side chain beta-carbonyl group, while losing the side chain lipid protecting group. Aspartyl imides are susceptible to base catalysis to produce stereoisomers and to ring opening reactions, resulting in the formation of various forms of by-products.
The amount of asparagine formed is largely determined by the time of contact with the base and the strength of the base, and the use of DBU is more likely to result in formation of asparagine than piperidine. Some methods for overcoming the side reaction exist, such as leading an amino acid before Asp to be protected by an Hmb, but the acylation reaction of the amino acid after coupling the amino acid protected by the Hmb is difficult, and the related raw materials are expensive, so that the method is unfavorable for large-scale production.
This also occurs during the synthesis of MT-II. MT-II (MelanotanII, chinese name: melanoputan II) peptide sequence: ac-Nle-cycle [ Asp-His-D-Phe-Arg-Trp-Lys ] -NH2, molecular weight 1024.2, the structure of which is cyclized to form side chain amide, and the structure diagram is shown in figure 1.
The conventional synthesis of MT-II is to first 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 melanoputan-II", is to synthesize linear peptide first and then cyclize on resin to obtain MT-II.
However, during linear peptide synthesis, when Asp is Fmoc free, a distinct hetero-peak (13.533) appears after coupling Nle, which is the peak at which Asp forms imide, as shown in FIG. 2.
After the linear peptide resin is coupled, OAll and Alloc protecting groups are removed by adopting tetraphenylphosphine palladium/phenylsilane, then a coupling agent is added for solid-phase cyclization, and finally two peaks with the molecular weight of 1024 are formed in the crude peptide, wherein the content of the hetero peak is about 25 percent (RT 13.318), and the content of the target peak is about 38 percent (RT13.4.653). It was demonstrated that Asp exhibited an asparagine phenomenon during linear peptide coupling due to the use of strong base during Fmoc removal.
Disclosure of Invention
The invention aims to provide a method for synthesizing cyclic peptide, which solves the problem that the quality of melanoputan II is affected by the peak of imide formed by Asp in the traditional 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 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 of the product obtained in the step A in sequence according to an Fmoc solid-phase synthesis strategy; fmoc-Asp (OAll) -OH;
Step C, removing Alloc and OAll groups on Lys and Asp of the product obtained in the step B by adopting tetraphenylphosphine palladium and phenylsilane at one time;
step D, carrying out solid-phase cyclization on the product obtained in the step C on resin;
E, removing Fmoc groups at the N end of the Asp of the product obtained in the step D, continuously coupling Fmoc-Nle-OH, and then performing acetylation to complete coupling;
Step F, cutting off the polypeptide from the resin by adopting a cleavage reaction on the product obtained in the step E to obtain MT-II (MelanotanII, chinese name: melanoputan II) crude peptide;
And G, purifying the MT-II obtained in the step F to obtain MT-II refined peptide.
Further, 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 0.2-0.6mmol/g.
Further, the amino Resin is RINK AMIDE-AM Resin; the amino acid substitution was 0.4mmol/g.
Further, fmoc-Trp (Boc) -OH, fmoc-Arg (pbf) -OH, fmoc-D-Phe-OH, fmoc-His (Trt) -OH are coupled in sequence as described in step B; during Fmoc-Asp (OAll) -OH, the completion of coupling was determined by ninhydrin detection; it is necessary to ensure that after Fmoc-Asp (OAll) -OH coupling, the N-terminal Fmoc group is not removed.
Further, the cyclizing conditions in step D are: when in coupling, the side chain coupling system of Lys and Asp is HATU/HOAt/DIPEA.
Further, in the step F, the cracking solution of the cracking reaction is a mixed solution of TFA, EDT and water, and the volume ratio is as follows: TFA: EDT: water = 90:8:2.
Further, in the step F, purification adopts a reversed-phase high-pressure liquid chromatography, reversed-phase octadecylsilane as a stationary phase, 0.1% acetic acid aqueous solution and acetonitrile as mobile phases, a target peak fraction is collected, concentrated and freeze-dried to obtain MT-II refined peptide.
The beneficial effects of the invention are as follows:
(1) The present invention is directed to the situation where asparagine is formed during the synthesis of MT-II, effectively avoiding formation of asparagine by altering the synthetic strategy. The strategy breaks the conventional thinking that linear full-chain synthesis is firstly carried out and then cyclization is carried out. After Asp synthesis, cyclization is carried out and synthesis is continued. Specifically, after coupling to Asp (OAll) through linear peptide resin, fmoc is not removed, tetraphenylphosphine palladium and phenylsilane are added to remove OAll and Alloc protecting groups, then a coupling agent is added to carry out solid-phase cyclization, fmoc protection on the Asp main chain is removed, and other amino acids are continuously coupled. This effectively solves the problem of the asparagine phenomenon from the root. Further solves the problem that Asp forms an imide hetero-peak in the coupling process of the linear peptide, so that the purity of the finally prepared product, namely the melanoputan II, is higher.
(2) The method does not adopt high-price raw materials in the whole processing process, but skillfully changes the synthesis sequence, reduces the exposure times and time of the Asp side chain in a strong alkali environment, reduces the risk of formation of the asparagine, and can greatly reduce the synthesis cost of the Meranolazine II.
Drawings
FIG. 1 is a block diagram of MT-II (MelanotanII, chinese name: melanoputan II);
FIG. 2 is a chromatogram of the product after coupling Nle when Asp is Fmoc removed and the product is synthesized by conventional linear coupling; wherein the distinct hetero peak (RT 13.533 min) is the peak of Asp forming imide;
FIG. 3 is a chromatogram of a crude peptide prepared by linear post-coupling cyclization; wherein the content of the miscellaneous peak is about 25% (RT 13.318 min), and the content of the target peak is about 38% (RT 14.653 min);
FIG. 4 is a chromatogram of the MT-II crude peptide resin obtained in step E in example 4.
Detailed Description
For the purpose of making 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 clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, based on the embodiments of the invention, which are apparent to those of ordinary skill in the art without inventive faculty, are intended to be within the scope of the invention.
To demonstrate the feasibility and effectiveness of the synthetic method of the invention, the steps were performed separately.
Example 1
Step A of a cyclic peptide synthesis method: the amino resin was selected and Fmoc-Lys (Alloc) -amino resin was prepared.
The preparation of resin with the substitution degree of 0.60mmolFmoc-Lys (Alloc) -RINK AMIDE RESIN is carried out by the following specific procedures:
Weighing RINK AMIDE RESIN resin 100g with substitution degree of 1.0mmol/g in a solid phase reaction column, adding DMF, and swelling for 60 minutes by nitrogen bubbling;
Fmoc-Lys (Alloc) -OH45.3 g (100 mmol) and HOBt16.2 g (120 mmol) were weighed, dissolved in DMF, added with 20.3mLDIC at 0deg.C, activated for 5min and added to the reaction column;
After two hours of reaction, 70mL acetic anhydride and 60mL 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 detection substitution of 0.58mmol/g.
Example 2
Step A of a cyclic peptide synthesis method: the amino resin was selected and Fmoc-Lys (Alloc) -amino resin was prepared.
The preparation of Resin with the selection substitution degree of 0.40mmolFmoc-Lys (Alloc) -RINK AMIDE-AM Resin comprises the following specific steps:
weighing RINK AMIDE-AM Resin 100g with substitution degree of 1.0mmol/g in a solid phase reaction column, adding DMF, and swelling by nitrogen bubbling for 60 minutes;
Fmoc-Lys (Alloc) -OH31.7 g (70 mmol) and HOBt11.3 g (84 mmol) were weighed, dissolved in DMF, added 14.2mLDIC at 0deg.C, activated for 5 min and added to the reaction column;
After two hours of reaction, 70mL acetic anhydride and 60mL 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 detection substitution of 0.40mmol/g.
Example 3
Step A of a cyclic peptide synthesis method: the amino resin was selected and Fmoc-Lys (Alloc) -amino resin was prepared.
The preparation method of the Resin with the selectivity of 0.20mmolFmoc-Lys (Alloc) -RINK AMIDE-MBHA Resin comprises the following specific steps:
weighing RINK AMIDE-AM Resin 100g with substitution degree of 1.0mmol/g in a solid phase reaction column, adding DMF, and swelling by nitrogen bubbling for 60 minutes;
Fmoc-Lys (Alloc) -OH15.9 g (35 mmol) and HOBt5.7 g (42 mmol) were weighed, dissolved in DMF, added 7.1mLDIC at 0deg.C, activated for 5min and added to the reaction column;
After two hours of reaction, 70mL acetic anhydride and 60mL 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 detection substitution of 0.22mmol/g.
The results of the detection substitution degree of Fmoc-Lys (Alloc) -Wang resin finally obtained in examples 1-3 were not out of tolerance with the amino acid substitution degree of each resin, confirming that the amino resin selected in the present invention and the amino acid substitution degree thereof were determined to be between 0.2 and 0.6mmol/g, which is feasible.
Since the Fmoc-Lys (Alloc) -amino Resin properties finally prepared in 3 examples were still similar, the Fmoc-Lys (Alloc) -RINK AMIDE-AM Resin prepared in final example 2 was arbitrarily selected for use in the subsequent synthesis in example 4.
Example 4
A method for synthesizing cyclic peptide comprises the following steps:
Step B, coupling Fmoc-Trp (Boc) -OH, fmoc-Arg (pbf) -OH, fmoc-D-Phe-OH and Fmoc-His (Trt) -OH of the product obtained in the step A in sequence according to an Fmoc solid-phase synthesis strategy; fmoc-Asp (OAll) -OH.
Specific:
100g of Fmoc-Lys (Alloc) -RINK AMIDE-AM Resin with substitution degree of 0.40mmol/g prepared in example 2 is weighed into a solid phase reaction column, DMF is added, and nitrogen bubbling is carried out for swelling for 60 minutes; then deprotected with DBLK for 6min+8min, and washed with DMF 6 times.
63.2G (120 mmol) of Fmoc-Trp (Boc) -OH and 22.5g (144 mmol) of HOBT were weighed and dissolved in DMF, 25mL (144 mmol) of DIPCDI was added under ice water bath to activate for 3min, and the mixture was added to the reaction column, reacted at room temperature for 2 hours, and the end of the reaction was detected with ninhydrin (if the resin was colorless and transparent, the reaction was terminated; if the resin was developed, the reaction was prolonged for 1 hour).
After the reaction, the resin was washed 3 times with DMF, deprotected by addition of DBLK for 6min+8min, washed 6 times with DMF, and the resin was checked for color.
Repeating the coupling operation, and sequentially coupling Fmoc-Arg (pbf) -OH, fmoc-D-Phe-OH and Fmoc-His (Trt) -OH according to the peptide sequence; fmoc-Asp (OAll) -OH.
And C, removing Alloc and OAll groups on Lys and Asp of the product obtained in the step B at one time by adopting tetraphenylphosphine palladium and phenylsilane.
Specific:
The peptide resin obtained in step B was swollen with DCM, 98.5ml of phenylsilane (800 mmol) was added, and 5.6g of tetrakis triphenylphosphine palladium (8 mmol) was added thereto and reacted at room temperature for 60min, and the removal of Alloc and OAll was confirmed by detection of ninhydrin.
And D, carrying out solid-phase cyclization on the product obtained in the step C on the resin.
Specific:
Fmoc-cycle [ Asp-His-D-Phe-Arg-Trp-Lys ] -RINK AMIDE-AM Resin preparation was performed:
15.2gHATU (40 mmol), 6.5gHOAt (48 mmol) were weighed, dissolved in 200mM MF, activated for 3 minutes by adding 13.9mLDIPEA (80 mmol) under ice water bath, and reacted for 2 hours by adding the reaction column, and the end point of the reaction was detected and judged by ninhydrin method.
Washing with DMF for 6 times, and shrinking with methanol to obtain
Fmoc-cyclo [ Asp-His-D-Phe-Arg-Trp-Lys ] -RINK AMIDE-AM Resin peptide Resin.
And E, removing Fmoc groups at the N end of the Asp of the product obtained in the step D, continuing to couple Fmoc-Nle-OH, and then performing acetylation to complete coupling.
Specific:
preparation of Ac-Nle-cycle [ Asp-His-D-Phe-Arg-Trp-Lys ] -RINK AMIDE-AM Resin:
Fmoc-cyclo [ Asp-His-D-Phe-Arg-Trp-Lys ] -RINK AMIDE-AM Resin in step D was treated with DBLK for 6+8min, fmoc groups were removed and washed 6 times with DMF.
42.4G (120 mmol) Fmoc-Nle-OH and 22.5g (144 mmol) HOBT were weighed and dissolved in DMF, 25mL (144 mmol) DIPCDI was added under ice water bath for 3min and then the mixture was added to the reaction column and reacted at room temperature for 2 hours, the end of the reaction was detected with ninhydrin (if the resin was colorless and transparent, the reaction was stopped; if the resin was developed, the reaction was prolonged for 1 hour). After the reaction, the resin was washed 3 times with DMF, deprotected by addition of DBLK for 6min+8min, washed 6 times with DMF, and the resin was checked for color.
76.1Ml (800 mmol) of acetic anhydride and 66.1ml (800 mmol) of pyridine were weighed and mixed into the resin, and reacted for 2 hours, and the ninhydrin detection resin was colorless and transparent. The resin was washed 6 times with DMF and then the resin was contracted with methanol and dried under vacuum to give MT-II peptide resin 182.4g.
After Nle coupling, small sample fragments are cleaved for analysis, and the chromatogram is shown in FIG. 4, and compared with the traditional linear peptide coupling method (shown in FIG. 3), the method can be seen as follows:
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% (RT 13.318 min) and the content of the target peak is about 38% (RT 14.653 min);
FIG. 4 is a chromatogram of a crude peptide prepared according to this example, with a purity of about 65% and a 27% improvement over the original method.
The method of the invention can effectively solve the phenomenon of the asparagine. Further solves the problem that Asp forms an imide hetero-peak in the coupling process of the linear peptide, so that the purity of the finally prepared product, namely the melanoputan II, is higher.
And F, cutting the polypeptide from the resin by adopting a cleavage reaction on the product obtained in the step E to obtain MT-II (MelanotanII, chinese name: melanopentan II) crude peptide.
The peptide resin 182.4 g from step E was added to a 3L three-port flask and pre-configured TFA pre-chilled to below 0 degrees celsius was added to EDT: h2o=90: 8:2 (V: V) 1.82L, reacted at room temperature for 2 hours, the resin was filtered, and the filtrate was collected. The resin was washed with a small amount of TFA and the filtrates were combined.
The filtrate was slowly added to 18.4L of glacial diethyl ether for precipitation, centrifugation, washing with glacial diethyl ether for 5 times, and drying under reduced pressure to obtain 39.7 g of crude peptide with a weight yield of 96.9%.
And G, purifying the MT-II obtained in the step F to obtain MT-II refined peptide.
39.7G of the solid obtained in step F was directly purified by high performance liquid chromatography.
The method comprises the steps of taking reversed-phase octadecylsilane as a stationary phase, taking 0.1% acetic acid aqueous solution and acetonitrile as mobile phases, collecting target peak fractions, concentrating and freeze-drying to obtain 18.8g of pure product, wherein the purity is more than 99.0%, and the yield is 45.9%.
Claims (2)
1. A method for synthesizing cyclic peptides, which is characterized by comprising the following steps:
Step A, selecting amino Resin, and preparing Fmoc-Lys (Alloc) -amino Resin, wherein the amino Resin is RINK AMIDE RESIN, RINK AMIDE-AM Resin, RINK AMIDE-MBHAResin; the substitution degree of the amino acid is 0.2-0.6mmol/g;
step B, coupling Fmoc-Trp (Boc) -OH, fmoc-Arg (pbf) -OH, fmoc-D-Phe-OH and Fmoc-His (Trt) -OH of the product obtained in the step A in sequence according to an Fmoc solid-phase synthesis strategy; fmoc-Asp (OAll) -OH;
The Fmoc-Trp (Boc) -OH, fmoc-Arg (pbf) -OH, fmoc-D-Phe-OH and Fmoc-His (Trt) -O H are coupled in sequence; during Fmoc-Asp (OAll) -OH, the completion of coupling was determined by ninhydrin detection; it is necessary to ensure that after Fmoc-Asp (OAll) -OH coupling is completed, the Fmoc group at the N end cannot be removed;
Step C, removing Alloc and OAll groups on Lys and Asp of the product obtained in the step B by adopting tetraphenylphosphine palladium and phenylsilane at one time;
Step D, carrying out solid-phase cyclization on the product obtained in the step C on resin; the cyclization conditions were: during coupling, the side chain coupling system of Lys and Asp is HATU/HOAt/DIPEA;
E, removing Fmoc groups at the N end of the Asp of the product obtained in the step D, continuously coupling Fmoc-Nle-OH, and then performing acetylation to complete coupling;
Step F, cutting off the polypeptide from the resin by adopting a cleavage reaction on the product obtained in the step E to obtain MT-II (MelanotanII, chinese name: melanoputan II) crude peptide; wherein, the cracking liquid of the cracking reaction is a mixed liquid of TFA, EDT and water, and the volume ratio is as follows: TFA: EDT: water = 90:8:2;
G, purifying the MT-II obtained in the step F to obtain MT-II refined peptide; the purification adopts a reversed phase high pressure liquid chromatography, uses reversed phase octadecylsilane as a stationary phase, uses 0.1% acetic acid aqueous solution and acetonitrile as mobile phases, collects target peak fractions, and concentrates and freeze-dries to obtain MT-II refined peptide.
2. The method of claim 1, wherein the method further comprises: the amino Resin is RINK AMIDE-AM Resin; the amino acid substitution was 0.4mmol/g.
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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 |
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