CN113135979A - Solid-phase synthesis method of peptide - Google Patents

Solid-phase synthesis method of peptide Download PDF

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CN113135979A
CN113135979A CN202010058802.3A CN202010058802A CN113135979A CN 113135979 A CN113135979 A CN 113135979A CN 202010058802 A CN202010058802 A CN 202010058802A CN 113135979 A CN113135979 A CN 113135979A
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fmoc
resin
peptide
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octreotide
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李新宇
马洪季
张利香
付玉清
李文静
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Shenzhen Jx Bio Pharmaceutical Co ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/06—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length using protecting groups or activating agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/04—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length on carriers
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
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    • C07K14/605—Glucagons
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    • 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
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    • 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

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Abstract

The invention discloses a solid-phase synthesis method of peptide, which comprises the steps of preparing linker-A-solid-phase resin, taking the linker-A-solid-phase resin as initial resin, and applying the resin to the solid-phase synthesis of the peptide, wherein A is one or more amino acids. According to the invention, the linker-A is introduced before the solid phase carrier and the target peptide, so that the space environment of target solid phase cyclization is greatly optimized, and the steric hindrance effect in solid phase synthesis is effectively reduced. The method is suitable for large-scale industrial production, can obviously improve the purity and yield of the target peptide, and has wide application prospect.

Description

Solid-phase synthesis method of peptide
Technical Field
The invention relates to the field of polypeptide synthesis, in particular to a method for performing solid-phase synthesis on polypeptide by using an amino acid extension linker.
Background
The polypeptide synthesis method mainly comprises two modes of solid-phase synthesis and liquid-phase synthesis. The liquid phase synthesis method is mainly used for short peptide synthesis and has the defects of complex intermediate purification operation, complex post-treatment operation, long synthesis period and the like. BruceMerrifield first proposed a solid-phase polypeptide synthesis method (SPPS) in 1963, which is convenient and rapid to synthesize, mild in reaction conditions, and widely used rapidly, and more than 85% of polypeptide drugs are obtained by solid-phase synthesis at present. The resin widely used in the solid-phase synthesis method is PS resin and is a resin ball of polystyrene copolymerized with 1% -2% divinylbenzene, and the three main factors influencing the solid-phase synthesis reaction are firstly the substitution value of the resin; secondly, the swelling coefficient of the resin; thirdly, due to the difference of the manufacturer process, the distribution of active sites in the resin is also uneven, and the synthetic effect of the resin is also affected. Due to the problems of the solid phase resin, the steric hindrance is too large, the amino acid coupling is difficult, and impurities such as missing peptide and the like are generated in the coupling process, so that the quality of a sample is influenced, and the yield of a product is reduced. For example, the cyclization of the cyclic peptide consisting of 7 amino acids is a difficult point in the synthesis of the cyclic peptide, and the existing cyclization methods comprise liquid phase synthesis and solid phase synthesis, wherein in the liquid phase synthesis, a large amount of intermolecular coupling byproducts are easily generated in the cyclization process, and the solid phase cyclization method causes the cyclization difficulty due to too large steric hindrance space. Currently, there are two main synthetic processes for octreotide, one is: using chloromethyl resin as a starting material, preparing cesium salt from Boc-Thr (tBu) -OH, sequentially connecting amino acids with protective groups according to a solid-phase synthesis method to obtain protected octapeptide resin, sequentially removing the Boc-protective groups by HCl/isopropanol, and carrying out a peptide-connecting reaction by using a condensing agent; reducing by using palladium carbon/hydrogen, and simultaneously cutting off a peptide chain to obtain reduced octreotide; and (3) reacting the reduced octreotide with an oxidant under alkaline conditions to generate a crude octreotide product with disulfide bonds forming rings. The other synthesis process comprises the following steps: using CTC resin as a starting material, preparing cesium salt from Fmoc-Thr (tBu) -OH, sequentially connecting amino acids with protective groups according to a solid-phase synthesis method to obtain protected octapeptide resin, sequentially removing the Fmoc-protective groups by using 20% piperidine, and performing coupling reaction by using a condensing agent; finally, cutting off the peptide chain by using TFA to obtain reduced octreotide; oxidizing the reduced octreotide under an acidic condition to generate a crude octreotide product with disulfide bonds forming rings. In the two processes, as the chloromethyl resin or the CTC resin is adopted, the coupling difficulty can be caused due to the overlarge steric hindrance of amino acid or the high density of active sites, so that the purity of the sample is low and the yield is not high.
In patent CN105601718B, Rinkamide Resin is used as a solid phase carrier, HO- (CH2) n-COOH is used as a Linker for connecting the solid phase carrier and a target peptide to synthesize the bumetanide, and the invention optimizes the space environment of solid phase cyclization of the target peptide by introducing long-chain fatty acid between the solid phase carrier and the bumetanide, thereby avoiding intermolecular coupling generated in liquid phase cyclization, however, as HO- (CH2) n-COOH Linker and bumetanide are condensed through ester bonds, HO- (CH2) n-COOH needs to be removed under alkaline conditions after sample cyclization, and the existence of alkali in the process can cause hydrolysis of the bumetanide, thereby generating unnecessary impurities. Patent application document CN106543269A discloses that Fmoc-Thr-x is used as a raw material to synthesize octreotide, which has the advantage of coupling Fmoc-Thr-x with AM resin, thereby avoiding the tedious steps of first preparing cesium salt from Boc-Thr (tbu) -OH or Fmoc-Thr (tbu) -OH and then coupling with chloromethyl resin or CTC resin, however, in the process of synthesizing Fmoc-Thr-x with AM resin, because Fmoc-Thr-x has larger steric hindrance relative to Fmoc-Thr-ol, the subsequent coupling efficiency is lower, and the purity and yield of the finished octreotide are not high.
Disclosure of Invention
Aiming at the problems that the synthesis process in the prior art is not suitable for large-scale industrial production, the purity of octreotide is not high, the yield is low and the like, the invention provides a solid-phase synthesis method of peptide, which takes AM resin or MBHA resin as a solid-phase carrier and takes linker-A as a linker for connecting the solid-phase carrier and target peptide. Compared with the prior art, the linker-A is introduced between the solid phase carrier and the target peptide, so that the space environment of solid phase cyclization of the target peptide is greatly optimized, and the steric hindrance effect in solid phase synthesis is effectively reduced.
In order to achieve the purpose, the technical scheme of the invention is as follows:
preparing linker-A-solid phase resin, and taking the linker-A-solid phase resin as starting resin to be applied to the solid phase synthesis of peptide, wherein A is one or more amino acids.
Preferably, the amino acid in the linker-A-solid phase resin is selected from one or more of Gly, Ala, Ava, Val, Leu and Ile with less steric hindrance.
Preferably, the number of amino acids in the linker-A-solid phase resin is not more than 5.
Preferably, the solid phase resin in the linker-A-solid phase resin can be selected from AM resin or MBHA resin, RINK AM resin, RINKMBHA resin. After the synthesis is finished, the cracking operation is carried out, and the linker-A can be left on the resin to be separated from the target peptide or can be simultaneously separated from the resin and the target peptide, so that the target peptide cannot be influenced.
The peptide synthesized by the scheme can be selected from the group consisting of brennol peptide, octreotide, procatide, liraglutide and somaglutide.
In a preferred scheme, when the scheme is adopted to synthesize octreotide, linker of the octreotide is preferably Fmoc-Thr-x, and the chemical structural general formula of the octreotide is as follows:
Figure BDA0002373722610000031
wherein the content of the first and second substances,
R1、R2、R3、R4selected from H, CH3、CH3O, OH, respectively, are provided, one or more of,
n is 0 to 5.
In the preferred scheme, when the scheme is adopted to synthesize octreotide, A is Gly, and the amino acid has the advantages of simplest structure, small space resistance and low price, and is most beneficial to industrial production.
Preferably, when the scheme is adopted to synthesize octreotide, because the octreotide chain is short, the solid-phase resin adopts AM resin or MBHA resin, and linker-A can remain on the resin in the operation step of cracking the peptide resin and cannot affect the target compound.
In a preferred scheme, when the scheme is adopted to synthesize octreotide, a cracking system of solid phase synthesis is as follows: and (3) mixed solution of TFA, TIS and Mpr, wherein the TIS can be used as a trapping agent for side chain protecting groups after cracking, and the MPR can effectively protect exposed mercapto groups after cracking and avoid oxidation.
The method is suitable for large-scale industrial production, can effectively solve the problem of space effect in the solid-phase synthesis process of the target peptide, improves the purity and yield of the target peptide, and has wide application prospect.
Drawings
Fig. 1 is an HPLC chromatogram of crude octreotide prepared in example 1.
Fig. 2 is an HPLC chromatogram of the crude bremelanotide prepared in example 3.
Fig. 3 is an HPLC chromatogram of the crude liraglutide prepared in example 4.
Fig. 4 is an HPLC chromatogram of crude octreotide prepared in comparative example 1.
Fig. 5 is an HPLC chromatogram of the crude bremelanotide prepared in comparative example 2.
Fig. 6 is an HPLC chromatogram of crude liraglutide prepared in comparative example 3.
Detailed Description
The present invention will be described in further detail with reference to specific examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.
Example 1 Synthesis of octreotide
Accurately weighing 10g of AM resin with a substitution value of 1.0mmol/g, placing the AM resin in a solid phase synthesis reaction kettle, washing with DMF for 2 times and 50 ml/time, and adding 100ml of DMF to swell for 30min after washing.
After swelling, a small sample was taken and subjected to ninhydrin detection, and the resin was dark blue. Weighing Fmoc-Gly-OH: 8.9g (30mmol, 3eq), HOBT: 4.4g (33mmol, 3.3eq), dissolved in 50ml DMF, pre-cooled at 0-5 ℃ for 10min, DIC: activating 5.6g (45mmol, 4.5eq) for 3-5min, putting into a solid phase synthesis reaction kettle, reacting at 20-30 deg.C for 3h, sampling ninhydrin after 3h, and detecting to obtain negative resin. Then, carrying out deprotection 2 times by adopting 20% piperidine/DMF, wherein the reaction time of the 1 st time is 5 min; the reaction time of the 2 nd time is 10min, and the resin is washed 6 times after two times of deprotection, 80ml each time and 1min each time. A small sample of ninhydrin was detected, and the resin was blue. Fmoc-Thr-x, Fmoc-Cys (Trt) -OH, Fmoc-Thr (tBu) -OH, Fmoc-Lys (Boc) -OH, Fmoc-D-Trp (Boc) -OH, Fmoc-Phe-OH, Fmoc-Cys (Trt) -OH and Fmoc-D-Phe-OH are coupled in sequence, and after the coupling is finished, the Fmoc protecting group is removed to obtain the octreotide peptide resin. Wherein the structure of Fmoc-Thr-x is as follows:
Figure BDA0002373722610000041
preparing a cleavage reagent which is composed of TFA/TIS/MPR (95/2.5/2.5) and precooled in a refrigerator at the temperature of 15 ℃ below zero, transferring the precooled cleavage reagent into a reaction kettle, adding the octreotide resin obtained in the step, and carrying out cleavage reaction for 2 hours at the temperature of 20-28 ℃.
After the cracking reaction is finished, filtering to remove resin, adding the obtained cracking filtrate into pre-frozen ether for settling, centrifuging and collecting; the resulting solid was centrifuged, washed with ether to a solution pH of 4-7 and dried in vacuo (30 ± 2 ℃, < -0.08MPa, 8h) to give crude octreotide linear peptide.
Dissolving the obtained linear crude octreotide in 30% acetic acid water solution, wherein the concentration of the linear peptide is 2mg/ml, slowly adding iodine/ethanol after the linear peptide is dissolved and clarified until the reaction solution is not discolored, sampling and detecting, wherein the purity of the crude octreotide is 90.35%, the synthesis yield is 79.1%, the maximum impurity close to the main peak is 0.16%, the maximum impurity is less than 1.0%, and an HPLC chromatogram is shown in figure 1.
Example 2 Synthesis of octreotide
10g of MBHA resin with the substitution value of 1.0mmol/g is accurately weighed and placed in a solid phase synthesis reaction kettle, DMF is used for washing for 2 times and 50 ml/time, and 100ml of DMF is added for swelling for 30min after washing is finished.
After swelling, a small sample was taken and subjected to ninhydrin detection, and the resin was dark blue. Weighing Fmoc-Ala-OH: 9.3g (30mmol, 3eq), HOBT: 4.4g (33mmol, 3.3eq), dissolved in 50ml DMF, pre-cooled at 0-5 ℃ for 10min, DIC: activating 5.6g (45mmol, 4.5eq) for 3-5min, putting into a solid phase synthesis reaction kettle, reacting at 20-30 deg.C for 3h, sampling ninhydrin after 3h, and detecting to obtain negative resin. Then, carrying out deprotection 2 times by adopting 20% piperidine/DMF, wherein the reaction time of the 1 st time is 5 min; the reaction time of the 2 nd time is 10min, and the resin is washed 6 times after two times of deprotection, 80ml each time and 1min each time. When the ninhydrin was removed, the resin appeared blue. Fmoc-Thr-x, Fmoc-Cys (Trt) -OH, Fmoc-Thr (tBu) -OH, Fmoc-Lys (Boc) -OH, Fmoc-D-Trp (Boc) -OH, Fmoc-Phe-OH, Fmoc-Cys (Trt) -OH and Fmoc-D-Phe-OH are coupled in sequence, and after the coupling is finished, the Fmoc protecting group is removed to obtain the octreotide peptide resin. Wherein the structure of Fmoc-Thr-x is as follows:
Figure BDA0002373722610000051
preparing a cleavage reagent, wherein the composition of TFA/TIS/MPR is 95/2.5/2.5, precooling for 1h in a refrigerator at the temperature of-15 ℃, transferring the cleavage reagent after precooling into a reaction kettle, adding the octreotide peptide resin obtained in the step (a), and carrying out cleavage reaction for 2h at the temperature of 20-28 ℃ according to the concentration of 10ml/g of the peptide resin.
After the cracking reaction is finished, filtering to remove resin, adding the obtained cracking filtrate into pre-frozen ether for settling, centrifuging and collecting; the resulting solid was centrifuged, washed with ether to a solution pH of 4-7 and dried in vacuo (30 ± 2 ℃, < -0.08MPa, 8h) to give crude octreotide linear peptide.
Dissolving the crude octreotide linear peptide in water solution with pH of 8.0, and slowly adding H2O2Stirring and reacting for 2h at room temperature, sampling and detecting, wherein the purity of crude octreotide is 89.2%, the synthesis yield is 74.1%, the maximum impurity close to the main peak is 0.19%, the maximum impurity is less than 1.0%, and the HPLC chromatogram is similar to that in figure 1.
Example 3 Synthesis of Blumenol peptide
10g of RinkMBHA resin with a substitution value of 1.0mmol/g is accurately weighed and placed in a solid phase synthesis reaction kettle, DMF is washed for 2 times and 50 ml/time, and 100ml of DMF is added to swell for 30min after the washing is finished. Then, 20% piperidine/DMF is adopted for deprotection for 2 times, and the reaction time of the 1 st time is 5 min; the reaction time of the 2 nd time is 10min, and the resin is washed 6 times after two times of deprotection, 80ml each time and 1min each time. At the end of the washing, a small sample was taken for ninhydrin detection and the resin was dark blue. Weighing Fmoc-Vla-OH: 10.3g (30mmol, 3eq), HOBT: 4.4g (33mmol, 3.3eq), dissolved in 50ml DMF, pre-cooled at 0-5 ℃ for 10min, DIC: activating 5.6g (45mmol, 4.5eq) for 3-5min, putting into a solid phase synthesis reaction kettle, reacting at 20-30 deg.C for 3h, sampling ninhydrin after 3h, and detecting to obtain negative resin. Then, carrying out deprotection 2 times by adopting 20% piperidine/DMF, wherein the reaction time of the 1 st time is 5 min; the reaction time of the 2 nd time is 10min, and the resin is washed 6 times after two times of deprotection, 80ml each time, and 1min each time. A small sample of ninhydrin was detected, and the resin was blue. Fmoc-Gly-OH, Fmoc-Ava-OH, HMP-linker, Fmoc-Lys (alloc) -OH, Fmoc-Trp (Boc) -OH, Fmoc-Arg (pbf) -OH, Fmoc-D-Phe-OH, Fmoc-His (Boc) -OH, Fmoc-Asp (oAll) -OH, Fmoc-Nle-OH are coupled in sequence. After Fmoc-Nle-OH coupling is completed, removing Fmoc protecting group, and acetylating with acetic anhydride/pyridine to obtain AC-Nle-Asp (oAll) -His (Boc) -D-Phe-Arg (pbf) -Trp (Boc) -Lys (alloc) -HMP linker-Ava-Gly-Val-RinkMBHA resin. Removing side chain protecting groups alloc and oall by adopting tetratriphenylphosphine palladium, and weighing HOBT: 4.4g (33mmol, 3.3eq), dissolved in 50ml DMF, precooled at 0-5 ℃ for 10min, added to the above resin and then added DIC: 5.6g (45mmol, 4.5eq), reacting at 20-30 ℃ for 4h, taking a small sample to detect that the resin is negative, and finishing the reaction to obtain Ac-Nle- [ Asp-His (Boc) -D-Phe-Arg (pbf) -Trp (Boc) -Lys ] -HMP linker-Ava-Gly-Val-RinkMBHA resin
Preparing a cracking reagent, wherein the composition of TFA/TIS/MPR/H2O is 92.5/2.5/2.5/2.5, precooling for 1H in a refrigerator at the temperature of-15 ℃, transferring the precooled cracking reagent into a reaction kettle, adding the obtained buminuo peptide resin, and carrying out cracking reaction for 3H at the temperature of 20-26 ℃ according to the concentration of 10ml/g of the peptide resin.
After the cracking reaction is finished, filtering to remove resin, adding the obtained cracking filtrate into pre-frozen ether for settling, centrifuging and collecting; the resulting solid was centrifuged, washed with ether to a solution pH of 4-7 and dried under vacuum (30 ± 2, < -0.08MPa, 8h) to give crude bremelanotide. Sampling and detecting, wherein the purity of the crude brennol peptide is 92.59%, the maximum single impurity is 1.14%, the purity is less than 2.0%, the synthesis yield is 70.3%, and an HPLC chromatogram is shown in figure 2.
Example 4 Synthesis of liraglutide
10g of MBHA resin with the substitution value of 1.0mmol/g is accurately weighed and placed in a solid phase synthesis reaction kettle, DMF is used for washing for 2 times and 50 ml/time, and 100ml of DMF is added for swelling for 30min after washing is finished.
After swelling, a small sample was taken and subjected to ninhydrin detection, and the resin was dark blue. Weighing Fmoc-Ava-OH: 10.1g (30mmol, 3eq), HOBT: 4.4g (33mmol, 3.3eq), dissolved in 50ml DMF, pre-cooled at 0-5 ℃ for 10min, DIC: activating 5.6g (45mmol, 4.5eq) for 3-5min, putting into a solid phase synthesis reaction kettle, reacting at 20-30 deg.C for 3h, sampling ninhydrin after 3h, and detecting to obtain negative resin. Then, carrying out deprotection 2 times by adopting 20% piperidine/DMF, wherein the reaction time of the 1 st time is 5 min; the reaction time of the 2 nd time is 10min, and the resin is washed 6 times after two times of deprotection, 80ml each time and 1min each time. When the ninhydrin was removed, the resin appeared blue. Coupling Fmoc-Ava-OH, HMPA-linker, Fmoc-Gly-OH, Fmoc-Arg (Pbf) -OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Trp (Boc) -OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Phe-OH, Fmoc-Glu (OtBu) -OH, Fmoc-Lys (Pla-Glu-OtBu) -OH, Fmoc-Ala-OH, Fmoc-Gln (Trt) -OH, Fmoc-Gly-OH, Fmoc-Glu (OtBu) -OH, Fmoc-Leu-OH, Fmoc-Tyr (tBu) -OH, Fmoc-Ser (tBu) -OH, Fmoc-Leu-OH, Fmoc-Tyr (Fmoc) -OH, Fmoc-Ser (tBu) -OH, Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Leu-Ala-OH, Fmoc-Ala-OH, Fmoc-Ala-Ser (Tab-OH, Fmoc-Ser (Tab-OH, Fmoc-Ser (Tab-Il) and/, Coupling of Fmoc-Ser (tBu) -OH, Fmoc-Val-OH, Fmoc-Asp (OtBu) -OH, Fmoc-Ser (tBu) -OH, Fmoc-Thr (tBu) -OH, Fmoc-Phe-OH, Fmoc-Thr (tBu) -OH, Fmoc-Gly-OH, Fmoc-Glu (OtBu) -OH, Fmoc-Ala-OH, Boc-His (Trt) -OH. After the reaction was complete, washed 6 times with DCM, MeOH was retracted and dried in vacuo to give the peptide resin.
Taking 13.94g of the obtained liraglutide resin, adding TFA: and Tis: water 94: 3: 3 (using amount of 10 mL/krause peptide resin), stirring uniformly, stirring at room temperature for reaction for 3 hours, filtering the reaction mixture by using a sand core funnel, collecting filtrate, washing the resin for 3 times by using a small amount of TFA, combining the filtrates, concentrating under reduced pressure, adding diethyl ether for precipitation, washing the precipitate for 6 times by using diethyl ether, drying under reduced pressure in vacuum to constant weight, obtaining 4.47g of white powder of the loratadine, wherein the yield is 79.44%, the purity of crude loratadine is 81.10%, the maximum single impurity close to the main peak is 0.41%, is less than 0.5%, and an HPLC chromatogram is shown in figure 3.
Example 5 Synthesis of Somalutide
10g of RinkAM resin with the substitution value of 1.0mmol/g is accurately weighed and placed in a solid phase synthesis reaction kettle, DMF is washed for 2 times and 50 ml/time, and 100ml of DMF is added for swelling for 30min after washing is completed.
After swelling, a small sample was taken and subjected to ninhydrin detection, and the resin was dark blue. Weighing Fmoc-Gly-OH: 8.9g (30mmol, 3eq), HOBT: 4.5g (33mmol, 3.3eq), dissolved in 50ml DMF, pre-cooled at 0-5 ℃ for 10min, DIC: activating 5.6g (45mmol, 4.5eq) for 3-5min, putting into a solid phase synthesis reaction kettle, reacting at 20-30 deg.C for 3h, sampling ninhydrin after 3h, and detecting to obtain negative resin. Then, carrying out deprotection 2 times by adopting 20% piperidine/DMF, wherein the reaction time of the 1 st time is 5 min; the reaction time of the 2 nd time is 10min, and the resin is washed 6 times after two times of deprotection, 80ml each time and 1min each time. When the ninhydrin was removed, the resin appeared blue. Sequentially coupling Fmoc-Gly-OH and HMP-linker to obtain HMP linker-Gly-Gly-RinkAM resin, and continuing to couple Fmoc-Gly-OH, Fmoc-Arg (Pbf) -OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Trp (Boc) -OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Phe-OH, Fmoc-Glu (OtBu) -OH, Fmoc-Lys (dde) -OH, Fmoc-Ala-OH, Fmoc-Gln (Trt) -OH, Fmoc-Gly-OH, Fmoc-Glu (OtBu) -OH, Fmoc-Leu-OH, Fmoc-Tyr-tBu) -OH, Fmoc-Ala-OH, Fmoc-Gln (Trt) -OH, Fmoc (Trn) (Pbf-Phe) -OH, Fmoc-Tyr-OH, Fmoc-Tyr-OH, Fmoc-Tyr-OH, and the like, Coupling of Fmoc-Ser (tBu) -OH, Fmoc-Val-OH, Fmoc-Asp (OtBu) -OH, Fmoc-Ser (tBu) -OH, Fmoc-Thr (tBu) -OH, Fmoc-Phe-OH, Fmoc-Thr (tBu) -OH, Fmoc-Gly-OH, Fmoc-Glu (OtBu) -OH, Fmoc-Ala-OH, Boc-His (Trt) -OH.
50ml of 4.0% hydrazine hydrate solution in DMF is added into the solid phase reaction column, the mixture is stirred and reacted for 1.0h under nitrogen, the reaction mixture is pumped out, washed for 6 times by DCM, and the ninhydrin test shows positive. And sequentially coupling Fmoc-AEEA-OH, Fmoc-Glu-OtBu and hexadecanoic acid, washing the resin by respectively adopting DCM, DMF and methanol after the coupling is finished, and drying in vacuum to obtain the Somali peptide resin.
Taking 14.68g of the obtained somalutide resin, adding TFA: and Tis: H2O, Mpr 92.5: 2.5: 2.5: 2.5 (10 mL/g Somaliptin resin), stirring uniformly, stirring at room temperature for reaction for 3 hours, filtering the reaction mixture by using a sand core funnel, collecting filtrate, washing the resin for 1 time by using a small amount of TFA, combining the filtrates, concentrating under reduced pressure, adding diethyl ether for precipitation, washing the precipitate for 3 times by using diethyl ether, pumping to obtain white-like powder, and drying under reduced pressure in vacuum to constant weight. 4.24g of crude somaglutide was obtained with a yield of 77.38%, a crude purity of 78.59% and a maximum single impurity of 1.3% and less than 1.5%.
Example 6 Synthesis of octreotide
10g of MBHA resin with the substitution value of 1.0mmol/g is accurately weighed and placed in a solid phase synthesis reaction kettle, DMF is used for washing for 2 times and 50 ml/time, and 100ml of DMF is added for swelling for 30min after washing is finished.
After swelling, a small sample was taken and subjected to ninhydrin detection, and the resin was dark blue. Weighing Fmoc-Ala-OH: 9.3g (30mmol, 3eq), HOBT: 4.4g (33mmol, 3.3eq), dissolved in 50ml DMF, pre-cooled at 0-5 ℃ for 10min, DIC: activating 5.6g (45mmol, 4.5eq) for 3-5min, putting into a solid phase synthesis reaction kettle, reacting at 20-30 deg.C for 3h, sampling ninhydrin after 3h, and detecting to obtain negative resin. Then, carrying out deprotection 2 times by adopting 20% piperidine/DMF, wherein the reaction time of the 1 st time is 5 min; the reaction time of the 2 nd time is 10min, and the resin is washed 6 times after two times of deprotection, 80ml each time and 1min each time. When the ninhydrin was removed, the resin appeared blue. Fmoc-Ava-OH, Fmoc-Gly-OH, Fmoc-Thr-x, Fmoc-Cys (Trt) -OH, Fmoc-Thr (tBu) -OH, Fmoc-Lys (Boc) -OH, Fmoc-D-Trp (Boc) -OH, Fmoc-Phe-OH, Fmoc-Cys (Trt) -OH and Fmoc-D-Phe-OH are coupled in sequence, and after the coupling is finished, the Fmoc protecting group is removed to obtain the octreotide peptide resin. Wherein the structure of Fmoc-Thr-x is as follows:
Figure BDA0002373722610000081
preparing a cleavage reagent, wherein the composition of TFA/TIS/MPR is 95/2.5/2.5, precooling for 1h in a refrigerator at the temperature of-15 ℃, transferring the cleavage reagent after precooling into a reaction kettle, adding the octreotide peptide resin obtained in the step (a), and carrying out cleavage reaction for 2h at the temperature of 20-28 ℃ according to the concentration of 10ml/g of the peptide resin.
After the cracking reaction is finished, filtering to remove resin, adding the obtained cracking filtrate into pre-frozen ether for settling, centrifuging and collecting; the resulting solid was centrifuged, washed with ether to a solution pH of 4-7 and dried in vacuo (30 ± 2, < -0.08MPa, 8h) to give crude octreotide linear peptide.
Dissolving the obtained linear crude octreotide in 30% acetic acid water solution, slowly adding iodine/ethanol until the reaction solution does not change color, sampling and detecting to obtain crude octreotide with purity of 90.5%, synthesis yield of 74.3%, maximum single impurity of 0.71%, less than 1.0%, and HPLC chromatogram similar to that of figure 1.
Example 7 Synthesis of octreotide
Accurately weighing 10g of AM resin with a substitution value of 1.0mmol/g, placing the AM resin in a solid phase synthesis reaction kettle, washing with DMF for 2 times and 50 ml/time, and adding 100ml of DMF to swell for 30min after washing.
After swelling, a small sample was taken and subjected to ninhydrin detection, and the resin was dark blue. Weighing Fmoc-Ava-OH: 10.2g (30mmol, 3eq), HOBT: 4.5g (33mmol, 3.3eq), dissolved in 50ml DMF, pre-cooled at 0-5 ℃ for 10min, DIC: activating 5.6g (45mmol, 4.5eq) for 3-5min, putting into a solid phase synthesis reaction kettle, reacting at 20-30 deg.C for 3h, sampling ninhydrin after 3h, and detecting to obtain negative resin. Then, carrying out deprotection 2 times by adopting 20% piperidine/DMF, wherein the reaction time of the 1 st time is 5 min; the reaction time of the 2 nd time is 10min, and the resin is washed 6 times after two times of deprotection, 80ml each time and 1min each time. When the ninhydrin was removed, the resin appeared blue. Fmoc-Ava-OH, Fmoc-Thr-x, Fmoc-Cys (Trt) -OH, Fmoc-Thr (tBu) -OH, Fmoc-Lys (Boc) -OH, Fmoc-D-Trp (Boc) -OH, Fmoc-Phe-OH, Fmoc-Cys (Trt) -OH and Fmoc-D-Phe-OH are coupled in sequence, and after the coupling is finished, the Fmoc protecting group is removed to obtain the octreotide peptide resin. Wherein the structure of Fmoc-Thr-x is as follows:
Figure BDA0002373722610000091
preparing a cleavage reagent which is composed of TFA/TIS/MPR (total soluble fraction of lysine/transferase/MPR) 94/3/3, precooling for 1h in a refrigerator at-15 ℃, transferring the cleavage reagent after precooling into a reaction kettle, adding the octreotide peptide resin obtained in the step, and carrying out cleavage reaction for 2h at 20-28 ℃ according to the concentration of 10ml/g of the peptide resin.
After the cracking reaction is finished, filtering to remove resin, adding the obtained cracking filtrate into pre-frozen ether for settling, centrifuging and collecting; the resulting solid was centrifuged, washed with ether to a solution pH of 4-7 and dried in vacuo (30 ± 2, < -0.08MPa, 8h) to give crude octreotide linear peptide.
The octreotide linear crude peptide obtained in the above step is dissolved in 10% DMSO/H2O solution, the reaction is carried out for 24H under stirring at room temperature, the sampling detection shows that the purity of the sample is 88.71%, the synthesis yield is 73.8%, the maximum single impurity is 0.91%, the maximum single impurity content is less than 1.0%, and the HPLC chromatogram is similar to that of the HPLC shown in the figure 1.
Example 8 Synthesis of octreotide
10g of MBHA resin with the substitution value of 1.0mmol/g is accurately weighed and placed in a solid phase synthesis reaction kettle, DMF is used for washing for 2 times and 50 ml/time, and 100ml of DMF is added for swelling for 30min after washing is finished.
After swelling, a small sample was taken and subjected to ninhydrin detection, and the resin was dark blue. Weighing Fmoc-Ava-OH: 10.1g (30mmol, 3eq), HOBT: 4.4g (33mmol, 3.3eq), dissolved in 50ml DMF, pre-cooled at 0-5 ℃ for 10min, DIC: activating 5.6g (45mmol, 4.5eq) for 3-5min, putting into a solid phase synthesis reaction kettle, reacting at 20-30 deg.C for 3h, sampling ninhydrin after 3h, and detecting to obtain negative resin. Then, carrying out deprotection 2 times by adopting 20% piperidine/DMF, wherein the reaction time of the 1 st time is 5 min; the reaction time of the 2 nd time is 10min, and the resin is washed 6 times after two times of deprotection, 80ml each time and 1min each time. When the ninhydrin was removed, the resin appeared blue. Fmoc-Gly-OH, Fmoc-Ava-OH, Fmoc-Thr-x, Fmoc-Cys (Trt) -OH, Fmoc-Thr (tBu) -OH, Fmoc-Lys (Boc) -OH, Fmoc-D-Trp (Boc) -OH, Fmoc-Phe-OH, Fmoc-Cys (Trt) -OH and Fmoc-D-Phe-OH are coupled in sequence, the coupling is finished, Fmoc protecting groups are removed, and the octreotide peptide resin is obtained. Wherein the structure of Fmoc-Thr-x is as follows:
Figure BDA0002373722610000101
preparing a cleavage reagent, wherein the composition of TFA/TIS/MPR is 95/2.5/2.5, precooling for 1h in a refrigerator at the temperature of-15 ℃, transferring the cleavage reagent after precooling into a reaction kettle, adding the octreotide peptide resin obtained in the step (a), and carrying out cleavage reaction for 2h at the temperature of 20-28 ℃ according to the concentration of 10ml/g of the peptide resin.
After the cracking reaction is finished, filtering to remove resin, adding the obtained cracking filtrate into pre-frozen ether for settling, centrifuging and collecting; the resulting solid was centrifuged, washed with ether to a solution pH of 4-7 and dried in vacuo (30 ± 2 ℃, < -0.08MPa, 8h) to give crude octreotide linear peptide.
Dissolving the obtained linear crude octreotide in a 15% acetic acid aqueous solution for dissolving and clarifying, slowly adding iodine/ethanol until the reaction solution does not change color, sampling and detecting, wherein the purity of the crude octreotide is 91.37%, the synthesis yield is 78.9%, the maximum single impurity content is 0.69%, and is less than 1.0%, and an HPLC chromatogram is similar to that of figure 1.
Example 9 Synthesis of octreotide
10g of MBHA resin with the substitution value of 1.0mmol/g is accurately weighed and placed in a solid phase synthesis reaction kettle, DMF is used for washing for 2 times and 50 ml/time, and 100ml of DMF is added for swelling for 30min after washing is finished.
After swelling, a small sample was taken and subjected to ninhydrin detection, and the resin was dark blue. Weighing Fmoc-Ava-OH: 10.1g (30mmol, 3eq), HOBT: 4.4g (33mmol, 3.3eq), dissolved in 50ml DMF, pre-cooled at 0-5 ℃ for 10min, DIC: activating 5.6g (45mmol, 4.5eq) for 3-5min, putting into a solid phase synthesis reaction kettle, reacting at 20-30 deg.C for 3h, sampling ninhydrin after 3h, and detecting to obtain negative resin. Then, carrying out deprotection 2 times by adopting 20% piperidine/DMF, wherein the reaction time of the 1 st time is 5 min; the reaction time of the 2 nd time is 10min, and the resin is washed 6 times after two times of deprotection, 80ml each time and 1min each time. When the ninhydrin was removed, the resin appeared blue. Fmoc-Ava-OH, Fmoc-Thr-x, Fmoc-Cys (Trt) -OH, Fmoc-Thr (tBu) -OH, Fmoc-Lys (Boc) -OH, Fmoc-D-Trp (Boc) -OH, Fmoc-Phe-OH, Fmoc-Cys (Trt) -OH and Fmoc-D-Phe-OH are coupled in sequence, the coupling is finished, the protecting group of the Fmoc is removed, and the octreotide resin is obtained. Wherein the structure of Fmoc-Thr-x is as follows:
Figure BDA0002373722610000111
preparing a cleavage reagent, wherein the composition of TFA/TIS/MPR is 95/2.5/2.5, precooling for 1h in a refrigerator at the temperature of-15 ℃, transferring the cleavage reagent after precooling into a reaction kettle, adding the octreotide peptide resin obtained in the step (a), and carrying out cleavage reaction for 2h at the temperature of 20-28 ℃ according to the concentration of 10ml/g of the peptide resin.
After the cracking reaction is finished, filtering to remove resin, adding the obtained cracking filtrate into pre-frozen ether for settling, centrifuging and collecting; the resulting solid was centrifuged, washed with ether to a solution pH of 4-7 and dried in vacuo (30 ± 2 ℃, < -0.08MPa, 8h) to give crude octreotide linear peptide.
Dissolving the obtained linear crude octreotide in 20% acetic acid water solution, slowly adding iodine/ethanol until the reaction solution does not change color, sampling and detecting to obtain crude octreotide with purity of 90.9%, synthesis yield of 77.5%, maximum single impurity of 0.51% and less than 1.0%, and HPLC chromatogram similar to that of figure 1.
Example 10 Synthesis of octreotide
Accurately weighing 20g of MBHA resin with a substitution value of 1.0mmol/g, placing the MBHA resin in a solid phase synthesis reaction kettle, washing with DMF for 2 times and 100 ml/time, and adding 200ml of DMF to swell for 30min after washing.
After swelling, a small sample was taken and subjected to ninhydrin detection, and the resin was dark blue. Weighing Fmoc-Ava-OH: 20.1g (60mmol, 3eq), HOBT: 8.7g (66mmol, 3.3eq), dissolved in 50ml DMF, pre-cooled at 0-5 ℃ for 10min, DIC: 11.3g (90mmol, 4.5eq) is activated for 3-5min, after the activation is finished, the mixture is put into a solid phase synthesis reaction kettle and reacts for 3h at the temperature of 20-30 ℃, and after 3h, ninhydrin is sampled for detection, and the resin is negative. Then, carrying out deprotection 2 times by adopting 20% piperidine/DMF, wherein the reaction time of the 1 st time is 5 min; the reaction time of the 2 nd time is 10min, and the resin is washed 6 times after two times of deprotection, 150ml each time and 1min each time. When the ninhydrin was removed, the resin appeared blue. Fmoc-Ava-OH, Fmoc-Thr-x, Fmoc-Cys (Trt) -OH, Fmoc-Thr (tBu) -OH, Fmoc-Lys (Boc) -OH, Fmoc-D-Trp (Boc) -OH, Fmoc-Phe-OH, Fmoc-Cys (Trt) -OH and Fmoc-D-Phe-OH are coupled in sequence, the coupling is finished, the protecting group of the Fmoc is removed, and the octreotide resin is obtained. Wherein the structure of Fmoc-Thr-x is as follows:
Figure BDA0002373722610000121
preparing a cleavage reagent, wherein the composition of TFA/TIS/MPR is 95/2.5/2.5, precooling for 1h in a refrigerator at the temperature of-15 ℃, transferring the cleavage reagent after precooling into a reaction kettle, adding the octreotide peptide resin obtained in the step (a), and carrying out cleavage reaction for 2h at the temperature of 20-28 ℃ according to the concentration of 10ml/g of the peptide resin.
After the cracking reaction is finished, filtering to remove resin, adding the obtained cracking filtrate into pre-frozen ether for settling, centrifuging and collecting; the resulting solid was centrifuged, washed with ether to a solution pH of 4-7 and dried in vacuo (30 ± 2 ℃, < -0.08MPa, 8h) to give crude octreotide linear peptide.
Dissolving the obtained linear crude octreotide in 20% acetic acid water solution, slowly adding iodine/ethanol until the reaction solution does not change color, sampling and detecting, wherein the purity of the crude octreotide is 88.91%, the synthesis yield is 74.5%, the maximum single impurity content is 0.68%, the maximum single impurity content is less than 1.0%, and the HPLC chromatogram is similar to that of figure 1.
Example 11 Synthesis of octreotide
Accurately weighing 11g of MBHA resin with a substitution value of 0.9mmol/g, placing the MBHA resin in a solid phase synthesis reaction kettle, washing with DMF for 2 times and 60 ml/time, and adding 110ml of DMF to swell for 30min after washing.
After swelling, a small sample was taken and subjected to ninhydrin detection, and the resin was dark blue. Weighing Fmoc-Ava-OH: 10.3g (30mmol, 3eq), HOBT: 4.5g (33mmol, 3.3eq), dissolved in 50ml DMF, pre-cooled at 0-5 ℃ for 10min, DIC: activating 5.6g (45mmol, 4.5eq) for 3-5min, putting into a solid phase synthesis reaction kettle, reacting at 20-30 deg.C for 3h, sampling ninhydrin after 3h, and detecting to obtain negative resin. Then, carrying out deprotection 2 times by adopting 20% piperidine/DMF, wherein the reaction time of the 1 st time is 5 min; the reaction time of the 2 nd time is 10min, and the resin is washed 6 times after two times of deprotection, 80ml each time and 1min each time. When the ninhydrin was removed, the resin appeared blue. Fmoc-Ava-OH, Fmoc-Thr-x, Fmoc-Cys (Trt) -OH, Fmoc-Thr (tBu) -OH, Fmoc-Lys (Boc) -OH, Fmoc-D-Trp (Boc) -OH, Fmoc-Phe-OH, Fmoc-Cys (Trt) -OH and Fmoc-D-Phe-OH are coupled in sequence, the coupling is finished, the protecting group of the Fmoc is removed, and the octreotide resin is obtained. Wherein the structure of Fmoc-Thr-x is as follows:
Figure BDA0002373722610000131
preparing a cleavage reagent, wherein the composition of TFA/TIS/MPR is 95/2.5/2.5, precooling for 1h in a refrigerator at the temperature of-15 ℃, transferring the cleavage reagent after precooling into a reaction kettle, adding the octreotide peptide resin obtained in the step (a), and carrying out cleavage reaction for 2h at the temperature of 20-28 ℃ according to the concentration of 10ml/g of the peptide resin.
After the cracking reaction is finished, filtering to remove resin, adding the obtained cracking filtrate into pre-frozen ether for settling, centrifuging and collecting; the resulting solid was centrifuged, washed with ether to a solution pH of 4-7 and dried in vacuo (30 ± 2 ℃, < -0.08MPa, 8h) to give crude octreotide linear peptide.
Dissolving the obtained linear crude octreotide in 20% acetic acid water solution, slowly adding iodine/ethanol until the reaction solution does not change color, sampling and detecting, wherein the purity of the crude octreotide is 89.1%, the synthesis yield is 75.6%, the maximum single impurity content is 0.87%, the maximum single impurity content is less than 1.0%, and the HPLC chromatogram is similar to that in figure 1.
Comparative example 1 Synthesis of octreotide
Accurately weighing 20g of AM resin with a substitution value of 0.5mmol/g, placing the AM resin in a solid phase synthesis reaction kettle, washing with DMF for 2 times and 50 ml/time, and adding 100ml of DMF to swell for 30min after washing.
After swelling, a small sample was taken and subjected to ninhydrin detection, and the resin was dark blue. Weighing Fmoc-Thr-x: 6.2g (30mmol, 3eq), HOBT: 4.4g (33mmol, 3.3eq), dissolved in 50ml DMF, pre-cooled at 0-5 ℃ for 10min, DIC: activating 5.6g (45mmol, 4.5eq) for 3-5min, and adding into solid phase for solid phase synthesisAnd (3) reacting in a reaction kettle at 20-30 ℃ for 3h, and sampling after 3h for ninhydrin detection, wherein the resin is negative. Then, carrying out deprotection 2 times by adopting 20% piperidine/DMF, wherein the reaction time of the 1 st time is 5 min; the reaction time of the 2 nd time is 10min, and the resin is washed 6 times after two times of deprotection, 80ml each time and 1min each time. The resin appeared blue when tested with ninhydrin. Fmoc-Cys (Trt) -OH, Fmoc-Thr (tBu) -OH, Fmoc-Lys (Boc) -OH, Fmoc-D-Trp (Boc) -OH, Fmoc-Phe-OH, Fmoc-Cys (Trt) -OH and Fmoc-D-Phe-OH are coupled in sequence, Fmoc protecting groups are removed after the coupling is finished, and the octreotide peptide resin is obtained. Wherein the structure of Fmoc-Thr-x is as follows:
Figure BDA0002373722610000141
preparing a cleavage reagent, wherein the composition of TFA/TIS/MPR is 95/2.5/2.5, precooling for 1h in a refrigerator at the temperature of-15 ℃, transferring the cleavage reagent after precooling into a reaction kettle, adding the octreotide peptide resin obtained in the step (a), and carrying out cleavage reaction for 2h at the temperature of 20-28 ℃ according to the concentration of 10ml/g of the peptide resin.
After the cracking reaction is finished, filtering to remove resin, adding the obtained cracking filtrate into pre-frozen ether for settling, centrifuging and collecting; the resulting solid was centrifuged, washed with ether to a solution pH of 4-7 and dried in vacuo (30 ± 2 ℃, < -0.08MPa, 8h) to give crude octreotide linear peptide.
Dissolving the obtained linear crude octreotide in 30% acetic acid water solution, slowly adding iodine/ethanol until the reaction solution does not change color, sampling and detecting, wherein the purity of the crude octreotide is 70.32%, the synthesis yield is 45.6%, the maximum single impurity close to the main peak is 3.34%, which is more than 1.0%, and the HPLC chromatogram is shown in figure 4.
Comparative example 2 Synthesis of Bremelanotide
Accurately weighing 100g of Rinkamide resin with the substitution degree of 0.52mmol/g, placing the Rinkamide resin in a solid phase synthesis kettle, washing twice with DMF (dimethyl formamide), 500 ml/time and 3 min/time, and adding 500ml of DCM (diethyl formamide) to swell the resin for 30min after washing. The Rinkamide resin is deprotected by 20% piperidine/DMF, 400 ml/time, the first reaction is 5min, and the second reaction is 15 min. After two deprotection, the resin was washed with DMF six times, 500 ml/time, 3 min/time. A small sample of the resin was tested with ninhydrin reagent, and the resin was dark blue.
Weighing HO- (CH2)15-COOH42.4g (156mmol, 3eq) and HOBt23.2g (171.6mmol, 3.3eq) in a dissolving bottle, dissolving with 200ml DMF, precooling for 10min at 0-5 ℃, adding DIC27.5ml (171.6mmol, 3.3eq) for activation for 5min, adding resin after activation, and stirring for reaction for 2h at 25 ℃. After 2h, the resin is taken out and detected by ninhydrin reagent, and the resin is colorless. Fmoc-Lys (alloc) -OH, Fmoc-Trp (Boc) -OH, Fmoc-Arg (pbf) -OH, Fmoc-D-Phe-OH, Fmoc-His (Boc) -OH, Fmoc-Asp (oAll) -OH, Fmoc-Nle-OH are coupled in sequence after Fmoc removal. After Fmoc-Nle-OH coupling is completed, removing Fmoc protecting group, and acetylating with acetic anhydride/pyridine to obtain AC-Nle-Asp (oAll) -His (Boc) -D-Phe-Arg (pbf) -Trp (Boc) -Lys (alloc) -Rink-Ava-Gly-Ala-MBHA resin. Removing side chain protecting groups Alloc and OAll by adopting tetratriphenylphosphine palladium, and then weighing HOBT: 2.2g, dissolved in 50ml DMF, precooled at 0-5 ℃ for 10min, added to the resin above, and then DIC: 2.8g, reacting at 20-30 ℃ for 4h, taking a small sample to detect that the resin is negative, and obtaining AC-Nle- [ Asp-His (Boc) -D-Phe-Arg (pbf) -Trp (Boc) -Lys ] -O- (CH2)15-CONH-Rinkamide resin after the reaction is finished.
Preparing a cracking reagent, wherein the composition of TFA/TIS/MPR/H2O is 92.5/2.5/2.5/2.5, precooling for 1H in a refrigerator at the temperature of-15 ℃, transferring the precooled cracking reagent into a reaction kettle, adding the obtained buminuo peptide resin, and carrying out cracking reaction for 3H at the temperature of 20-26 ℃ according to the concentration of 10ml/g of the peptide resin.
After the cracking reaction is finished, filtering to remove resin, adding the obtained cracking filtrate into pre-frozen ether for settling, centrifuging and collecting; the resulting solid was centrifuged, washed with ether to a solution pH of 4-7 and dried under vacuum (30 ± 2 ℃, < -0.08MPa, 8h) to give crude bremelanotide. Sampling and detecting, wherein the purity of the crude product of the brennol peptide is 66.59%, the synthesis yield is 38.6%, the maximum single impurity content is 11.76%, and the maximum single impurity content is more than 2.0%, and an HPLC chromatogram is shown in figure 5.
Comparative example 3 Synthesis of liraglutide
Accurately weighing 20g of wang resin with a substitution value of 1.0mmol/g, placing the wang resin in a solid phase synthesis reaction kettle, washing with DMF for 2 times and 50 ml/time, and adding 100ml of DMF to swell for 30min after washing.
After swelling, weighing Fmoc-Gly-OH: 11.8g (40mmol, 2eq), HOBT: 5.9g (44mmol, 2.2eq), dissolved in 50ml DMF, pre-cooled at 0-5 ℃ for 10min, DIC: 7.5g (60mmol, 3eq), adding DMAP (4mmol, 0.2eq) to activate for 3-5min, activating, putting into a solid phase synthesis reaction kettle, reacting for 5h at 20-30 ℃, capping with acetic anhydride/DIEA after 5h, sequentially coupling Fmoc-Arg (Pbf) -OH, Fmoc-Gly-OH, Fmoc-Arg (Pbf) -OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Trp (Boc) -OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Phe-OH, Fmoc-Glu (OtBu) -OH, Fmoc-Lys (Pla-Glu-OtBu) -OH, Fmoc-Ala-OH, Fmoc-Gln (Trt) -OH, Fmoc-Gly-OH, Fmoc-Gln (Trt) -OH, Fmoc-Gly-OH, Fmoc-Arg-OH, Fmoc-Arg-OH, Fmoc-Arg-OH, Fmoc-Arg-OH, Fmoc-Arg-OH, Fmoc-Arg-OH, and/Ala-Arg-OH, Fmoc-Arg-OH, Fmoc-Arg-OH, Fmoc-Arg-OH, Fmoc-Arg-OH, Coupling of Fmoc-Glu (OtBu) -OH, Fmoc-Leu-OH, Fmoc-Tyr (tBu) -OH, Fmoc-Ser (tBu) -OH, Fmoc-Val-OH, Fmoc-Asp (OtBu) -OH, Fmoc-Ser (tBu) -OH, Fmoc-Thr (tBu) -OH, Fmoc-Phe-OH, Fmoc-Thr (tBu) -OH, Fmoc-Gly-OH, Fmoc-Glu (OtBu) -OH, Fmoc-Ala-OH, Boc-His (Trt) -OH. After the reaction was complete, washed 6 times with DCM, MeOH was retracted and dried in vacuo to give the peptide resin.
Taking 13.94g of the obtained liraglutide resin, adding TFA: and Tis: water 95: 2.5: 2.5 (using amount of 10mL/g liraglutide resin), stirring uniformly, stirring at room temperature for reaction for 3 hours, filtering the reaction mixture by using a sand core funnel, collecting the filtrate, washing the resin for 3 times by using a small amount of TFA, combining the filtrates, concentrating under reduced pressure, adding diethyl ether for precipitation, washing the precipitate for 6 times by using diethyl ether, and drying under reduced pressure in vacuum to constant weight, wherein 3.47g of the white powder of the liraglutide is obtained, the yield is 29.44%, the purity is 50.79%, the maximum impurity close to the main peak is 1.55%, the maximum impurity is more than 0.5%, and the HPLC chromatogram is shown in fig. 6.

Claims (9)

1. A solid-phase synthesis method of peptide is characterized in that linker-A-solid-phase resin is prepared and used as initial resin for solid-phase synthesis of peptide, wherein A is one or more amino acids.
2. The method of claim 1, wherein the amino acids in the linker-A-solid phase resin are selected from one or more of Gly, Ala, Ava, Val, Leu, and Ile.
3. The method of claim 1, wherein the number of amino acids in the linker-A-solid phase resin is not more than 5.
4. The method of claim 1, wherein the solid phase resin of the linker-A-solid phase resin is selected from the group consisting of AM resin, MBH resin, RINKAM resin, and RINKMBHA resin.
5. The method of claim 1, wherein the peptide is selected from the group consisting of brennol, octreotide, exenatide, pramlintide, liraglutide, and somaglutide.
6. The method of claim 5, wherein when the peptide is octreotide, the linker of the linker-A-solid phase resin is Fmoc-Thr-x, and the general chemical structure is:
Figure FDA0002373722600000011
wherein the content of the first and second substances,
R1、R2、R3、R4selected from H, CH3、CH3O, OH, respectively, are provided, one or more of,
n is 0 to 5.
7. The method of claim 5, wherein the solid phase resin of the linker-A-solid phase resin is AM resin or MBHA resin when the peptide is octreotide.
8. The method for the solid-phase synthesis of a peptide according to any one of claims 6 or 7, wherein when the peptide is octreotide, the cleavage system for the solid-phase synthesis of the peptide is: mixed solution of TFA, TIS and Mpr.
9. The method of claim 8, wherein the amino acid in the linker-A-solid phase resin is Gly when the peptide is octreotide.
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