CN116789768A - Preparation method of F1 polypeptide - Google Patents
Preparation method of F1 polypeptide Download PDFInfo
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- CN116789768A CN116789768A CN202310953906.4A CN202310953906A CN116789768A CN 116789768 A CN116789768 A CN 116789768A CN 202310953906 A CN202310953906 A CN 202310953906A CN 116789768 A CN116789768 A CN 116789768A
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- 102000004196 processed proteins & peptides Human genes 0.000 title claims abstract description 122
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- 125000004213 tert-butoxy group Chemical group [H]C([H])([H])C(O*)(C([H])([H])[H])C([H])([H])[H] 0.000 claims abstract description 25
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- 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 abstract description 13
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- PSACHCMMPFMFAJ-UHFFFAOYSA-N nmm n-methylmorpholine Chemical compound CN1CCOCC1.CN1CCOCC1 PSACHCMMPFMFAJ-UHFFFAOYSA-N 0.000 description 1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/001—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof by chemical synthesis
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- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Biochemistry (AREA)
- Gastroenterology & Hepatology (AREA)
- Life Sciences & Earth Sciences (AREA)
- General Chemical & Material Sciences (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention belongs to the technical field of pharmacy, and particularly relates to a preparation method of F1 polypeptide, which comprises the following steps: preparing Rink Amide MBHA resin; preparing Gly-Leu-Leu-Ser (tBu) -Val-Leu-Gly-Ser (tBu) -Val-Ala-Lys (Boc) -His (Trt) -Val-Leu-Pro-His (Trt) -Val-Leu-Pro-His (Trt) -Val-Val-Pro-Val-Ile-Ala-Glu (OtBu) -His (Trt) -Leu-Rin kAmide MBHA Resin resin in two steps; adding into cutting liquid to cut side chain protecting group, depositing, washing, drying and cracking; purifying, changing salt and freeze-drying. The invention improves the purity of the F1 polypeptide.
Description
Technical Field
The invention belongs to the technical field of pharmacy, and particularly relates to a preparation method of F1 polypeptide.
Background
Almost all cells in an organism are regulated by polypeptides such as: cell differentiation, neurohormonal transmitter regulation, immune regulation and the like, the polypeptide has the dual effects of regulating physiological functions of organisms and providing nutrition for the organisms,therefore, the research and development of the polypeptide medicine have important research significance and application prospect. The F1 polypeptide Chinese sequence is: glycyl-L-leucyl-L-seryl-L-valyl-L-alanyl-L-lysyl-L-histidyl-L-valyl-L-leucyl-L-prolyl-L group aminoacyl-L-valyl-L-leucyl-L-prolyl-L-histidyl-L-valyl-L-prolyl-L-valyl-L-isoleucyl-L-alanyl-L-glutamyl-L-histidyl-L-leucinamide acetate. The English sequence is: H-Gly-Leu-Leu-Ser-Val-Leu-Gly-Ser-Val-Ala-Lys-His-Val-Leu-Pro-His-Val-Leu-Pro-His-Val-Val-Pro-Val-Ile-Ala-Glu-His-Leu-NH 2 . The molecular formula is: c (C) 143 H 237 N 39 O 33 。
The structure of the F1 polypeptide is as follows:
people can obtain various active peptides by utilizing methods such as in vivo separation, biotechnology, chemical synthesis and the like, and particularly, the advantages of conveniently changing the primary structure of the polypeptide, adding special amino acid, modifying the tail end of the polypeptide and the like in the chemical synthesis process are paid attention to. The existing preparation method of the F1 polypeptide is to connect Fmoc-amino acids in sequence according to the polypeptide sequence, but for the sequence with continuous hydrophobic amino acid at the N end, polycondensation is easy to occur in the synthesis process, so that the yield is low, the impurity content is high, and the preparation method is not beneficial to large-scale high-purity production in the pharmaceutical industry.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a preparation method of F1 polypeptide. The invention improves the yield of the F1 polypeptide, greatly reduces the impurity content, obviously improves the purity of the F1 polypeptide, and is beneficial to large-scale high-purity production in the pharmaceutical industry.
The technical scheme of the invention is as follows:
a method for preparing an F1 polypeptide, comprising the steps of:
s1, preparing Rink Amide MBHA resin;
s2 preparing full-protection peptide resin in two steps
Gly-Leu-Leu-Ser (tBu) -Val-Leu-Gly-Ser (tBu) -Val-Ala-Lys (Boc) -His (Trt) -Val-Leu-Pro-His (Trt) -Val-Leu-Pro-His (Trt) -Val-Val-Val-Pro-Val-Ile-Ala-Glu (OtBu) -His (Trt) -Leu-Rink Amide MBHA Resin resin, comprising: (1) Sequentially coupling corresponding amino acids from the C end to the N end according to the F1 polypeptide sequence, and synthesizing to obtain Fmoc-Val-Ala-Lys (Boc) -His (Trt) -Val-Leu-Pro-His (Trt) -Val-Leu-Pro-His (Trt) -Val-Val-Val-Pro-Val-Ile-Ala-Glu (OtBu) -His (Trt) -Leu-Rink Amide MBHA Resin resin; (2) Coupling the product obtained in the step (1) with 8 subsequent amino acid sites to obtain Fmoc-Gly-Leu-Leu-Ser (tBu) -Val-Leu-Gly-Ser (tBu) -Val-Ala-Lys (Boc) -His (Trt) -Val-Leu-Pro-His (Trt) -Val-Leu-Pro-His (Trt) -Val-Val-Pro-Val-Ile-Ala-Glu (OtBu) -His (Trt) -Leu-Rink Amide MBHA Resin resin, removing Fmoc protection to obtain full protection peptide resin Gly-Leu-Leu-Ser (tBu) -Val-Leu-Gly-Ser (tBu) -Val-Ala-Lys (Boc) -His (Trt) -Val-Leu-Pro-His (Trt) -Val-Leu-Pro-His (Trt) -Val-Ile-Ala-His (OtBu) -Leu-Rink Amide MBHA Resin resin;
S3, adding the full-protection peptide resin obtained in the step S2 into a cutting fluid to cut a side chain protecting group, precipitating, washing and drying to obtain a crude F1 polypeptide;
s4, purifying the crude product of the F1 polypeptide obtained in the step S3, purifying by salt exchange, and freeze-drying to obtain the F1 polypeptide.
Further, the Rink Amide MBHA resin in the step S1 is prepared from MBHA resin and Rink Amide Linker serving as main raw materials, and the initial substitution degree of the MBHA resin is 0.1-0.8 mmol/g. Because the peptide chain sequence is longer, the substitution degree is too high, and the connection is more difficult; the substitution degree is too low, which causes waste of reagents, and the production equipment has too high requirements on the scale and performance, so that the large-scale industrial production cannot be performed.
Further, the specific steps for preparing the Rink Amide MBHA resin in the step S1 are as follows:
(1) Weighing MBHA resin, placing the MBHA resin into a solid phase synthesis reactor with a sieve plate, adding 5% NMM/DMF, swelling for 60-120 minutes at 20-30 ℃, pumping, washing the resin for 2 times by using DMF with the volume of 1.0 times of the resin bed layer, pumping for 2-4 minutes each time, taking a small amount of resin as ninhydrin for detection, and leading the result to be positive;
(2) Weighing 3eq Rink Amide Linker and 2.85eq HBTU, adding into a container, dissolving DMF with the volume of 0.5-1.0 times of resin, cooling under the condition of a cold bath, slowly adding NMM, pre-activating for 5-15 minutes, adding the pre-activated reaction liquid into the reactor containing resin in the step (1), reacting for 90-120 minutes at 20-30 ℃, detecting ninhydrin every 30+/-5 minutes in the reaction process, and pumping out the reaction liquid, washing resin 3 times with DMF with the volume of 1.0 times of resin bed layer for 2-4 minutes each time if the continuous two ninhydrin detections are negative and the prescribed reaction time is reached, and pumping out the reaction liquid to obtain the product.
Preferably, the MBHA resin has an initial degree of substitution of 0.47mmol/g.
Further, the step S2 is to couple corresponding amino acids from the C end to the N end according to the F1 polypeptide sequence, and synthesize the amino acids first
The specific steps of Fmoc-Val-Ala-Lys (Boc) -His (Trt) -Val-Leu-Pro-His (Trt) -Val-Leu-Pro-His (Trt) -Val-Val-Pro-Val-Ile-Ala-Glu (OtBu) -His (Trt) -Leu-Rink Amide MBHA Resin resin are as follows:
(a) Placing Rink Amide MBHA resin in a reactor, adding 20% (v/v) piperidine/DMF solution with the volume of 1.0 times of the resin bed layer into the reactor, stirring for 10+/-1 minutes, pumping, adding 20% (v/v) piperidine/DMF solution with the volume of 1.0 times of the resin bed layer into the reactor, stirring for 20+/-1 minutes, pumping, and obtaining Fmoc-removed protective resin; washing resin with DMF (dimethyl formamide) with 1.0 times of the volume of the resin bed layer for 8 times, pumping for 2-4 minutes each time, taking a small amount of resin for ninhydrin detection, and leading the result to be positive;
(b) And (3) weighing Fmoc-AA-OH and a coupling agent, adding the Fmoc-AA-OH and the coupling agent into a container, dissolving DMF with the volume of 0.5-1.0 times of the resin, cooling under a cold bath condition, slowly adding DIC, pre-activating for 5-15 minutes, adding the pre-activated reaction liquid into a reactor, reacting for 120-180 minutes at 20-30 ℃, detecting ninhydrin every 30+/-5 minutes in the reaction process, and if the ninhydrin detection is negative continuously twice, and the specified reaction time is reached, pumping out the reaction liquid, washing the resin 3 times with DMF with the volume of 1.0 times of the resin bed, and pumping out for 2-4 minutes each time, and circulating in this way, so as to connect all amino acids.
Further, fmoc-AA-OH in the step (b) is Fmoc-Leu-OH, fmoc-His (Trt) -OH, fmoc-Glu (OtBu) -OH, fmoc-Ala-OH, fmoc-Ile-OH, fmoc-Val-OH, fmoc-Pro-OH, fmoc-Lys (Boc) -OH.
Further, when Fmoc-AA-OH is Fmoc-His (Trt) -OH, the coupling agent is HOOBt; when Fmoc-AA-OH is Fmoc-Leu-OH, fmoc-Glu (OtBu) -OH, fmoc-Ala-OH, fmoc-Ile-OH, fmoc-Val-OH, fmoc-Pro-OH, fmoc-Lys (Boc) -OH, the coupling agent is HOBt.
The invention selects Fmoc-AA-OH which is a commercially available Fmoc protected amino acid raw material, and can reduce the production cost.
Furthermore, when the fully-protected peptide resin is prepared in the step S2, fmoc-Ser (tBu) -OH is adopted to couple Ser amino acid sites, and when Val amino acid at the 5 th position is connected, the liquid exchange operation is carried out to obtain the best product effect, because the liquid exchange leads the concentration of reactants in the reaction kettle to be improved, and simultaneously the concentration of byproducts to be reduced, the reaction is moved towards the direction of the production product, and the higher yield can be obtained;
further, the coupling system used for coupling the Ser amino acid site with Fmoc-Ser (tBu) -OH is selected from HOBt/DIC.
Further, the step S3 is to add the full-protection peptide resin obtained in the step S2 into a cutting fluid to cut a side chain protecting group, and the specific steps of precipitation, washing and drying are as follows:
1) Adding the full-protection peptide resin obtained in the step S3 into cutting fluid, stirring while adding, controlling the reaction temperature to be 25-35 ℃, stirring and reacting for 2.5-3.5 hours, filtering the resin by using a sand core funnel after cutting is completed, collecting filtrate, washing the resin by using TFA, combining the filtrate, and recording the volume of the filtrate;
2) Slowly adding the collected filtrate into frozen diethyl ether with the volume of 10 times, stirring while adding, centrifuging or filtering to obtain crude product after peptide chain is separated out, and washing with diethyl ether for 3 times;
3) Transferring the crude product obtained in the step 2) into a container for drying, and vacuum drying until the weight change rate of the crude product is less than 1.0% in 2 hours.
Further, the cutting fluid in the step S3 comprises trifluoroacetic acid, anisole sulfide, 1, 2-ethanedithiol, phenol and water. Further, in the step S3, the cutting fluid is: trifluoroacetic acid, anisole, 1, 2-ethanedithiol, phenol, water=87.5-90:1-5:2.5-3:2.5-3:2.5-3.
Further, in the step S3, the cutting fluid is: trifluoroacetic acid: anisole: 1, 2-ethanedithiol: phenol: water=87.5:5:2.5:2.5:2.5.
The cleavage liquid of the invention can effectively separate the polypeptide from the carrier for further purification and analysis, wherein the anisole can increase the surface tension and viscosity to promote the separation and precipitation of the polypeptide, thereby facilitating the separation and purification of the polypeptide. In addition, the anisole also has a certain antioxidation effect, and can prevent the polypeptide from being damaged by oxidation in the cutting fluid. The 1, 2-ethanedithiol protects the polypeptide from oxidation or other adverse factors, and can reduce the surface tension in aqueous solution to promote the separation and purification of the polypeptide. The primary function of phenol is to increase the viscosity and surface tension of the cutting fluid, which aids in the separation of the polypeptide from the solid support. In addition, phenol can neutralize the acidity in the cutting fluid, so that the pH value of the cutting fluid is adjusted, and the separation and purification of the polypeptide are facilitated. When the side chain protecting group is cut into the fully protected peptide resin, incomplete removal of the amino acid side chain protecting group, cleavage of peptide bond, oxidation and the like are easily generated, and the impurities generated in the step are more and more complex. The components in the cutting fluid of the invention act together to effectively separate the polypeptide from the carrier, reduce the generation of impurities, and improve the purity of the crude polypeptide so as to further purify and analyze.
Further, the step S4 comprises the specific steps of purifying the crude product of the F1 polypeptide obtained in the step S3:
(A) Dissolving a crude product: taking a crude F1 polypeptide product, and using acetic acid: acetonitrile: purified water = 1:2:7 (V: V) dissolving the crude F1 polypeptide to 50mg/mL, and then filtering with a filter membrane with a pore diameter of 1.5 μm to obtain a crude solution;
(B) Loading the chromatographic column in a balance way: balancing the chromatographic column with a mixed solution of 95% mobile phase A and 5% mobile phase B for at least 8 min, and loading the crude solution at 100%;
(C) Gradient elution: after sample loading, a mixed solution equilibrium chromatographic column of 95% of mobile phase A and 5% of mobile phase B is used until an acetic acid peak is completely eluted, and then a mixed solution equilibrium chromatographic column of 62% of mobile phase A and 38% of mobile phase B is used, wherein the equilibrium time is not less than 8 minutes, and the sample is subjected to gradient elution;
(D) Sample collection: collecting eluent corresponding to a target peak of a product into numbered collecting bottles in a segmented manner, ensuring the consistency of the volumes of each bottle, and marking the corresponding collecting bottle number in a detection map;
(E) And (3) collecting liquid detection: and detecting and analyzing the purified collection liquid by adopting an HPLC method, integrating the spectrum after the detection is finished, analyzing the purity and impurity content of the sample according to an area normalization method, and collecting the sample meeting the standard.
Further, the crude product in the step S4 is purified by using a reversed-phase high performance liquid chromatography, C18 filler with the particle size of 3 mu m is used as a stationary phase of a chromatographic column, triethylamine, phosphoric acid and water with the volume ratio of 1:1:100 are used as mobile phase A phase, and the mobile phase B phase consists of the mobile phase A phase and acetonitrile according to the volume ratio of 2:8.
Further, the column temperature of the chromatographic column is kept at 50-55 ℃ in the purification process, and the impurity separation degree before and after the main peak can be improved under the column temperature condition of the chromatographic column; preferably, the column temperature of the chromatographic column is kept at 55 ℃ during the purification process, and the impurity separation degree before and after the main peak is optimal under the column temperature condition of the chromatographic column. Neither the temperature is too high nor too low to allow good separation of the main peak from the impurities using HPLC. Further, in the step (C), gradient elution is carried out on the sample according to a linear gradient from 38% to 58% in the proportion of the mobile phase B phase within 90 minutes, and under the purification gradient, the pre-impurity separation degree can be obviously improved, the post-impurity can be separated out, and the product purity is improved.
Further, the specific steps of the salt replacement purification in the step S5 are as follows:
and (I) equilibrium loading: balancing the chromatographic column with a mixed solution of 95% acetic acid system mobile phase A and 5% acetic acid system mobile phase B for at least 8 min, and mixing the collected liquid and purified water according to a ratio of 1:1, sampling in proportion, and replacing salt balance chromatographic column with 32g/L acetic acid solution for at least 30min;
(II) gradient elution: after salt replacement and balancing, balancing the chromatographic column by using a mixed solution of 75% of mobile phase A and 25% of mobile phase B, wherein the balancing time is not less than 8 minutes, and carrying out gradient elution on a sample according to a linear gradient that the proportion of the mobile phase B of an acetic acid system is increased from 25% to 55% within 60 minutes;
(III) sample collection: collecting eluent corresponding to a target peak of a product into numbered collecting bottles in a segmented manner, ensuring the consistency of the volumes of each bottle, and marking the corresponding collecting bottle number in a detection map;
and (IV) detecting and analyzing the purified collection liquid by adopting an HPLC method, integrating the spectrum after the detection is finished, analyzing the purity and impurity content of the sample according to an area normalization method, and collecting the sample meeting the standard, wherein the detection method is consistent with the detection of the purified collection liquid in the first step.
Because the part of the sequence close to the N end is continuous hydrophobic amino acid, polycondensation is easy to occur in the production process, so that the production is difficult, the invention adopts segmented synthesis, and uses Fmoc-Ser (tBu) -OH to couple serine sites, thereby inhibiting the occurrence of polycondensation, reducing the production difficulty, improving the yield of the F1 polypeptide, greatly reducing the impurity content and obviously improving the purity of the F1 polypeptide.
Compared with the prior art, the invention has the following advantages:
(1) The invention develops and optimizes the synthesis, cutting and purification methods and processes of the F1 polypeptide, can reduce the production cost, improve the yield of the F1 polypeptide, greatly reduce the impurity content, remarkably improve the purity of the F1 polypeptide, and has the purity as high as 99.77 percent, thereby being beneficial to large-scale high-purity production in the pharmaceutical industry;
(2) In the synthesis process of the F1 polypeptide, only the Val at the 5 th position is subjected to forced liquid exchange operation, and liquid exchange is not needed in each step, so that the yield is improved, and the cost of industrial production is saved.
Drawings
FIG. 1 is an IR spectrum diagram of an F1 polypeptide prepared in example 1 of the present invention;
FIG. 2 is a mass spectrum of the F1 polypeptide prepared in example 1 of the present invention;
FIG. 3 is a first-order mass spectrum of the F1 polypeptide prepared in example 1 of the present invention;
FIG. 4 is a primary mass spectrum deconvolution spectrum of the F1 polypeptide prepared in example 1 of the present invention;
FIG. 5 is a secondary mass spectrum of the F1 polypeptide prepared in example 1 of the present invention;
FIG. 6 is a HPLC chart of F1 polypeptide prepared in example 1 of the present invention.
Detailed Description
The invention is further illustrated by the following description of specific embodiments, which are not intended to be limiting, and various modifications or improvements can be made by those skilled in the art in light of the basic idea of the invention, but are within the scope of the invention without departing from the basic idea of the invention.
The relevant nouns in the present invention are explained in the following table 1:
table 1: noun interpretation in connection with the present invention
| His | Histidine |
| Gly | Glycine (Gly) |
| Glu | Glutamic acid |
| Ala | Alanine (Ala) |
| Pro | Proline (proline) |
| Lys | Lysine |
| Leu | Leucine (leucine) |
| Ser | Serine (serine) |
| Val | Valine (valine) |
| Ile | Isoleucine (Ile) |
| Fmoc | 9-fluorenylmethoxycarbonyl |
| HOOBt | 3-hydroxy-1, 2, 3-benzotriazin-4 (3H) -one |
| HOBT | 1-hydroxybenzotriazoles |
| DIC | N, N-diisopropylcarbodiimide |
| TFA | Trifluoroacetic acid |
| EDT | 1, 2-ethanedithiol |
| TEA | Triethylamine |
| NMM | N-methyl morpholine |
| DMF | N, N-dimethylformamide |
| HBTU | benzotriazol-N, N, N ', N' -tetramethylurea hexafluorophosphate |
| ACN | Acetonitrile |
。
Unless otherwise indicated, all terms of art used herein are intended to be related to the same meaning as commonly understood by one of ordinary skill in the art.
Example 1 preparation of F1 Polypeptides
Step S1: preparation of Rink Amide MBHA resin
(1) Preparing 5mmol of MBHA resin (substitution degree is 0.47 mmol/g), placing the resin in a solid phase synthesis reactor with a sieve plate, adding 5% of NMM/DMF (NMM DMF solution with volume fraction of 5%) and swelling the resin at room temperature for 1h, pumping, washing the resin for 2 times by using DMF with 2.5 times of resin bed volume for 4 minutes, pumping, taking a small amount of resin for ninhydrin detection, and leading the result to be positive;
(2) Weighing 15mmol Rink Amide Linker and 14mmol HBTU, adding into a container, dissolving 1.0 times of DMF (dimethyl formamide) of resin volume of MBHA, cooling under a cold bath condition (solution temperature is 15 ℃), slowly adding 45mmol NMM, pre-activating for 10 minutes, adding the pre-activated reaction liquid into the reactor containing the resin in the step (1), reacting for 2.5 hours at room temperature, sampling to detect ninhydrin, and if the ninhydrin is negative in two continuous ninhydrin detections and reaches a specified reaction time, pumping out the reaction liquid, washing the resin for 4 times by using 2.5 times of DMF of resin bed volume, and pumping out each time for 3 minutes, thus obtaining the final product.
Step S2: preparation of fully protected peptide resin in two steps
Gly-Leu-Leu-Ser (tBu) -Val-Leu-Gly-Ser (tBu) -Val-Ala-Lys (Boc) -His (Trt) -Val-Leu-Pro-His (Trt) -Val-Leu-Pro-His (Trt) -Val-Val-Val-Pro-Val-Ile-Ala-Glu (OtBu) -His (Trt) -Leu-Rink Amide MBHAResin resin
(1) Coupling corresponding amino acids sequentially from C terminal to N terminal according to F1 polypeptide sequence, and synthesizing to obtain
Fmoc-Val-Ala-Lys (Boc) -His (Trt) -Val-Leu-Pro-His (Trt) -Val-Leu-Pro-His (Trt) -Val-Val-Pro-Val-Ile-Ala-Glu (OtBu) -His (Trt) -Leu-Rink Amide MBHAResin resin, comprising the following specific steps:
(a) Placing the Rink Amide MBHA resin obtained in the step S1 into a reactor, adding 20% (v/v) piperidine/DMF solution (20% piperidine DMF solution by volume fraction) of 2.5 times of resin bed volume into the reactor, stirring for 10 minutes, pumping, adding 20% (v/v) piperidine/DMF solution (20% piperidine DMF solution by volume fraction) of 2.5 times of resin bed volume into the reactor, stirring for 20 minutes, pumping, and obtaining Fmoc-removed protective resin; washing resin with DMF (dimethyl formamide) with 2.5 times of resin bed volume for 3 minutes each time, pumping, and taking a small amount of resin for ninhydrin detection, wherein the result is positive;
(b) Weighing 15mmol Fmoc-AA-OH and 15mmol HOBT, adding into a container, using DMF to dissolve, cooling under a cold bath condition, slowly adding 15mmol DIC, pre-activating for 10 minutes, adding the pre-activating reaction liquid into the reactor, reacting for 2.5 hours at room temperature, sampling to detect ninhydrin, if the detection of the ninhydrin is negative twice continuously, and the specified reaction time is reached, pumping out the reaction liquid, washing the resin with DMF with 2.5 times of the volume of the resin bed for 4 times, pumping out for 3 minutes each time, and circulating in such a way, and connecting all amino acids.
The Fmoc-AA-OH in the step (b) is Fmoc-Leu-OH, fmoc-His (Trt) -OH, fmoc-Glu (OtBu) -OH, fmoc-Ala-OH, fmoc-Ile-OH, fmoc-Val-OH, fmoc-Pro-OH, fmoc-Lys (Boc) -OH.
When Fmoc-AA-OH is Fmoc-His (Trt) -OH, the coupling agent is HOOBt; when Fmoc-AA-OH is Fmoc-Leu-OH, fmoc-Glu (OtBu) -OH, fmoc-Ala-OH, fmoc-Ile-OH, fmoc-Val-OH, fmoc-Pro-OH, fmoc-Lys (Boc) -OH, the coupling agent is HOBt.
Wherein Fmoc-AA-OH/HOBt (HOOBt)/DIC=3/3/3 or 5/5, his was HOOBt, and 5eq was used after polycondensation of the resin. The amino acid materials, coupling reagents and reactions used are shown in Table 2.
Table 2: amino acid material, coupling reagent and reaction condition
(2) Coupling the product obtained in the step (1) with the subsequent 8 amino acid sites to obtain Fmoc-Gly-Leu-Leu-Ser (tBu) -Val-Leu-Gly-Ser (tBu) -Val-Ala-Lys (Boc) -His (Trt) -Val-Leu-Pro-His (Trt) -Val-Leu-Pro-His (Trt) -Val-Val-Pro-Val-Ile-Ala-Glu (OtBu) -His (Trt) -Leu-Rink Amide MBHA Resin resin, wherein the preparation method comprises the following specific steps of:
the subsequent amino acid site ligation was performed using 20% of Fmoc-Val-Ala-Lys (Boc) -His (Trt) -Val-Leu-Pro-His (Trt) -Val-Leu-Pro-His (Trt) -Val-Val-Val-Pro-Val-Ile-Ala-Glu (OtBu) -His (Trt) -Leu-Rink Amide MBHA Resin resin (i.e., 20% of the first-step product was used in the second-step coupling reaction) and the amino acid materials, coupling reagents and reaction conditions were as shown in Table 3.
Table 3: amino acid material, coupling reagent and reaction condition
After all amino acids are connected, adding 20% (v/v) piperidine/DMF solution with 1.0 times of resin bed volume into a reactor to remove Fmoc protection, stirring for 20 minutes, pumping, washing resin with 1.0 times of resin bed volume of DMF for 2 times, washing for 10 minutes for the first time, washing for 20 minutes for the second time, pumping, taking a small amount of resin as ninhydrin for detection, and obtaining the full-protection peptide resin with positive result
Gly-Leu-Leu-Ser (tBu) -Val-Leu-Gly-Ser (tBu) -Val-Ala-Lys (Boc) -His (Trt) -Val-Leu-Pro-His (Trt) -Val-Leu-Pro-His (Trt) -Val-Val-Val-Pro-Val-Ile-Ala-Glu (OtBu) -His (Trt) -Leu-Rink Amide MBHA Resin resin.
Step S3: adding the full-protection peptide resin obtained in the step S2 into a cutting fluid to cut a side chain protecting group, precipitating, washing and drying to obtain a crude F1 polypeptide product, wherein the method comprises the following specific steps of:
(1) Adding the full-protection peptide resin obtained in the step S2 into a cutting fluid, wherein the volume-mass ratio of the cutting fluid to the full-protection peptide resin is 10mL/g, and the cutting fluid is as follows: trifluoroacetic acid, anisole and phenol, wherein the ratio of the anisole to the 1, 2-ethanedithiol to the phenol is equal to 87.5:5:2.5:2.5:2.5, stirring is carried out while the reaction temperature is controlled to 25 ℃, stirring is carried out for 3 hours, after cutting, a sand core funnel is used for filtering out resin, the filtrate is collected, the resin is washed by TFA, the filtrate is combined, and the volume of the filtrate is recorded;
(2) Slowly adding the collected filtrate into frozen diethyl ether with the volume of 10 times, stirring while adding, centrifuging to obtain a crude product after peptide chains are separated out, and washing with diethyl ether for 4 times;
(3) And (3) transferring the crude product obtained in the step (2) into a container for drying, and vacuum drying until the weight change rate of the crude product is less than 1.0% in 2 hours.
Step S4: purifying the crude product of the F1 polypeptide obtained in the step S3, purifying by salt exchange, and freeze-drying to obtain the F1 polypeptide
Purifying the crude F1 polypeptide product obtained in step S3 by reverse phase high performance liquid chromatography, wherein C18 filler with particle size of 3 μm is used as stationary phase of chromatographic column with Luna 3 μm C18LC Column 150X 3mm in a volume ratio of 1:1:100, wherein triethylamine, phosphoric acid and water are taken as a mobile phase A phase, a mobile phase B phase consists of the mobile phase A phase and acetonitrile according to the volume ratio of 2:8, and the column temperature of the chromatographic column is kept at 55 ℃ in the purification process, and the specific steps are as follows:
(A) Dissolving a crude product: taking F1 polypeptide crude product, and using acetic acid, wherein the loading amount is 18.3mg of sample per g of filler: acetonitrile: purified water = 1:2:7 (V: V) dissolving the crude F1 polypeptide to 50mg/mL, and then filtering with a filter membrane with a pore diameter of 1.5 μm to obtain a crude solution;
(B) Loading the chromatographic column in a balance way: the column was equilibrated with a mixed solution of 95% mobile phase A and 5% mobile phase B, mobile phase A being TEA/H 3 PO 4 /H 2 O=1/1/100 (V/V), mobile phase B is mobile phase a/acn=2/8 (V/V), equilibration time is not less than 8 minutes, then the crude solution is loaded in 100% ratio;
(C) Gradient elution: after sample loading, firstly balancing the chromatographic column by using a mixed solution of 95% of mobile phase A and 5% of mobile phase B until the acetic acid peak is completely eluted, then balancing the chromatographic column by using a mixed solution of 62% of mobile phase A and 38% of mobile phase B for at least 8 minutes, and then carrying out gradient elution on the sample according to a linear gradient that the proportion of the mobile phase B is increased from 38% to 58% within 90 minutes;
(D) Sample collection: collecting eluent corresponding to a target peak of a product into numbered collecting bottles in a segmented manner, ensuring the consistency of the volumes of each bottle, and marking the corresponding collecting bottle number in a detection map;
(E) And (3) collecting liquid detection: the purified collection liquid is detected and analyzed by adopting an HPLC method, the detection conditions and the method are set as shown in table 4, after the detection is finished, the spectrum is subjected to integral treatment, the purity and the impurity content of the sample are analyzed according to an area normalization method, and the sample meeting the standard is collected.
Table 4: detection conditions and methods
The specific steps of salt replacement purification in the step S4 are as follows:
and (I) equilibrium loading: the chromatographic column is balanced by using a mixed solution of 95% acetic acid system mobile phase A and 5% acetic acid system mobile phase B, wherein the acetic acid system mobile phase A comprises acetic acid and purified water according to the volume ratio of 5:1000, wherein the mobile phase A of the acetic acid system is formed by mixing acetic acid and purified water according to a volume ratio of 5:1000, wherein the mobile phase B of the acetic acid system is formed by mixing acetic acid, purified water and acetonitrile according to the volume ratio of 5:500:800, and balancing for not less than 8 minutes, and mixing the collected liquid and purified water according to a ratio of 1:1, sampling in proportion, and replacing salt balance chromatographic column with 32g/L acetic acid solution for at least 30min; (II) gradient elution: after salt replacement and balancing, balancing the chromatographic column by using a mixed solution of 75% of mobile phase A and 25% of mobile phase B, wherein the balancing time is not less than 8 minutes, and carrying out gradient elution on a sample according to a linear gradient that the proportion of the mobile phase B of an acetic acid system is increased from 25% to 55% within 60 minutes;
(III) sample collection: collecting eluent corresponding to a target peak of a product into numbered collecting bottles in a segmented manner, ensuring the consistency of the volumes of each bottle, and marking the corresponding collecting bottle number in a detection map;
And (IV) detecting and analyzing the purified collection liquid by adopting an HPLC method, integrating the spectrum after the detection is finished, analyzing the purity and impurity content of the sample according to an area normalization method, and collecting the sample meeting the standard, wherein the detection method is consistent with the detection of the purified collection liquid in the first step.
Example 2 preparation of F1 Polypeptides
Step S1: preparation of Rink Amide MBHA resin
(1) Weighing 5mmol of MBHA resin (substitution degree is 0.54 mmol/g), placing the resin in a solid phase synthesis reactor with a sieve plate, adding 5% NMM/DMF (NMM DMF solution with volume fraction of 5%) and swelling the resin for 1h at room temperature, pumping out, washing the resin for 2 times by using DMF with 2.5 times of the resin bed volume for 3 minutes, pumping out, taking a small amount of resin as ninhydrin for detection, and leading the result to be positive;
(2) Weighing 15mmol Rink Amide Linker and 15mmol HOBT, adding into a container, dissolving 1.0 times of DMF (dimethyl formamide) of resin volume of MBHA, cooling under a cold bath condition, slowly adding 15mmol DIC, pre-activating for 5 minutes, adding the pre-activated reaction liquid into the reactor containing the resin in the step (1), reacting for 2 hours at room temperature, sampling, detecting ninhydrin, and if the detection of the ninhydrin is negative twice continuously, and reaching a specified reaction time, pumping out the reaction liquid, washing the resin for 4 times by using 2.5 times of DMF of resin bed volume, and pumping out each time for 3 minutes.
Step S2: preparation of fully protected peptide resin in two steps
Gly-Leu-Leu-Ser (tBu) -Val-Leu-Gly-Ser (tBu) -Val-Ala-Lys (Boc) -His (Trt) -Val-Leu-Pro-His (Trt) -Va l-Leu-Pro-His (Trt) -Val-Val-Pro-Val-Ile-Ala-Glu (OtBu) -His (Trt) -Leu-Rink Amide MBHA Resin resin
(1) Coupling corresponding amino acids sequentially from C terminal to N terminal according to F1 polypeptide sequence, and synthesizing to obtain
Fmoc-Val-Ala-Lys (Boc) -His (Trt) -Val-Leu-Pro-His (Trt) -Val-Leu-Pro-His (Trt) -Val-Val-Pro-Val-Ile-Ala-Glu (OtBu) -His (Trt) -Leu-Rink Amide MBHA Resin resin, comprising the following specific steps:
(a) Placing the Rink Amide MBHA resin obtained in the step S1 into a reactor, adding 20% (v/v) piperidine/DMF solution (20% piperidine DMF solution by volume fraction) with 2.5 times of resin bed volume into the reactor, stirring for 10 minutes, pumping, adding 20% (v/v) piperidine/DMF solution with 2.5 times of resin bed volume into the reactor, stirring for 20 minutes, pumping, and obtaining Fmoc-removed protective resin; washing resin with DMF (dimethyl formamide) with 2.5 times of resin bed volume for 3 minutes each time, pumping, and taking a small amount of resin for ninhydrin detection, wherein the result is positive;
(b) Weighing 15mmol Fmoc-AA-OH and 15mmol HOBT, adding into a container, using DMF to dissolve, cooling under a cold bath condition, slowly adding 15mmol DIC, pre-activating for 5 minutes, adding the pre-activating reaction liquid into the reactor, reacting for 2 hours at room temperature, sampling to detect ninhydrin, if the detection of the ninhydrin is negative twice continuously, and the specified reaction time is reached, pumping out the reaction liquid, washing the resin with DMF with 2.5 times of the volume of the resin bed layer for 4 times, pumping out for 3 minutes each time, and circulating in such a way, and connecting all amino acids.
The Fmoc-AA-OH in the step (b) is Fmoc-Leu-OH, fmoc-His (Trt) -OH, fmoc-Glu (OtBu) -OH, fmoc-Ala-OH, fmoc-Ile-OH, fmoc-Val-OH, fmoc-Pro-OH, fmoc-Lys (Boc) -OH.
When Fmoc-AA-OH is Fmoc-His (Trt) -OH, the coupling agent is HOOBt; when Fmoc-AA-OH is Fmoc-Leu-OH, fmoc-Glu (OtBu) -OH, fmoc-Ala-OH, fmoc-Ile-OH, fmoc-Val-OH, fmoc-Pro-OH, fmoc-Lys (Boc) -OH, the coupling agent is HOBt.
Wherein Fmoc-AA-OH/HOBt (HOOBt)/DIC=3/3/3 or 5/5, his was HOOBt, and 5eq was used after polycondensation of the resin. The amino acid materials, coupling reagents and reactions used were the same as in Table 2 of example 1.
(2) Coupling the product obtained in the step (1) with the subsequent 8 amino acid sites to obtain Fmoc-Gly-Leu-Leu-Ser (tBu) -Val-Leu-Gly-Ser (tBu) -Val-Ala-Lys (Boc) -His (Trt) -Val-Leu-Pro-His (Trt) -Val-Leu-Pro-His (Trt) -Val-Val-Pro-Val-Ile-Ala-Glu (OtBu) -His (Trt) -Leu-Rink Amide MBHA Resin resin, wherein the preparation method comprises the following specific steps of:
the subsequent amino acid site ligation was performed using 20% of Fmoc-Val-Ala-Lys (Boc) -His (Trt) -Val-Leu-Pro-His (Trt) -Val-Leu-Pro-His (Trt) -Val-Val-Pro-Val-Ile-Ala-Glu (OtBu) -His (Trt) -Leu-Rink Amide MBHA Resin resin (i.e., 20% of the first-step product was used in the second-step coupling reaction) using the same amino acid materials, coupling reagents and reactions as in Table 3 of example 1.
After all amino acids are connected, adding 20% (v/v) piperidine/DMF solution with 1.0 times of resin bed volume into a reactor to remove Fmoc protection, stirring for 20 minutes, pumping, washing resin with 1.0 times of resin bed volume of DMF for 2 times, washing for 10 minutes for the first time, washing for 20 minutes for the second time, pumping, taking a small amount of resin as ninhydrin for detection, and obtaining the full-protection peptide resin with positive result
Gly-Leu-Leu-Ser (tBu) -Val-Leu-Gly-Ser (tBu) -Val-Ala-Lys (Boc) -His (Trt) -Val-Leu-Pro-His (Trt) -Val-Leu-Pro-His (Trt) -Val-Val-Val-Pro-Val-Ile-Ala-Glu (OtBu) -His (Trt) -Leu-Rink Amide MBHA Resin resin.
Step S3: adding the full-protection peptide resin obtained in the step S2 into a cutting fluid to cut a side chain protecting group, precipitating, washing and drying to obtain a crude F1 polypeptide product, wherein the method comprises the following specific steps of:
(1) Adding the full-protection peptide resin obtained in the step S2 into a cutting fluid, wherein the volume-mass ratio of the cutting fluid to the full-protection peptide resin is 10mL/g, and the cutting fluid is as follows: trifluoroacetic acid, anisole, 1, 2-ethanedithiol, phenol, water=90:2.5:2.5:2.5:2.5, stirring while controlling the reaction temperature to 20 ℃, stirring and reacting for 3 hours, filtering out resin by a sand core funnel after cutting, collecting filtrate, washing the resin by TFA, combining the filtrates, and recording the volume of the filtrate;
(2) Slowly adding the collected filtrate into frozen diethyl ether with the volume of 10 times, stirring while adding, centrifuging to obtain a crude product after peptide chains are separated out, and washing with diethyl ether for 4 times;
(3) And (3) transferring the crude product obtained in the step (2) into a container for drying, and vacuum drying until the weight change rate of the crude product is less than 1.0% in 2 hours.
Step S4: purifying the crude F1 polypeptide obtained in the step S3, purifying with salt exchange, and lyophilizing to obtain F1 polypeptide, purifying the crude F1 polypeptide obtained in the step S3 by reverse phase high performance liquid chromatography, and purifying with C18 filler with particle size of 3 μm as stationary phase of chromatographic column with Luna 3 μm C18LC Column150×3mm, with triethylamine, phosphoric acid and water as mobile phase A phase at volume ratio of 1:1:100, mobile phase B phase is composed of mobile phase A phase and acetonitrile at volume ratio of 2:8, and the Column temperature of chromatographic Column is kept at 50deg.C during purification process, and comprises the following steps:
(A) Dissolving a crude product: taking F1 polypeptide crude product, and using acetic acid, wherein the loading amount is 18.3mg of sample per g of filler: acetonitrile: purified water = 1:2:7 (V: V) dissolving the crude F1 polypeptide to 50mg/mL, and then filtering with a filter membrane with a pore diameter of 1.5 μm to obtain a crude solution;
(B) Loading the chromatographic column in a balance way: the column was equilibrated with a mixed solution of 95% mobile phase A and 5% mobile phase B, mobile phase A being TEA/H 3 PO 4 /H 2 O=1/1/100 (V/V), mobile phase B is mobile phasePhase a/acn=2/8 (V/V), equilibration time is not less than 8 minutes, then the crude solution is loaded in 100% proportions;
(C) Gradient elution: after sample loading, firstly balancing the chromatographic column by using a mixed solution of 95% of mobile phase A and 5% of mobile phase B until the acetic acid peak is completely eluted, then balancing the chromatographic column by using a mixed solution of 62% of mobile phase A and 38% of mobile phase B for at least 8 minutes, and then carrying out gradient elution on the sample according to a linear gradient that the proportion of the mobile phase B is increased from 38% to 58% within 90 minutes;
(D) Sample collection: collecting eluent corresponding to a target peak of a product into numbered collecting bottles in a segmented manner, ensuring the consistency of the volumes of each bottle, and marking the corresponding collecting bottle number in a detection map;
(E) And (3) collecting liquid detection: the purified collection liquid is detected and analyzed by an HPLC method, the detection conditions and the method are set to be the same as those in table 4 in the embodiment 1, after the detection is finished, the spectrum is subjected to integral treatment, the purity and the impurity content of the sample are analyzed by an area normalization method, and the sample meeting the standard is collected.
The specific procedure for the purification of the salt change in step S4 is the same as in example 1.
Example 3 preparation of F1 Polypeptides
Step S1: preparation of Rink Amide MBHA resin
(1) Weighing 5mmol of MBHA resin (substitution degree is 0.62 mmol/g), placing the resin in a solid phase synthesis reactor with a sieve plate, adding 5% NMM/DMF (NMM DMF solution with volume fraction of 5%) and swelling the resin for 1h at room temperature, pumping out, washing the resin for 2 times by using DMF with 2.5 times of the resin bed volume for 3 minutes, pumping out, taking a small amount of resin as ninhydrin for detection, and leading the result to be positive;
(2) Weighing 15mmol Rink Amide Linker and 15mmol HOBT, adding into a container, dissolving 1.0 times of DMF (dimethyl formamide) of resin volume of MBHA, cooling under a cold bath condition, slowly adding 15mmol DIC, pre-activating for 15 minutes, adding the pre-activated reaction liquid into the reactor containing the resin in the step (1), reacting for 3 hours at room temperature, sampling, detecting ninhydrin, and if the detection of the ninhydrin is negative twice continuously, and reaching a specified reaction time, pumping out the reaction liquid, washing the resin for 4 times by using 2.5 times of DMF of resin bed volume, and pumping out each time for 3 minutes.
Step S2: preparation of fully protected peptide resin in two steps
Gly-Leu-Leu-Ser (tBu) -Val-Leu-Gly-Ser (tBu) -Val-Ala-Lys (Boc) -His (Trt) -Val-Leu-Pro-His (Trt) -Val-Leu-Pro-His (Trt) -Val-Val-Val-Pro-Val-Ile-Ala-Glu (OtBu) -His (Trt) -Leu-Rink Amide MBHA Resin resin
(1) Coupling corresponding amino acids sequentially from C terminal to N terminal according to F1 polypeptide sequence, and synthesizing to obtain
Fmoc-Val-Ala-Lys (Boc) -His (Trt) -Val-Leu-Pro-His (Trt) -Val-Leu-Pro-His (Trt) -Val-Val-Pro-Val-Ile-Ala-Glu (OtBu) -His (Trt) -Leu-Rink Amide MBHA Resin resin, comprising the following specific steps:
(a) Placing the Rink Amide MBHA resin obtained in the step S1 into a reactor, adding a 20% (v/v) piperidine/DMF solution with the volume of 2.5 times of the resin bed layer into the reactor, stirring for 10 minutes, pumping, adding a 20% (v/v) piperidine/DMF solution with the volume of 2.5 times of the resin bed layer into the reactor (a 20% piperidine DMF solution with the volume fraction), stirring for 20 minutes, and pumping to obtain Fmoc-removed protective resin; washing resin with DMF (dimethyl formamide) with 2.5 times of resin bed volume for 3 minutes each time, pumping, and taking a small amount of resin for ninhydrin detection, wherein the result is positive;
(b) Weighing 15mmol Fmoc-AA-OH and 15mmol HOBT, adding into a container, dissolving by using DMF, cooling under a cold bath condition, slowly adding 15mmol DIC, pre-activating for 15 minutes, adding the pre-activating reaction liquid into the reactor, reacting for 3 hours at room temperature, sampling, detecting ninhydrin, if the detection of the ninhydrin is negative twice continuously, and the specified reaction time is reached, pumping out the reaction liquid, washing the resin with DMF with 2.5 times of the volume of the resin bed layer for 4 times, pumping out for 3 minutes each time, and circulating in such a way, and connecting all amino acids.
The Fmoc-AA-OH in the step (b) is Fmoc-Leu-OH, fmoc-His (Trt) -OH, fmoc-Glu (OtBu) -OH, fmoc-Ala-OH, fmoc-Ile-OH, fmoc-Val-OH, fmoc-Pro-OH, fmoc-Lys (Boc) -OH.
When Fmoc-AA-OH is Fmoc-His (Trt) -OH, the coupling agent is HOOBt; when Fmoc-AA-OH is Fmoc-Leu-OH, fmoc-Glu (OtBu) -OH, fmoc-Ala-OH, fmoc-Ile-OH, fmoc-Val-OH, fmoc-Pro-OH, fmoc-Lys (Boc) -OH, the coupling agent is HOBt.
Wherein Fmoc-AA-OH/HOBt (HOOBt)/DIC=3/3/3 or 5/5, his was HOOBt, and 5eq was used after polycondensation of the resin. The amino acid materials, coupling reagents and reactions used were the same as in Table 2 of example 1.
(2) Coupling the product obtained in the step (1) with the subsequent 8 amino acid sites to obtain Fmoc-Gly-Leu-Leu-Ser (tBu) -Val-Leu-Gly-Ser (tBu) -Val-Ala-Lys (Boc) -His (Trt) -Val-Leu-Pro-His (Trt) -Val-Leu-Pro-His (Trt) -Val-Val-Pro-Val-Ile-Ala-Glu (OtBu) -His (Trt) -Leu-Rink Amide MBHA Resin resin, wherein the preparation method comprises the following specific steps of:
the subsequent amino acid site ligation was performed using 20% of Fmoc-Val-Ala-Lys (Boc) -His (Trt) -Val-Leu-Pro-His (Trt) -Val-Leu-Pro-His (Trt) -Val-Val-Pro-Val-Ile-Ala-Glu (OtBu) -His (Trt) -Leu-Rink Amide MBHA Resin resin (i.e., 20% of the first-step product was used in the second-step coupling reaction) using the same amino acid materials, coupling reagents and reactions as in Table 3 of example 1.
After all amino acids are connected, adding 20% (v/v) piperidine/DMF solution with 1.0 times of resin bed volume into a reactor to remove Fmoc protection, stirring for 20 minutes, pumping, washing resin with 1.0 times of resin bed volume of DMF for 2 times, washing for 10 minutes for the first time, washing for 20 minutes for the second time, pumping, taking a small amount of resin as ninhydrin for detection, and obtaining the full-protection peptide resin with positive result
Gly-Leu-Leu-Ser (tBu) -Val-Leu-Gly-Ser (tBu) -Val-Ala-Lys (Boc) -His (Trt) -Val-Leu-Pro-His (Trt) -Val-Leu-Pro-His (Trt) -Val-Val-Val-Pro-Val-Ile-Ala-Glu (OtBu) -His (Trt) -Leu-Rink Amide MBHA Resin resin.
Step S3: adding the full-protection peptide resin obtained in the step S2 into a cutting fluid to cut a side chain protecting group, precipitating, washing and drying to obtain a crude F1 polypeptide product, wherein the method comprises the following specific steps of:
(1) Adding the full-protection peptide resin obtained in the step S2 into a cutting fluid, wherein the proportion of the cutting fluid is 10mL/g, and the cutting fluid is as follows: trifluoroacetic acid, anisole, 1, 2-ethanedithiol, phenol and water=90:1:3:3:3, stirring while controlling the reaction temperature to 30 ℃, stirring and reacting for 3 hours, filtering out resin by a sand core funnel after cutting, collecting filtrate, washing the resin by TFA, combining the filtrates, and recording the volume of the filtrate;
(2) Slowly adding the collected filtrate into frozen diethyl ether with the volume of 10 times, stirring while adding, centrifuging to obtain a crude product after peptide chains are separated out, and washing with diethyl ether for 4 times;
(3) And (3) transferring the crude product obtained in the step (2) into a container for drying, and vacuum drying until the weight change rate of the crude product is less than 1.0% in 2 hours.
Step S4: purifying the crude F1 polypeptide obtained in the step S3, purifying with salt exchange, and lyophilizing to obtain F1 polypeptide, purifying the crude F1 polypeptide obtained in the step S3 by reverse phase high performance liquid chromatography, and purifying with C18 filler with particle size of 3 μm as stationary phase of chromatographic column with Luna 3 μm C18LC Column150×3mm, with triethylamine, phosphoric acid and water as mobile phase A phase at volume ratio of 1:1:100, mobile phase B phase is composed of mobile phase A phase and acetonitrile at volume ratio of 2:8, and the Column temperature of chromatographic Column is kept at 55deg.C during purification process, and comprises the following steps:
(A) Dissolving a crude product: taking F1 polypeptide crude product, and using acetic acid, wherein the loading amount is 18.3mg of sample per g of filler: acetonitrile: purified water = 1:2:7 (V: V) dissolving the crude F1 polypeptide to 50mg/mL, and then filtering with a filter membrane with a pore diameter of 1.5um to obtain a crude solution;
(B) Loading the chromatographic column in a balance way: the column was equilibrated with a mixed solution of 95% mobile phase A and 5% mobile phase B, mobile phase A being TEA/H 3 PO 4 /H 2 O=1/1/100 (V/V), mobile phase B is mobile phase a/acn=2/8 (V/V), equilibration time is not less than 8 minutes, then the crude solution is loaded in 100% ratio;
(C) Gradient elution: after sample loading, firstly balancing the chromatographic column by using a mixed solution of 95% of mobile phase A and 5% of mobile phase B until the acetic acid peak is completely eluted, then balancing the chromatographic column by using a mixed solution of 62% of mobile phase A and 38% of mobile phase B for at least 8 minutes, and then carrying out gradient elution on the sample according to a linear gradient that the proportion of the mobile phase B is increased from 38% to 58% within 90 minutes;
(D) Sample collection: collecting eluent corresponding to a target peak of a product into numbered collecting bottles in a segmented manner, ensuring the consistency of the volumes of each bottle, and marking the corresponding collecting bottle number in a detection map;
(E) And (3) collecting liquid detection: the purified collection liquid is detected and analyzed by an HPLC method, the detection conditions and the method are set to be the same as those in table 4 in the embodiment 1, after the detection is finished, the spectrum is subjected to integral treatment, the purity and the impurity content of the sample are analyzed by an area normalization method, and the sample meeting the standard is collected.
The specific procedure for the purification of the salt change in step S4 is the same as in example 1.
Preparation of comparative example 1, F1 Polypeptides MBHA Resin having a degree of substitution of 0.47mmol/g was used as a starting material, corresponding amino acids were coupled in sequence from C-terminus to N-terminus according to the F1 polypeptide sequence, and the amino acid materials, coupling reagents and reaction conditions used at positions 29 to 9 were the same as those in Table 2 of example 1. In the case of the subsequent amino acid connection, the coupling reaction was completed only with the reaction times listed in Table 5, using the amino acid materials of Table 5, as well as the coupling reagents, and the feed ratios. The connection of the peptide resin of example 1 of the present invention to that of comparative example 1 is shown in Table 6.
Table 5: amino acid material, coupling reagent and reaction condition used in comparative example 1
Table 6: comparison of the connection of the peptide resins of example 1 and comparative example 1 of the present invention
As can be seen from the comparison of the two times of peptide resin connection of the example 1 and the comparative example 1 in Table 6, the amino acid connection mode of the invention is obviously superior to that of the comparative example 1, the amino acid sites needing repeated connection are obviously reduced, and the reaction time is greatly shortened.
Preparation of comparative example 2, F1 Polypeptides
The preparation of the F1 polypeptide is similar to that of example 1.
The difference from example 1 is that the cutting fluid is: trifluoroacetic acid, anisole, 1, 2-ethanedithiol, triisopropylsilane, water=87.5:5:2.5:2.5:2.5.
Preparation of comparative example 3, F1 Polypeptides
The preparation of the F1 polypeptide is similar to that of example 1.
The difference from example 1 is that the cutting fluid is: trifluoroacetic acid, anisole, 1, 2-ethanedithiol, phenol, water=85:5:5:3:2.
Preparation of comparative example 4, F1 Polypeptides
The preparation of the F1 polypeptide is similar to that of example 1.
The difference from example 1 is that the column temperature was kept at 45℃during the purification.
Preparation of comparative example 5, F1 Polypeptides
The preparation of the F1 polypeptide is similar to that of example 1.
The difference from example 1 is that the sample is subjected to gradient elution according to a linear gradient in which the proportion of mobile phase B phase is increased from 35% to 65% in 60 minutes when the crude F1 polypeptide obtained in step S3 is subjected to gradient elution in the specific step (C) of crude purification.
Test example one, characterization of F1 Polypeptides
1.1 Infrared Spectrometry detection
And recording a Fourier transform infrared spectrogram of the sample by adopting a Fourier transform infrared spectrometer. An IR spectrum of the F1 polypeptide prepared in example 1 of the present invention is shown in fig. 1. The IR spectrum peak assignment table is shown in Table 7.
Table 7: infrared spectrum peak assignment table
| Wave number (cm) -1 ) | Corresponding radicals |
| 3299.6 | Stretching vibration of N-H, O-H |
| 2963.2，2875.6 | Telescopic vibration of saturated C-H |
| 1654.0 | C=O stretching vibration (amide I band) |
| 1540.3 | Flexural vibration of N-H and telescopic vibration of C-N (amide II band) |
| 1468.6，1449.0 | Skeletal vibration of benzene ring |
| 1235.6 | Flexural vibration of N-H and telescopic vibration of C-N (amide III band) |
。
As can be seen from FIG. 1 and Table 7, in the F1 polypeptideIn the IR spectrum, 3299.6cm -1 The characteristic peak mainly comes from the stretching vibration of N-H and O-H, 2963.2 and 2875.6cm -1 The characteristic peak mainly comes from the stretching vibration of saturated C-H, 1654.0cm -1 The characteristic peak mainly comes from the stretching vibration (amide I band) of C=O, 1540.3cm -1 The characteristic peaks mainly originate from bending vibration of N-H and stretching vibration of C-N (amide II band), 1468.6, 1449.0cm -1 The characteristic peak at 1235.6 is mainly derived from the bending vibration of N-H and the stretching vibration of C-N (amide III band).
1.2 Mass Spectrometry analysis
The mass spectrum of the F1 polypeptide prepared in the embodiment 1 of the invention is shown in figure 2, which shows the multi-charge peak of the F1 polypeptide, and the average molecular weight of the F1 polypeptide of the invention is 3030.85 and is consistent with the theoretical average molecular weight 3030.65 by weighting calculation.
1.3 amino acid ratio detection
The amino acid ratio of the F1 polypeptide obtained in example 1 of the present invention was detected by a release detection method, and the detection results are shown in Table 8.
Table 8: amino acid ratio detection results
The results in Table 8 show that the amino acid ratio of the F1 polypeptide obtained in example 1 of the present invention is consistent with the theoretical ratio.
1.4 sequence analysis
The full sequence detection is carried out on the F1 polypeptide obtained in the embodiment 1 of the invention by a Q-TOF flight mass spectrometry method, the primary mass spectrum test is carried out on the F1 polypeptide prepared in the invention, the primary mass spectrum of the F1 polypeptide prepared in the embodiment 1 of the invention is shown in figure 3, and the primary mass spectrum deconvolution spectrum of the F1 polypeptide prepared in the embodiment 1 of the invention is shown in figure 4. And then carrying out a secondary mass spectrum test on the target peak of the test sample, wherein the secondary mass spectrum of the F1 polypeptide prepared in the embodiment 1 of the invention is shown in figure 5. The secondary test data was subjected to matching analysis by using a MassHunter, and secondary retrieval analysis was performed on the secondary mass spectrum data of the target peaks of the test sample, and the MS/MS spectrum peak list is shown in table 9.
Table 9: MS/MS Spectrum peak List
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From the results shown in FIGS. 3 to 5 and Table 9, it can be confirmed that the fragment peak sequence obtained by QTOF mass spectrometry of the F1 polypeptide obtained in example 1 of the present invention matches with the theoretical fragment peak sequence, and the error is small, which is sufficient to confirm that the amino acid sequence of the F1 polypeptide obtained in example 1 of the present invention matches with the theoretical sequence.
Test example two, determination of related substances and purity of F1 polypeptide
2.1 determination of crude purity
The purity of the crude F1 polypeptide obtained in step S4 of examples 1, 2, 3, 2 and 3 was measured by HPLC method and the results are shown in Table 10.
The F1 polypeptides obtained in example 1, example 2, example 3, comparative example 4 and comparative example 5 of the present invention were measured by HPLC method, and the results are shown in Table 11. The HPLC spectrum of the F1 polypeptide prepared in example 1 of the present invention is shown in FIG. 6, and the information of each peak of the HPLC spectrum of the F1 polypeptide is shown in Table 12.
Table 10: purity measurement result of F1 polypeptide crude product
| Project | Example 1 | Example 2 | Example 3 | Comparative example 2 | Comparative example 3 |
| Purity (%) | 67.81 | 65.01 | 64.40 | 64.31 | 60.17 |
。
Table 11: purity measurement of F1 polypeptide
| Project | Example 1 | Example 2 | Example 3 | Comparative example 4 | Comparative example 5 |
| Purity (%) | 99.77 | 98.80 | 98.42 | 95.80 | 96.90 |
| Maximum mono-hetero (%) | 0.11 | 0.44 | 0.50 | 1.55 | 0.58 |
| Total impurity (%) | 0.23 | 1.20 | 1.58 | 4.20 | 3.10 |
。
Table 12: HPLC spectrogram of F1 polypeptide
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As can be seen from Table 10, the crude F1 polypeptide obtained in step S4 of the present invention has a high purity, and the crude F1 polypeptide obtained in the present invention has a higher purity than those of comparative examples 2 and 3. As can be seen from Table 11, the F1 polypeptides obtained in the present invention were high in purity, and the F1 polypeptides obtained in the present invention were higher in purity than those obtained in comparative examples 4 and 5. As can be seen from FIG. 6 and Table 12, the F1 polypeptide obtained in example 1 of the present invention has a purity as high as 99.77%. The invention greatly reduces the impurity content, obviously improves the purity of the F1 polypeptide, and is beneficial to large-scale high-purity production in the pharmaceutical industry.
Claims (10)
1. A method for preparing an F1 polypeptide, comprising the steps of:
s1, preparing Rink Amide MBHA resin;
s2 preparing full-protection peptide resin in two steps
Gly-Leu-Leu-Ser (tBu) -Val-Leu-Gly-Ser (tBu) -Val-Ala-Lys (Boc) -His (Trt) -Val-Leu-Pro-His (Trt) -Val-Leu-Pro-His (Trt) -Val-Val-Val-Pro-Val-Ile-Ala-Glu (OtBu) -His (Trt) -Leu-Rin k Amide MBHA Resin resin,
comprising the following steps: (1) Sequentially coupling corresponding amino acids from the C end to the N end according to the F1 polypeptide sequence, and synthesizing to obtain Fmoc-Val-Ala-Lys (Boc) -His (Trt) -Val-Leu-Pro-His (Trt) -Val-Leu-Pro-His (Trt) -Val-Val-Val-Pro-Val-Ile-Ala-Glu (OtBu) -His (Trt) -Leu-RinkAmideMBHA Resin resin; (2) Coupling the product obtained in the step (1) with 8 subsequent amino acid sites to obtain Fmoc-Gly-Leu-Leu-Ser (tBu) -Val-Leu-Gly-Ser (tBu) -Val-Ala-Lys (Boc) -His (Trt) -Val-Leu-Pro-His (Trt) -Val-Leu-Pro-His (Trt) -Val-Val-Pro-Val-Ile-Ala-Glu (OtBu) -His (Trt) -Leu-Rink Amide MBHA Resin resin, removing Fmoc protection to obtain full protection peptide resin Gly-Leu-Leu-Leu-Ser (tBu) -Val-Leu-Gly-Ser (Boc) -Val-Ala-Lys (Trt) -Val-Leu-Pro-His (Trt) -Val-Leu-Pro-His (Trt) -Val-Ile-Ala-His (OtBu) -Leu-Rin k Amide MBHA Resin resin;
S3, adding the full-protection peptide resin obtained in the step S2 into a cutting fluid to cut a side chain protecting group, precipitating, washing and drying to obtain a crude F1 polypeptide;
s4, purifying the crude product of the F1 polypeptide obtained in the step S3, purifying by salt exchange, and freeze-drying to obtain the F1 polypeptide.
2. The method of claim 1, wherein the Rink Amide MBHA resin in step S1 is prepared from MBHA resin and Rink Amide Linker as main raw materials, and the initial substitution degree of the MBHA resin is 0.1-0.8 mmol/g.
3. The method of claim 2, wherein the MBHA resin has an initial degree of substitution of 0.47mmol/g.
4. The method of claim 1, wherein the step S2 is performed by Fmoc-Ser (tBu) -OH for coupling Ser amino acid sites.
5. The method of producing F1 polypeptide according to claim 4, wherein the coupling system used for coupling the Ser amino acid site with Fmoc-Ser (tBu) -OH is selected from HOBt/DIC.
6. The method of producing an F1 polypeptide according to claim 1, wherein the cleavage liquid in step S3 comprises trifluoroacetic acid, anisole, 1, 2-ethanedithiol, phenol and water.
7. The method of producing F1 polypeptide as defined in claim 6, wherein the cleavage liquid in step S3 is: trifluoroacetic acid, anisole, 1, 2-ethanedithiol, phenol, water=87.5-90:1-5:2.5-3:2.5-3:2.5-3.
8. The method for preparing F1 polypeptide according to claim 1, wherein the crude product of step S4 is purified by reverse phase high performance liquid chromatography, wherein a C18 packing with a particle size of 3 μm is used as a stationary phase of a chromatographic column, triethylamine, phosphoric acid and water with a volume ratio of 1:1:100 are used as mobile phase a phase, and mobile phase B phase is composed of mobile phase a phase and acetonitrile with a volume ratio of 2:8.
9. The method for producing an F1 polypeptide according to claim 8, wherein the column temperature is maintained at 50 to 55℃during the purification.
10. The method of claim 8, wherein the step S4 is performed with a linear gradient of increasing the proportion of mobile phase B phase from 38% to 58% in 90 minutes.
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