CN110835365B - Preparation method of procatide - Google Patents
Preparation method of procatide Download PDFInfo
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- CN110835365B CN110835365B CN201810929802.9A CN201810929802A CN110835365B CN 110835365 B CN110835365 B CN 110835365B CN 201810929802 A CN201810929802 A CN 201810929802A CN 110835365 B CN110835365 B CN 110835365B
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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
- C07K1/04—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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- 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
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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
- C07K1/14—Extraction; Separation; Purification
- C07K1/16—Extraction; Separation; Purification by chromatography
- C07K1/20—Partition-, reverse-phase or hydrophobic interaction chromatography
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
Abstract
A method for preparing procatide, comprising the following steps: 1) sequentially coupling 16 th-7 th amino acids on solid-phase synthetic resin according to an Fmoc solid-phase synthesis strategy; 2) after removing the Fmoc protecting group, treating by using a reagent for removing the Mmt protecting group to remove the side chain protecting groups Mmt of Cys at the 7 th site and the 15 th site; 3) forming a disulfide bond between Cys at positions 7 and 15 by a bonding reaction; 4) sequentially coupling 6-1 th amino acid 5) according to an Fmoc solid-phase synthesis strategy to remove an Fmoc protecting group, and completing a disulfide bond between 4 th and 12 th Cys through an oxidation reaction; 6) all side chain protecting groups are removed, the resin is cracked by a cracking reagent, and the procatide is obtained by ether precipitation. The method is simple and has high efficiency.
Description
Technical Field
The invention belongs to the field of polypeptide synthesis, and particularly relates to a preparation method of procainatide.
Background
Procatide is a guanylate cyclase C (GC-C) receptor agonist developed by synergetics Pharmaceuticals, Inc, under the trade name Trulance. The effect is similar to that of the natriuretic peptide uroguanylin, and can induce secretion of liquid into gastrointestinal tract, thereby increasing gastrointestinal motility and treating constipation. Approval by the U.S. Food and Drug Administration (FDA) was obtained on 19/01/2017 for marketing.
The procatide is named as Plectanatide in English, has CAS number of 467426-54-6, and consists of 16L-type amino acid residues, wherein Cys4、Cys12And Cys7、Cys15Are respectively connected by disulfide bonds. The peptide sequence is as follows:
CN 103694320A and CN 104211777A are both issued patent by the present company, and mainly refer to the introduction of different methods for positioning two pairs of disulfide bonds by solid-liquid combination of procatide. CN 103694320A introduces the use of tBu and Acm as protection of sulfydryl on two pairs of disulfide bonds respectivelyGroup through H2O2And I2A method for forming two pairs of disulfide bonds by oxidation respectively. CN 104211777A is formed into a first pair and a second pair of disulfide bonds by different oxidation methods by using any two of StBu, Acm, Mmt and Trt as protecting groups of sulfhydryl groups on two pairs of disulfide bonds of procapsipeptide.
CN104628827A describes a method of full solid phase synthesis to position two disulfide bonds, which is the biggest difference from the former two patents in Cys (Cys4, Cys) of the first disulfide bond12) The thiol protecting groups are selected from StBu and Mmt, and Cys (Cys) of the second disulfide bond7、Cys15) The thiol protecting group is Acm. After the synthesis of the prophase peptide resin is finished, StBu is firstly removed on a solid phase by mercaptoethanol, then DTNP is added to react with the exposed sulfhydryl, Mmt is removed by TFA with certain concentration, and simultaneously, the first pair of disulfide bonds (Cys) is carried out4、Cys12) And a second pair of disulfide bonds (Cys)7、Cys15) Is cyclized by using2And (4) oxidizing. This is the oxidation of the two disulfide bonds that is accomplished on the solid phase.
CN107313870A and CN107313871A are both applications of Suzhou university of science and technology, the former introduces a solid phase synthesis of peptide resin, and the linear crude peptide obtained by cracking the peptide resin freely forms a ring in a liquid phase. The latter introduction is a conventional all solid phase synthetic peptide resin strategy.
Patents CN 103694320A, CN 104211777A and CN104628827A are different experimental protocols for solid phase synthesis of linear polypeptides followed by different formation around disulfide bond bonding. These several solutions all have a significant drawback: neglecting the difficulty of synthesizing the proca peptide linear peptide, according to the scheme, the linear peptide has serious resin shrinkage and difficult coupling caused by the influence of the secondary structure of the linear peptide, the purity of the linear peptide is 40-55%, and the yield of the final product obtained by bonding the disulfide bond after the linear peptide is synthesized is lower, generally 20-25%.
Disclosure of Invention
In view of the above problems, it is an object of the present invention to provide an Fmoc/tBu solid phase synthesis route which can greatly improve the swellability of resin, facilitate coupling, and improve the purity of peptide, thereby improving the yield. In order to achieve the purpose, the invention adopts the following technical scheme:
through Cys7And Cys15Protected with Mmt or Trt, and Cys4And Cys12Adopting Acm protection and Fmoc/tBu solid phase synthesis method to synthesize polypeptide, coupling Cys7Thereafter, the Mmt or Trt protecting group is removed with a concentration of TFA, followed by H2O2First solid phase oxidation to Cys7And Cys15Then continuing to couple subsequent amino acid residues, and adopting I after all residues are coupled2A second pair of disulfide bonds is oxidized. Due to the formation of disulfide bonds, the swelling property of the resin is greatly enhanced by the change of the secondary structure of peptide sequences, the coupling difficulty of subsequent amino acid residues is greatly reduced, the purity of the final crude peptide is greatly improved to more than 80 percent, and the yield of the purified refined peptide is improved>40 percent. Meanwhile, the whole operation process is completed in a solid phase, and the operation is simple and feasible.
The reaction process is as follows:
one aspect of the present invention provides a preparation method of procatide, which comprises the following steps:
1) Fmoc-Leu are sequentially coupled on solid-phase synthetic resin according to Fmoc solid-phase synthesis strategy16–OH，Fmoc-Cys15(Mmt)-OH，Fmoc-Gly14-OH、Fmoc-Thr13(tBu)-OH、Fmoc-Cys12(Acm)-OH、Fmoc-Ala11-OH、Fmoc-Val10-OH、Fmoc-Asn9(Trt)-OH、Fmoc-Val8-OH、Fmoc-Cys7(Mmt)-OH；
2) After removing the Fmoc protecting group, treating by using a reagent for removing the Mmt protecting group to remove the side chain protecting groups Mmt of Cys at the 7 th site and the 15 th site;
3) forming a disulfide bond between Cys at positions 7 and 15 by a bonding reaction;
4) according to Fmoc solid phase synthesis strategyCoupling Fmoc-Leu6-OH、Fmoc-Glu5(OtBu)-OH、Fmoc-Cys4(Acm)-OH、Fmoc-Glu3(OtBu)-OH、Fmoc-Asp2(OtBu)-OH、Fmoc-Asn1(Trt)-OH；
5) Completing disulfide bond between Cys4 and Cys 12 by oxidation reaction, and then removing Fmoc protecting group;
6) cracking the resin with a cracking reagent to simultaneously remove all side chain protecting groups, and precipitating with diethyl ether to obtain procatide;
optionally, 7) purification by reverse phase chromatography.
In the technical scheme of the invention, the step 1) and the step 4) of Fmoc solid-phase synthesis strategy coupling refer to coupling Fmoc-protected amino acid residues, then removing Fmoc protecting groups, and then coupling the next Fmoc-protected amino acid residue.
In the technical scheme of the invention, the method for coupling Fmoc-protected amino acid residues comprises the step of coupling Fmoc-AA-OH with solid-phase synthetic resin or polypeptide-solid-phase synthetic resin with Fmoc protecting groups removed under the action of a coupling reagent.
The Fmoc protecting group is removed by using 20% piperidine/DMF solution
The coupling reagent used is a combination of DIPCDI and compound A or a combination of DIPEA and compound A and compound B, wherein compound A is HOAt or HOBt, and compound B is PyAOP, PyBOP, HATU, HBTU or TBTU, preferably a combination of DIPCDI and compound A. Further, the ratio of each component in the coupling agent to the protected Amino Acid (AA) is, in terms of molar ratio, DIPCDI: a: AA ═ 1.3:1.2:1.0, DIPEA: a: B: AA ═ 2.0:1.2:1.0: 1.0.
The solvent selected includes tetrahydrofuran, dimethyl sulfoxide, dimethylformamide, dioxane, acetone, diethyl ether, acetonitrile, carbon tetrachloride, carbon disulfide, benzene, toluene, hexane, chloroform, dichloromethane, etc., preferably a mixed solution of dimethylformamide and dichloromethane at a ratio of 1: 1.
In the technical scheme of the invention, the solid phase synthetic resin is selected from wang resin and 2CTC resin. The resin substitution degree is 0.2 to 0.5mmol/g, preferably 0.25 to 0.40 mmol/g.
The resin is swollen with a solvent before use, and the solvent selected includes DMF, NMP, dichloromethane, etc., preferably DMF.
AA in Fmoc-AA-OH is various protected amino acids.
The time for the coupling reaction of each amino acid is usually 1.5 to 4 hours, preferably 2 to 3 hours; the temperature is preferably room temperature (i.e., 20. + -. 5 ℃ C.), and may be suitably elevated or reduced.
The Mmt protective group in the step 2) is removed by adopting a method of reacting a mixed solution of TFA, TIS, DCM (1-5) and (2-10) with resin for 15-30 times and 1-5 minutes each time, and the preferable solution ratio is TFA, TIS, DCM (2: 5: 93) and the reaction times are 20 times and 2 minutes each time.
The bonding reaction of the disulfide bond in the step 3) adopts H2O2Carrying out a bonding reaction, preferably 1-30 equivalents of H based on the amount of substrate material2O2The reaction with the polypeptide-solid phase synthesis resin is carried out for 0.5 to 3 hours, and more preferably 10 equivalents for 1 hour.
The reaction solvent in step 3) is selected from one or more of tetrahydrofuran, dimethyl sulfoxide, dimethylformamide, dioxane, acetone, diethyl ether, acetonitrile, carbon tetrachloride, carbon disulfide, benzene, toluene, hexane, chloroform and dichloromethane, and is preferably dimethylformamide.
The oxidation reaction in the step 5) adopts I2Bonding of the second pair of disulfide bonds is performed. I is2The amount of (B) is 0.8 to 10 equivalents, preferably 3 equivalents, based on the amount of resin-polypeptide (product obtained in step 4) material. The reaction time is 0.5 to 3 hours, preferably 1 hour. And washing and shrinking after the reaction is finished to obtain the peptide resin. I is2The side chain protecting groups of cysteine can be removed and the disulfide bond linkage can be completed at the same time.
The solvent in the step 5) is one or more of tetrahydrofuran, dimethyl sulfoxide, dimethylformamide, dioxane, acetone, diethyl ether, acetonitrile, carbon tetrachloride, carbon disulfide, benzene, toluene, hexane, chloroform and dichloromethane, preferably dimethylformamide.
The lysis solution used in the step 6) is TFA, TIS and H2O, anisole, thioanisole, phenolA mixture of one or more; preferred are TFA, TIS and H2Mixtures of O, more preferably TFA TIS H2O＝92:4:4。
Step 7) the purification step may employ reversed phase high pressure liquid chromatography. Further, the reversed-phase high-pressure liquid chromatography comprises: using reverse octadecylsilane as stationary phase and 0.1% trifluoroacetic acid water solution/acetonitrile as mobile phase, collecting target peak fraction, concentrating, and lyophilizing.
Abbreviations and English meanings
Advantageous effects
The method only changes the synthetic sequence, greatly enhances the swelling property of the resin due to the change of the secondary structure of the peptide sequence, greatly reduces the difficulty of the subsequent amino acid residue coupling, and finally greatly improves the purity of the crude peptide. Meanwhile, the whole operation process is completed in a solid phase, and the operation is simple and feasible.
Detailed Description
The following examples are provided to better understand the present invention, not to limit the embodiments, and not to limit the content and the scope of the present invention, and any method similar or similar to the present invention, which is obtained by combining the features of the present invention and other prior art, with the present invention, falls within the scope of the present invention.
Example 1: preparation of Fmoc-Leu-Wang Resin
Weighing 150g of Wang resin with the substitution degree of 1.03mmol/g into a solid phase reaction column, washing the Wang resin with DMF for 2 times, swelling the resin with DMF for 30 minutes, dissolving 37.5g of Fmoc-Leu-OH, 15.6g of HOBt and 1.29g of DMAP in 400ml of mixed solution of DCM and DMF with the volume ratio of 1:1, stirring the solution in a bath of ice salt water, dropwise adding 17.4g of DIPCDI after controlling the temperature to be 0-10 ℃, activating the solution for 2-3 minutes, adding the solution into the solid phase reaction column, and reacting for 1.5 hours at room temperature. After the reaction is finished, washing the mixture for 4 times by using DMF, adding 140mL of acetic anhydride, 120mL of pyridine and a proper amount of DMF, mixing and sealing the mixture for reaction for more than 8 hours. After the reaction was complete, the reaction was washed 4 times with DMF and 2 times with DCM. After shrinking with methanol for 2 times, the resin was drained to obtain 160.5g of Fmoc-Leu-Wang resin, the detected substitution was 0.21 mmol/g.
Example 2: preparation of peptide resins
47.6g (10mmol) of Fmoc-Leu-Wang resin with substitution 0.21mmol/g prepared in example 1 was weighed into a solid phase reaction column, washed 2 times with DMF and then the resin was swelled with DMF for 30 min, then deprotected with 150ml of DBLK for 5min +7min and washed 6 times with 150ml of DBLK.
30.8g (50mmol) of Fmoc-Cys (Mmt) -OH and 8.1g (60mmol) of HOBt are weighed and dissolved in 100mL of DMF/DCM (1:1), 10mL (65mmol) of DIPCDI is added under an ice-water bath for activation for 2-3min, the mixture is added into a reaction column and reacted for 2h at room temperature, and the reaction end point is detected by ninhydrin (the reaction is stopped if the resin is colorless and transparent; the reaction is prolonged for 1h if the resin is colored). After the reaction is finished, washing the resin for 3 times by using 150mLDMF, adding 150mLDBLK for deprotection for 6min +8min, washing the resin for 6 times by using 150mLDMF, and detecting the color of the resin by ninhydrin.
The above coupling operation was repeated to successively couple Fmoc-Gly-OH, Fmoc-Thr (tBu) -OH, Fmoc-Cys (Acm) -OH, Fmoc-Ala-OH, Fmoc-Val-OH, Fmoc-Asn (Trt) -OH, Fmoc-Val-OH, Fmoc-Cys (Mmt) -OH in the order of peptide sequence, each protected amino acid was charged in an amount of 50mmol, and the charge ratios of the other materials corresponded to the protected amino acids.
After the removal of Fmoc protection the resin was washed 6 times with DMF, the peptide resin was washed 3 additional times with DCM and drained, then the peptide resin was treated with Mmt removal using TFA, TIS, DCM 2:5:93 for 20 reactions and 2 minutes each, and after the completion the resin was washed 6 times with DMF.
10.2ml (100mmol) of 30% hydrogen peroxide is weighed and added into 150ml of DMF, the mixture is added into a solid phase reaction column to carry out bonding reaction of a first pair of disulfide bonds with resin, air blowing reaction is carried out for 1h, and DMF washing is continued for 6 times after the reaction is finished.
Repeating the above coupling procedure, continuing to couple the remaining amino acid residues in sequence in peptide sequence order: Fmoc-Leu-OH, Fmoc-Glu (OtBu) -OH, Fmoc-Cys (Acm) -OH, Fmoc-Glu (OtBu) -OH, Fmoc-Asp (OtBu) -OH, Fmoc-Asn (Trt) -OH. 50mmol of protected amino acid is added, and the adding ratio of other materials corresponds to the protected amino acid.
And bonding a second pair of disulfide bonds after the coupling of the residues is completed. 7.6g (30mmol) of I are weighed2Dissolved in 150ml of DMF and then introduced into a reaction column. The reaction time was 1 h. After the reaction was completed, 150mL of DBLK was used for deprotection for 5min +7min, 150mL of DMF was used for washing 5 times, DCM was used for washing 2 times, 150mL of methanol was used for shrinking the resin 2 times for 5min each time, and the peptide resin was dried under reduced pressure to obtain 78.6g of peptide resin.
Example 3: cleavage of peptide resins
78.6g (10mmol) of the peptide resin (1) obtained in example 2 was placed in its entirety in a cleavage reactor, and 780ml of a cleavage reagent (TFA: TIS: H) was added at a ratio of 10ml/g of the peptide resin2O92: 4:4(V/V)), and stirred at room temperature for 2 h. The reaction was filtered through a sand funnel, after the filtration was complete a small amount of TFA was added to wash the resin, the filtrates were collected and combined and concentrated to volume under reduced pressure. The solution was precipitated by addition of frozen dry ether (100ml/g peptide resin), centrifuged, the supernatant removed and the precipitate washed 3 times with dry ether and dried under vacuum to give 17.2g of crude peptide as a white solid powder. The crude peptide was 81.3% pure (HPLC assay conditions: Waters C18300A 1.7.7 um 2.0 × 100mM, buffer 50mM ammonium phosphate buffer (pH 6.2 adjusted with phosphoric acid), phase A acetonitrile: buffer 10:90(V: V), phase B: buffer 50:50(V: V), gradient: 5-45% (20 min)/45.1-95% (10min), flow rate: 0.4ml/min detection wavelength: 220 nm.
Example 4: preparation of procatide
17.2g of the crude peptide obtained in example 3 was subjected to reverse phase liquid phase preparative purification using octadecylsilane as a stationary phase and 0.1% trifluoroacetic acid aqueous solution/acetonitrile as a mobile phase, and the desired peak fraction was collected, concentrated and lyophilized. 6.8g of refined peptide with the purity of 99.5 percent and the total yield of the procatide of 40.4 percent is obtained.
Example 5: preparation of peptide resin (conventional comparative method 1)
47.6g (10mmol) of Fmoc-Leu-Wang resin with substitution 0.21mmol/g prepared in example 1 was weighed into a solid phase reaction column, washed 2 times with DMF and then the resin was swelled with DMF for 30 min, then deprotected with 150ml of DBLK for 5min +7min and washed 6 times with 150ml of DBLK. 30.8g (50mmol) of Fmoc-Cys (Mmt) -OH and 8.1g (60mmol) of HOBt are weighed and dissolved in 100mL of DMF/DCM (1:1), 10mL (65mmol) of DIPCDI is added under an ice-water bath for activation for 2-3min, the mixture is added into a reaction column and reacted for 2h at room temperature, and the reaction end point is detected by ninhydrin (the reaction is stopped if the resin is colorless and transparent; the reaction is prolonged for 1h if the resin is colored). After the reaction is finished, washing the resin for 3 times by using 150mLDMF, adding 100mLDBLK for deprotection for 6min +8min, washing the resin for 6 times by using 100mLDMF, and detecting the color of the resin by ninhydrin. The above coupling operation was repeated to continue coupling Fmoc-Gly-OH, Fmoc-Thr (tBu) -OH, Fmoc-Cys (Acm) -OH, Fmoc-Ala-OH, Fmoc-Val-OH, Fmoc-Asn (Trt) -OH, Fmoc-Val-OH, Fmoc-Cys (Mmt) -OH, Fmoc-Leu-OH, Fmoc-Glu (OtBu) -OH, Fmoc-Cys (Acm) -OH, Fmoc-Glu (OtBu) -OH, Fmoc-Asp (OtBu) -OH, Fmoc-Asn (Trt) -OH in the order of peptide sequence as described in step 2 above. 50mmol of protected amino acid is added, and the adding ratio of other materials corresponds to the protected amino acid.
After the coupling of the above residues was complete, the peptide resin was subjected to a demamt treatment with appropriate amounts of TFA, TIS, DCM 2:5:93, 20 times and 2 min each, after which the resin was washed 6 times with DMF. 10.2ml (100mmol) of 30% hydrogen peroxide is weighed and added into 150ml of DMF, the mixture is added into a solid phase reaction column to carry out bonding reaction of a first pair of disulfide bonds with resin, the DMF is continuously washed for 6 times after air blowing reaction for 1 hour, and then bonding of a second pair of disulfide bonds is carried out. 7.6g (30mmol) of I are weighed2Dissolved in 150ml of DMF and then introduced into a reaction column. The reaction time was 1 h. After the reaction was completed, 150mL of DBLK was used for deprotection for 5min +7min, 150mL of DMF was used for 5 times, DCM was used for 2 times, 150mL of methanol was used for shrinking the resin for 2 times, each time for 5min, and the peptide resin was dried under reduced pressure to obtain 76.8g of peptide resin.
Example 6: cleavage of peptide resin (conventional comparative method 1)
76.8g (10mmol) of the peptide resin obtained in example 5 was placed in its entirety in a cleavage reactor, and 770ml of a cleavage reagent (TFA: TIS: H) was added at a ratio of 10ml/g of the peptide resin2O92: 4:4(V/V)), and stirred at room temperature for 2 h. The reaction was filtered through a sand funnel, after the filtration was complete a small amount of TFA was added to wash the resin, the filtrates were collected and combined and concentrated to volume under reduced pressure. The solution was precipitated by addition of frozen dry ether (100ml/g peptide resin), centrifuged, the supernatant removed and the precipitate washed 3 times with dry ether and dried in vacuo to give 16.8g of crude peptide as a white solid powder. The crude peptide was 42.3% pure.
Example 7: preparation of procapsipeptide (conventional comparative method 1)
16.8g of the crude peptide obtained in example 6 was subjected to reverse phase liquid phase preparative purification using octadecylsilane as a stationary phase and 0.1% trifluoroacetic acid aqueous solution/acetonitrile as a mobile phase, and the desired peak fraction was collected, concentrated and lyophilized. 3.5g of refined peptide with the purity of 99.3 percent and the total yield of the procatide of 20.8 percent is obtained.
Example 8: preparation of peptide resin (conventional comparative method 2)
47.6g (10mmol) of Fmoc-Leu-Wang resin with substitution 0.21mmol/g prepared in example 1 was weighed into a solid phase reaction column, washed 2 times with DMF and then the resin was swelled with DMF for 30 min, then deprotected with 150ml of DBLK for 5min +7min and washed 6 times with 150ml of DBLK. 20.7g (50mmol) of Fmoc-Cys (Acm) -OH and 8.1g (60mmol) of HOBt are weighed and dissolved in 100mL of DMF/DCM (1:1), 10mL (65mmol) of DIPCDI is added under ice-water bath for activation for 2-3min, the mixture is added into a reaction column and reacted for 2h at room temperature, and the reaction end point is detected by ninhydrin (the reaction is stopped if the resin is colorless and transparent; the reaction is prolonged for 1h if the resin is colored). After the reaction is finished, washing the resin for 3 times by using 150mLDMF, adding 100mLDBLK for deprotection for 6min +8min, washing the resin for 6 times by using 100mLDMF, and detecting the color of the resin by ninhydrin. The above coupling operation was repeated to continue coupling Fmoc-Gly-OH, Fmoc-Thr (tBu) -OH, Fmoc-Cys (Mmt) -OH, Fmoc-Ala-OH, Fmoc-Val-OH, Fmoc-Asn (Trt) -OH, Fmoc-Val-OH, Fmoc-Cys (Acm) -OH, Fmoc-Leu-OH, Fmoc-Glu (OtBu) -OH, Fmoc-Cys (Mmt) -OH, Fmoc-Glu (OtBu) -OH, Fmoc-Asp (OtBu) -OH, Fmoc-Asn (Trt) -OH in the order of peptide sequence as described in step 2 above. 50mmol of protected amino acid is added, and the adding ratio of other materials corresponds to the protected amino acid.
After the coupling of the above-mentioned residues is completed, the above-mentioned residues are adoptedThe peptide resin was subjected to a treatment of demamt with appropriate amounts of TFA, TIS, DCM 2:5:93, 20 times and 2 minutes each, after which the resin was washed 6 times with DMF. 10.2ml (100mmol) of 30% hydrogen peroxide is weighed and added into 150ml of DMF, the mixture is added into a solid phase reaction column to carry out bonding reaction of a first pair of disulfide bonds with resin, the DMF is continuously washed for 6 times after air blowing reaction for 1 hour, and then bonding of a second pair of disulfide bonds is carried out. 7.6g (30mmol) of I are weighed2Dissolved in 150ml of DMF and then introduced into a reaction column. The reaction time was 1 h. After the reaction was completed, 150mL of DBLK was used for deprotection for 5min +7min, 150mL of DMF was used for 5 times, DCM was used for 2 times, 150mL of methanol was used for shrinking the resin for 2 times, each time for 5min, and the peptide resin was dried under reduced pressure to obtain 75.9g of peptide resin.
Example 9: cleavage of peptide resin (conventional comparative method 2)
75.9g (10mmol) of the peptide resin obtained in example 8 were placed in its entirety in a cleavage reactor, and 760ml of a cleavage reagent (TFA: TIS: H) was added at a ratio of 10ml/g of the peptide resin2O92: 4:4(V/V)), and stirred at room temperature for 2 h. The reaction was filtered through a sand funnel, after the filtration was complete a small amount of TFA was added to wash the resin, the filtrates were collected and combined and concentrated to volume under reduced pressure. The solution was precipitated by addition of frozen dry ether (100ml/g peptide resin), centrifuged, the supernatant removed and the precipitate washed 3 times with dry ether and dried in vacuo to give 16.4g of crude peptide as a white solid powder. The crude peptide was 44.7% pure.
Example 10: preparation of procapsipeptide (conventional comparative method 1)
16.4g of the crude peptide obtained in example 9 was subjected to reverse phase liquid phase preparative purification using octadecylsilane as a stationary phase and 0.1% trifluoroacetic acid aqueous solution/acetonitrile as a mobile phase, and the desired peak fraction was collected, concentrated and lyophilized. 3.5g of refined peptide with the purity of 99.5 percent and the total yield of the procatide of 24.8 percent is obtained.
Claims (17)
1. A method for preparing procatide, comprising the following steps:
1) Fmoc-Leu are sequentially coupled on solid-phase synthetic resin according to Fmoc solid-phase synthesis strategy16–OH、Fmoc-Cys15(Mmt)-OH、Fmoc-Gly14-OH、Fmoc-Thr13(tBu)-OH、Fmoc-Cys12(Acm)-OH、Fmoc-Ala11-OH、Fmoc-Val10-OH、Fmoc-Asn9(Trt)-OH、Fmoc-Val8-OH、Fmoc-Cys7(Mmt)-OH；
2) After removing the Fmoc protecting group, treating by using a reagent for removing the Mmt protecting group to remove the side chain protecting groups Mmt of Cys at the 7 th site and the 15 th site;
3) forming a disulfide bond between Cys at positions 7 and 15 by a bonding reaction;
4) sequentially coupling Fmoc-Leu according to Fmoc solid phase synthesis strategy6-OH、Fmoc-Glu5(OtBu)-OH、Fmoc-Cys4(Acm)-OH、Fmoc-Glu3(OtBu)-OH、Fmoc-Asp2(OtBu)-OH、Fmoc-Asn1(Trt)-OH；
5) Completing disulfide bond between Cys at positions 4 and 12 through oxidation reaction, and then removing Fmoc protecting group;
6) cracking the resin with a cracking reagent to simultaneously remove all side chain protecting groups, and precipitating with diethyl ether to obtain procatide;
optionally, 7) purification by reverse phase chromatography.
2. The method of claim 1, wherein the Mmt protecting group is removed in step 2) by reacting the resin with a mixed solution of TFA, TIS, DCM (1-5), DCM (2-10), and (97-85) for 1-5 min 15-30 times.
3. The preparation method of claim 2, wherein the Mmt protecting group is removed in step 2) by reacting the resin with a mixed solution of TFA, TIS, DCM 2:5:93 for 20 times and 2 minutes each.
4. The method according to claim 1, wherein the disulfide bond bonding reaction in step 3) is performed using H2O2A bonding reaction is performed.
5. The method according to claim 4, wherein the disulfide bond bonding reaction in step 3) is carried out using 1 to 30 equivalents of H2O2Reacting with polypeptide-solid phase synthetic resin for 0.5-3 hr.
6. The method according to claim 5, wherein the disulfide bond bonding reaction in step 3) is carried out using 10 equivalents of H2O2And reacting with the polypeptide-solid phase synthetic resin for 1 hour.
7. The method according to claim 1, wherein the oxidation reaction in step 5) is carried out by using I2Bonding a second pair of disulfide bonds; i is2The amount of (B) is 0.8 to 10 equivalents based on the amount of the resin substance.
8. The process according to claim 7, step 5) I2The amount of (B) is 3 equivalents of the amount of the resin substance.
9. The method of claim 1, wherein the coupling step 1) and 4) Fmoc solid phase synthesis strategy comprises coupling the Fmoc-protected amino acid residue, removing the Fmoc protecting group, and coupling the next Fmoc-protected amino acid residue.
10. The method of claim 1, wherein the Fmoc-protected amino acid residues are coupled by coupling Fmoc-AA-OH to the solid-phase synthetic resin or the Fmoc-deprotected polypeptide-solid-phase synthetic resin with a coupling reagent.
11. The process according to claim 10, wherein the coupling reagent used in the process for coupling Fmoc-protected amino acid residues is a combination of DIPCDI and Compound A or a combination of DIPEA and Compound A and Compound B, wherein Compound A is HOAt or HOBt and Compound B is PyAOP, PyBOP, HATU, HBTU or TBTU.
12. The method of claim 11, wherein the coupling reagent used in the step of coupling the Fmoc-protected amino acid residue is a combination of DIPCDI and compound a.
13. The method of claim 11, wherein the coupling agent is used in the coupling of Fmoc-protected amino acid residues in a molar ratio of DIPCDI: a: AA: 1.3:1.2:1.0, DIPEA: a: B: AA: 2.0:1.2:1.0:1.0 to the protected amino acid.
14. The method of claim 1, wherein the Fmoc protecting group is removed by a 20% piperidine/DMF solution.
15. The method according to claim 1, wherein the lysis solution used in step 6) is TFA or H2O, anisole, thioanisole and phenol.
16. The method according to claim 1, wherein the purification step of step 7) is performed by reversed-phase high-pressure liquid chromatography.
17. The preparation method according to claim 16, the reverse-phase high-pressure liquid chromatography comprising: using reverse octadecylsilane as stationary phase and 0.1% trifluoroacetic acid water solution/acetonitrile as mobile phase, collecting target peak fraction, concentrating, and lyophilizing.
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| CN114195855A (en) * | 2020-09-17 | 2022-03-18 | 深圳翰宇药业股份有限公司 | Solid-phase synthesis method for polypeptide containing three pairs of disulfide bonds by introducing one pair of disulfide bonds |
| CN114276411B (en) * | 2020-09-27 | 2024-02-09 | 北京大学深圳研究生院 | Solid phase synthesis method of cyclic peptide compound containing trithioether bond |
| CN113512091B (en) * | 2021-05-08 | 2023-06-27 | 漳州未名博欣生物医药有限公司 | Method for synthesizing procalcitonin |
| CN113444150B (en) * | 2021-08-10 | 2023-02-28 | 礼济生物医药科技(南京)有限公司 | Solid-phase preparation method of procatide |
| CN114573662A (en) * | 2021-09-30 | 2022-06-03 | 深圳翰宇药业股份有限公司 | Preparation method of procainatide ammonium salt |
| CN114044813B (en) * | 2022-01-13 | 2022-04-15 | 浙江湃肽生物有限公司南京分公司 | Preparation and purification method of procatide |
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| CN105017387A (en) * | 2015-07-31 | 2015-11-04 | 济南康和医药科技有限公司 | Method for preparing linaclotide |
| CN105504012A (en) * | 2014-09-30 | 2016-04-20 | 深圳翰宇药业股份有限公司 | Preparation method of polypeptide |
| CN107383171A (en) * | 2017-08-15 | 2017-11-24 | 苏州科技大学 | A kind of method by secondary cyclization synthesis in solid state Pu Kana peptides |
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| US20150273013A1 (en) * | 2014-03-25 | 2015-10-01 | Synergy Pharmaceuticals, Inc. | Agonists of guanylate cyclase useful for the treatment of gastrointestinal disorders, inflammation, cancer and other disorders |
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| CN105017387A (en) * | 2015-07-31 | 2015-11-04 | 济南康和医药科技有限公司 | Method for preparing linaclotide |
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