CN107903305B - Method for synthesizing dessorrel by solid-liquid combination - Google Patents
Method for synthesizing dessorrel by solid-liquid combination Download PDFInfo
- Publication number
- CN107903305B CN107903305B CN201711415994.3A CN201711415994A CN107903305B CN 107903305 B CN107903305 B CN 107903305B CN 201711415994 A CN201711415994 A CN 201711415994A CN 107903305 B CN107903305 B CN 107903305B
- Authority
- CN
- China
- Prior art keywords
- fmoc
- val
- ile
- gln
- trt
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
Abstract
The invention discloses a solid-liquid combined synthesis method of Desomotay, which comprises the steps of synthesizing Fmoc-Ile-COOH, methionine and aspartic acid by adopting a liquid phase synthesis method, synthesizing Fmoc-Ile-Met-Asp-OH by adopting Fmoc-Gln (Trt) -COOH, valine and proline as raw materials, synthesizing Fmoc-Gln (Trt) -Val-Pro-OH by adopting Fmoc-Gln (Trt) -COOH, Fmoc-Ser (tBu) -OH, Fmoc-Phe-OH, Fmoc-Gln (Trt) -Val-Pro-OH and Fmoc-Ile-Met-Asp-OH are sequentially connected to 2-CTC Resin by adopting a solid phase synthesis method, obtaining Fmoc-Ile-Met-Asp-Gln (Trt) -Val-Pro-Phe-Ser (tBu) -Val-2-CTC Resin, removing Fmoc-protecting group, cutting, separating and purifying to obtain the dessorocat peptide product. The method has the advantages of low synthesis cost, high synthesis efficiency and simple synthesis method, and can be used for large-scale synthesis of dessorrel.
Description
Technical Field
The invention belongs to the technical field of polypeptide synthesis, and particularly relates to solid-liquid phase combined synthesis of dessorrel.
Background
Melanoma, which is generally referred to as Malignant Melanoma (MM), is one of the malignant melanomas of the skin, and constitutes the most common three malignant tumors of the skin together with squamous cell carcinoma of the skin and basal cell carcinoma of the skin. Melanoma is mostly due to malignant changes in nevi or plaques formed by melanocytes. Melanoma has become the most rapidly growing malignant tumor among all malignant tumors in recent years, with an annual growth rate of 3% to 5%. Moreover, once malignant melanoma enters a rapid growth phase, the prognosis of a patient is very poor, and the fatality rate is high.
Desomot is an active polypeptide containing nine amino acids, the sequence of which is H-Ile-Met-Asp-Gln-Val-Pro-Phe-Ser-Val-OH, and the Desomot is a polypeptide for treating melanoma. The existing method for synthesizing the dessorrel is mainly traditional solid phase synthesis and has the defects of low synthesis efficiency, large impurity accumulation and the like. Therefore, the research on the synthetic method of the dessorrel has important significance for preventing and treating the melanoma.
Disclosure of Invention
The invention aims to solve the technical problem of providing a method for synthesizing dessorrel by solid-liquid combination, which has the advantages of low synthesis cost, high synthesis efficiency and simple synthesis method.
The technical scheme adopted for solving the technical problems comprises the following steps:
1. synthesis of Fmoc-Ile-Met-Asp-OH
(1) Taking tetrahydrofuran as a solvent, carrying out stirring reaction on Fmoc-Ile-COOH, N-hydroxysuccinimide and N, N' -dicyclohexylcarbodiimide at room temperature for 2-3 hours, and filtering to remove solid insoluble substances to obtain a tetrahydrofuran solution of Fmoc-Ile-COOSu; dropwise adding an aqueous solution containing methionine and sodium bicarbonate into an Fmoc-Ile-COO tetrahydrofuran solution, stirring and reacting at room temperature for 18-20 hours after dropwise adding, concentrating under reduced pressure, adjusting the pH value of a concentrated solution to 2-3 by using a citric acid aqueous solution with the mass fraction of 10%, extracting by using ethyl acetate, washing an organic phase by using water, washing by using saturated salt water, drying by using anhydrous sodium sulfate, filtering under reduced pressure, and concentrating under reduced pressure to obtain Fmoc-Ile-Met-COOH.
(2) Fmoc-Ile-Met-Asp-OH was synthesized using Fmoc-Ile-Met-COOH and aspartic acid according to the procedure of step (1) above.
2. Synthesis of Fmoc-Gln (Trt) -Val-Pro-OH
Synthesizing Fmoc-Gln (Trt) -Val-COOH by using Fmoc-Gln (Trt) -COOH and valine as raw materials, and then synthesizing Fmoc-Gln (Trt) -Val-COOH by using Fmoc-Gln (Trt) -Val-COOH and proline to obtain Fmoc-Gln (Trt) -Val-Pro-OH;
3. synthesis of Fmoc-Ser (tBu) -Val-2-CTC Resin
(1) Taking dichloromethane as a solvent, and reacting the dichloromethane-swollen 2-CTC Resin with Fmoc-Val-OH and N, N' -diisopropylethylamine at room temperature for 1-2 hours under the protection of nitrogen to obtain Fmoc-Val-2-CTC Resin;
(2) removing Fmoc protecting groups from Fmoc-Val-2-CTC Resin by using mixed liquid of piperidine and N, N-dimethylformamide in a volume ratio of 1:4, adding the mixture into N, N-dimethylformamide, adding Fmoc-Ser (tBu) -OH, 1-hydroxy phenylpropyl triazole, benzotriazole-N, N, N ', N ' -tetramethylurea hexafluorophosphate and N, N ' -diisopropylethylamine, and reacting at room temperature for 1-2 hours under the protection of nitrogen to obtain Fmoc-Ser (tBu) -Val-2-CTC Resin;
4. synthesis of Fmoc-Ile-Met-Asp-Gln (Trt) -Val-Pro-Phe-Ser (tBu) -Val-2-CTC Resin
Connecting Fmoc-Phe-OH, Fmoc-Gln (Trt) -Val-Pro-OH and Fmoc-Ile-Met-Asp-OH to Fmoc-Ser (tBu) -Val-2-CTC Resin in sequence according to the method in the step 3 to obtain Fmoc-Ile-Met-Asp-Gln (Trt) -Val-Pro-Phe-Ser (tBu) -Val-2-CTC Resin;
5. synthetic dessoracetat
And (3) removing the Fmoc-protecting group from the Fmoc-Ile-Met-Asp-Gln (Trt) -Val-Pro-Phe-Ser (tBu) -Val-2-CTC Resin obtained in the step (4) by using a mixed solution of piperidine and N, N-dimethylformamide in a volume ratio of 1:4, and then cutting, separating and purifying the mixture to obtain H-Ile-Met-Asp-Gln-Val-Pro-Phe-Ser-Val-OH, namely the Desomoto product.
In the step 1 (1), the molar ratio of Fmoc-Ile-COOH to N-hydroxysuccinimide, N' -dicyclohexylcarbodiimide, methionine and sodium bicarbonate is preferably 1: 1-2: 1.1-2.
In the step (2) of the step 1, the molar ratio of N-hydroxysuccinimide, N' -dicyclohexylcarbodiimide, aspartic acid, sodium bicarbonate and Fmoc-Ile-Met-COOH is preferably 1-2: 1.1-2: 1.
In the step 2, the molar ratio of Fmoc-Gln (Trt) -COOH to valine and proline is preferably 1: 1.1-2.
In the step 3 (1), the molar ratio of the 2-CTC Resin to Fmoc-Val-OH and N, N' -diisopropylethylamine is preferably 1: 1-3: 2-4.
In the step (2) of the step 3, the molar ratio of Fmoc-Ser (tBu) -OH, 1-hydroxy benzotriazole, benzotriazole-N, N, N ', N ' -tetramethylurea hexafluorophosphate, N, N ' -diisopropylethylamine to 2-CTC Resin is preferably 1-3: 2-4: 1.
In the step 4, the molar ratio of Fmoc-Phe-OH, Fmoc-Gln (Trt) -Val-Pro-OH, Fmoc-Ile-Met-Asp-OH and 2-CTC Resin is preferably 1-3: 1.
In the step 5, the cutting fluid used for cutting comprises the following components in percentage by mass: trifluoroacetic acid 83%, phenol 5%, thioanisole 4%, water 3%, and triisopropylsilane 5%.
The method combines the traditional solid-phase synthesis method and the liquid-phase synthesis method, adopts the fragment method to synthesize the desomopitae, overcomes the defects of low synthesis efficiency and large impurity accumulation of the traditional classical solid-phase synthesis of the desomopitae, and also overcomes the defects of complex process, long synthesis period and low efficiency of the liquid-phase synthesis of the desomopitae, has the advantages of low synthesis cost, high synthesis efficiency and simple synthesis method, and can be used for large-scale synthesis of the desomopitae.
Detailed Description
The present invention will be described in further detail with reference to examples, but the scope of the present invention is not limited to these examples.
Example 1
1. Synthesis of Fmoc-Ile-Met-COOH
(1) 17.67g (0.05mol) of Fmoc-Ile-COOH was added to 170mL of tetrahydrofuran, and after stirring and dissolving at room temperature, 6.33g (0.055mol) of N-hydroxysuccinimide was added and stirred until dissolving, and then 23mL of a tetrahydrofuran solution containing 11.35g (0.055mol) of N, N' -dicyclohexylcarbodiimide was slowly added dropwise thereto, and after completion of the dropwise addition, the reaction was stirred at room temperature for 3 hours, and the solid insoluble matter was removed by filtration under reduced pressure to obtain a tetrahydrofuran solution of Fmoc-Ile-COOSu. Dissolving 8.21g (0.055mol) of methionine and 4.62g (0.055mol) of sodium bicarbonate in 250mL of distilled water, dropwise adding the obtained solution into a tetrahydrofuran solution of Fmoc-Ile-COOSu, stirring at room temperature for reaction for 20 hours, concentrating under reduced pressure, adjusting the pH value of the concentrated solution to 2-3 by using a citric acid aqueous solution with the mass fraction of 10%, extracting by using ethyl acetate, washing an organic phase by water, washing by using saturated salt water, drying by using anhydrous sodium sulfate, filtering under reduced pressure, and concentrating under reduced pressure to obtain Fmoc-Ile-Met-COOH.
In the reaction, the molar ratio of Fmoc-Ile-COOH to N-hydroxysuccinimide, N' -dicyclohexylcarbodiimide, methionine and sodium bicarbonate is 1:1.1:1.1: 1.1.
(2) And (2) adding the Fmoc-Ile-Met-COOH obtained in the step (1) into 200mL of tetrahydrofuran, stirring at room temperature to dissolve, adding 6.33g (0.055mol) of N-hydroxysuccinimide, stirring to dissolve, slowly dropwise adding 23mL of tetrahydrofuran solution containing 11.35g (0.055mol) of N, N' -dicyclohexylcarbodiimide, stirring at room temperature to react for 3 hours after dropwise adding, and filtering under reduced pressure to remove solid insoluble substances to obtain the tetrahydrofuran solution of Fmoc-Ile-Met-COOSu. Dissolving 7.35g (0.055mol) of aspartic acid and 4.62g (0.055mol) of sodium bicarbonate in 250mL of distilled water, dropwise adding the obtained solution into a tetrahydrofuran solution of Fmoc-Ile-Met-COOSu, stirring at room temperature for reaction for 20 hours, concentrating under reduced pressure, adjusting the pH value of the concentrated solution to 2-3 by using a citric acid aqueous solution with the mass fraction of 10%, extracting by using ethyl acetate, washing the organic phase by using water, washing the organic phase by using saturated salt water, drying by using anhydrous sodium sulfate, filtering under reduced pressure, and concentrating under reduced pressure to obtain 19.18g of Fmoc-Ile-Met-Asp-OH, wherein the yield is 58.2%.
In the reaction, the molar ratio of N-hydroxysuccinimide, N' -dicyclohexylcarbodiimide, aspartic acid, sodium bicarbonate and Fmoc-Ile-Met-COOH is 1.1:1.1:1.1:1.
2. Synthesis of Fmoc-Gln (Trt) -Val-Pro-OH
According to the method of step 1, Fmoc-Gln (Trt) -Val-COOH was synthesized from 30.54g (0.05mol) of Fmoc-Gln (Trt) -COOH and 6.44g (0.055mol) of valine, and then 23.22g of Fmoc-Gln (Trt) -Val-Pro-OH was synthesized from Fmoc-Gln (Trt) -Val-COOH and 6.33g (0.055mol) of proline, with a yield of 52.3%.
In the reaction of the step, the molar ratio of Fmoc-Gln (Trt) -COOH to valine and proline is 1:1.1: 1.1.
3. Synthesis of Fmoc-Ser (tBu) -Val-2-CTC Resin
(1) Adding 7.15g of 2-CTC Resin (with the substitution degree of 1.75mmol/g) into 70mL of dichloromethane for swelling for 10min, carrying out suction filtration, adding the swelled 2-CTC Resin into 70mL of dichloromethane, adding 8.49g (0.025mol) of Fmoc-Val-OH and 6.5mL (0.0375mol) of N, N' -diisopropylethylamine, and carrying out stirring reaction at room temperature for 2 h under the protection of nitrogen to obtain the Fmoc-Val-2-CTC Resin.
In the reaction, the molar ratio of 2-CTC Resin to Fmoc-Val-OH and N, N' -diisopropylethylamine is 1:2: 3.
(2) Adding Fmoc-Val-2-CTC Resin into 70mL of mixed solution of piperidine and N, N-dimethylformamide in a volume ratio of 1:4, stirring at room temperature for reaction for 20 minutes to remove Fmoc-protecting groups, sequentially washing with isopropanol and N, N-dimethylformamide twice, then adding into 70mL of N, N-dimethylformamide, adding 9.59g (0.025mol) of Fmoc-Ser (tBu) -OH, 5.07g (0.0375mol) of 1-hydroxy phenylpropriazole, 14.22g (0.0375mol) of benzotriazole-N, N, N ', N ' -tetramethylurea hexafluorophosphate and 6.5mL (0.0375mol) of N, N ' -diisopropylethylamine, and stirring at room temperature for reaction for 2 hours under the protection of nitrogen to obtain Fmoc-Ser (tBu) -Val-2-CTC Resin.
In the reaction, the molar ratio of Fmoc-Ser (tBu) -OH, 1-hydroxy benzotriazole, benzotriazole-N, N, N ', N ' -tetramethylurea hexafluorophosphate, N, N ' -diisopropylethylamine and 2-CTC Resin is 2:3:3:3: 1.
4. Synthesis of Fmoc-Ile-Met-Asp-Gln (Trt) -Val-Pro-Phe-Ser (tBu) -Val-2-CTC Resin
According to the method of step 3 (2), Fmoc-Phe-OH, Fmoc-Gln (Trt) -Val-Pro-OH, Fmoc-Ile-Met-Asp-OH, Fmoc-Phe-OH, Fmoc-Gln (Trt) -Val-Pro-OH and Fmoc-Ile-Met-Asp-OH are sequentially added to Fmoc-Ser (tBu) -Val-2-CTC Resin in an amount of 9.68g (0.025mol), 20.17g (0.025mol) and 14.99g (0.025mol) to obtain Fmoc-Ile-Met-Asp-Gln (Trt) -Val-Pro-Phe-Ser (tBu) -Val-2-CTC Resin.
In the reaction of the step, the molar ratio of Fmoc-Phe-OH, Fmoc-Gln (Trt) -Val-Pro-OH, Fmoc-Ile-Met-Asp-OH and 2-CTC Resin is 2:1.
5. Synthetic dessoracetat
Removing Fmoc protecting groups from the Fmoc-Ile-Met-Asp-Gln (Trt) -Val-Pro-Phe-Ser (tBu) -Val-2-CTC Resin obtained in the step 4 by using 70mL of mixed solution of piperidine and N, N-dimethylformamide in the volume ratio of 1:4 to obtain desomourtal peptide Resin, adding the desortal peptide Resin into 70mL of cutting solution consisting of 83% of trifluoroacetic acid, 5% of phenol, 4% of benzylsulfide, 3% of water and 5% of triisopropylsilane by mass percent, stirring at room temperature for 2 hours, filtering, adding the filtrate into 70mL of cold diethyl ether to separate out precipitates, purifying the precipitates by reverse phase chromatography (the purification conditions are that the column specification is GLP-ID50mm x 450mm, the detection wavelength is 215nm, the flow rate is 30mL/min, the mobile phase A is a trifluoroacetic acid aqueous solution with the volume fraction of 0.1%, the mobile phase B is a trifluoroacetic acid methanol solution with the volume fraction of 0.1%, the elution gradient was: 20-30% of phase B for 30min, 30-40% of phase B for 30min), and freeze drying to obtain H-Ile-Met-Asp-Gln-Val-Pro-Phe-Ser-Val-OH, namely, 9.06g of Desomot, with the yield of 69.9%.
Example 2
1. Synthesis of Fmoc-Ile-Met-COOH
(1) 17.67g (0.05mol) of Fmoc-Ile-COOH was added to 170mL of tetrahydrofuran, and after the mixture was dissolved by stirring at room temperature, 8.63g (0.075mol) of N-hydroxysuccinimide was added and stirred until the mixture was dissolved, and then 35mL of a tetrahydrofuran solution containing 15.47g (0.075mol) of N, N' -dicyclohexylcarbodiimide was slowly added dropwise thereto, and after the completion of the dropwise addition, the mixture was reacted by stirring at room temperature for 3 hours, and then the solid insoluble matter was removed by filtration under reduced pressure to obtain a tetrahydrofuran solution of Fmoc-Ile-COOSu. Dissolving 11.19g (0.075mol) of methionine and 6.30g (0.075mol) of sodium bicarbonate in 250mL of distilled water, dropwise adding the obtained solution into a tetrahydrofuran solution of Fmoc-Ile-COOSu, stirring at room temperature for reaction for 20 hours, concentrating under reduced pressure, adjusting the pH value of the concentrated solution to 2-3 by using a citric acid aqueous solution with the mass fraction of 10%, extracting by using ethyl acetate, washing an organic phase by using water, washing by using saturated salt water, drying by using anhydrous sodium sulfate, filtering under reduced pressure, and concentrating under reduced pressure to obtain Fmoc-Ile-Met-COOH.
In the reaction, the molar ratio of Fmoc-Ile-COOH to N-hydroxysuccinimide, N' -dicyclohexylcarbodiimide, methionine and sodium bicarbonate is 1:1.5:1.5:1.5: 1.5.
(2) Adding Fmoc-Ile-Met-COOH obtained in the step (1) into 200mL of tetrahydrofuran, stirring at room temperature to dissolve, adding 8.63g (0.075mol) of N-hydroxysuccinimide, stirring to dissolve, slowly dropwise adding 35mL of tetrahydrofuran solution containing 15.47g (0.075mol) of N, N' -dicyclohexylcarbodiimide, stirring at room temperature to react for 3 hours after dropwise adding, and filtering under reduced pressure to remove solid insoluble substances to obtain the tetrahydrofuran solution of Fmoc-Ile-Met-COOSu. Dissolving 10.02g (0.075mol) of aspartic acid and 6.30g (0.075mol) of sodium bicarbonate in 250mL of distilled water, dropwise adding the obtained solution into a tetrahydrofuran solution of Fmoc-Ile-Met-COOSu, stirring at room temperature for reaction for 20 hours, concentrating under reduced pressure, adjusting the pH value of the concentrated solution to 2-3 by using a citric acid aqueous solution with the mass fraction of 10%, extracting by using ethyl acetate, washing an organic phase by using water, washing the organic phase by using saturated salt water, drying by using anhydrous sodium sulfate, filtering under reduced pressure, and concentrating under reduced pressure to obtain 18.38g of Fmoc-Ile-Met-Asp-OH, wherein the yield is 55.9%.
In the reaction, the molar ratio of N-hydroxysuccinimide, N' -dicyclohexylcarbodiimide, aspartic acid, sodium bicarbonate and Fmoc-Ile-Met-COOH is 1.5:1.5:1.5:1.
2. Synthesis of Fmoc-Gln (Trt) -Val-Pro-OH
According to the method of step 1, Fmoc-Gln (Trt) -Val-COOH was synthesized from 30.54g (0.05mol) of Fmoc-Gln (Trt) -COOH and 8.78g (0.075mol) of valine, and then 24.65g of Fmoc-Gln (Trt) -Val-Pro-OH was synthesized from Fmoc-Gln (Trt) -Val-COOH and 8.63g (0.075mol) of proline, with a yield of 55.5%.
In the reaction of the step, the molar ratio of Fmoc-Gln (Trt) -COOH to valine and proline is 1:1.5: 1.5.
3. Synthesis of Fmoc-Ser (tBu) -Val-2-CTC Resin
(1) Adding 17.5g of 2-CTC Resin (with the substitution degree of 1.75mmol/g) into 170mL of dichloromethane for swelling for 10min, carrying out suction filtration, adding the swelled 2-CTC Resin into 170mL of dichloromethane, adding 10.18g (0.03mol) of Fmoc-Val-OH and 10.39mL (0.06mol) of N, N' -diisopropylethylamine, and carrying out stirring reaction at room temperature for 2 h under the protection of nitrogen to obtain the Fmoc-Val-2-CTC Resin.
In the reaction, the molar ratio of 2-CTC Resin to Fmoc-Val-OH and N, N' -diisopropylethylamine is 1:1: 2.
(2) Adding Fmoc-Val-2-CTC Resin into 170mL of mixed solution of piperidine and N, N-dimethylformamide in a volume ratio of 1:4, stirring at room temperature for reaction for 20 minutes to remove Fmoc-protecting groups, sequentially washing with isopropanol and N, N-dimethylformamide twice, then adding into 170mL of N, N-dimethylformamide, adding 11.50g (0.03mol) of Fmoc-Ser (tBu) -OH, 8.11g (0.06mol) of 1-hydroxy phenylpropriazole, 22.76g (0.06mol) of benzotriazole-N, N, N ', N ' -tetramethylurea hexafluorophosphate and 10.39mL (0.06mol) of N, N ' -diisopropylethylamine, and stirring at room temperature for reaction for 2 hours under the protection of nitrogen to obtain Fmoc-Ser (tBu) -Val-2-CTC Resin.
In the reaction, the molar ratio of Fmoc-Ser (tBu) -OH, 1-hydroxy benzotriazole, benzotriazole-N, N, N ', N ' -tetramethylurea hexafluorophosphate, N, N ' -diisopropylethylamine and 2-CTC Resin is 1:2:2:2: 1.
4. Synthesis of Fmoc-Ile-Met-Asp-Gln (Trt) -Val-Pro-Phe-Ser (tBu) -Val-2-CTC Resin
According to the method of step 3 (2), Fmoc-Phe-OH, Fmoc-Gln (Trt) -Val-Pro-OH, Fmoc-Ile-Met-Asp-OH, Fmoc-Phe-OH, Fmoc-Gln (Trt) -Val-Pro-OH and Fmoc-Ile-Met-Asp-OH are sequentially added to Fmoc-Ser (tBu) -Val-2-CTC Resin in an amount of 11.62g (0.03mol), 24.21g (0.03mol) and 17.99g (0.03mol) to obtain Fmoc-Ile-Met-Asp-Gln (Trt) -Val-Pro-Phe-Ser (tBu) -Val-2-CTC Resin.
In the reaction of the step, the molar ratio of Fmoc-Phe-OH, Fmoc-Gln (Trt) -Val-Pro-OH, Fmoc-Ile-Met-Asp-OH and 2-CTC Resin is 1:1.
5. Synthetic dessoracetat
Removing Fmoc protecting groups from the Fmoc-Ile-Met-Asp-Gln (Trt) -Val-Pro-Phe-Ser (tBu) -Val-2-CTC Resin obtained in the step 4 by using 170mL of mixed solution of piperidine and N, N-dimethylformamide in the volume ratio of 1:4 to obtain desomourtal peptide Resin, adding the desortal peptide Resin into 170mL of cutting solution consisting of 83% of trifluoroacetic acid, 5% of phenol, 4% of benzylsulfide, 3% of water and 5% of triisopropylsilane by mass percent, stirring at room temperature for 2 hours, filtering, adding the filtrate into 170mL of cold diethyl ether to separate out precipitates, purifying the precipitates by reverse phase chromatography (the purification conditions are that a column specification is GLP-ID50mm x 450mm, the detection wavelength is 215nm, the flow rate is 30mL/min, the mobile phase A is a trifluoroacetic acid aqueous solution with the volume fraction of 0.1%, the mobile phase B is a trifluoroacetic acid methanol solution with the volume fraction of 0.1%, the elution gradient was: 20-30% of phase B for 30min, 30-40% of phase B for 30min), and freeze drying to obtain 20.33g of dessorrel with a yield of 64.0%.
Example 3
1. Synthesis of Fmoc-Ile-Met-COOH
(1) 17.67g (0.05mol) of Fmoc-Ile-COOH was added to 170mL of tetrahydrofuran, and after stirring and dissolving at room temperature, 11.51g (0.10mol) of N-hydroxysuccinimide was added and stirred until dissolving, and then 50mL of a tetrahydrofuran solution containing 20.63g (0.10mol) of N, N' -dicyclohexylcarbodiimide was slowly added dropwise thereto, and after completion of the dropwise addition, the reaction was stirred at room temperature for 3 hours, and solid insoluble matter was removed by filtration under reduced pressure to obtain a tetrahydrofuran solution of Fmoc-Ile-COOSu. Dissolving 14.92g (0.10mol) of methionine and 8.40g (0.10mol) of sodium bicarbonate in 250mL of distilled water, dropwise adding the obtained solution into a tetrahydrofuran solution of Fmoc-Ile-COOSu, stirring at room temperature for reaction for 20 hours, concentrating under reduced pressure, adjusting the pH value of the concentrated solution to 2-3 by using a citric acid aqueous solution with the mass fraction of 10%, extracting by using ethyl acetate, washing an organic phase by using water, washing by using saturated salt water, drying by using anhydrous sodium sulfate, filtering under reduced pressure, and concentrating under reduced pressure to obtain Fmoc-Ile-Met-COOH.
In the reaction, the molar ratio of Fmoc-Ile-COOH to N-hydroxysuccinimide, N' -dicyclohexylcarbodiimide, methionine and sodium bicarbonate is 1:2:2:2: 2.
(2) Adding the Fmoc-Ile-Met-COOH obtained in the step (1) into 200mL of tetrahydrofuran, stirring at room temperature to dissolve, adding 11.51g (0.10mol) of N-hydroxysuccinimide, stirring to dissolve, slowly dropwise adding 50mL of tetrahydrofuran solution containing 20.63g (0.10mol) of N, N' -dicyclohexylcarbodiimide, stirring at room temperature to react for 3 hours after dropwise adding, and filtering under reduced pressure to remove solid insoluble substances to obtain the tetrahydrofuran solution of Fmoc-Ile-Met-COOSu. Dissolving 13.36g (0.10mol) of aspartic acid and 8.40g (0.10mol) of sodium bicarbonate in 250mL of distilled water, dropwise adding the obtained solution into a tetrahydrofuran solution of Fmoc-Ile-Met-COOSu, stirring at room temperature for reaction for 20 hours, concentrating under reduced pressure, adjusting the pH value of the concentrated solution to 2-3 by using a citric acid aqueous solution with the mass fraction of 10%, extracting by using ethyl acetate, washing an organic phase by using water, washing by using saturated salt water, drying by using anhydrous sodium sulfate, filtering under reduced pressure, and concentrating under reduced pressure to obtain 21.62g of Fmoc-Ile-Met-Asp-OH, wherein the yield is 65.7%.
In the reaction, the molar ratio of N-hydroxysuccinimide, N' -dicyclohexylcarbodiimide, Fmoc-Asp-OH, sodium bicarbonate and Fmoc-Ile-Met-COOH is 2:2:2:2: 1.
2. Synthesis of Fmoc-Gln (Trt) -Val-Pro-OH
According to the method of step 1, Fmoc-Gln (Trt) -Val-COOH was synthesized from 30.54g (0.05mol) of Fmoc-Gln (Trt) -COOH and 11.71g (0.10mol) of valine, and then 23.22g of Fmoc-Gln (Trt) -Val-Pro-OH was synthesized from Fmoc-Gln (Trt) -Val-COOH and 11.51g (0.10mol) of proline, and the yield was 57.6%.
In the reaction of the step, the molar ratio of Fmoc-Gln (Trt) -COOH to valine and proline is 1:2: 2.
3. Synthesis of Fmoc-Ser (tBu) -Val-2-CTC Resin
(1) 6.86g of 2-CTC Resin (with the substitution degree of 1.75mmol/g) is added into 70mL of dichloromethane for swelling for 10min, suction filtration is carried out, the swelled 2-CTC Resin is added into 70mL of dichloromethane, 12.22g (0.036mol) of Fmoc-Val-OH and 8.31mL (0.048mol) of N, N' -diisopropylethylamine are added, and the mixture is stirred and reacted for 2 hours at room temperature under the protection of nitrogen, so as to obtain the Fmoc-Val-2-CTC Resin.
In the reaction, the molar ratio of 2-CTC Resin to Fmoc-Val-OH and N, N' -diisopropylethylamine is 1:3: 4.
(2) Adding Fmoc-Val-2-CTC Resin into 70mL of mixed solution of piperidine and N, N-dimethylformamide in a volume ratio of 1:4, stirring at room temperature for reaction for 20 minutes to remove Fmoc-protecting groups, sequentially washing with isopropanol and N, N-dimethylformamide twice, then adding into 70mL of N, N-dimethylformamide, adding 13.80g (0.036mol) of Fmoc-Ser (tBu) -OH, 6.49g (0.048mol) of 1-hydroxy phenylpropyl triazole, 17.92g (0.048mol) of benzotriazole-N, N, N ', N ' -tetramethylurea hexafluorophosphate and 8.31mL (0.048mol) of N, N ' -diisopropylethylamine, and stirring at room temperature for reaction for 2 hours under the protection of nitrogen to obtain Fmoc-Ser (tBu) -Val-2-CTC Resin.
In the reaction, the molar ratio of Fmoc-Ser (tBu) -OH, 1-hydroxy benzotriazole, benzotriazole-N, N, N ', N ' -tetramethylurea hexafluorophosphate, N, N ' -diisopropylethylamine and 2-CTC Resin is 3:4:4:4: 1.
4. Synthesis of Fmoc-Ile-Met-Asp-Gln (Trt) -Val-Pro-Phe-Ser (tBu) -Val-2-CTC Resin
According to the method in step 3 (2), Fmoc-Phe-OH, Fmoc-Gln (Trt) -Val-Pro-OH, Fmoc-Ile-Met-Asp-OH, Fmoc-Phe-OH, Fmoc-Gln (Trt) -Val-Pro-OH and Fmoc-Ile-Met-Asp-OH are sequentially added to Fmoc-Ser (tBu) -Val-2-CTC Resin in an amount of 13.95g (0.036mol), 29.05g (0.036mol) and 23.97g (0.036mol) to obtain Fmoc-Ile-Met-Asp-Gln (Trt) -Val-Pro-Phe-Ser (tBu) -Val-2-CTC Resin.
In the reaction of the step, the molar ratio of Fmoc-Phe-OH, Fmoc-Gln (Trt) -Val-Pro-OH, Fmoc-Ile-Met-Asp-OH and 2-CTC Resin is 3: 1.
5. Synthetic dessoracetat
Removing Fmoc protecting groups from the Fmoc-Ile-Met-Asp-Gln (Trt) -Val-Pro-Phe-Ser (tBu) -Val-2-CTC Resin obtained in the step 4 by using 70mL of mixed solution of piperidine and N, N-dimethylformamide in the volume ratio of 1:4 to obtain desomourtal peptide Resin, adding the desortal peptide Resin into 70mL of cutting solution consisting of 83% of trifluoroacetic acid, 5% of phenol, 4% of benzylsulfide, 3% of water and 5% of triisopropylsilane by mass percent, stirring at room temperature for 2 hours, filtering, adding the filtrate into 70mL of cold diethyl ether to separate out precipitates, purifying the precipitates by reverse phase chromatography (the purification conditions are that the column specification is GLP-ID50mm x 450mm, the detection wavelength is 215nm, the flow rate is 30mL/min, the mobile phase A is a trifluoroacetic acid aqueous solution with the volume fraction of 0.1%, the mobile phase B is a trifluoroacetic acid methanol solution with the volume fraction of 0.1%, the elution gradient was: 20-30% of phase B for 30min, 30-40% of phase B for 30min), and freeze drying to obtain H-Ile-Met-Asp-Gln-Val-Pro-Phe-Ser-Val-OH, namely, 9.32g of Desomot, with the yield of 74.9%.
Claims (8)
1. A method for synthesizing dessorrel by solid-liquid combination is characterized by comprising the following steps:
(1) synthesis of Fmoc-Ile-Met-Asp-OH
Taking tetrahydrofuran as a solvent, carrying out stirring reaction on Fmoc-Ile-COOH, N-hydroxysuccinimide and N, N' -dicyclohexylcarbodiimide for 2-3 hours at room temperature, and filtering to remove solid insoluble substances to obtain a tetrahydrofuran solution of Fmoc-Ile-COOSu; dropwise adding an aqueous solution containing methionine and sodium bicarbonate into an Fmoc-Ile-COO tetrahydrofuran solution, stirring and reacting at room temperature for 18-20 hours after dropwise adding, concentrating under reduced pressure, adjusting the pH value of a concentrated solution to 2-3 by using a citric acid aqueous solution with the mass fraction of 10%, extracting by using ethyl acetate, washing an organic phase by using water, washing by using saturated salt water, drying by using anhydrous sodium sulfate, filtering under reduced pressure, and concentrating under reduced pressure to obtain Fmoc-Ile-Met-COOH;
secondly, synthesizing Fmoc-Ile-Met-Asp-OH by using Fmoc-Ile-Met-COOH and aspartic acid according to the method of the first step;
(2) synthesis of Fmoc-Gln (Trt) -Val-Pro-OH
Synthesizing Fmoc-Gln (Trt) -Val-COOH by taking Fmoc-Gln (Trt) -COOH and valine as raw materials, and then synthesizing Fmoc-Gln (Trt) -Val-COOH by using Fmoc-Gln (Trt) -Val-COOH and proline to obtain Fmoc-Gln (Trt) -Val-Pro-OH;
(3) synthesis of Fmoc-Ser (tBu) -Val-2-CTC Resin
Taking dichloromethane as a solvent, and reacting the dichloromethane-swollen 2-CTC Resin with Fmoc-Val-OH and N, N' -diisopropylethylamine at room temperature for 1-2 hours under the protection of nitrogen to obtain Fmoc-Val-2-CTC Resin;
removing Fmoc protecting groups from Fmoc-Val-2-CTC Resin by using mixed solution of piperidine and N, N-dimethylformamide in a volume ratio of 1:4, adding the mixture into N, N-dimethylformamide, adding Fmoc-Ser (tBu) -OH, 1-hydroxy phenylpropyl triazole, benzotriazole-N, N, N ', N ' -tetramethylurea hexafluorophosphate and N, N ' -diisopropylethylamine, and reacting at room temperature for 1-2 hours under the protection of nitrogen to obtain Fmoc-Ser (tBu) -Val-2-CTC Resin;
(4) synthesis of Fmoc-Ile-Met-Asp-Gln (Trt) -Val-Pro-Phe-Ser (tBu) -Val-2-CTC Resin
According to the method in the step (3), Fmoc-Phe-OH, Fmoc-Gln (Trt) -Val-Pro-OH and Fmoc-Ile-Met-Asp-OH are sequentially connected to Fmoc-Ser (tBu) -Val-2-CTC Resin to obtain Fmoc-Ile-Met-Asp-Gln (Trt) -Val-Pro-Phe-Ser (tBu) -Val-2-CTC Resin;
(5) synthetic dessoracetat
And (3) removing the Fmoc-protecting group from the Fmoc-Ile-Met-Asp-Gln (Trt) -Val-Pro-Phe-Ser (tBu) -Val-2-CTC Resin obtained in the step (4) by using a mixed solution of piperidine and N, N-dimethylformamide in a volume ratio of 1:4, and then cutting, separating and purifying the mixture to obtain H-Ile-Met-Asp-Gln-Val-Pro-Phe-Ser-Val-OH, namely the Desomoto product.
2. The method for synthesizing dessomototal by solid-liquid combination according to claim 1, characterized in that: in the step (1), the molar ratio of Fmoc-Ile-COOH to N-hydroxysuccinimide, N' -dicyclohexylcarbodiimide, methionine and sodium bicarbonate is 1: 1-2: 1.1-2.
3. The method for synthesizing dessomototal by solid-liquid combination according to claim 1, characterized in that: in the step (1), the molar ratio of the N-hydroxysuccinimide, the N, N' -dicyclohexylcarbodiimide, the aspartic acid, the sodium bicarbonate and the Fmoc-Ile-Met-COOH is 1-2: 1.1-2: 1.
4. The method for synthesizing dessomototal by solid-liquid combination according to claim 1, characterized in that: in the step (2), the molar ratio of Fmoc-Gln (Trt) -COOH to valine to proline is 1: 1.1-2.
5. The method for synthesizing dessomototal by solid-liquid combination according to claim 1, characterized in that: in the step (3), the molar ratio of the 2-CTC Resin to Fmoc-Val-OH and N, N' -diisopropylethylamine is 1: 1-3: 2-4.
6. The method for synthesizing dessomototal by solid-liquid combination according to claim 1, characterized in that: in the step (3), the molar ratio of Fmoc-Ser (tBu) -OH, 1-hydroxy benzotriazole, benzotriazole-N, N, N ', N ' -tetramethylurea hexafluorophosphate, N, N ' -diisopropylethylamine and 2-CTC Resin is 1-3: 2-4: 1.
7. The method for synthesizing dessomototal by solid-liquid combination according to claim 1, characterized in that: in the step (4), the molar ratio of Fmoc-Phe-OH, Fmoc-Gln (Trt) -Val-Pro-OH, Fmoc-Ile-Met-Asp-OH and 2-CTC Resin is 1-3: 1.
8. The method for synthesizing dessomototal by solid-liquid combination according to claim 1, characterized in that: in the step (5), the cutting fluid used for cutting comprises the following components in percentage by mass: trifluoroacetic acid 83%, phenol 5%, thioanisole 4%, water 3%, and triisopropylsilane 5%.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711415994.3A CN107903305B (en) | 2017-12-25 | 2017-12-25 | Method for synthesizing dessorrel by solid-liquid combination |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711415994.3A CN107903305B (en) | 2017-12-25 | 2017-12-25 | Method for synthesizing dessorrel by solid-liquid combination |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN107903305A CN107903305A (en) | 2018-04-13 |
| CN107903305B true CN107903305B (en) | 2020-11-13 |
Family
ID=61870976
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201711415994.3A Active CN107903305B (en) | 2017-12-25 | 2017-12-25 | Method for synthesizing dessorrel by solid-liquid combination |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107903305B (en) |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2012027379A2 (en) * | 2010-08-24 | 2012-03-01 | University Of Pittsburgh - Of The Commonwealth System Of Higher Education | Interleukin-13 receptor alpha 2 peptide-based brain cancer vaccines |
| CN105294853A (en) * | 2015-11-13 | 2016-02-03 | 中肽生化有限公司 | Preparation method for liraglutide |
| CN105384799A (en) * | 2015-12-30 | 2016-03-09 | 济南康和医药科技有限公司 | Method for preparing sinapultide by combining solid phase and liquid phase |
| CN106632655A (en) * | 2016-12-29 | 2017-05-10 | 陕西慧康生物科技有限责任公司 | Method for preparing exenatide and product thereof |
-
2017
- 2017-12-25 CN CN201711415994.3A patent/CN107903305B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2012027379A2 (en) * | 2010-08-24 | 2012-03-01 | University Of Pittsburgh - Of The Commonwealth System Of Higher Education | Interleukin-13 receptor alpha 2 peptide-based brain cancer vaccines |
| CN105294853A (en) * | 2015-11-13 | 2016-02-03 | 中肽生化有限公司 | Preparation method for liraglutide |
| CN105384799A (en) * | 2015-12-30 | 2016-03-09 | 济南康和医药科技有限公司 | Method for preparing sinapultide by combining solid phase and liquid phase |
| CN106632655A (en) * | 2016-12-29 | 2017-05-10 | 陕西慧康生物科技有限责任公司 | Method for preparing exenatide and product thereof |
Non-Patent Citations (1)
| Title |
|---|
| Antigen Presentation of a Modified Tumor-Derived Peptide by Tumor Infiltrating Lymphocytes;Sara O. Dionne etal;《CELLULAR IMMUNOLOGY》;20011215;第214卷(第2期);第139-144页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN107903305A (en) | 2018-04-13 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106632609B (en) | Preparation method of hexapeptide and product thereof | |
| CN110684077B (en) | Large-scale synthesis method of achirelin | |
| CN107778349B (en) | Method for synthesizing GHK acetate | |
| CN111620928B (en) | Large-scale synthesis method of hexapeptide | |
| CN106243214B (en) | Preparation method of melanotan I | |
| CN114315538B (en) | Benzyl alcohol phenolic ether compound serving as polypeptide liquid phase synthesis carrier and preparation method and application thereof | |
| CN112111001B (en) | Method for synthesizing thymosin T alpha-1 | |
| CN106478805A (en) | A kind of preparation method of GLP-1 derivant | |
| CN111875668A (en) | Synthetic method of cyclic dipeptide containing glutamine and asparagine | |
| CN107903305B (en) | Method for synthesizing dessorrel by solid-liquid combination | |
| AU2017101705A4 (en) | Preparation method of argireline and argireline product | |
| CN107629111B (en) | Liquid phase synthesis method of acetyl tetrapeptide-2 | |
| CN107857796B (en) | Synthesis method of trifluoroacetyl tripeptide-2 | |
| CN116143867A (en) | Method for preparing GHK tripeptide and blue copper peptide without condensing agent | |
| CN110461843B (en) | Improved synthesis of NIR fluorescent probes | |
| CN103665109A (en) | Synthesis method of C-terminal pentapeptide of osteogenic growth peptide | |
| CN108047305A (en) | A kind of synthetic method of myristyl aminobutyryl valyl amido butyric acid urea trifluoroacetate | |
| WO2021103458A1 (en) | Solid-phase synthesis method for degarelix | |
| CN110642936A (en) | Method for preparing teriparatide | |
| CN107722108B (en) | Liquid phase synthesis method of acetyl tetrapeptide-9 | |
| CN109988062B (en) | Liquid phase spherical carrier and preparation method and application thereof | |
| CN104447963B (en) | A kind of method for preparing aviptadil | |
| CN112175067B (en) | Preparation method of teduglutide | |
| CN114014912B (en) | Method for preparing leuprorelin by combining solid phase and liquid phase | |
| CN113880921B (en) | Synthetic method of bremer langdan acetate |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |