Method for synthesizing oxytocin by taking modified natural polysaccharide as solid-phase carrier

Abstract

The invention provides a method for synthesizing oxytocin by taking modified natural polysaccharide as a solid-phase carrier. The method comprises the following steps: 1) modifying and synthesizing the carrier; (2) synthesizing an oxytocin linear peptide-modified carrier; (3) splitting the oxytocin linear peptide-modified carrier; and 4) oxidizing the oxytocin linear peptide to obtain the oxytocin crude peptide. Firstly, according to the method, common amino resin in traditional solid-phase synthesis is avoided, common natural polysaccharide chitin is used as a carrier, the cost is greatly reduced, the carrier is easier to degrade after being discarded, and pollution to the environment is reduced. Secondly, in the synthesis process of the carrier, the substitution degree of the modified carrier can be controlled by controlling the reaction time and the feeding multiple, and synthesis of polypeptides with different lengths is facilitated. Finally, solid-phase synthesis is adopted in the synthesis method, operation is easy and convenient, the synthesis time can be effectively shortened, and the purity of the obtained crude peptide is high.

Description

Technical Field

[0001] The invention relates to the technical field of drug synthesis, and in particular to a method for synthesizing oxytocin by using modified natural polysaccharide as a solid phase carrier. Background Art

[0002] Oxytocin is a drug sold under the trade name Oxytocin, marketed as an OXTR agonist, is a drug that has been lauded for its clinical efficacy in the field of obstetrics and gynecology. Oxytocin is manufactured by Novartis, and it was first approved in the United States in 1980. The drug is primarily used in antenatal and postpartum care as it has been shown to be effective in inducing or enhancing uterine contractions. Its recombinant hormonal nature has made it a preferred drug by obstetricians and gynecologists around the world who seek a reliable and trustworthy option for women who require antenatal and postpartum care. In addition, oxytocin has been found to be a commonly prescribed drug for pregnant women to help them with labor and delivery or to control postpartum bleeding.

[0003] Its structure is as Figure 1 The existing synthesis processes of oxytocin are roughly divided into three categories: liquid phase synthesis, Fmoc solid phase synthesis and solid-liquid combination.

[0004] The operation of liquid phase synthesis is relatively complicated and the yield is not high, while the operation of solid phase synthesis is convenient but the cost is relatively high. Summary of the invention

[0005] The purpose of the present invention is to provide a method for solid phase synthesis of oxytocin, which avoids the use of amino resin and uses natural polysaccharides with a wider source as carriers. The process is simple and the material cost is greatly reduced. At the same time, the crude oxytocin peptide prepared by the method has high purity and high reaction yield.

[0006] In order to solve the above technical problems, the present invention provides a method for synthesizing oxytocin by using modified natural polysaccharide as a solid phase carrier.

[0007] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0008] The present invention provides a method for synthesizing oxytocin by using modified natural polysaccharide as solid phase carrier, the method comprising the following steps: 1) modification and synthesis of the carrier; 2) synthesis of oxytocin linear peptide-modified carrier; 3) cleavage of oxytocin linear peptide-modified carrier; 4) oxidation of oxytocin linear peptide to obtain crude oxytocin peptide.

[0009] Furthermore, the step 1) specifically includes: subjecting the carrier to an acetylation reaction with a sodium hydroxide solution to obtain chitosan, and subjecting the chitosan to a deacetylation treatment to obtain completely deacetylated chitosan; subjecting the completely deacetylated chitosan and the modification material to an activation reaction in a modification reaction solvent to obtain an activation solution; and after washing the activation solution, adding acetic anhydride and pyridine to react until the Kaiser Test is colorless to obtain a modified carrier.

[0010] Furthermore, in the step 1), the carrier source can be shrimp shells, crab shells, etc., preferably crab shells; the sodium hydroxide solution is 60% to 100%, preferably 80%; the acetyl reaction temperature is 120 to 160°C, preferably 140°C; the materials used for modification are Fmoc-(4-aminomethyl)benzoic acid, Fmoc-(3-aminomethyl)benzoic acid, Fmoc-(2-aminomethyl)benzoic acid, preferably Fmoc-(4-aminomethyl)benzoic acid; the modification reaction solvent is preferably DMF; the activation reaction temperature is 20 to 60°C, preferably 25°C.

[0011] Furthermore, the step 2) specifically includes: swelling the modified carrier with a reaction solvent, and then removing the Fmoc of the modified carrier with a DBLK solution to obtain a reaction system; then adding an amino acid raw material and a coupling reagent to the reaction system for a coupling reaction; repeating the steps of removing the Fmoc protection and coupling the next amino acid raw material until the coupling is completed, and then washing or drying to obtain an oxytocin linear peptide-modified carrier.

[0012] Furthermore, in the step 2), the coupling reagent is DICPDI / HOBt; the reaction solvent is one or more of DMF and DCM; the reaction time is 1 to 4 hours, preferably 2 hours, and the reaction temperature is 20 to 30° C., preferably 25° C.

[0013] Further, in the step 2), the amino acid raw materials coupled in sequence with the coupling reagent are Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-Pro-OH, Fmoc-Cys(Trt)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ile-OH, Fmoc-Tyr(tBu)-OH, and Boc-Cys(Trt)-OH; the ratio of the synthesis scale (the product of the degree of substitution of the modified carrier and the mass, representing the theoretical molar amount of the synthesized oxytocin linear peptide) to the amino acid raw material is 1:2 to 5, preferably 1:3,

[0014] Furthermore, the step 3) specifically includes: adding a lysis solution to the oxytocin linear peptide-modified carrier to perform a lysis reaction to obtain a lysis system; and filtering, settling, centrifuging, washing and drying the lysis system in sequence to obtain a solid.

[0015] Furthermore, in the step 3), the cleavage reagent is preferably TFA:H2O=95:5, the cleavage time is 1 to 3 hours, preferably 2 hours, and the reaction temperature is 20 to 30°C, preferably 25°C.

[0016] Furthermore, the step 4) specifically includes: dissolving the solid into a solution, adjusting the pH to 8.0, adding an oxidizing agent to the solution for reaction to obtain a crude oxytocin peptide solution, and adjusting the pH to 3.5 to obtain a crude oxytocin peptide.

[0017] Furthermore, in the step 4), the concentration of the crude oxytocin linear peptide solution is 0.3 mg / mL to 1.0 mg / ML, preferably 0.5 mg / ML; the oxidizing agent is 30% H2O2, I2 / methanol, DMSO, air, preferably 30% H2O2.

[0018] Compared with the prior art, the technical solution provided by the present invention has at least the following advantages:

[0019] The present invention provides a method for synthesizing oxytocin by using modified natural polysaccharides as solid phase carriers. First, the method avoids using amino resins commonly used in traditional solid phase synthesis, and uses common natural polysaccharide chitin as the carrier, which greatly reduces the cost, and the carrier is more easily degraded after being discarded, reducing pollution to the environment. Secondly, during the carrier synthesis process, the degree of substitution of the modified carrier can be controlled by controlling the reaction time and the feed multiple, which is conducive to the synthesis of polypeptides of different lengths. Finally, the synthesis method uses solid phase synthesis, which is easy to operate, can effectively reduce the time used for synthesis, and the resulting crude peptide has a high purity.

[0020] In summary, the synthesis method provided by the present invention shortens the synthesis time, reduces the cost, improves the purity of the crude peptide, and reduces the pollution to the environment. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 It is the molecular structure of oxytocin;

[0022] Figure 2 It is the liquid chromatogram of oxytocin crude peptide solution. DETAILED DESCRIPTION

[0023] The inventors have found that the existing synthesis processes of oxytocin can be roughly divided into the following three categories:

[0024] The first type is a liquid phase synthesis method: CN101914136B: Liquid phase reaction to obtain benzyloxycarbonylbenzylcysteinyltyrosylisoleucine, liquid phase coupling in sequence to obtain benzyloxycarbonylbenzylcysteinyltyrosylisoleucylglutamylaminoacetylaspartylbenzylcysteinylprolylleucylglutamylamine, and then the protecting group is removed by the sodium ammonia method to obtain a crude product, and the crude product is liquid phase oxidized to obtain an oxytocin solution.

[0025] The second type is the Fmoc solid phase synthesis method: this type of synthesis method is reported more frequently, mainly based on the standard Fmoc solid phase coupling in sequence. Patents CN110746491B and CN102850441B: Rink Amide AM Resin is used as a carrier, and the corresponding protected amino acids are coupled in sequence to obtain oxytocin linear peptide resin; after cleavage, sedimentation, washing, and drying, a crude linear oxytocin product is obtained; the aqueous solution of the crude linear oxytocin product is oxidized for cyclization reaction, and after the reaction is complete, it is purified to obtain the oxytocin.

[0026] The third type is solid-liquid combination: Patent CN106589069A: Using 2-CTC as a carrier, Fmoc-Cys(Trt)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ile-OH, Fmoc-Tyr(tBu)-OH, Boc-Cys(Trt)-OH are coupled in sequence to obtain the first intermediate Boc-Cys(Trt)-Tyr(tBu)-Ile-Gln(Trt)-Asn(Trt)-Cys(Trt)-OH. Liquid phase synthesis obtains the second intermediate Pro-Leu-Gly-NH2. Then, in a liquid phase reaction, the first intermediate and the second intermediate are condensed to obtain Boc-Cys(Trt)-Tyr(tBu)-Ile-Gln(Trt)-Asn(Trt)-Cys(Trt)-Pro-Leu-Gly-NH2, which is oxidized with iodine to form a disulfide bond, and then oxytocin is obtained by peptide cleavage.

[0027] The present invention is described in detail below in conjunction with specific implementation modes.

[0028] (I) Synthesis of carrier:

[0029]

[0030] (II) Synthesis of oxytocin linear peptide-modified carrier

[0031]

[0032] (III) Cleavage of oxytocin linear peptide-modified carrier

[0033]

[0034] (IV) Oxidation of oxytocin linear peptide

[0035]

[0036] The present invention comprises the following steps:

[0037] 1. Modification and synthesis of carriers;

[0038] 2. Synthesis of oxytocin linear peptide-modified carrier;

[0039] 3. Cleavage of oxytocin linear peptide-modified carrier;

[0040] 4. Oxidation of oxytocin linear peptide to obtain oxytocin crude peptide;

[0041] In the step 1), the carrier source can be shrimp shells, crab shells, etc., preferably crab shells; the sodium hydroxide solution is 60% to 100%, preferably 80%, and the reaction temperature is 120 to 160°C, preferably 140°C. The materials used for modification are Fmoc-(4-aminomethyl)benzoic acid, Fmoc-(3-aminomethyl)benzoic acid, Fmoc-(2-aminomethyl)benzoic acid, etc., preferably Fmoc-(4-aminomethyl)benzoic acid, the modification reaction solvent is preferably DMF, and the reaction temperature is 20 to 60°C, preferably 25°C.

[0042] In the step 2), the coupling reagent is DICPDI / HOBt; the reaction solvent is one or more of DMF and DCM; according to the coupling reagent, the amino acids coupled in sequence are Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-Pro-OH, Fmoc-Cys(Trt)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ile-OH, Fmoc-Tyr(tBu)-OH, Boc-Cys(Trt)-OH, the ratio of the synthesis scale (the product of the degree of substitution of the modified carrier and the mass, representing the theoretical molar amount of the synthesized oxytocin linear peptide) to the amino acid is 1:2-5, preferably 1:3, the reaction time is 1-4h, preferably 2h, and the reaction temperature is 20-30°C, preferably 25°C.

[0043] In the step 3), the cleavage reagent is preferably TFA:H2O=95:5, the cleavage time is 1 to 3 hours, preferably 2 hours, and the reaction temperature is 20 to 30°C, preferably 25°C.

[0044] In the step 4), the concentration of the crude oxytocin linear peptide solution is 0.3 mg / mL to 1.0 mg / mL, preferably 0.5 mg / mL, and the oxidizing agent is 30% H2O2, I2 / methanol, DMSO, air, preferably 30% H2O2.

[0045] Examples

[0046] 1. Modification and synthesis of carrier

[0047] Example 1-1

[0048] (1) Wash the crab shell, dry it, grind it into powder and sieve it to obtain crab shell chitin. Weigh 20g of chitin and 200mL of 80% sodium hydroxide aqueous solution in a 500mL three-necked flask, react at 140℃ for 4h, and obtain chitosan with about 98% acetylation after the reaction. Pour the reaction system into water, cool it, filter it, collect the obtained chitosan, and dry it with air at 60℃. Deacetylate the dried chitosan again, collect the almost completely deacetylated chitosan, and obtain 15g of white solid.

[0049] (2) Add 5 g of chitosan (42 mmol in terms of monomer molar weight) to the solid phase reactor, swell with DMF for 30 min, weigh 4.5 g (12 mmol) of Fmoc-(4-aminomethyl)benzoic acid and 1.7 g (12.6 mmol) of HOBt, add an appropriate amount of DMF to dissolve, and cool to 5 °C. Slowly add 2 mL (12.6 mmol) of DICPDI to the solution to obtain an activated solution, pour the solution into the solid phase reactor, and react for 2 h. Wash with DMF 3 times, then add 20 mL (200 mmol) of acetic anhydride and 16 mL (200 mmol) of pyridine to the system, react for 4 h, and the Kaiser Test is colorless, proving that all unmodified amino sites on the chitosan have been blocked. Wash with DCM 3 times and methanol 3 times. Collect and dry to obtain a modified carrier with a degree of substitution of 0.81 mmol / g.

[0050] Example 1-2

[0051] The preparation of completely deacetylated chitosan is shown in Example 1-1 (1).

[0052] Add 5g chitosan (42mmol in molar weight based on monomer) to the solid phase reactor, swell with DMF for 30min, weigh 4.5g (12mmol) of Fmoc-(4-aminomethyl)benzoic acid and 1.7g (12.6mmol) of HOBt, add appropriate amount of DMF to dissolve, and cool to 5°C, slowly drop 2mL (12.6mmol) of DICPDI to the solution to obtain an activated solution, pour the solution into the solid phase reactor, and react for 1.5h. Wash with DMF 3 times, then add 20mL (200mmol) of acetic anhydride and 16mL (200mmol) of pyridine to the system, react for 4h, and Kaiser Test is colorless, proving that all the unmodified amino sites on chitosan have been blocked. Wash with DCM 3 times and methanol 3 times. Collect and dry to obtain a modified carrier with a degree of substitution of 0.64mmol / g.

[0053] Examples 1-3

[0054] The preparation of completely deacetylated chitosan is shown in Example 1-1 (1).

[0055] Add 5g chitosan (42mmol in terms of molar weight of monomer) to the solid phase reactor, swell with DMF for 30min, weigh 6g (16mmol) of Fmoc-(4-aminomethyl)benzoic acid and 2.3g (16.8mmol) of HOBt, add appropriate amount of DMF to dissolve, and cool to 5°C, slowly drop 2.6mL (16.8mmol) of DICPDI to the solution to obtain an activated solution, pour the solution into the solid phase reactor, and react for 1h. Wash with DMF 3 times, then add 20mL (200mmol) of acetic anhydride and 16mL (200mmol) of pyridine to the system, react for 4h, and Kaiser Test is colorless, proving that all unmodified amino sites on chitosan have been blocked. Wash with DCM 3 times and methanol 3 times. Collect and dry to obtain a modified carrier with a degree of substitution of 0.71mmol / g.

[0056] 2. Synthesis of oxytocin linear peptide-modified carrier

[0057] Example 2-1

[0058] Weigh 4.8 g of the modified carrier (substitution degree 0.64 mmol / g) and add it to the solid phase reactor. Swell with DMF for 30 min. Add DBLK solution (20% piperidine / DMF solution) to remove the Fmoc of the modified carrier. React twice for 5 min + 7 min. Wash with DMF 6 times. Weigh 2.7 g (9 mmol) of Fmoc-Gly-OH and 1.3 g (9.45 mmol) of HOBt. Dissolve them in DMF. Add 1.6 mL (9.45 mmol) of DIC. React at room temperature for 2 h. After the Kaiser Test shows colorless, continue to remove the Fmoc protection and couple the next amino acid.

[0059] The order of coupling amino acids is: Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-Pro-OH, Fmoc-Cys(Trt)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ile-OH, Fmoc-Tyr(tBu)-OH, Boc-Cys(Trt)-OH.

[0060] After the coupling was completed, the mixture was washed with DCM for 3 times and with methanol for 3 times, with the methanol washing time being 5+5+10 min. The mixture was blown dry with nitrogen to obtain 11 g of a white solid, which was the oxytocin linear peptide-modified carrier.

[0061] 3. Cleavage of oxytocin linear peptide-modified carrier

[0062] Weigh 11 g of oxytocin linear peptide-modified carrier into a 250 mL single-necked bottle, prepare 110 mL of lysis solution (TFA:H2O=95:5), pour into the single-necked bottle, and react for 2 h.

[0063] The cracking system was filtered, and the filtrate was poured into 1100 mL of pre-cooled tert-methyl ether for sedimentation. The precipitate was centrifuged and washed three times by centrifugation. The solid was then dried with dry nitrogen to obtain 3.1 g of solid.

[0064] 4. Oxidation of oxytocin linear peptide

[0065] 6.2 L of purified water was added to the obtained 3.1 g solid to dissolve it, and the pH was adjusted to 8.0 with 10% ammonia water. Then 1.5 mL of 30% H2O2 was added to the solution, and the reaction was carried out at room temperature for 30 min. The reaction was monitored by HPLC until the oxytocin linear peptide was basically completely oxidized to obtain a crude oxytocin peptide solution. Acetic acid aqueous solution was added to adjust the pH to 3.5. Samples were taken for testing, and the purity was 85.31%. Figure 2 shown.

[0066] Abbreviations and English meanings

[0067]

[0068]

[0069] Those skilled in the art can understand that the above-mentioned embodiments are specific examples for implementing the present application, and in practical applications, various changes can be made to them in form and detail without departing from the spirit and scope of the present application. Any person skilled in the art can make their own changes and modifications without departing from the spirit and scope of the present application, so the scope of protection of the present application shall be based on the scope defined in the claims.

Claims

1. A method for synthesizing oxytocin using modified natural polysaccharides as solid phase carriers, characterized in that: The method comprises the following steps: 1) Modification and synthesis of carriers; 2) Synthesis of oxytocin linear peptide-modified carriers; 3) cleavage of the oxytocin linear peptide-modified carrier; 4) Oxidation of oxytocin linear peptide to obtain crude oxytocin peptide.

2. The method for synthesizing oxytocin using modified natural polysaccharides as solid phase carriers according to claim 1, characterized in that: The step 1) specifically comprises: subjecting the carrier to an acetylation reaction with a sodium hydroxide solution to obtain chitosan, and subjecting the chitosan to a deacetylation treatment to obtain completely deacetylated chitosan; subjecting the completely deacetylated chitosan and the materials used for modification to an activation reaction in a modification reaction solvent to obtain an activation solution; and washing the activation solution, adding acetic anhydride and pyridine to react until the Kaiser Test is colorless to obtain a modified carrier.

3. The method for synthesizing oxytocin using modified natural polysaccharides as solid phase carriers according to claim 2, characterized in that: In the step 1), the carrier source can be shrimp shells, crab shells, etc., preferably crab shells; the sodium hydroxide solution is 60% to 100%, preferably 80%; the acetyl reaction temperature is 120 to 160° C., preferably 140° C.; the materials used for modification are Fmoc-(4-aminomethyl)benzoic acid, Fmoc-(3-aminomethyl)benzoic acid, Fmoc-(2-aminomethyl)benzoic acid, preferably Fmoc-(4-aminomethyl)benzoic acid; the modification reaction solvent is preferably DMF; the activation reaction temperature is 20 to 60° C., preferably 25° C.

4. The method for synthesizing oxytocin using modified natural polysaccharides as solid phase carriers according to claim 1, characterized in that: The step 2) specifically includes: swelling the modified carrier with a reaction solvent, and then removing the Fmoc of the modified carrier with a DBLK solution to obtain a reaction system; then adding an amino acid raw material and a coupling reagent to the reaction system for a coupling reaction; repeating the steps of removing the Fmoc protection and coupling the next amino acid raw material until the coupling is completed, and then washing, that is, drying, to obtain an oxytocin linear peptide-modified carrier.

5. The method for synthesizing oxytocin using modified natural polysaccharides as solid phase carriers according to claim 4, characterized in that: In the step 2), the coupling reagent is DICPDI / HOBt; the reaction solvent is one or more of DMF and DCM; the reaction time is 1 to 4 hours, preferably 2 hours, and the reaction temperature is 20 to 30° C., preferably 25° C.

6. The method for synthesizing oxytocin using modified natural polysaccharides as solid phase carriers according to claim 4, characterized in that: In the step 2), the amino acid raw materials coupled in sequence with the coupling reagent are Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-Pro-OH, Fmoc-Cys(Trt)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ile-OH, Fmoc-Tyr(tBu)-OH, and Boc-Cys(Trt)-OH; the ratio of the synthesis scale to the amino acid raw material is 1:2 to 5, preferably 1:

3.

7. The method for synthesizing oxytocin using modified natural polysaccharides as solid phase carriers according to claim 1, characterized in that: The step 3) specifically comprises: adding a lysis solution to the oxytocin linear peptide-modified carrier to carry out a lysis reaction to obtain a lysis system; and filtering, settling, centrifuging, washing and drying the lysis system in sequence to obtain a solid.

8. The method for synthesizing oxytocin using modified natural polysaccharides as solid phase carriers according to claim 7, characterized in that: In the step 3), the cleavage reagent is preferably TFA:H2O=95:5, the cleavage time is 1 to 3 hours, preferably 2 hours, and the reaction temperature is 20 to 30°C, preferably 25°C.

9. The method for synthesizing oxytocin using modified natural polysaccharides as solid phase carriers according to claim 1, characterized in that: The step 4) specifically comprises: dissolving the solid into a solution, adjusting the pH to 8.0, adding an oxidizing agent to the solution for reaction to obtain a crude oxytocin peptide solution, and adjusting the pH to 3.5 to obtain a crude oxytocin peptide.

10. The method for synthesizing oxytocin using modified natural polysaccharides as solid phase carriers according to claim 9, characterized in that: In the step 4), the concentration of the crude oxytocin linear peptide solution is 0.3 mg / mL to 1.0 mg / mL, preferably 0.5 mg / mL; the oxidizing agent is 30% H2O2, I2 / methanol, DMSO, air, preferably 30% H2O2.

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