CN107698663B - Liquid phase synthesis method of vilospiritide - Google Patents

Abstract

The invention discloses a liquid phase synthesis method of vilofibrutin, which adopts a fragment method of 2 plus 1 plus 2, and firstly Fmoc-Arg (Pbf) -OH and H-Pro-Ala-NH₂Or hydrochloride or trifluoroacetate thereof to generate Fmoc-Arg (Pbf) -Pro-Ala-NH₂Then reacting with piperidine to remove Fmoc protecting group, reacting with Boc-Gly-Pro-OMe to generate full-protection pentapeptide, deprotecting with trifluoroacetic acid, and salifying with hydrochloric acid to obtain H-Gly-Pro-Arg-Pro-Ala-NH₂HCl, and ion-exchanged to obtain vilospilatide. The vilofibrin is synthesized by adopting a one-pot method through deprotection amine ester exchange, compared with the traditional solid phase synthesis, the method can obviously improve the reaction yield by gradually prolonging the peptide chain, avoids the use of a large amount of high-cost and high-toxicity solvents, and has the characteristics of easy scale, high intermediate purity, no need of column chromatography, less raw and auxiliary material consumption, low cost, environmental friendliness and the like.

Description

Liquid phase synthesis method of vilospiritide

Technical Field

The invention belongs to the technical field of polypeptide liquid phase synthesis, and particularly relates to a liquid phase synthesis method of vilofilapeptide (pentapeptide-3).

Background

The vilofibrine is a pentapeptide, also called pentapeptide-3 or Vialox, which is formed by connecting glycine, proline, arginine and alanyl ammonia, belongs to a neurotransmitter inhibiting peptide, and can locally block the nerve transmission muscle contraction information by inhibiting the excessive release of acetylcholine receptors, relax facial muscles, and fulfill the aims of smoothing wrinkles and smoothing and tightening skin. The successful development of the vilofibrutin is a great breakthrough of anti-wrinkle active peptide compounds, has wide application, and is particularly suitable for being applied to parts with concentrated expression muscles, such as canthus, face, forehead and the like.

After decades of development, with the increasing of market demand year by year and the development of polypeptide products with great potential, great economic and social benefits are brought. China has made good development in the aspects of production and sale of cosmetics, and although great progress is made, the cosmetics still face the problems of laggard production process, lack of innovation in research and development and the like. Therefore, the development of a novel high-efficiency liquid-phase large-scale synthesis method becomes a technical problem which needs to be solved urgently.

Disclosure of Invention

The invention aims to solve the technical problem of providing a low-cost, environment-friendly and easily-scaled liquid-phase synthesis method of vilofilapeptide.

The technical method for solving the technical problems comprises the following steps:

1. reacting Boc-Gly-OH, N-hydroxysuccinimide and N, N' -dicyclohexylcarbodiimide at 0-15 ℃ for 3-5 hours by using tetrahydrofuran as a solvent, filtering to remove insoluble substances, adding H-Pro-OMe HCl and an aqueous solution of sodium bicarbonate into the filtrate, reacting at 15-25 ℃ for 8-12 hours, and separating and purifying to obtain Boc-Gly-Pro-OMe.

2. Using dichloromethane or tetrahydrofuran as solvent, reacting Boc-Pro-OH or Fmoc-Pro-OH with isobutyl chloroformate and organic base at-15-10 ℃ for 10-45 minutes, and reacting with H-Ala-NH₂Reacting for 1 hour, then heating to room temperature for 6-8 hours, separating and purifying to obtain Boc-Pro-Ala-NH₂Or Fmoc-Pro-Ala-NH₂Then removing Boc protecting group with ethyl acetate solution of hydrogen chloride to obtain H-Pro-Ala-NH₂Removal of the Boc protecting group with HCl or trifluoroacetic acid (TFA) in tetrahydrofuran to give H-Pro-Ala-NH₂TFA, or removal of the Fmoc protecting group with piperidine in tetrahydrofuran or concentrated aqueous ammonia to give H-Pro-Ala-NH₂。

3. Using dichloromethane as solvent, reacting Fmoc-Arg (Pbf) -OH, 1-hydroxybenzotriazole, organic base and N-ethyl-N' - (3-dimethylaminopropyl) carbodiimide hydrochloride at-15 to-5 ℃ for 30 to 60 minutes, and reacting with H-Pro-Ala-NH₂Or H-Pro-Ala-NH₂HCl or H-Pro-Ala-NH₂TFA is reacted for 30-60 minutes, then the temperature is raised to room temperature for reaction for 6-8 hours, and the reaction product is separated and purified to obtain Fmoc-Arg (Pbf) -Pro-Ala-NH₂。

4. Removal of Fmoc-Arg (Pbf) -Pro-Ala-NH with piperidine in N, N-dimethylformamide₂Reacting the Fmoc protecting group with Boc-Gly-Pro-OMe at room temperature for 12-24 hours, separating and purifying to obtain Boc-Gly-Pro-Arg (Pbf) -Pro-Ala-NH₂Then deprotecting with trifluoroacetic acid solution, salifying with hydrochloric acid to obtain H-Gly-Pro-Arg-Pro-Ala-NH₂HCl, ion exchange to obtain vilofilapeptide.

In the step 1, preferably, the molar ratio of Boc-Gly-OH, N-hydroxysuccinimide, N' -dicyclohexylcarbodiimide, H-Pro-OMe & HCl and sodium bicarbonate is 1: 1.1-1.3: 1.5-2.0: 1.2-1.5: 2.5-4.0.

In the above step 2, preferably Boc-Pro-OH or Fmoc-Pro-OH, isobutyl chloroformate, organic base, H-Ala-NH₂The molar ratio of (A) to (B) is 1: 1.05-1.2: 2.5-4.0: 1.1-1.3.

In the step 3, Fmoc-Arg (Pbf) -OH, 1-hydroxybenzotriazole, organic base, N-ethyl-N' - (3-dimethylaminopropyl) carbodiimide hydrochloride, H-Pro-Ala-NH are preferable₂Or H-Pro-Ala-NH₂HCl or H-Pro-Ala-NH₂The molar ratio of TFA is 1: 1.0-1.05: 2.5-5.0: 1.0-1.05: 1.1-1.3, wherein the organic base is preferably any one of N-methylmorpholine, N-diisopropylethylamine and N-methylpyrrolidone.

Fmoc-Arg (Pbf) -Pro-Ala-NH is preferred in step 4 above₂And the Boc-Gly-Pro-OMe is in a molar ratio of 1: 1.3-1.6.

The invention adopts a fragment method of '2 +' 1+2₂Or H-Pro-Ala-NH₂HCl or H-Pro-Ala-NH₂TFA condensation to Fmoc-Arg (Pbf) -Pro-Ala-NH₂Then reacting with piperidine to remove Fmoc protecting group, reacting with Boc-Gly-Pro-OMe to generate full-protection pentapeptide, deprotecting with trifluoroacetic acid, and salifying with hydrochloric acid to obtain H-Gly-Pro-Arg-Pro-Ala-NH₂HCl, by ion exchange to giveVilofibrutin. The invention synthesizes the vilofibrin peptide by one-pot method through deprotection amine ester exchange, compared with the traditional solid phase synthesis, the method can obviously improve the reaction yield by gradually prolonging the peptide chain, avoid the use of a large amount of high-cost and high-toxicity solvents such as N, N-dimethylformamide, dimethyl sulfoxide and the like, reduce the use of a large amount of solvents such as methanol, isopropanol and the like, and has the characteristics of easy scale production, high intermediate purity, no need of column chromatography, less raw and auxiliary material consumption, low cost, environmental friendliness and the like.

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. Adding 17.5g (0.1mol) of Boc-Gly-OH and 200mL of tetrahydrofuran into a 500mL three-necked flask, stirring to dissolve, cooling to 0-5 ℃, adding 13.8g (0.12mol) of N-hydroxysuccinimide, stirring for 10 minutes, adding 32.8g (0.15mol) of N, N' -dicyclohexylcarbodiimide, reacting at 5-15 ℃ for 4 hours, filtering, adding 19.8g (0.12mol) of H-Pro-OMe & HCl and 100mL of 3mol/L sodium bicarbonate aqueous solution into the filtrate, stirring at 15-25 ℃ for 8 hours, stopping the reaction, removing the solvent from the reaction solution under reduced pressure, pouring into 200mL of water, extracting with ethyl acetate (100 mL. times.3), washing the organic phase with 0.5mol/L of hydrochloric acid until the pH of the aqueous phase is 3-4, washing the organic phase with 200mL of saturated common salt water, drying anhydrous sodium sulfate for 12 hours, filtering, removing the solvent under reduced pressure, 27.2g of a white solid, i.e. Boc-Gly-Pro-OMe, was obtained in a yield of 95%.

2. Adding 21.5g (0.1mol) of Boc-Pro-OH and 150mL of dichloromethane into a 500mL three-necked bottle, cooling to-15-10 ℃ after stirring and dissolving, adding 33mL (0.30mol) of N-methylmorpholine, slowly dropwise adding 14mL (0.108mol) of isobutyl chloroformate, and keeping the temperature for reacting for 30 minutes; 11g (0.124mol) of H-Ala-NH₂Dissolving in 50mL of dichloromethane, slowly adding into the three-neck flask, reacting at-15-10 deg.C for 1 hr, naturally heating to room temperature, reacting for 6 hr, stopping reaction, pouring the reaction solution into 300mL of water, standing, separating, extracting the water phase with 100mL of dichloromethane, washing the extracted dichloromethane phase with 0.5mol/L hydrochloric acidThen washed with 100mL of saturated aqueous sodium chloride solution and the solvent removed under reduced pressure to give 25g of a white solid, i.e., Boc-Pro-Ala-NH₂The yield thereof was found to be 87.7%. The Boc-Pro-Ala-NH obtained₂Adding the mixture into 200mL of ethyl acetate solution of 4mol/L hydrogen chloride, stirring the mixture at 0-10 ℃ for reaction for 2 hours, filtering the reaction product, washing the solid with ethyl acetate, and drying the solid in vacuum at 40 ℃ to obtain H-Pro-Ala-NH₂·HCl。

3. Adding 32.4g (0.05mol) of Fmoc-Arg (Pbf) -OH and 300mL of dichloromethane into a 1000mL three-necked bottle, cooling to-15 ℃ after stirring and dissolving, adding 6.8g (0.05mol) of 1-hydroxybenzotriazole and 10g (0.10mol) of N-methylmorpholine, adding 9.6g (0.05mol) of N-ethyl-N' - (3-dimethylaminopropyl) carbodiimide hydrochloride after stirring for 15 minutes, and reacting for 45 minutes at-15 to-10 ℃; 13.3g (0.06mol) of H-Pro-Ala-NH₂HCl and 15g (0.15mol) of N-methylmorpholine are dissolved in 100mL of dichloromethane and then are dripped into a three-necked bottle, the mixture reacts at the temperature of-10 to-5 ℃ for 1 hour, then the temperature is naturally raised to room temperature, the mixture is stirred and reacts for 6 hours, the reaction is stopped, the reaction solution is poured into 500mL of water, the mixture is kept stand and separated, the water layer is extracted by 200mL of dichloromethane, the extracted dichloromethane phase is washed by 1mol/L hydrochloric acid, washed by saturated sodium chloride solution, dried by anhydrous sodium sulfate and filtered, the filtrate is decompressed and the solvent is removed, 34.7g of white solid, namely Fmoc-Arg (Pbf) -Pro-Ala-NH is obtained, and the white solid is Fmoc-₂The yield thereof was found to be 85%.

4. Into a 1000mL three-necked flask was added 20g (0.03mol) of Fmoc-Arg (Pbf) -Pro-Ala-NH₂And 100mL of N, N-dimethylformamide solution of piperidine with the volume concentration of 20%, stirring at room temperature for reaction for 1 hour, adding 50mL of N, N-dimethylformamide solution containing 12.9g (0.045mol) of Boc-Gly-Pro-OMe, stirring at room temperature for reaction for 24 hours, stopping the reaction, pouring the reaction solution into 300mL of water, stirring, adding citric acid to separate out a large amount of white solid in the system, filtering, washing with water, pulping with ethyl acetate twice after vacuum drying, filtering the solid, and drying in vacuum to obtain 15g of white solid Boc-Gly-Pro-Arg (Pbf) -Pro-Ala-NH₂The yield thereof was found to be 72.2%. Boc-Gly-Pro-Arg (Pbf) -Pro-Ala-NH₂Adding 200mL of mixed solution of trifluoroacetic acid, triisopropylsilane and water in a volume ratio of 90:5:5, stirring at room temperature for reaction for 1 hour, and removing under reduced pressurePouring the solvent and the concentrated solution into 200mL of acetone, dropwise adding concentrated hydrochloric acid while stirring until a large amount of white solid is separated out, filtering, washing the solid with acetone, and drying in vacuum to obtain a white solid H-Gly-Pro-Arg-Pro-Ala-NH₂·HCl。

Reacting H-Gly-Pro-Arg-Pro-Ala-NH₂Adding HCl into 50mL of water, removing hydrochloric acid by ion exchange (constant flow of 2% volume of acetic acid aqueous solution, detection wavelength 210nm, flow rate 10mL/min, chromatographic column specification: 5cm × 45cm, filler DEAE), and finally freeze-drying to obtain 8g of white solid, namely vilos peptide, with yield of 66% and purity of more than 85%.

Example 2

1. Adding 17.5g (0.1mol) of Boc-Gly-OH and 250mL of tetrahydrofuran into a 500mL three-necked flask, stirring to dissolve, cooling to 0-10 ℃, adding 14.9g (0.13mol) of N-hydroxysuccinimide, stirring for 10-15 minutes, adding 39.4g (0.18mol) of N, N' -dicyclohexylcarbodiimide, reacting at 5-10 ℃ for 3 hours, filtering, adding 21.45g (0.13mol) of H-Pro-OMe & HCl and 120mL of 3mol/L sodium bicarbonate aqueous solution into the filtrate, stirring at 15-25 ℃ for 11 hours to stop the reaction, removing the solvent from the reaction solution under reduced pressure, pouring into 200mL of water, extracting with ethyl acetate (100 mL. times.3 times), washing the organic phase with 0.5mol/L of hydrochloric acid until the pH of the aqueous phase is 3-4, washing the organic phase with 200mL of saturated common salt solution, drying with anhydrous sodium sulfate for 12 hours, filtering, removing the solvent under reduced pressure, 27.0g of a white solid, i.e. Boc-Gly-Pro-OMe, was obtained in a yield of 94.3%.

2. Adding 16.85g (50mmol) of Fmoc-Pro-OH and 200mL of dichloromethane into a 500mL three-necked flask, cooling to-15-10 ℃ after stirring and dissolving, adding 25.8g (200mmol) of N, N-diisopropylethylamine, slowly dropwise adding 7.45mL (57.5mmol) of isobutyl chloroformate, and keeping the temperature for reacting for 45 minutes; 5g (56.5mmol) of H-Ala-NH₂Dissolving in 30mL of dichloromethane, slowly adding into the three-neck flask, reacting at-15-10 deg.C for 1 hr, naturally heating to room temperature, reacting for 8 hr, stopping reaction, pouring the reaction solution into 300mL of water, standing, separating, extracting the water phase with 100mL of dichloromethane, washing the extracted dichloromethane phase with 150mL of saturated sodium chloride water solution, removing solvent under reduced pressure to obtain 16.6g of white solid, namely Fmoc-Pro-Ala-NH₂. Adding the obtained white solid into 100mL of tetrahydrofuran solution, adding 100mL of concentrated ammonia water at 0-10 ℃, stirring for reaction for 12 hours, then decompressing and removing tetrahydrofuran, separating out solid, filtering, and drying in vacuum to obtain 6.8g of white solid H-Pro-Ala-NH₂The yield thereof was found to be 75.6%.

3. 30g (0.046mol) of Fmoc-Arg (Pbf) -OH and 300mL of dichloromethane are added into a 1000mL three-necked flask, the mixture is cooled to-15 ℃ after being stirred and dissolved, 6.45g (0.047mol) of 1-hydroxybenzotriazole and 9.7g (75mmol) of N, N-diisopropylethylamine are added, 9.1g (0.047mol) of N-ethyl-N' - (3-dimethylaminopropyl) carbodiimide hydrochloride are added after being stirred for 15 minutes, and the mixture is reacted for 45 minutes at-15 to-10 ℃; 11.1g (0.06mol) of H-Pro-Ala-NH₂9.7g (75mmol) of N, N-diisopropylethylamine is dissolved in 100mL of dichloromethane and then is dripped into a three-necked bottle, the mixture reacts at the temperature of-10 to-5 ℃ for 1 hour, then the temperature naturally rises to room temperature, the reaction is stirred for 8 hours, the reaction is stopped, the reaction solution is poured into 500mL of water, the mixture is kept stand and separated, the water layer is extracted by 200mL of dichloromethane, the extracted dichloromethane phase is washed by 1mol/L hydrochloric acid, washed by saturated sodium chloride solution, dried by anhydrous sodium sulfate and filtered, the filtrate is decompressed and the solvent is removed, 31.6g of white solid, namely Fmoc-Arg (Pbf) -Pro-Ala-NH is obtained, the white solid is Fmoc-Arg (Pb₂The yield thereof was found to be 77.4%.

4. Into a 1000mL three-necked flask was added 16.7g (0.025mol) of Fmoc-Arg (Pbf) -Pro-Ala-NH₂And 100mL of N, N-dimethylformamide solution of piperidine with the volume concentration of 20%, stirring at room temperature for reaction for 2 hours, adding 50mL of N, N-dimethylformamide solution containing 12.0g (0.042mol) of Boc-Gly-Pro-OMe, stirring at room temperature for reaction for 24 hours, stopping the reaction, pouring the reaction liquid into 600mL of water, stirring, adding citric acid to separate out a large amount of white solid in the system, filtering, washing with water, pulping twice with ethyl acetate after vacuum drying, filtering the solid, and drying in vacuum to obtain 15.3g of white solid Boc-Gly-Pro-Arg (Pbf) -Pro-Ala-NH₂. Boc-Gly-Pro-Arg (Pbf) -Pro-Ala-NH₂Adding 200mL of mixed solution of trifluoroacetic acid, triisopropylsilane and water in a volume ratio of 90:5:5, stirring at room temperature for reaction for 1 hour, removing the solvent under reduced pressure, pouring the concentrated solution into 200mL of acetone, dropwise adding concentrated hydrochloric acid while stirring until a large amount of white solid is separated out, filtering, and solidifyingWashing with acetone, and vacuum drying to obtain white solid H-Gly-Pro-Arg-Pro-Ala-NH₂·HCl。

Reacting H-Gly-Pro-Arg-Pro-Ala-NH₂HCl is added into 50mL water, ion-exchanged to remove hydrochloric acid (constant flow with 2% volume of acetic acid solution, detection wavelength 210nm, flow rate 10mL/min, column specification: 5cm × 45cm, filler DEAE), and finally freeze-dried to obtain 8.2g of white solid, i.e. vilos peptide, yield 67.5% and purity greater than 85%.

Boc represents t-butyloxycarbonyl, Fmoc represents 9-fluorenyl-methoxycarbonyl, and Pbf represents 2,2,4,6, 7-pentamethylbenzodihydrofuran-5-sulfonyl.

Claims (6)

1. A liquid phase synthesis method of vilospiritide is characterized by comprising the following steps:

(1) reacting Boc-Gly-OH, N-hydroxysuccinimide and N, N' -dicyclohexylcarbodiimide at 0-15 ℃ for 3-5 hours by taking tetrahydrofuran as a solvent, filtering to remove insoluble substances, adding H-Pro-OMe HCl and an aqueous solution of sodium bicarbonate into the filtrate, reacting at 15-25 ℃ for 8-12 hours, and separating and purifying to obtain Boc-Gly-Pro-OMe;

(2) using dichloromethane or tetrahydrofuran as solvent, reacting Boc-Pro-OH or Fmoc-Pro-OH with isobutyl chloroformate and organic base at-15-10 ℃ for 10-45 minutes, and reacting with H-Ala-NH₂Reacting for 1 hour, then heating to room temperature for 6-8 hours, separating and purifying to obtain Boc-Pro-Ala-NH₂Or Fmoc-Pro-Ala-NH₂Then removing Boc protecting group with ethyl acetate solution of hydrogen chloride to obtain H-Pro-Ala-NH₂Removal of the Boc protecting group by HCl or in tetrahydrofuran with trifluoroacetic acid to give H-Pro-Ala-NH₂TFA, or removal of the Fmoc protecting group with piperidine in tetrahydrofuran or concentrated aqueous ammonia to give H-Pro-Ala-NH₂；

(3) Using dichloromethane as a solvent, reacting Fmoc-Arg (Pbf) -OH, 1-hydroxybenzotriazole, organic base and N-ethyl-N' - (3-dimethylaminopropyl) carbodiimide hydrochloride at-15 to-5 ℃ for 30 to 60 minutes, and then reacting with H-Pro-Ala-NH₂Or H-Pro-Ala-NH₂·HCl or H-Pro-Ala-NH₂TFA is reacted for 30-60 minutes, then the temperature is raised to room temperature for reaction for 6-8 hours, and the reaction product is separated and purified to obtain Fmoc-Arg (Pbf) -Pro-Ala-NH₂；

(4) Removal of Fmoc-Arg (Pbf) -Pro-Ala-NH with piperidine in N, N-dimethylformamide₂Reacting the Fmoc protecting group with Boc-Gly-Pro-OMe at room temperature for 12-24 hours, separating and purifying to obtain Boc-Gly-Pro-Arg (Pbf) -Pro-Ala-NH₂Then deprotecting with trifluoroacetic acid solution, salifying with hydrochloric acid to obtain H-Gly-Pro-Arg-Pro-Ala-NH₂HCl, ion exchange to obtain vilofilapeptide.

2. The liquid phase synthesis method of vilospthread peptide according to claim 1, wherein: in the step (1), the mole ratio of Boc-Gly-OH, N-hydroxysuccinimide, N' -dicyclohexylcarbodiimide, H-Pro-OMe & HCl and sodium bicarbonate is 1: 1.1-1.3: 1.5-2.0: 1.2-1.5: 2.5-4.0.

3. The liquid phase synthesis method of vilospthread peptide according to claim 1, wherein: in step (2), the Boc-Pro-OH or Fmoc-Pro-OH, isobutyl chloroformate, organic base, H-Ala-NH₂The molar ratio of (A) to (B) is 1: 1.05-1.2: 2.5-4.0: 1.1-1.3.

4. The liquid phase synthesis method of vilospthread peptide according to claim 1, wherein: in step (3), the Fmoc-Arg (Pbf) -OH, 1-hydroxybenzotriazole, organic base, N-ethyl-N' - (3-dimethylaminopropyl) carbodiimide hydrochloride, H-Pro-Ala-NH₂The molar ratio of (A) to (B) is 1: 1.0-1.05: 2.5-5.0: 1.0-1.05: 1.1-1.3.

5. The liquid phase synthesis method of vilospiritide according to claim 3 or 4, wherein: the organic base is any one of N-methylmorpholine, N-diisopropylethylamine and N-methylpyrrolidone.

6. Liquid phase synthesis of vilospiritide according to claim 1The method is characterized in that: in step (4), the Fmoc-Arg (Pbf) -Pro-Ala-NH₂And the Boc-Gly-Pro-OMe is in a molar ratio of 1: 1.3-1.6.