Preparation method of Fmoc-Arg (Pbf)-OH

Abstract

The invention discloses a preparation method of Fmoc-Arg (Pbf)-OH, which comprises the following steps of: reacting cheap and easily available cyanamide serving as an initial raw material with PbfCl to prepare Pbf cyanamide, reacting the Pbf cyanamide with a hydrogen chloride methanol solution to prepare N-Pbf-oxymethylisourea hydrochloride, preparing arginine Pbf copper salt from the N-Pbf-oxymethylisourea hydrochloride and L-ornithine under the protection condition of copper salt, and preparing the Fmoc-Arg (Pbf)-OH from the arginine Pbf copper salt. The preparation method comprises the following steps of: purifying a copper salt, removing copper to obtain arginine Pbf, reacting the arginine Pbf with Fmoc-OSu to obtain a Fmoc-Arg (Pbf)-OH crude product, and refining and drying the crude product to obtain a Fmoc-Arg (Pbf)-OH finished product. The method has the advantages of simple route, cheap and easily available raw materials, good reaction selectivity, easy operation and mild reaction conditions, and is a simple and feasible preparation method.

Description

Technical Field

[0001] The present invention relates to the technical field of polypeptide drug synthesis, and in particular to a method for preparing Fmoc-Arg(Pbf)-OH. Background Art

[0002] Arginine is a basic amino acid and one of the 20 basic amino acids. Arginine can be synthesized in the human liver and is a non-essential amino acid. On the one hand, it participates in the ornithine cycle in the human body and can convert the ammonia produced in the body into non-toxic urea, which is excreted from the body through urine, thereby reducing the concentration of ammonia in the blood; on the other hand, it also participates in important processes such as protein anabolism, polyamine and nitric oxide synthesis. Its unique chemical structure and biological functions enable it to play an important role in life activities. In peptide synthesis, in order to avoid unnecessary side reactions, the guanidine and amino groups of arginine must be protected. Fmoc-Arg(Pbf)-OH is a commonly used protected amino acid and a key protected amino acid widely used in the field of peptide synthesis. Its unique protecting group characteristics make it play an important role in both liquid and solid phase peptide synthesis. The present application describes a method for synthesizing Fmoc-Arg(Pbf)-OH.

[0003] Hong Yongyu et al. reported a method for synthesizing Fmoc-Arg(Pbf)-OH in Chemical Reagents [2006, 28 (1), 57-58]. This method uses Cbz-Arg-OH as a raw material, and the guanidine group of the arginine side chain is protected with Pbf-Cl in acetone-water two-phase conditions. Then, Cbz is removed by palladium carbon hydrogenation, and the amino group is protected with Fmoc. The above method is to protect the guanidine group with Pbf in the aqueous phase. However, Pbf-Cl decomposes severely, and the reaction of the substrate Cbz-Arg-OH is not complete. At the same time, the expensive palladium carbon hydrogenation is used to remove the protecting group, which is too expensive and limits its industrial application.

[0004] Patents CN200810034390.9 and CN20180694250.8 describe a method using Boc-Arg-OH as a raw material, protecting the arginine side chain guanidine group with 1.6-2 equivalents of Pbf-Cl in different solvent systems, followed by acidolysis to remove the Boc protecting group and Fmoc protection on the free amino group. The above two methods avoid the use of expensive palladium-carbon catalysts and dangerous hydrogenation processes, reducing costs to a certain extent, achieving a conventional reaction, and facilitating production operations. However, there are also problems with Pbf protection on the guanidine group in aqueous systems, and the relatively expensive reagent Pbf-Cl decomposes severely and consumes a large amount of it; the substrate Boc-Arg-OH does not react completely, and the substrate decomposes the raw materials, producing certain citrulline and ornithine byproducts, making separation and purification difficult and the economic benefits poor.

[0005] Patent CN201710305099.X uses Boc-Arg-OMe (OEt) as the starting material, replacing the Boc-Arg-OH used in the aforementioned patent. The purpose is to enhance the solubility of the raw protected amino acid in the organic phase, enabling Pbf protection of the arginine guanidine group under non-aqueous conditions and reducing the decomposition of the relatively expensive protecting reagent Pbf-Cl under strong aqueous pH conditions, thereby reducing costs. However, experimental verification revealed that the reaction conditions used by the applicant resulted in two unfavorable consequences. First, the protected amino acid reacted under Pbf protection on the guanidine group, resulting in significant racemization. Second, the protecting reagent also decomposed severely, causing the reaction to almost stall halfway through. Increasing the amount of protecting reagent, changing the reaction temperature, extending the reaction time, and optimizing the reaction solvent did not significantly improve the reaction.

[0006] In summary, the existing process scheme has the disadvantages of harsh process conditions, high cost of precious metal catalysts, low conversion rate, large amount of reagents, high cost of raw and auxiliary materials, and low operability. Summary of the Invention

[0007] The present invention aims to provide a green and economical method for synthesizing Fmoc-Arg(Pbf)-OH with low raw material cost, high conversion rate, mild reaction conditions, simple operation, and certain versatility. Furthermore, using the common reaction substrate, N-Pbf-oxymethylisourea hydrochloride, this method is also applicable to the derivatization of similar basic amino acids with different carbon chain lengths, such as lysine, 4-aminobutyric acid, and 3-aminoalanine, to prepare terminal guanidino-protected amino acids.

[0008] To achieve the above objectives, the present invention provides the following technical solutions: The present invention provides a method for preparing Fmoc-Arg(Pbf)-OH, comprising the following steps: S1, cyanamide and protective reagent Pbf-Cl react to prepare Pbf cyanamide; S2, Pbf cyanamide and hydrogen chloride methanol solution to prepare N-Pbf-oxymethyl isourea hydrochloride; Arginine Pbf was prepared from S3, N-Pbf-oxymethylisourea hydrochloride and L-ornithine; S4. Fmoc protection of arginine Pbf was used to prepare Fmoc-Arg(Pbf)-OH.

[0009] The route is as follows: .

[0010] Furthermore, in S1, cyanamide and protective reagent Pbf-Cl are reacted in a solution of triethylamine and dichloromethane at a temperature of 0-5°C.

[0011] Furthermore, in S2, the Pbf cyanamide obtained in S1 is treated with a saturated hydrogen chloride methanol solution, and the reaction is post-treated to obtain N-Pbf-oxymethylisourea hydrochloride.

[0012] Furthermore, the S3 is specifically prepared by reacting N-Pbf-oxymethylisourea hydrochloride and L-ornithine in a sodium hydroxide solution and a copper sulfate solution, and post-treating the copper salt to obtain arginine Pbf.

[0013] Furthermore, the post-treatment of the copper salt in S3 is specifically as follows: The obtained copper salt was added to 400 mL of water and stirred thoroughly, and then sodium sulfide nonahydrate was added and stirred thoroughly until all the copper salt in the system turned dark brown. Injectable activated carbon was added and stirred for 15 minutes. The mixture was filtered and cooled to 0-5°C. Hydrochloric acid was added to the filtrate to adjust the pH to 6-7. A large amount of solid gradually precipitated. The mixture was stirred until the pH stabilized at 6-7. The temperature was maintained at 0-5°C and stirred slowly for 10-12 hours. The mixture was filtered and the filter cake was washed with 200 mL of water. The solid TLC showed a single point. The filtrate was concentrated to a volume of 500 L. A large amount of solid precipitated. The filter cake was filtered and washed twice with 50 mL of water. The two filter cakes were combined and dried. The dried solid was recrystallized with ethyl acetate-ethanol in a volume ratio of 10:1 to obtain arginine Pbf.

[0014] Furthermore, the reagents and conditions in S4 are: sodium carbonate, acetone, Fmoc-OSu, and react at 20-25° C. until a trace amount of Fmoc-Osu remains.

[0015] Based on the above technical solution, the embodiments of the present invention can produce at least the following technical effects: (1) The starting raw cyanamide of the present invention is an industrial product, cheap and easy to obtain, and has low cost; (2) The process route used in the invention to prepare the common intermediate N-Pbf-oxymethylisourea hydrochloride avoids the disadvantages of poor solubility of amino acids and easy decomposition of the expensive protective reagent Pbf-Cl under alkaline conditions. The reaction conditions are mild and the yield is high. (3) The synthesis of the intermediate N-Pbf-oxymethylisourea hydrochloride is simple, with mild reaction conditions, and is easy to produce on an industrial scale; (4) The intermediate N-Pbf-oxymethylisourea hydrochloride used in the present invention can be applied to the derivatization of other basic amino acids to prepare terminal guanidine-protected amino acids, and has a certain degree of universality.

[0016] (5) The method for synthesizing Fmoc-Arg(Pbf)-OH of the present invention has the advantages of simple process, mild reaction conditions, easy operation, and suitability for industrial large-scale production. DETAILED DESCRIPTION

[0017] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0018] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each smaller range between any intermediate value within a stated value or stated range and any other stated value or intermediate value within the stated range is also encompassed by the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0019] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0020] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be exemplary only.

[0021] Example 1 A method for preparing Fmoc-Arg(Pbf)-OH, specifically: S1. Add cyanamide (25.2 g, 0.6 mol, 1.2 eq), triethylamine (60.6 g, 0.6 mol, 1.2 eq), and dichloromethane (300 ml) to a reaction flask, cool to 0-5°C, and add a freshly prepared dichloromethane (100 ml) solution of Pbf-Cl (144.5 g, 0.5 mol, 1 eq) dropwise. After the addition is complete, react at 0-5°C for half an hour, naturally raise the temperature to 20-25°C, and react for 2-3 hours. Monitor the complete reaction of Pbf-Cl by TLC, and stop the reaction. The reaction solution is washed with 1N dilute hydrochloric acid, 1N sodium bicarbonate solution, and saturated brine, respectively, dried, filtered, and concentrated to obtain a slightly yellow powdery solid. The solid is recrystallized from ethyl acetate-petroleum ether (1:1) and dried at 50°C to obtain 140 g of Pbf cyanamide as a white solid with a yield of 95%.

[0022] S2. Pbf cyanamide (117.6 g, 0.4 mol, 1 eq) was added to a reaction flask and treated with saturated methanolic hydrogen chloride solution (30%, 500 ml) at room temperature. After reacting for 5-6 hours, TLC was monitored to complete conversion of the raw material. The reaction was stopped and concentrated to a slurry. Ethyl acetate (200 ml) was added for beating, and the filter cake was filtered. The filter cake was dried at 50°C to obtain 130.7 g of white solid powder of N-Pbf-oxymethylisourea hydrochloride in a yield of 90%.

[0023] S3. Sodium hydroxide (11.2 g, 0.28 mol) and water (100 mL) were added to the reaction flask, stirred to dissolve, and then L-ornithine hydrochloride (46.4 g, 0.275 mol) was added to the system. The mixture was stirred and dissolved for later use. Copper sulfate pentahydrate (36.45 g, 0.146 mol) was dissolved in 150 L of water and added to the ornithine system at one time. 10 mol / L sodium hydroxide solution was added to maintain the pH at about 9. The mixture was stirred for 1 hour. N-Pbf-oxymethylisourea hydrochloride (90.75 g, 0.25 mol) was added to the system in batches at a temperature of 20-30 ° C. 10 mol / L sodium hydroxide solution was added to maintain the pH at about 9. After the addition was completed, the mixture was reacted at 20-30 ° C for 12 hours. TLC showed that the raw material N-Pbf-oxymethylisourea hydrochloride was basically reacted. The reaction solution was filtered and the solid copper salt was washed thoroughly with 500 mL of water for later use. The copper salt obtained in the previous step was added to 400 mL of water and stirred thoroughly. Sodium sulfide nonahydrate (33.6 g, 0.14 mol) was then added and stirred thoroughly until the copper salt in the system turned dark brown. 20 g of activated carbon for injection was added, stirred for 15 minutes, filtered, and cooled to 0–5°C. Hydrochloric acid was added to the filtrate to adjust the pH to 6–7, causing a large amount of solid to precipitate. The mixture was stirred until the pH stabilized at 6–7. The temperature was maintained at 0–5°C and stirred slowly for 10–12 hours. Filtered, the filter cake was washed with 200 mL of water, and a single spot was observed by TLC (411, ninhydrin). The filtrate was concentrated to 500 L, resulting in a large amount of solid precipitation. Filtered, the filter cake was washed twice with 50 mL of water. The two filter cakes were combined and air-dried. The air-dried solid was recrystallized from ethyl acetate-ethanol (10:1) to obtain 90.7 g of arginine Pbf as a white solid with a single spot by TLC. The purity was greater than 99%, and the yield was 85%.

[0024] S4. Add water and sodium carbonate (26.8 g, 0.26 mol) to the reaction flask and stir to dissolve. Then add arginine Pbf (85.4 g, 0.2 mol) and acetone (400 mL) and stir to mix. Then add Fmoc-OSu (70.8 g, 0.21 mol) slowly in batches and react at 20-25°C for 4 hours. Monitor the reaction of the raw materials by TLC to ensure that the reaction is complete and there is a trace amount of Fmoc-Osu remaining. 250 mL of water was added to the reaction system for dilution, and then ethyl acetate-petroleum ether (1:1) was added to extract small polar impurities (150 mL x 3). 600 mL of ethyl acetate was added to the aqueous phase, and 5 mol / L hydrochloric acid was slowly added to a pH of 3-4. The mixture was stirred thoroughly and allowed to stand for stratification. The organic phase was washed with saturated brine (100 mL x 3), dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. 300 mL of ethyl acetate was added and stirred to dissolve. The product gradually precipitated. The product was slowly stirred to crystallize for more than 10 hours. The product was filtered, washed with ethyl acetate, and dried to obtain 115.5 g of a white solid product with a yield of 89% and a purity greater than 99%.

[0025] The above description is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this field, several variations and improvements can be made without departing from the creative concept of the present invention, which all fall within the scope of protection of the present invention.

Claims

1. A method for preparing Fmoc-Arg(Pbf)-OH, characterized in that: The preparation process includes four steps and the preparation route is as follows: 。 2. The method for preparing Fmoc-Arg(Pbf)-OH according to claim 1, wherein In the S1, cyanamide and a protecting reagent Pbf-Cl are reacted in a solution of triethylamine and dichloromethane with the temperature lowered to 0-5°C.

3. The method for preparing Fmoc-Arg(Pbf)-OH according to claim 1, wherein In the step S2, the Pbf cyanamide obtained in the step S1 is treated with a saturated methanolic hydrogen chloride solution, and the reaction is followed by post-treatment to obtain N-Pbf-oxymethylisourea hydrochloride.

4. The method for preparing Fmoc-Arg(Pbf)-OH according to claim 1, wherein Specifically, S3 comprises preparing a copper salt by reacting N-Pbf-oxymethylisourea hydrochloride and L-ornithine in a sodium hydroxide solution and a copper sulfate solution, and then post-treating the copper salt to obtain arginine Pbf.

5. The method for preparing Fmoc-Arg(Pbf)-OH according to claim 4, wherein The post-treatment of the copper salt in S3 is specifically as follows: The obtained copper salt was added to 400 mL of water and stirred thoroughly, and then sodium sulfide nonahydrate was added and stirred thoroughly until all the copper salt in the system turned dark brown. Injectable activated carbon was added and stirred for 15 minutes. The mixture was filtered and cooled to 0-5°C. Hydrochloric acid was added to the filtrate to adjust the pH to 6-7. A large amount of solid gradually precipitated. The mixture was stirred until the pH stabilized at 6-7. The temperature was maintained at 0-5°C and stirred slowly for 10-12 hours. The mixture was filtered and the filter cake was washed with 200 mL of water. The solid TLC showed a single point. The filtrate was concentrated to a volume of 500 L. A large amount of solid precipitated. The filter cake was filtered and washed twice with 50 mL of water. The two filter cakes were combined and dried. The dried solid was recrystallized with ethyl acetate-ethanol in a volume ratio of 10:1 to obtain arginine Pbf.

6. The method for preparing Fmoc-Arg(Pbf)-OH according to claim 1, wherein The reagents and conditions in S4 are: sodium carbonate, acetone, Fmoc-OSu, and the reaction is carried out at 20-25° C. until a trace amount of Fmoc-Osu remains.

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