Process for the synthesis of glycinyl-beta-alanyl-l-histidine

Abstract

The application discloses a synthesis method of glycyl-beta-alanyl-L-histidine and belongs to the technical field of organic synthesis. The method comprises the following steps: (1) reacting L-muscle peptide, a silane protecting agent and an acid applying agent in a solvent A at 0-25 DEG C to obtain a trisilane-muscle peptide, wherein the silane protecting agent is selected from trimethylchlorosilane or hexamethyldisilazane, the acid applying agent is selected from triethylamine or diisopropylethylamine, and the molar ratio of the L-muscle peptide to the acid applying agent is 1:4.0-6.5; (2) condensing the trisilane-muscle peptide with phthalylglycine chloride to obtain phthalylglycyltrisilane-muscle peptide; (3) quenching the phthalylglycyltrisilane-muscle peptide by adding water, adjusting the pH value of the aqueous phase to 2-3 by adding acid, and hydrolyzing to obtain phthalylglycylmuscle peptide; and (4) hydrazinolysis of the phthalylglycylmuscle peptide to obtain glycyl-beta-alanyl-L-histidine. The method has the advantages of high yield, simple process and high purity.

Description

Technical Field

[0001] The present invention belongs to the technical field of organic synthesis, and in particular relates to a method for synthesizing glycyl-β-alanyl-L-histidine. Background Art

[0002] Carnosine (L-carnosine) is a naturally occurring dipeptide, chemically known as β-alanyl-L-histidine. Carnosine possesses strong antioxidant properties and is beneficial to the human body. Carnosine exhibits anti-inflammatory, anti-glycation, and antioxidant properties, making it an over-the-counter dietary supplement with a role in the prevention and adjunctive treatment of chronic diseases such as cardiovascular disease and neurodegeneration. In animal studies, carnosine's neuroprotective mechanisms have been shown to prevent permanent cerebral ischemia. Carnosine supplementation is believed to help alleviate some age-related neurological disorders, such as Alzheimer's disease and Parkinson's disease. Carnosine is also an important intracellular antioxidant. Its non-toxicity and strong antioxidant properties have attracted widespread attention as a novel food additive and pharmaceutical agent. Medically, carnosine can also prevent Alzheimer's disease and neurological and brain degeneration. Glycine is the simplest amino acid. Despite its simple structure, glycine is an important inhibitory neurotransmitter in the central nervous system, playing a crucial role in controlling neuronal excitability. Glycine-β-alanyl-L-histidine (glycyl-L-carnosine) is a tripeptide composed of glycine and carnosine linked by a peptide chain. It is highly water-soluble and exhibits significant biological activity. Its neuroprotective properties are significantly stronger than those of carnosine or glycine, and it has broad application prospects in areas such as stroke, brain injury, Alzheimer's disease, and Parkinson's disease (Li Jianxiong, Patent Publication No.: CN113150065A, July 23, 2021, A Synthetic Peptide and Its Application, US20240002435). This patent develops a liquid-phase synthesis method for glycyl-carnosine (glycyl-β-alanyl-L-histidine). Summary of the Invention

[0003] To address the above issues, the present invention provides a method for synthesizing glycyl-β-alanyl-L-histidine. This method has the advantages of high yield, simple process, and high purity. The tripeptide glycyl-β-alanyl-L-histidine (glycyl-L-carnosine) can be obtained in only four steps, two of which do not require post-processing and separation, significantly reducing the production cycle and production costs. The first three steps can basically be carried out at room temperature under mild conditions. The synthetic route is shown below:

[0004] .

[0005] The present invention provides a method for synthesizing glycyl-β-alanyl-L-histidine, which comprises:

[0006] (1) In solvent A, L-carnosine, a silane protective agent, and an acidifying agent react at 0-25°C to obtain trisilane-carnosine, wherein the silane protective agent is selected from trimethylchlorosilane or hexamethyldiazosilane, the acidifying agent is selected from triethylamine or diisopropylethylamine, the molar ratio of L-carnosine to trimethylchlorosilane is 1:3.0-4.5, the molar ratio of L-carnosine to hexamethyldiazosilane is 1:1.5-2.5, and the molar ratio of L-carnosine to the acidifying agent is 1:4.0-6.5. In this step, L-carnosine is converted into trisilane-carnosine by protecting the carboxyl group and two amino groups with silane. Among them, under the conditions of this patent, only one of the two hydrogens of the amino group on alanine is protected by silane, and the other hydrogen does not react to carry out the peptide grafting reaction. The reason is that L-carnosine has steric hindrance and only replaces one hydrogen in the presence of a weak base. If the base is very strong, such as n-butyl lithium, two hydrogens can be replaced. This patent only requires replacing one hydrogen, and the other hydrogen forms a peptide.

[0007] (2) Condensation of trisilane-carnosine with phthalylglycyl chloride to obtain phthalylglycyltrisilanecarnosine. In this step, no condensing agent is required.

[0008] (3) The reaction solution of step (2) is quenched with water, and the aqueous phase is acidified to adjust the pH to 2-3, and hydrolyzed to obtain phthalylglycylcarnosine. In this step, all three silane protecting groups are removed simultaneously.

[0009] (4) Phthaloylglycylcarnosine is hydrazinolyzed to obtain glycyl-β-alanyl-L-histidine, which is a conventional hydrazinolysis process.

[0010] Wherein, solvent A is selected from dichloromethane, tetrahydrofuran or 1,2-dichloroethane, etc., preferably dichloromethane. Specifically, solvent A is selected from dichloromethane. In step (1), under nitrogen protection, L-carnosine and an acidifying agent are added to dichloromethane, and a silane protective agent is added dropwise in a suspension state. The reaction is carried out at 0-25°C. During the addition process, L-carnosine slowly dissolves. After the reaction is completed, a trisilane-carnosine solution is obtained.

[0011] Wherein, step (2) specifically comprises: dissolving phthalylglycine and catalyst DMF in solvent B, then adding an acylating agent dropwise, and reacting at 0-25°C to obtain a phthalylglycyl chloride solution. The phthalylglycyl chloride solution is directly reacted with a trisilane-carnosine solution without treatment, adding a catalyst DMAP to the trisilane-carnosine solution, then adding the phthalylglycyl chloride solution dropwise, and reacting at 0-25°C to obtain phthalylglycyltrisilanecarnosine. Wherein, the molar ratio of phthalylglycine, DMF, and acylating agent is 1:0.01-0.05:1-2, and the molar ratio of trisilane-carnosine to phthalylglycyl chloride is 1:1.1-1.5. Wherein, solvent B is selected from dichloromethane, tetrahydrofuran, or 1,2-dichloroethane, and is preferably dichloromethane. The acylating agent is selected from thionyl chloride or oxalyl chloride, and is preferably oxalyl chloride.

[0012] Wherein, step (3) comprises: adding the reaction of step (2) dropwise into ice water for quenching, separating the liquids after quenching, extracting the organic phase with water, combining the aqueous phases, adding acid to adjust the pH value to 2-3 for hydrolysis, concentrating after the hydrolysis (separating most of the water, such as 80% of the water), adding a precipitant, and performing solid-liquid separation to obtain phthalylglycylcarnosine. Wherein, the precipitant is selected from ethanol, isopropanol, methanol or acetone, preferably isopropanol. The amount of the precipitant is 15-25 times the weight of L-carnosine. The acid can be a common acid, specifically concentrated hydrochloric acid.

[0013] Wherein, step (4) comprises: hydrazinolysis of phthaloylglycylcarnosine and hydrazine hydrate alcohol solution at 70-80° C., and after the hydrazinolysis is completed, recovering the solvent and purifying to obtain glycyl-β-alanyl-L-histidine. The volume concentration of the hydrazine hydrate alcohol solution is 20-60%, specifically, the volume concentration of the hydrazine hydrate alcohol solution is 30%. The hydrazine hydrate alcohol solution is selected from hydrazine hydrate methanol solution, hydrazine hydrate ethanol solution or hydrazine hydrate isopropanol solution, and is preferably 90% hydrazine hydrate ethanol solution.

[0014] The purification process of step (4) is as follows: water is added to the reaction product, impurities are removed by extraction, the aqueous phase is decolorized by activated carbon, solid-liquid separation is performed after decolorization (specifically, filtration is performed while hot), the water is evaporated to obtain a paste, and glycyl-β-alanyl-L-histidine is obtained by recrystallization. The amount of activated carbon used is 1-10% of the weight of phthalylglycylcarnosine, the extraction agent is selected from dichloromethane or toluene, and preferably dichloromethane. The recrystallization solvent is selected from ethanol, isopropanol, methanol or acetone, and preferably ethanol. The decolorization reaction time is 10-60 minutes, and specifically can be 20 minutes.

[0015] Specifically, the present invention provides a method for synthesizing glycyl-β-alanyl-L-histidine, the method comprising:

[0016] (1) under nitrogen protection, adding diisopropyl ethylamine and L-muscle peptide in dichloromethane, adding trimethylsilyl chloride dropwise in the state of suspension, reacting at 0-25 DEG C, obtaining a trisilane-muscle peptide solution after the reaction is completed. The molar ratio of L-muscle peptide, trimethylsilyl chloride and diisopropyl ethylamine is 1:3.0-4.5:4.0-6.5.

[0017] (2) dissolving phthaloylglycine and catalyst DMF in dichloromethane, then adding oxalyl chloride dropwise, obtaining a phthaloylglycyl chloride solution after reacting at 0-25 DEG C; adding catalyst DMAP in the trisilane-muscle peptide solution, then adding the phthaloylglycyl chloride solution dropwise, obtaining a phthaloylglycyl trisilane-muscle peptide after reacting at 0-25 DEG C. The molar ratio of phthaloylglycine, DMF and oxalyl chloride is 1:0.01-0.05:1-2, and the molar ratio of trisilane-muscle peptide and phthaloylglycyl chloride is 1:1.1-1.5.

[0018] (3) adding the reaction of step (2) to ice water for quenching, separating the organic phase after the quenching is completed, extracting the water phase, adding hydrochloric acid in the water phase to adjust the pH value to 2-3 for hydrolysis, concentrating after the hydrolysis is completed, adding isopropyl alcohol, and separating the solid and liquid to obtain phthaloylglycyl muscle peptide.

[0019] (4) adding phthaloylglycyl muscle peptide to hydrazine hydrate ethanol solution, hydrazinolysis at 70-80 DEG C, recovering ethanol after the hydrazinolysis is completed; adding water in the reaction product, then extracting impurities by dichloromethane, then decolorizing the water phase by activated carbon, separating the solid and liquid after the decolorization is completed, evaporating water to obtain a paste, and finally recrystallizing by ethanol solution to obtain glycyl-β-alanyl-L-histidine. The amount of activated carbon is 1-10% of the weight of phthaloylglycyl muscle peptide.

[0020] The synthesis method provided by the present invention has the following advantages: it directly uses commercially available L-carnosine as the starting material, eliminating the need to consider the peptide chain connection between alanine and histidine during the reaction. Excess trimethylsilyl chloride is directly used in dichloromethane for silane protection. The alanine amino group of L-carnosine and the amino and carboxyl groups on the histidine ring are all protected with silane. After the reaction is completed, it is directly condensed with phthaloylglycyl chloride. The remaining activated hydrogen on the alanine amino group of L-carnosine condenses with the acyl chloride to form a peptide bond. Post-treatment hydrolysis yields phthaloylglycylcarnosine. After crystallization and purification, it is simply hydrazinolyzed with ethanol hydrate to obtain glycyl-L-carnosine (glycyl-β-alanyl-L-histidine). Traditional methods cannot avoid the reaction between the amino groups on the glycine and histidine rings, typically requiring multiple protection and deprotection treatments, resulting in very low yields (typically 10-20%) and failing to meet production needs. The present invention has a simple process. The two-step reaction products, trisilylcarnosine and phthalylglycyl chloride, are directly condensed, hydrolyzed, and crystallized to yield phthalylglycylcarnosine (phthalylglycyl-β-alanyl-L-histidine) without purification. After further purification, the product is simply hydrolyzed with alcohol hydrate, extracted and decontaminated, and the isoelectric point is adjusted (specifically, to neutral). The solid is then crystallized from the alcohol to yield the tripeptide glycyl-L-carnosine (glycyl-β-alanyl-L-histidine). The tripeptide glycyl-L-carnosine (glycyl-β-alanyl-L-histidine) can also be synthesized via solid-phase synthesis, but this is costly and unsuitable for large-scale industrial production (Li Jianxiong, Patent Publication No. CN113150065A, July 23, 2021, "A Synthetic Peptide and Its Application," US20240002435). BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is the liquid chromatography mass spectrum of phthaloylglycylcarnosine (phthaloylglycyl-β-alanyl-L-histidine);

[0022] Figure 2 This is a mass spectrometry analysis of phthaloylglycylcarnosine (phthaloylglycyl-β-alanyl-L-histidine);

[0023] Figure 3 This is the liquid chromatography mass spectrum of glycyl-β-alanyl-L-histidine (glycyl-L-carnosine);

[0024] Figure 4 This is a mass spectrometry analysis of glycyl-β-alanyl-L-histidine (glycyl-L-carnosine). DETAILED DESCRIPTION

[0025] In order to make the objectives, technical solutions and advantages of the present invention more clear, the present invention is described in further detail below.

[0026] Example 1 Synthesis of trisilane-protected carnosine

[0027] Add 113 g (0.5 mol) of carnosine to 1000 ml of dichloromethane, and then add 300 g (2.3 mol) of diisopropylethylamine (DIPEA). Protect with nitrogen, cool to 0°C, and dropwise add 200 g (1.8 mol) of trimethylchlorosilane. Stir to react. During the addition, carnosine can be seen to slowly dissolve and gas (from overflowed hydrochloric acid) can be released. Stir overnight. After the reaction, the carnosine is completely dissolved to obtain a red solution for use.

[0028] Example 2 Synthesis of Phthaloylglycyl Chloride

[0029] To 125 g (0.61 mol) of phthaloylglycine, add 800 ml of dichloromethane and 6 ml of DMF, cool to 0-5°C, and slowly add 88 g (0.7 mol) of oxalyl chloride dropwise. After the addition is complete, stir and react for 8 hours to obtain a red dichloromethane solution of phthaloylglycine chloride.

[0030] Example 3 Synthesis of Phthaloylglycylcarnosine (Phthaloylglycyl-Alanyl-L-Histidine)

[0031] The red trisilane-protected carnosine dichloromethane solution was maintained at 0-5°C, 5 grams of DMAP was added, and a dichloromethane solution of phthalylglycyl chloride was slowly added dropwise. The mixture was stirred and insulated for 6 hours to complete the reaction. 200g of purified water was taken, cooled to 0°C, and the reaction solution was slowly added dropwise to quench the mixture. The quenching was completed and the dichloromethane phase was extracted twice with 200ml*2 pure water. The combined aqueous phases were added, and 50ml of concentrated hydrochloric acid was stirred until the mixture was acidic. The mixture was stirred for 5 hours to complete the hydrolysis. After concentrating most of the water, 2000ml of isopropyl alcohol was added and stirred to obtain a yellow solid. Filtering gave 160g of phthalylglycylcarnosine, a yield of 77.5%.

[0032] Example 4 Synthesis of Glycyl-L-Carnosine (Glycyl-β-Alanyl-L-Histidine)

[0033] 103g (0.25mol) of phthalylglycylcarnosine and 600ml of ethanol were added dropwise with 200ml of hydrazine hydrate. After addition, the mixture was heated to 78°C and stirred under reflux for 4 hours. The reaction was monitored by thin layer chromatography and the solvent was recovered to obtain a yellow oil. 200ml of water was added and impurities were removed by dichloromethane extraction. 5g of activated carbon was added to the aqueous phase and the mixture was heated to 60°C for half an hour for decolorization. The solution was filtered to obtain a colorless solution. The aqueous phase was dried to obtain a white paste. The mixture was stirred and beaten with 90% ethanol for 10 hours. The solid of the white tripeptide (glycyl-β-alanyl-L-histidine) was filtered to obtain 60g, with a yield of 86% and a purity greater than 99%.

[0034] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for synthesizing glycyl-β-alanyl-L-histidine, characterized in that: The method comprises: (1) In solvent A, L-carnosine, a silane protective agent and an acid dressing agent react at 0-25° C. to obtain trisilane-L-carnosine, wherein the silane protective agent is selected from trimethylchlorosilane or hexamethyldisilazane, the acid dressing agent is selected from triethylamine or diisopropylethylamine, the solvent A is selected from dichloromethane, tetrahydrofuran or 1,2-dichloroethane, the molar ratio of L-carnosine to trimethylchlorosilane is 1:3.0-4.5, the molar ratio of L-carnosine to hexamethyldisilazane is 1:1.5-2.5, and the molar ratio of L-carnosine to the acid dressing agent is 1:4.0-6.5; (2) condensation of trisilane-L-carnosine with phthalylglycyl chloride to obtain phthalylglycyltrisilane-L-carnosine; (3) the reaction solution of step (2) is quenched by adding water, the aqueous phase is acidified to adjust the pH value to 2-3, and hydrolyzed to obtain phthalylglycyl L-carnosine; (4) Phthaloylglycyl L-carnosine is hydrazinolyzed to obtain glycyl-β-alanyl-L-histidine; The structural formula of trisilane-L-carnosine is as follows: ； The structural formula of phthaloylglycyltrisilane L-carnosine is as follows: ； The structural formula of phthaloylglycyl L-carnosine is as follows: ； The structural formula of glycyl-β-alanyl-L-histidine is as follows: 。 2. The method for synthesizing glycyl-β-alanyl-L-histidine according to claim 1, wherein The solvent A is selected from dichloromethane. In step (1), L-carnosine and an acidifying agent are added to dichloromethane under nitrogen protection, and a silane protective agent is added dropwise in a suspension state. The reaction is carried out at 0-25° C. After the reaction is completed, a trisilane-L-carnosine solution is obtained.

3. The method for synthesizing glycyl-β-alanyl-L-histidine according to claim 2, wherein Step (2) specifically comprises: dissolving phthalylglycine and catalyst DMF in solvent B, wherein the solvent B is selected from dichloromethane, tetrahydrofuran or 1,2-dichloroethane, then adding an acylating agent dropwise, and reacting at 0-25° C. to obtain a phthalylglycyl chloride solution; adding catalyst DMAP to the trisilane-L-carnosine solution, then adding the phthalylglycyl chloride solution dropwise, and reacting at 0-25° C. to obtain phthalylglycyltrisilane L-carnosine, wherein the molar ratio of the phthalylglycine, DMF and acylating agent is 1:0.01-0.05:1-2, and the molar ratio of the trisilane-L-carnosine to the phthalylglycyl chloride is 1:1.1-1.

5.

4. The method for synthesizing glycyl-β-alanyl-L-histidine according to claim 3, wherein The acylating agent is selected from thionyl chloride or oxalyl chloride.

5. The method for synthesizing glycyl-β-alanyl-L-histidine according to claim 3, wherein Step (3) comprises: adding the reaction of step (2) dropwise into ice water for quenching, separating the liquids after quenching, extracting the organic phase with water, combining the aqueous phases, adding acid to adjust the pH value to 2-3 for hydrolysis, concentrating after the hydrolysis is completed, adding a precipitant, and performing solid-liquid separation to obtain phthalylglycyl L-carnosine.

6. The method for synthesizing glycyl-β-alanyl-L-histidine according to claim 5, wherein The precipitant is selected from ethanol, isopropanol, methanol or acetone, and the amount of the precipitant is 15-25 times the weight of L-carnosine.

7. The method for synthesizing glycyl-β-alanyl-L-histidine according to claim 1, wherein Step (4) comprises: hydrazinolysis of phthalylglycyl L-carnosine and hydrazine hydrate alcohol solution at 70-80° C., and after the hydrazinolysis is completed, recovering the solvent and purifying to obtain glycyl-β-alanyl-L-histidine; the volume concentration of the hydrazine hydrate alcohol solution is 20-60%, and the hydrazine hydrate alcohol solution is selected from hydrazine hydrate methanol solution, hydrazine hydrate ethanol solution or hydrazine hydrate isopropanol solution.

8. The method for synthesizing glycyl-β-alanyl-L-histidine according to claim 7, wherein The purification process of step (4) is as follows: water is added to the reaction product, impurities are removed by extraction, the aqueous phase is decolorized by activated carbon, solid-liquid separation is performed after decolorization, water is evaporated to obtain a paste, and glycrystallization is performed to obtain glycyl-β-alanyl-L-histidine, wherein the amount of the activated carbon is 1-10% by weight of the phthaloylglycyl L-carnosine, the extractant is selected from dichloromethane or toluene, and the recrystallization solvent is selected from ethanol, isopropanol, methanol or acetone.

9. The method for synthesizing glycyl-β-alanyl-L-histidine according to claim 1, wherein The method comprises: (1) under nitrogen protection, diisopropylethylamine and L-carnosine are added to dichloromethane, trimethylchlorosilane is added dropwise in a suspension state, and the mixture is reacted at 0-25° C. After the reaction is completed, a trisilane-L-carnosine solution is obtained; the molar ratio of L-carnosine, trimethylchlorosilane and diisopropylethylamine is 1:3.0-4.5:4.0-6.5; (2) dissolving phthalylglycine and a catalyst, DMF, in dichloromethane, adding oxalyl chloride dropwise, and reacting at 0-25° C. to obtain a phthalylglycyl chloride solution; adding a catalyst, DMAP, to a trisilane-L-carnosine solution, adding the phthalylglycyl chloride solution dropwise, and reacting at 0-25° C. to obtain phthalylglycyltrisilane-L-carnosine, wherein the molar ratio of the phthalylglycine, DMF, and oxalyl chloride is 1:0.01-0.05:1-2, and the molar ratio of the trisilane-L-carnosine to the phthalylglycyl chloride is 1:1.1-1.5; (3) adding the reaction of step (2) dropwise into ice water for quenching, separating the liquids after quenching, extracting the organic phase with water, combining the aqueous phases, adding hydrochloric acid to the aqueous phase to adjust the pH value to 2-3 for hydrolysis, concentrating, adding isopropyl alcohol, and performing solid-liquid separation to obtain phthalylglycyl L-carnosine; (4) adding phthalylglycyl L-carnosine to a hydrazine hydrate ethanol solution, performing hydrazinolysis at 70-80° C., and recovering ethanol after the hydrazinolysis is completed; adding water to the reaction product, extracting and removing impurities with dichloromethane, and then decolorizing the aqueous phase with activated carbon. After the decolorization is completed, solid-liquid separation is performed, and the water is evaporated to obtain a paste, and finally recrystallizing from an ethanol solution to obtain glycyl-β-alanyl-L-histidine, wherein the amount of the activated carbon used is 1-10% of the weight of the phthalylglycyl L-carnosine.

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