Synthesis method of glycyl-beta-alanyl-L-histidine

Abstract

The invention discloses a synthesis method of glycyl-beta-alanyl-L-histidine, and belongs to the technical field of organic synthesis. The method comprises the steps that 1, L-carnosine, a silane protective agent and an acid applying agent react in a solvent A at the temperature of 0-25 DEG C to obtain trisilane-carnosine, the silane protective agent is selected from trimethylchlorosilane or hexamethyldiazo silane, the acid applying agent is selected from triethylamine or diisopropylethylamine, and the molar ratio of the L-carnosine to the acid applying agent is 1: (4.0-6.5); (2) carrying out condensation on the trisilane-carnosine and the phthaloyl glycyl chloride to obtain phthaloyl glycyl trisilane carnosine; (3) adding water into the phthaloyl glycyl trisilane carnosine to quench, adding acid into the water to adjust the pH value to 2-3, and hydrolyzing to obtain phthaloyl glycyl carnosine; and (4) carrying out hydrazinolysis on the phthaloyl glycyl carnosine to obtain the glycyl-beta-alanyl-L-histidine. The method has the advantages of high yield, simple process, high purity and the like.

Description

Technical Field

[0001] The 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 natural dipeptide, and its chemical name is β-alanyl-L-histidine. Carnosine has strong antioxidant capacity and is beneficial to the human body. Carnosine has anti-inflammatory, anti-glycation and antioxidant effects. It can be used as an over-the-counter food supplement and has a certain role in the prevention and adjuvant treatment of chronic diseases such as cardiovascular disease and neurodegeneration. In animal experiments, the neuroprotective mechanism of carnosine can prevent permanent brain ischemia. Carnosine supplementation is believed to help alleviate some age-related neurological diseases, such as Alzheimer's disease and Parkinson's disease. Carnosine is also an important intracellular antioxidant. Carnosine is not only non-toxic, but also has strong antioxidant properties. Therefore, it has attracted widespread attention as a new type of food additive and medicinal agent. In medicine, carnosine can also prevent Alzheimer's disease and prevent nerve and brain degeneration. Glycine is the amino acid with the simplest chemical structure. Despite its simple structure, glycine is an important inhibitory neurotransmitter in the central nervous system and plays an important role in controlling neuronal excitability. Glycine-β-alanyl-L-histidine (glycyl-L-carnosine) is a tripeptide composed of glycine and carnosine connected by a peptide chain. It is highly water-soluble and has significant biological activity. Its neuroprotective function is much stronger than that of carnosine or glycine. It has broad application prospects in the fields of stroke, brain injury, Alzheimer's disease, Parkinson's disease, etc. (Li Jianxiong, patent publication number: CN113150065A, 2021 / 07 / 23, 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] In order to solve the above problems, the embodiment of the present invention provides a method for synthesizing glycyl-β-alanyl-L-histidine. The method has the advantages of high yield, simple process and high purity. Only four steps of reaction can obtain the tripeptide glycyl-β-alanyl-L-histidine (glycyl-L-carnosine), two of which do not require post-treatment separation, which greatly reduces the production cycle and production cost. The first three steps can basically be carried out at room temperature under mild conditions. The synthetic route is as follows: .

[0004] The present invention provides a method for synthesizing glycyl-β-alanyl-L-histidine, the method comprising: (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, of the two hydrogens of the amino group on alanine, only one hydrogen is protected by silane under the conditions of this patent, and the other hydrogen does not react to carry out the peptide grafting reaction. The reason is: L-carnosine has steric hindrance, and only replaces one hydrogen in a weak base. If the base is very strong, such as n-butyl lithium, it can replace two. This patent just needs to replace one, and the other one becomes a peptide.

[0005] (2) condensation of trisilane-carnosine with phthalylglycyl chloride to obtain phthalylglycyl trisilane-carnosine. In this step, no condensation agent is required.

[0006] (3) The reaction solution of step (2) is quenched by adding water, and the aqueous phase is acidified to adjust the pH value to 2-3, and hydrolyzed to obtain phthalyl glycyl carnosine. In this step, the three silane protecting groups are removed simultaneously.

[0007] (4) Phthaloylglycylcarnosine is hydrazinolyzed to obtain glycyl-β-alanyl-L-histidine, which is a conventional hydrazinolysis process.

[0008] 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, and the reaction is carried out at 0-25° C. During the addition, L-carnosine slowly dissolves, and after the reaction is completed, a trisilane-carnosine solution is obtained.

[0009] Wherein, step (2) specifically comprises: dissolving phthalylglycine and catalyst DMF in solvent B, then dropping an acylating agent, 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 dropping a phthalylglycyl chloride solution, 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, etc., preferably dichloromethane. The acylating agent is selected from thionyl chloride or oxalyl chloride, and is preferably oxalyl chloride.

[0010] Wherein, step (3) comprises: adding the reaction of step (2) dropwise into ice water for quenching, separating the liquid after quenching, extracting the organic phase with water, combining the aqueous phase, adding acid to adjust the pH value to 2-3 for hydrolysis, and concentrating after the hydrolysis is completed (separating most of the water, such as 80% of the water), adding a precipitant, and performing solid-liquid separation to obtain phthalyl glycyl carnosine. Wherein, the precipitant is selected from ethanol, isopropanol, methanol or acetone, etc., 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.

[0011] Wherein, step (4) comprises: hydrazinolysis of phthalylglycylcarnosine and hydrazine hydrate alcohol solution at 70-80°C, and after the hydrazinolysis is completed, the solvent is recovered and purified 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, etc., preferably 90% hydrazine hydrate ethanol solution.

[0012] Wherein, the purification process of step (4) is: adding water to the reaction product, then removing impurities by extraction, decolorizing the aqueous phase by activated carbon, separating the solid and liquid after decolorization (specifically filtering while hot), evaporating the water to obtain a paste, and recrystallizing to obtain glycyl-β-alanyl-L-histidine. Wherein, the amount of activated carbon used is 1-10% of the weight of phthalylglycylcarnosine, and the extractant is selected from dichloromethane or toluene, preferably dichloromethane. The recrystallization solvent is selected from ethanol, isopropanol, methanol or acetone, preferably ethanol. Wherein, the decolorization reaction time is 10-60 minutes, specifically 20 minutes.

[0013] Specifically, the present invention provides a method for synthesizing glycyl-β-alanyl-L-histidine, the method comprising: (1) Under nitrogen protection, diisopropylethylamine and L-carnosine are added to dichloromethane, trimethylsilyl chloride is added dropwise in a suspension state, and the mixture is reacted at 0-25° C. After the reaction is completed, a trisilane-carnosine solution is obtained. The molar ratio of L-carnosine, trimethylsilyl chloride and diisopropylethylamine is 1:3.0-4.5:4.0-6.5.

[0014] (2) dissolving phthalylglycine and catalyst DMF in dichloromethane, then dropping oxalyl chloride, and reacting at 0-25°C to obtain phthalylglycyl chloride solution; adding catalyst DMAP to trisilane-carnosine solution, then dropping phthalylglycyl chloride solution, and reacting at 0-25°C to obtain phthalylglycyltrisilanecarnosine. The molar ratio of phthalylglycine, DMF and oxalyl chloride is 1:0.01-0.05:1-2, and the molar ratio of trisilane-carnosine to phthalylglycyl chloride is 1:1.1-1.5.

[0015] (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 after the hydrolysis is complete, adding isopropanol, and performing solid-liquid separation to obtain phthalylglycylcarnosine.

[0016] (4) adding phthalyl glycyl carnosine to hydrazine hydrate ethanol solution, performing hydrazinolysis at 70-80° C., 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, separating the solid and liquid after the decolorization is completed, evaporating the water to obtain a paste, and finally recrystallizing from an ethanol solution to obtain glycyl-β-alanyl-L-histidine. The amount of activated carbon used is 1-10% of the weight of the phthalyl glycyl carnosine.

[0017] The synthesis method provided by the present invention has the following advantages: directly using commercial L-carnosine as a starting material, no need to consider the peptide chain connection problem of alanine and histidine in the reaction, directly using excessive trimethylsilyl chloride for silane protection in dichloromethane, the alanine amino group of L-carnosine and the amino group and carboxyl group on the histidine ring are all protected by silane, directly condensing with phthalylglycyl chloride after the reaction is completed, 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 to obtain phthalylglycylcarnosine, crystallization and purification, simple ethanol hydrazine hydrazine hydrolysis to obtain glycyl-L-carnosine (glycyl-β-alanyl-L-histidine). The traditional method cannot avoid the reaction of the amino groups on the glycine and histidine rings, usually requires multiple protection and deprotection treatments, and the yield is very low (the yield is usually 10-20%), which cannot meet the production needs. The process of the present invention is simple. The two-step reaction products trisilane carnosine and phthalylglycyl chloride do not need to be purified and taken out, and directly condensed, hydrolyzed and crystallized to obtain phthalylglycyl carnosine (phthalylglycyl-β-alanyl-L-histidine). After further purification, simple alcohol hydrazine hydrazine hydrazine hydrolysis is performed, and after extraction and impurity removal, the isoelectric point is adjusted (specifically neutral) and the alcohol crystallizes into a solid to obtain the tripeptide glycyl-L-carnosine (glycyl-β-alanyl-L-histidine). The tripeptide glycyl-L-carnosine (glycyl-β-alanyl-L-histidine) can also be synthesized by a solid phase synthesis method, but the cost is high and it is not suitable for large-scale industrial production (Li Jianxiong, Patent Publication No.: CN113150065A, 2021 / 07 / 23, A Synthetic Peptide and Its Application, US20240002435). BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 It is the liquid chromatography mass spectrum of phthaloylglycylcarnosine (phthaloylglycyl-β-alanyl-L-histidine); Figure 2 It is the mass spectrometry analysis of phthaloylglycylcarnosine (phthaloylglycyl-β-alanyl-L-histidine); Figure 3 It is the liquid chromatography mass spectrum of glycyl-β-alanyl-L-histidine (glycyl-L-carnosine); Figure 4 This is the mass spectrometry analysis of glycyl-β-alanyl-L-histidine (glycyl-L-carnosine). DETAILED DESCRIPTION

[0019] In order to make the purpose, technical solutions and advantages of the present invention more clear, the present invention is described in further detail below.

[0020] Example 1 Synthesis of trisilane-protected carnosine Add 113g (0.5mol) of carnosine to 1000ml of dichloromethane, add 300g (2.3mol) of diisopropylethylamine (DIPEA), protect with nitrogen, cool to 0℃, add 200g (1.8mol) of trimethylchlorosilane dropwise, stir to react. During the addition process, carnosine can be seen to dissolve slowly and gas (overflowing hydrochloric acid) is released. Stir overnight. After the reaction, carnosine is completely dissolved to obtain a red solution for use.

[0021] Example 2 Synthesis of Phthaloyl Glycyl Chloride Add 800 ml of dichloromethane and 6 ml of DMF to 125 g (0.61 mol) of phthalylglycine, cool to 0-5°C, slowly drop 88 g (0.7 mol) of oxalyl chloride, and stir the mixture for 8 hours to obtain a red dichloromethane solution of phthalylglycine chloride.

[0022] Example 3 Synthesis of Phthaloylglycylcarnosine (Phthaloylglycyl-Alanyl-L-Histidine) The above-mentioned red trisilane protected carnosine dichloromethane solution was kept at 0-5°C, DMAP5 grams were added, the dichloromethane solution of phthalyl glycyl chloride was slowly dripped in, and the reaction was stirred for 6 hours after dripping and insulated. 200g of purified water was taken, cooled to 0°C, the reaction solution was slowly dripped in to quench, and the quenching was completed. The dichloromethane phase was extracted twice with 200ml*2 pure water, the aqueous phase was combined, and 50ml of concentrated hydrochloric acid was added and stirred to be acidic, and the hydrolysis was completed in 5 hours. After concentrating most of the water, 2000ml of isopropanol was added and stirred to obtain a yellow solid, and 160g of phthalyl glycyl carnosine was obtained by filtration, with a yield of 77.5%.

[0023] Example 4 Synthesis of Glycyl-L-Carnosine (Glycyl-β-Alanyl-L-Histidine) Phthaloylglycylcarnosine 103g (0.25mol), ethanol 600ml, add hydrazine hydrate 200ml, heat to 78℃, stir and reflux for 4 hours, monitor the reaction by thin layer, and recover the solvent to obtain a yellow oil. Add 200ml of water, extract with dichloromethane to remove impurities, add 5g of activated carbon to the aqueous phase, heat to 60℃ for half an hour to decolorize, filter to obtain a colorless solution, spin dry the aqueous phase to obtain a white paste, stir and beat with 90% ethanol for 10 hours, and filter to obtain 60g of white tripeptide (glycyl-β-alanyl-L-histidine) solid, with a yield of 86% and a purity greater than 99%.

[0024] 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 principle of the present invention should be included in the protection scope 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-carnosine, wherein the silane protective agent is selected from trimethylchlorosilane or hexamethyldiazosilane, the acid dressing 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 acid dressing agent is 1:4.0-6.5; (2) condensing trisilane-carnosine with phthalylglycyl chloride to obtain phthalylglycyltrisilanecarnosine; (3) the reaction solution of step (2) is quenched by adding water, and the aqueous phase is acidified to adjust the pH value to 2-3, and hydrolyzed to obtain phthalylglycylcarnosine; (4) Phthaloylglycylcarnosine is hydrolyzed to give glycyl-β-alanyl-L-histidine.

2. The method for synthesizing glycyl-β-alanyl-L-histidine according to claim 1, characterized in that: The solvent A is selected from dichloromethane, tetrahydrofuran or 1,2-dichloroethane.

3. The method for synthesizing glycyl-β-alanyl-L-histidine according to claim 2, characterized in that: 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-carnosine solution is obtained.

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

5.

5. The method for synthesizing glycyl-β-alanyl-L-histidine according to claim 4, characterized in that: The solvent B is selected from dichloromethane, tetrahydrofuran or 1,2-dichloroethane, and the acylating agent is selected from thionyl chloride or oxalyl chloride.

6. The method for synthesizing glycyl-β-alanyl-L-histidine according to claim 4, characterized in that: Step (3) comprises: adding the reaction of step (2) dropwise into ice water for quenching, separating the liquids after the quenching is completed, 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 phthalylglycylcarnosine.

7. The method for synthesizing glycyl-β-alanyl-L-histidine according to claim 6, characterized in that: 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.

8. The method for synthesizing glycyl-β-alanyl-L-histidine according to claim 1, characterized in that: Step (4) comprises: hydrazinolysis of phthalylglycylcarnosine 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.

9. The method for synthesizing glycyl-β-alanyl-L-histidine according to claim 8, characterized in that: The purification process of step (4) is as follows: adding water to the reaction product, removing impurities by extraction, decolorizing the aqueous phase by activated carbon, separating the solid and liquid after decolorization, evaporating the water to obtain a paste, and recrystallizing to obtain glycyl-β-alanyl-L-histidine, wherein the amount of the activated carbon is 1-10% of the weight of phthalylglycylcarnosine, the extractant is selected from dichloromethane or toluene, and the recrystallization solvent is selected from ethanol, isopropanol, methanol or acetone.

10. The method for synthesizing glycyl-β-alanyl-L-histidine according to claim 1, characterized in that: The method comprises: (1) under nitrogen protection, adding diisopropylethylamine and L-carnosine to dichloromethane, adding trimethylsilyl chloride dropwise in a suspension state, reacting at 0-25° C., and obtaining a trisilane-carnosine solution after the reaction is completed; the molar ratio of L-carnosine, trimethylsilyl chloride and diisopropylethylamine is 1:3.0-4.5:4.0-6.5; (2) dissolving phthalylglycine and a catalyst DMF in dichloromethane, then dropping oxalyl chloride, and reacting at 0-25° C. to obtain a phthalylglycyl chloride solution; adding a catalyst DMAP to a trisilane-carnosine solution, then dropping the phthalylglycyl chloride solution, and reacting at 0-25° C. to obtain phthalylglycyltrisilanecarnosine, 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-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 isopropanol, and performing solid-liquid separation to obtain phthalylglycylcarnosine; (4) adding phthalyl glycyl 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 recrystallization is performed with 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 phthalyl glycyl carnosine.

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