Preparation method and application of small molecule ketone peptide

Abstract

The present invention belongs to the technical field of synthesis and utilization of ketone peptide products, and specifically relates to a method for preparing a small molecule ketone peptide and its application. 3-Hydroxybutyrate, amino acid and solvent pyridine are evenly mixed and placed in a reaction vessel, and reacted at 60-100°C for 5-24h to obtain a small molecule ketone peptide. The present invention uses pyridine as a solvent and catalyst, and the solvent pyridine can be recovered by simple vacuum distillation; in the subsequent separation process, the environmentally friendly solvent ethanol is used to prepare a high-purity small molecule ketone peptide by 0°C refrigerated centrifugation and drying or vacuum distillation. The present invention has a one-step reaction, a simple process, easy operation, and a green and environmentally friendly process, and has broad prospects for industrial application.

Description

Technical Field

[0001] The present invention belongs to the technical field of synthesis and utilization of ketone peptide products, and in particular relates to a preparation method and application of a small molecule ketone peptide. Background Art

[0002] Ketogenic peptides are small peptide molecules composed of 3-hydroxybutyrate and amino acids. In the human body, ketogenic peptides serve as precursors to 3-hydroxybutyrate, transported through the circulatory system to the brain and various tissues and organs. After enzymatic cleavage, 3-hydroxybutyrate molecules and amino acids are obtained. While exerting the effects of 3-hydroxybutyrate in the body, they also enhance the function of amino acids. Numerous studies have shown that 3-hydroxybutyrate is not only an intermediate metabolite but also an important metabolic regulator that can influence gene expression, lipid metabolism, neuronal function, and overall metabolic rate.

[0003] Amino acids are a general term for a class of organic compounds containing amino and carboxyl groups. They are the fundamental building blocks of biologically functional macromolecules and the essential substances for the formation of proteins required for animal nutrition. They play a vital role in the human body. For example, lysine is an essential amino acid for humans and mammals; the body cannot synthesize it on its own and must be supplemented through food. Lysine has positive nutritional benefits in promoting growth and development, enhancing immunity, fighting viruses, promoting fat oxidation, and alleviating anxiety. It also promotes the absorption of certain nutrients and can synergize with other nutrients to enhance the physiological functions of various nutrients. Glycine is widely present in organs and tissues such as connective tissue, muscle tissue, and epidermal tissue, and has positive effects on the human digestive system, metabolic circulatory system, and nervous system. Glycine accelerates the breakdown and conversion of nutrients such as glycogen, fat, and protein in the digestive system, providing the body with energy and essential nutrients.

[0004] Based on the principle of ketone peptide utilization, small-molecule ketone peptides containing 3-hydroxybutyric acid and amino acid structures have the potential to simultaneously exert the therapeutic effects of 3-hydroxybutyric acid and amino acids in the human body. However, there are few reports on the synthesis technology of small-molecule ketone peptides, which severely limits the production and application of ketone peptide products. Patent CN109734575A discloses a method for preparing a 3-hydroxybutyric acid amino acid salt complex. First, 3-hydroxybutyric acid ester is catalytically hydrolyzed to 3-hydroxybutyric acid using an inorganic base catalyst. The prepared 3-hydroxybutyric acid and natural amino acids are then further reacted to obtain the 3-hydroxybutyric acid amino acid salt complex. This patented process involves a two-step reaction, requiring two post-processing operations, multiple process steps, and complex conditions. Therefore, the development of new green technologies and simple processes to prepare small-molecule ketone peptides is of great significance and has broad application value. Summary of the Invention

[0005] To address the current shortage of small-molecule ketone peptide synthesis technologies, the present invention proposes a method for preparing small-molecule ketone peptides and their applications. The prepared small-molecule ketone peptides contain both 3-hydroxybutyric acid and amino acid structures, potentially exerting the effects of both 3-hydroxybutyric acid and amino acids in the human body. Furthermore, the preparation process of the small-molecule ketone peptides presented in the present invention is simple, easy to operate, and environmentally friendly, offering broad prospects for industrial application.

[0006] In order to achieve the above object, the technical solution of the present invention is implemented as follows:

[0007] The 3-hydroxybutyrate, amino acid and pyridine solvent were mixed evenly and placed in a reactor for reaction. After the reaction was completed, the mixture was cooled to room temperature and post-treated to obtain a small molecule ketone peptide.

[0008] Furthermore, the 3-hydroxybutyrate is methyl 3-hydroxybutyrate or ethyl 3-hydroxybutyrate.

[0009] Furthermore, the amino acid is selected from lysine, glycine or arginine.

[0010] Furthermore, the mass ratio of the 3-hydroxybutyrate to the amino acid is (1-2): (1-2).

[0011] Furthermore, the concentration of the amino acid in the solvent pyridine is 0.05-0.2 g / mL.

[0012] Furthermore, the reaction temperature is 60-100° C., and the reaction time is 5-24 hours.

[0013] Furthermore, post-treatment includes the following two methods. One post-treatment method is: after the reaction is completed, the mixture is cooled to room temperature to obtain a solid-liquid mixture, and the solvent pyridine is removed by vacuum distillation at 80°C to obtain a solid product; ethanol solvent is added to the solid product, stirred and dispersed, and the mixture is kept at 0-5°C for 1 hour; the mixture is then centrifuged in a 0°C refrigerated centrifuge, the solid layer is removed, and the ethanol washing and refrigerated centrifugation are repeated twice to remove the solid layer, and the small molecule ketone peptide is obtained after drying at 70°C. The other post-treatment method is: after the reaction is completed, the mixture is cooled to room temperature to obtain a solid-liquid mixture, and the solvent pyridine is removed by vacuum distillation at 80°C to obtain a solid product; ethanol solvent is added to the solid product, stirred and dispersed, and the mixture is kept at 0-5°C for 1 hour; the upper ethanol solution is removed by vacuum rotary evaporation at 60°C to obtain the small molecule ketone peptide. When the obtained product is insoluble in ethanol solvent, the first post-treatment method is used; when the obtained product is soluble in ethanol solvent, the second post-treatment method is used.

[0014] The small molecule ketone peptide prepared by the above preparation method.

[0015] Application of the above-mentioned small molecule ketone peptides in health products and medicines.

[0016] The beneficial effects produced by the present invention are:

[0017] The present invention provides a method for preparing small-molecule ketone peptides. In this process, 3-hydroxybutyrate, an amino acid, and a pyridine solvent are uniformly mixed and placed in a reactor. The small-molecule ketone peptide is prepared through a simple one-step reaction at a temperature of 60-100°C. Pyridine serves as a solvent, capable of dissolving both 3-hydroxybutyrate and an amino acid (such as lysine) at the reaction temperature. It also acts as an organic base catalyst, as the nitrogen in pyridine has a lone pair of electrons, catalyzing the acylation reaction between 3-hydroxybutyrate and the amino acid, thereby preparing the small-molecule ketone peptide. Furthermore, the pyridine solvent can be recovered by simple rotary evaporation. In the subsequent product isolation process, high-purity small-molecule ketone peptide products can be obtained using the environmentally friendly solvent ethanol through refrigerated centrifugation, drying, or further vacuum rotary evaporation. The present invention features simple processes, convenient operation, and an environmentally friendly process, promising broad prospects for industrial application. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0019] Figure 1 This is a physical picture of the product obtained in Example 1 of the present invention.

[0020] Figure 2 This is a high-resolution mass spectrometry analysis spectrum (positive ion mode and negative ion mode) of the product obtained in Example 1 of the present invention.

[0021] Figure 3 This is an infrared spectrum of methyl 3-hydroxybutyrate, lysine, and the resulting product in Example 1 of the present invention.

[0022] Figure 4 This is the H NMR spectrum of the product obtained in Example 1 of the present invention.

[0023] Figure 5 This is the NMR carbon spectrum of the product obtained in Example 1 of the present invention.

[0024] Figure 6 This is the structural formula of the product obtained in Example 1 of the present invention.

[0025] Figure 7 This is a physical picture of the product obtained in Example 6 of the present invention.

[0026] Figure 8The chromatogram of the liquid chromatography-mass spectrometry analysis of the product obtained in Example 6 of the present invention is shown.

[0027] Figure 9 This is an infrared spectrum of methyl 3-hydroxybutyrate, glycine, and the resulting product in Example 6 of the present invention.

[0028] Figure 10 This is the H NMR spectrum of the product obtained in Example 6 of the present invention.

[0029] Figure 11 is the structural formula of the product obtained in Example 6 of the present invention. DETAILED DESCRIPTION

[0030] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without creative work are within the scope of protection of the present invention.

[0031] Example 1

[0032] The preparation method of a small molecule ketone peptide in this embodiment comprises the following steps:

[0033] (1) 8 g of methyl 3-hydroxybutyrate, 4 g of lysine, and 40 mL of pyridine were added to a 100 mL reactor. The mechanical stirring rate was controlled at 250 rpm, and the temperature was raised to 90°C and the reaction was carried out at this temperature for 8 h. After the reaction was completed, the mixture was cooled to room temperature and the product was transferred to a rotary evaporator for rotary evaporation. The solvent pyridine was recovered by rotary distillation at 80°C under reduced pressure to obtain a solid product.

[0034] (2) Add 15 mL of anhydrous ethanol to the solid product obtained in step (1), shake well and store in a refrigerator for 1 hour. Then centrifuge the sample in a refrigerated centrifuge at 0°C to obtain two phases of solid-liquid separation: the lower layer is the solid product and the upper liquid is the ethanol solution. Add 15 mL of anhydrous ethanol to the solid product obtained, repeat the ethanol washing-refrigerated centrifugation process twice, and dry the lower solid product obtained after three centrifugations at 70°C to obtain 5.9 g of light yellow solid (see Figure 1 ).

[0035] (3) The light yellow solid obtained in step (2) is easily soluble in water. The aqueous solution is diluted to 1 ppm and analyzed by high-resolution liquid chromatography-mass spectrometry (see Figure 2 ), in both positive and negative ion modes, there was only one main product peak, and the molecular weight of the product was 232 (the recommended molecular formula was C 10 H 20 N2O4).

[0036] Further comparative analysis of the obtained product with the raw materials 3-hydroxybutyric acid methyl ester and lysine was performed by infrared spectroscopy (see Figure 3 ), the obtained product retained the main functional groups of 3-hydroxybutyric acid methyl ester and lysine, and formed a new amide bond (such as peak 1640cm -1 and 3320cm -1 ); After the obtained product was subjected to nuclear magnetic hydrogen spectrum (see Figure 4 ) and NMR carbon spectroscopy (see Figure 5 ) analysis, it was found to contain functional groups such as carboxyl, hydroxyl, peptide bond, methyl and amino groups. Based on the structural characteristics of the reaction raw materials 3-hydroxybutyric acid methyl ester and lysine, comprehensive high-resolution liquid chromatography-mass spectrometry analysis, infrared spectroscopy, nuclear magnetic resonance and other analyses, it was determined that the product was 2-amino-6-[(3-hydroxy-1-oxyylidenebutyl)amino]hexanoic acid (see Figure 6 ).

[0037] Example 2

[0038] The preparation method of a small molecule ketone peptide in this embodiment comprises the following steps:

[0039] (1) 6 g of methyl 3-hydroxybutyrate, 6 g of lysine, and 30 mL of pyridine were added to a 100 mL reactor. The mechanical stirring rate was controlled at 250 rpm, and the temperature was raised to 100°C and the reaction was carried out at this temperature for 5 h. After the reaction was completed, the mixture was cooled to room temperature and the product was transferred to a rotary evaporator for rotary evaporation. The solvent pyridine was recovered by rotary distillation at 80°C under reduced pressure to obtain a solid product.

[0040] (2) 15 mL of anhydrous ethanol was added to the solid product obtained in step (1), and the mixture was thoroughly shaken and stored in a refrigerator for 1 hour. The sample was then centrifuged in a refrigerated centrifuge at 0°C to obtain two phases of solid-liquid separation: the lower layer was the solid product, and the upper liquid layer was the ethanol solution. 15 mL of anhydrous ethanol was further added to the obtained solid product, and the ethanol washing-refrigerated centrifugation process was repeated twice. The lower solid product obtained after the three centrifugations was dried at 70°C to obtain 7.1 g of 2-amino-6-[(3-hydroxy-1-oxyylidenebutyl)amino]hexanoic acid.

[0041] Example 3

[0042] The preparation method of a small molecule ketone peptide in this embodiment comprises the following steps:

[0043] (1) 3 g of methyl 3-hydroxybutyrate, 3 g of lysine, and 60 mL of pyridine were added to a 100 mL reactor. The mechanical stirring rate was controlled at 250 rpm, and the temperature was raised to 60°C and the reaction was carried out at this temperature for 24 h. After the reaction was completed, the mixture was cooled to room temperature and the product was transferred to a rotary evaporator for rotary evaporation. The solvent pyridine was recovered by rotary distillation at 80°C under reduced pressure to obtain a solid product.

[0044] (2) 15 mL of anhydrous ethanol was added to the solid product obtained in step (1), and the mixture was thoroughly shaken and stored in a refrigerator for 1 hour. The sample was then centrifuged in a refrigerated centrifuge at 0°C to obtain two phases of solid-liquid separation: the lower layer was the solid product, and the upper liquid layer was the ethanol solution. 15 mL of anhydrous ethanol was added to the obtained solid product, and the ethanol washing-refrigerated centrifugation process was repeated twice. The lower solid product obtained after the three centrifugations was dried at 70°C to obtain 3.3 g of 2-amino-6-[(3-hydroxy-1-oxyylidenebutyl)amino]hexanoic acid.

[0045] Example 4

[0046] The preparation method of a small molecule ketone peptide in this embodiment comprises the following steps:

[0047] (1) 6 g of methyl 3-hydroxybutyrate, 4 g of lysine, and 60 mL of pyridine were added to a 100 mL reactor. The mechanical stirring rate was controlled at 250 rpm, and the temperature was raised to 70°C and the reaction was carried out at this temperature for 12 h. After the reaction was completed, the mixture was cooled to room temperature and the product was transferred to a rotary evaporator for rotary evaporation. The solvent pyridine was recovered by rotary distillation at 80°C under reduced pressure to obtain a solid product.

[0048] (2) 15 mL of anhydrous ethanol was added to the solid product obtained in step (1), and the mixture was thoroughly shaken and stored in a refrigerator for 1 hour. The sample was then centrifuged in a refrigerated centrifuge at 0°C to obtain two phases of solid-liquid separation: the lower layer was the solid product, and the upper liquid layer was the ethanol solution. 15 mL of anhydrous ethanol was further added to the obtained solid product, and the ethanol washing-refrigerated centrifugation process was repeated twice. The lower solid product obtained after the three centrifugations was dried at 70°C to obtain 4.4 g of 2-amino-6-[(3-hydroxy-1-oxyylidenebutyl)amino]hexanoic acid.

[0049] Example 5

[0050] The preparation method of a small molecule ketone peptide in this embodiment comprises the following steps:

[0051] (1) 8 g of ethyl 3-hydroxybutyrate, 4 g of lysine, and 60 mL of pyridine were added to a 100 mL reactor. The mechanical stirring rate was controlled at 250 rpm, and the temperature was raised to 80°C and the reaction was carried out at this temperature for 12 h. After the reaction was completed, the mixture was cooled to room temperature and the product was transferred to a rotary evaporator for rotary evaporation. The solvent pyridine was recovered by rotary distillation at 80°C under reduced pressure to obtain a solid product.

[0052] (2) 15 mL of anhydrous ethanol was added to the solid product obtained in step (1), and the mixture was thoroughly shaken and stored in a refrigerator for 1 hour. The sample was then centrifuged in a refrigerated centrifuge at 0°C to obtain two phases of solid-liquid separation: the lower layer was the solid product, and the upper liquid layer was the ethanol solution. 15 mL of anhydrous ethanol was further added to the obtained solid product, and the ethanol washing-refrigerated centrifugation process was repeated twice. The lower solid product obtained after the three centrifugations was dried at 70°C to obtain 4.3 g of 2-amino-6-[(3-hydroxy-1-oxyylidenebutyl)amino]hexanoic acid.

[0053] Example 6

[0054] The preparation method of a small molecule ketone peptide in this embodiment comprises the following steps:

[0055] (1) 4 g of methyl 3-hydroxybutyrate, 8 g of glycine, and 40 mL of pyridine were added to a 100 mL reactor. The mechanical stirring rate was controlled at 250 rpm, and the temperature was raised to 90°C and the reaction was carried out at this temperature for 9 h. After the reaction was completed, the mixture was cooled to room temperature and the product was transferred to a rotary evaporator for rotary evaporation. The solvent pyridine was recovered by rotary distillation at 80°C under reduced pressure to obtain a viscous solid product.

[0056] (2) Add 30 mL of anhydrous ethanol to the viscous solid product obtained in step (1), shake well, and store in a refrigerator for 1 hour. Then centrifuge the sample in a refrigerated centrifuge at 0°C to obtain two phases of solid-liquid separation: the lower solid is glycine that has not reacted completely, and the upper liquid is an ethanol solution containing the solid product. The obtained ethanol solution is subjected to vacuum rotary evaporation at 60°C to finally obtain 4.6 g of a yellow viscous solid (see Figure 7 ).

[0057] (3) The viscous solid obtained in step (2) is easily soluble in water. High-resolution liquid chromatography-mass spectrometry analysis revealed that there is only one major product, zinc, and the molecular weight of the product is 161 (the recommended molecular formula is C6H 11 NO4) (see Figure 8 )

[0058] Further comparative analysis of the obtained product with the raw materials 3-hydroxybutyric acid methyl ester and glycine was performed by infrared spectroscopy (see Figure 9 ), the obtained product retained the main functional groups of 3-hydroxybutyric acid methyl ester and glycine, and formed a new amide bond (such as the peak at 1600cm -1 and 3300cm -1 ); After the obtained product was subjected to nuclear magnetic hydrogen spectrum (see Figure 10) analysis, it was found to contain functional groups such as carboxyl, hydroxyl, peptide bond, methyl and methylene groups. Based on the structural characteristics of the reaction raw materials 3-hydroxybutyric acid methyl ester and glycine, comprehensive high-resolution liquid chromatography-mass spectrometry analysis, infrared spectroscopy, nuclear magnetic hydrogen spectrum analysis, etc., it was determined that N-(3-hydroxy-1-oxyylidenebutyl)glycine (see Figure 11 ).

[0059] Example 7

[0060] The preparation method of a small molecule ketone peptide in this embodiment comprises the following steps:

[0061] (1) 4 g of methyl 3-hydroxybutyrate, 4 g of glycine, and 20 mL of pyridine were added to a 100 mL reactor. The mechanical stirring rate was controlled at 250 rpm, and the temperature was raised to 100°C and the reaction was carried out at this temperature for 6 h. After the reaction was completed, the mixture was cooled to room temperature and the product was transferred to a rotary evaporator for rotary evaporation. The solvent pyridine was recovered by rotary distillation at 80°C under reduced pressure to obtain a viscous solid product.

[0062] (2) 30 mL of anhydrous ethanol was added to the viscous solid product obtained in step (1), and the mixture was thoroughly shaken and stored in a refrigerator for 1 hour. The sample was then centrifuged in a refrigerated centrifuge at 0°C to obtain two phases of solid-liquid separation: the lower solid phase was glycine that had not reacted completely, and the upper liquid phase was an ethanol solution containing the obtained product. The obtained ethanol solution was subjected to vacuum rotary evaporation at 60°C to obtain 3.9 g of N-(3-hydroxy-1-oxyylidenebutyl)glycine.

[0063] Example 8

[0064] The preparation method of a small molecule ketone peptide in this embodiment comprises the following steps:

[0065] (1) 4 g of methyl 3-hydroxybutyrate, 6 g of glycine, and 80 mL of pyridine were added to a 100 mL reactor. The mechanical stirring rate was controlled at 250 rpm, and the temperature was raised to 60°C and the reaction was carried out at this temperature for 24 h. After the reaction was completed, the mixture was cooled to room temperature and the product was transferred to a rotary evaporator for rotary evaporation. The solvent pyridine was recovered by rotary distillation at 80°C under reduced pressure to obtain a viscous solid product.

[0066] (2) 30 mL of anhydrous ethanol was added to the viscous solid product obtained in step (1), and the mixture was thoroughly shaken and stored in a refrigerator for 1 hour. The sample was then centrifuged in a refrigerated centrifuge at 0°C to obtain two phases of solid-liquid separation: the lower solid phase was glycine that had not reacted completely, and the upper liquid phase was an ethanol solution containing the obtained product. The obtained ethanol solution was subjected to vacuum rotary evaporation at 60°C to obtain 4.3 g of N-(3-hydroxy-1-oxyylidenebutyl)glycine.

[0067] Example 9

[0068] The preparation method of a small molecule ketone peptide in this embodiment comprises the following steps:

[0069] (1) 4 g of methyl 3-hydroxybutyrate, 8 g of glycine, and 60 mL of pyridine were added to a 100 mL reactor. The mechanical stirring rate was controlled at 250 rpm, and the temperature was raised to 80°C and the reaction was carried out at this temperature for 15 h. After the reaction was completed, the mixture was cooled to room temperature and the product was transferred to a rotary evaporator for rotary evaporation. The solvent pyridine was recovered by rotary distillation at 80°C under reduced pressure to obtain a viscous solid product.

[0070] (2) 30 mL of anhydrous ethanol was added to the viscous solid product obtained in step (1), and the mixture was thoroughly shaken and stored in a refrigerator for 1 hour. The sample was then centrifuged in a refrigerated centrifuge at 0°C to obtain two phases of solid-liquid separation: the lower solid phase was glycine that had not reacted completely, and the upper liquid phase was an ethanol solution containing the obtained product. The obtained ethanol solution was subjected to vacuum rotary evaporation at 60°C to obtain 4.4 g of N-(3-hydroxy-1-oxyylidenebutyl)glycine.

[0071] Example 10

[0072] The preparation method of a small molecule ketone peptide in this embodiment comprises the following steps:

[0073] (1) 4 g of ethyl 3-hydroxybutyrate, 8 g of glycine, and 80 mL of pyridine were added to a 100 mL reactor. The mechanical stirring rate was controlled at 250 rpm, and the temperature was raised to 90°C and the reaction was carried out at this temperature for 15 h. After the reaction was completed, the mixture was cooled to room temperature and the product was transferred to a rotary evaporator for rotary evaporation. The solvent pyridine was recovered by rotary distillation at 80°C under reduced pressure to obtain a viscous solid product.

[0074] (2) 30 mL of anhydrous ethanol was added to the viscous solid product obtained in step (1), and the mixture was thoroughly shaken and stored in a refrigerator for 1 hour. The sample was then centrifuged in a refrigerated centrifuge at 0°C to obtain two phases of solid-liquid separation: the lower solid phase was glycine that had not reacted completely, and the upper liquid phase was an ethanol solution containing the obtained product. The obtained ethanol solution was subjected to vacuum rotary evaporation at 60°C to obtain 4.3 g of N-(3-hydroxy-1-oxyylidenebutyl)glycine.

[0075] Example 11

[0076] The preparation method of a small molecule ketone peptide in this embodiment comprises the following steps:

[0077] (1) 3 g of methyl 3-hydroxybutyrate, 3 g of arginine, and 60 mL of pyridine were added to a 100 mL reactor. The mechanical stirring rate was controlled at 250 rpm, and the temperature was raised to 100°C and the reaction was carried out at this temperature for 24 h. After the reaction was completed, the mixture was cooled to room temperature and the product was transferred to a rotary evaporator for rotary evaporation. The solvent pyridine was recovered by rotary distillation at 80°C under reduced pressure to obtain a solid product.

[0078] (2) Add 15 mL of anhydrous ethanol to the solid product obtained in step (1), shake well, and freeze for 1 hour. Then centrifuge the sample in a refrigerated centrifuge at 0°C to obtain two phases of solid-liquid separation: the lower layer is the solid product, and the upper liquid layer is the ethanol solution. Add 15 mL of anhydrous ethanol to the obtained solid product, repeat the ethanol washing-refrigerated centrifugation process twice, and dry the lower solid product obtained after three centrifugations at 70°C to obtain the arginine-based ketone peptide small molecule 2-amino-5-({nitrogen subunit [(3-hydroxy-1-oxygen subunit butyl) amino] methyl} amino) pentanoic acid.

[0079] Example 12

[0080] The preparation method of a small molecule ketone peptide in this embodiment comprises the following steps:

[0081] (1) 6 g of methyl 3-hydroxybutyrate, 3 g of arginine, and 60 mL of pyridine were added to a 100 mL reactor. The mechanical stirring rate was controlled at 250 rpm, and the temperature was raised to 60°C and the reaction was carried out at this temperature for 24 h. After the reaction was completed, the mixture was cooled to room temperature and the product was transferred to a rotary evaporator for rotary evaporation. The solvent pyridine was recovered by rotary distillation at 80°C under reduced pressure to obtain a solid product.

[0082] (2) Add 15 mL of anhydrous ethanol to the solid product obtained in step (1), shake well, and freeze for 1 hour. Then centrifuge the sample in a refrigerated centrifuge at 0°C to obtain two phases of solid-liquid separation: the lower layer is the solid product, and the upper liquid layer is the ethanol solution. Add 15 mL of anhydrous ethanol to the obtained solid product, repeat the ethanol washing-refrigerated centrifugation process twice, and dry the lower solid product obtained after three centrifugations at 70°C to obtain the arginine-based ketone peptide small molecule 2-amino-5-({nitrogen subunit [(3-hydroxy-1-oxygen subunit butyl) amino] methyl} amino) pentanoic acid.

[0083] 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 preparing a small molecule ketone peptide, characterized in that: The method comprises the following steps: adding 3-hydroxybutyrate and amino acid in a mass ratio of (1-2): (1-2) to a solvent pyridine, wherein the concentration of the amino acid in pyridine is 0.05-0.2 g / mL; mixing the mixture evenly; placing the mixture in a reaction vessel; reacting the mixture at a temperature of 60-100° C. for 5-24 hours; cooling the mixture to room temperature after the reaction is completed; and removing the solvent pyridine by vacuum distillation to obtain a solid product; adding an ethanol solvent to the solid product, stirring and dispersing the mixture thoroughly, and keeping the mixture warm at 0-5° C. for 1 hour; centrifuging the mixture in a 0° C. refrigerated centrifuge, and then drying the lower solid or vacuum distilling the upper ethanol solution to obtain a small molecule ketone peptide, depending on whether the product is soluble in the ethanol solvent; wherein the amino acid is selected from lysine, glycine, or arginine.

2. The method for preparing a small molecule ketone peptide according to claim 1, characterized in that: The 3-hydroxybutyrate is methyl 3-hydroxybutyrate or ethyl 3-hydroxybutyrate.

3. The method for preparing a small molecule ketone peptide according to claim 2, characterized in that: The temperature of the reduced pressure distillation during the process of removing the pyridine solvent is 80°C, the drying temperature is 70°C, and the temperature of the reduced pressure distillation during the process of removing the ethanol solution is 60°C.

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