Amino acid dipeptide

Abstract

Disclosed in the present invention is an amino acid dipeptide. The amino acid dipeptide provided by the present invention has good bioavailability, shows a good comprehensive effect, and is particularly suitable for being used in dietary or nutritional supplements; in addition, the present invention involves a controllable preparation process and low cost, and is environmentally friendly.

Description

Amino acid dipeptides TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological chemical industry, and particularly relates to amino acid dipeptides. BACKGROUND

[0002] With the rapid development of economy and science, people's demand for dietary or nutritional supplements is growing, aiming to improve the health of individuals and reduce the risk of disease.

[0003] People have realized the numerous health benefits of amino acids, including enhancing immunity, enhancing muscle strength, etc. Citrulline can increase vasodilation, lower blood pressure, improve performance and endurance, remove harmful compounds, and maintain muscle strength. Arginine participates in the ornithine cycle in the human body, promotes the formation of urea, converts the ammonia produced in the human body into non-toxic urea through the ornithine cycle, and is excreted from the urine, thereby reducing the blood ammonia concentration; having a higher concentration of hydrogen ions helps to correct the acid-base balance in hepatic encephalopathy. Ornithine mainly participates in the urea cycle and plays an important role in the excretion of ammonia nitrogen in the body; it can also help sleep and reduce fatigue. Hydroxyisoleucine has the effects of promoting insulin secretion and anti-diabetic activity. The deficiency of using amino acids alone is that arginine has strong bitter, astringent and fishy odor, which is difficult to mask by perfuming and flavoring, and reduces the consumer's use pleasure. Some amino acids have strong hygroscopicity, which also affects their application.

[0004] Two amino acid molecules can condense to form an amino acid dipeptide, which can be hydrolyzed by dipeptidase in the body to form two free amino acids, thereby supplementing amino acids. At present, there are few researches and reports on amino acid dipeptides, and how to effectively prepare amino acid dipeptides and make them widely used in the fields of dietary supplements, food, nutritional products, health products, etc. is the focus of research for those skilled in the art. SUMMARY

[0005] In one aspect, the present application provides an amino acid dipeptide formed by an amino acid I and an amino acid II, wherein the amino acid I and the amino acid II are independently selected from one of the following: citrulline, arginine, ornithine, hydroxyisoleucine.

[0006] In some embodiments, the carboxyl group of the amino acid I is connected to the amino group of the amino acid II, the amino acid I is one of citrulline, arginine, ornithine, and hydroxyisoleucine, and the amino acid II is one of citrulline, ornithine, and hydroxyisoleucine.

[0007] In some embodiments, the amino acid dipeptide is a citrulline dipeptide, an arginine-citrulline dipeptide, an ornithine dipeptide, or a hydroxyisoleucine dipeptide.

[0008] In some embodiments, the amino acid dipeptide has the following structure:

[0009] In some embodiments, the citrulline dipeptide has the following NMR data: 1 H NMR (400 MHz, DMSO-d6 + 2d hydrochloric acid): δ 8.90 (d, 1H), 8.33 (d, 3H), 4.14-4.19 (m, 1H), 3.83-3.86 (m, 1H), 3.04-3.08 (m, 4H), 1.46-1.75 (m, 8H).

[0010] In some embodiments, the arginine-citrulline dipeptide has the following NMR data: 1 H NMR (400 MHz, D2O): δ 4.14-4.19 (m, 1H), 3.82-3.85 (m, 1H), 3.02-3.08 (m, 4H), 1.49-1.77 (m, 8H).

[0011] In some embodiments, the ornithine dipeptide has the following NMR data: 1 H NMR (400 MHz, D2O): δ 4.53-4.57 (m, 1H), 3.18-3.25 (m, 1H), 2.58-2.67 (m, 4H), 1.73-1.82 (m, 4H), 1.49-1.61 (m, 4H).

[0012] In some embodiments, the hydroxyisoleucine dipeptide has the following NMR data: 1 H NMR (400 MHz, D2O): δ 4.18-4.27 (m, 1H), 3.48-3.53 (m, 2H), 3.33-3.42 (m, 1H), 2.38-2.47 (m, 1H), 1.96-2.01 (m, 1H), 1.11 (dd, 6H), 0.93 (dd, 6H).

[0013] In some embodiments, the amino acid dipeptide is prepared as a food, beverage, supplement, nutraceutical.

[0014] In some embodiments, the amino acid dipeptide is prepared as a solid formulation or a liquid formulation.

[0015] In another aspect, the present application provides a method for preparing the amino acid dipeptide as described above, comprising the following steps: a. reacting amino acid I with Boc anhydride under alkaline conditions to obtain Boc-amino acid I; b. preparing amino acid II to obtain amino acid II carboxylate hydrochloride; c. reacting Boc-amino acid I, a condensing agent, amino acid II carboxylate hydrochloride, a base in a reaction solvent to obtain Boc-amino acid I-amino acid II carboxylate; d. hydrolyzing Boc-amino acid I-amino acid II carboxylate by reacting with a base, and then removing Boc to obtain the amino acid dipeptide.

[0016] In some embodiments, the condensing agent in step c is selected from one or more of the following: EDCI, DCC, HATU, HOSU, HOBt, DMAP; the base is selected from one or more of the following: triethylamine, DIPEA, N-methylmorpholine, pyridine; and the reaction solvent is selected from one or more of the following: dichloromethane, N,N-dimethylformamide, THF, dioxane, NMP.

[0017] In some embodiments, when amino acid I is arginine, the arginine is first nitrolyzed to nitroarginine, and then step a is performed to react nitroarginine with Boc anhydride under alkaline conditions to obtain Boc-nitroarginine as Boc-amino acid I; and step d is performed to reduce after removing Boc to obtain the amino acid dipeptide.

[0018] In some embodiments, when amino acid II is ornithine, the ornithine is first prepared to obtain Cbz-ornithine, and then step b is performed to obtain Cbz-ornithine methyl ester hydrochloride as amino acid II carboxylate hydrochloride; and step d is performed to reduce after removing Boc to obtain the amino acid dipeptide.

[0019] In some embodiments, when amino acid II is hydroxyisoleucine, step b is to react hydroxyisoleucine with hydrochloric acid to obtain cyclo-hydroxyisoleucine hydrochloride as amino acid II carboxylate hydrochloride.

[0020] In some embodiments, when amino acid I is hydroxyisoleucine, the hydroxyisoleucine is first reacted with hydrochloric acid to obtain cyclo-hydroxyisoleucine hydrochloride, and then step a is performed to react cyclo-hydroxyisoleucine hydrochloride with Boc anhydride under alkaline conditions to obtain hydroxy Boc-isoleucine as Boc-amino acid I.

[0021] The present application can efficiently and stably produce amino acid dipeptides, has the advantages of controllable preparation process, low energy consumption and material consumption, etc. In addition, the amino acid dipeptides of the present application have good bioavailability and exhibit good comprehensive effects when applied to subjects, and can have wide application prospects in the field of diet or nutritional supplements or food. DETAILED DESCRIPTION

[0022] Reference will now be made in detail to the preferred embodiments of the application, examples of which are illustrated in the accompanying drawings. While the application will be described in conjunction with the preferred embodiments, it will be understood that they are not intended to limit the application to these embodiments. On the contrary, the application is intended to cover alternatives, modifications, and equivalents, which can be included within the spirit and scope of the application as defined by the claims. Furthermore, in the following detailed description of the application, numerous specific details are set forth in order to provide a thorough understanding of the application. However, it will be apparent to one of ordinary skill in the art that the application can be practiced without these specific details. In other instances, well-known methods, procedures, components, and other features are not described in detail in order to avoid unnecessarily obscuring aspects of the application.

[0023] The following abbreviations or shorthand are used in the present application: Boc anhydride: di-tert-butyl dicarbonate; Boc: tert-butyloxycarbonyl; EDCI: l-(3-dimethylaminopropyl)-3- ethylcarbodiimide; DCC: dicyclohexylcarbodiimide; HATU: 2-(7-azobenzotriazol)-N,N,N',N'- tetramethyluronium hexafluorophosphate; HOSU: N-hydroxysuccinimide; HOBt: 1-hydroxybenzotriazole; DMAP: 4-dimethylaminopyridine; DIPEA: N,N-diisopropylethylamine; THF: tetrahydrofuran; NMP: N-methylpyrrolidone; Cbz: benzyloxycarbonyl; Cbz-Cl: benzyl chloroformate; DMF: N,N-dimethylformamide; TLC: thin layer chromatography; DCM: dichloromethane; MS: mass spectrometry.

[0024] As used herein, the term "or" is intended to mean "and / or," unless otherwise indicated by context. In other words, the term "or" as used herein shall not be interpreted as being exclusive.

[0025] As used herein, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0026] As used herein, the terms "comprises," "comprising," "includes," "including" and the like are meant to be inclusive, not exclusive, unless explicitly indicated otherwise. They also include the phrase "consisting essentially of. They further include the phrase "consisting of.

[0027] As used herein, the term "physiologically acceptable" refers to pharmaceutically, physiologically, dietary, nutritionally acceptable, and refers to those compositions or agents, materials or combinations of compositions and / or dosage forms thereof that are within the scope of sound medical judgment, suitable for contact with human and animal tissues, compatible with other ingredients of the composition, without excessive toxicity, irritation, allergic response or other problems or complications, and commensurate with a reasonable benefit / risk ratio.

[0028] In some embodiments, the amino acid dipeptides of the present invention can be prepared as compositions together with physiologically acceptable carriers. In the present invention, the administration form of the composition involves a liquid or solid filler, diluent, excipient, solvent or encapsulating material. Each carrier must be "acceptable" in the sense that it is compatible with the other ingredients of the composition and is not harmful to the subject, i.e., suitable for consumption or nutritionally acceptable.

[0029] In some embodiments, the amino acid dipeptides of the present invention can be administered with other supplements, such as vitamins, minerals, nootropics, and other supplements known in the art. The amino acid dipeptides of the present invention can be prepared into food and beverage products and nutritional supplements for human consumption.

[0030] The following examples are illustrative of selected embodiments of the present invention and are not intended to limit the scope of the invention.

[0031] The experimental methods described in the examples are conventional methods unless otherwise specified; the reagents and materials used are commercially available unless otherwise specified.

[0032] The amino acid dipeptide of the present invention can be prepared by the following method: a. reacting amino acid I with Boc anhydride under alkaline conditions to obtain Boc-amino acid I; b. preparing amino acid II carboxylate hydrochloride from amino acid II; c. reacting Boc-amino acid I, a condensing agent, amino acid II carboxylate hydrochloride, and a base in a reaction solvent to obtain Boc-amino acid I-amino acid II carboxylate; d. hydrolyzing the Boc-amino acid I-amino acid II carboxylate with a base, and then removing the Boc to obtain the amino acid dipeptide. The above reactions can be carried out in a suitable solvent at an appropriate temperature.

[0033] wherein amino acid I and amino acid II are independently selected from one of the following: citrulline, arginine, ornithine, hydroxyisoleucine. The basic condition of step a can be selected from one or more of the following: sodium hydroxide, sodium bicarbonate, sodium carbonate, potassium hydroxide, potassium carbonate, triethylamine, etc. In step b, amino acid II can be reacted with methanol and a chlorinating agent, which can be selected from one or more of the following: thionyl chloride, oxalyl chloride, phosphorus pentachloride, etc. In step c, the condensing agent can be selected from one or more of the following: EDCI, DCC, HATU, HOSU, HOBt, DMAP; the base can be selected from one or more of the following: triethylamine, DIPEA, N-methylmorpholine, pyridine; the reaction solvent can be selected from one or more of the following: dichloromethane, N,N-dimethylformamide, THF, dioxane, NMP. The base in step d can be lithium hydroxide, sodium hydroxide, potassium hydroxide, etc.

[0034] In addition, when amino acid I is arginine, arginine can be first nitrolyzed to nitroarginine, and then step a is performed to react the nitroarginine with Boc anhydride under basic condition to obtain Boc-nitroarginine as Boc-amino acid I; after removal of Boc in step d, a reduction reaction can be performed with hydrogen in the presence of palladium on carbon to obtain the amino acid dipeptide of the present application. When amino acid II is ornithine, ornithine can be first prepared, such as reacting with Cbz-Cl in the presence of a base to obtain Cbz-ornithine, and then step b is performed to react amino acid II with methanol and a chlorinating agent to obtain Cbz-ornithine methylester hydrochloride as amino acid II carboxylic acid ester hydrochloride; after removal of Boc in step d, a reduction reaction is performed with hydrogen in the presence of palladium on carbon to obtain the amino acid dipeptide of the present application. When amino acid II is hydroxyisoleucine, step b is to react hydroxyisoleucine with hydrochloric acid to obtain cyclo-hydroxyisoleucine hydrochloride as amino acid II carboxylic acid ester hydrochloride. When amino acid I is hydroxyisoleucine, hydroxyisoleucine is first reacted with hydrochloric acid to obtain cyclo-hydroxyisoleucine hydrochloride, and then step a is performed to react the cyclo-hydroxyisoleucine hydrochloride with Boc anhydride under basic condition to obtain hydroxy Boc-isoleucine as Boc-amino acid I.

[0035] To L-citrulline (50 g, 1.0 eq) in 200 mL water, add 100 mL tetrahydrofuran, add sodium hydroxide (22.8 g, 2.0 eq), stir to dissolve, control temperature 0-10 °C, dropwise add Boc anhydride (74.7 g, 1.2 eq). After dropwise addition, warm to 20-25 °C and react for 6-8 h, TLC shows reaction is complete. Add 50 mL heptane, stir for 15 min, allow to separate into layers, discard organic layer. Adjust pH of aqueous layer to 3-4 with citric acid, extract with n-butanol 200 mL x 3, combine organic layers, concentrate under reduced pressure to give the target product 72.3 g, 92% yield. L-citrulline methyl ester hydrochloride preparation

[0036] To L-citrulline (50 g, 1.0 eq) in 200 mL water, add 100 mL tetrahydrofuran, add sodium hydroxide (22.8 g, 2.0 eq), stir to dissolve, control temperature 0-10 °C, dropwise add Boc anhydride (74.7 g, 1.2 eq). After dropwise addition, warm to 20-25 °C and react for 6-8 h, TLC shows reaction is complete. Add 50 mL heptane, stir for 15 min, allow to separate into layers, discard organic layer. Adjust pH of aqueous layer to 3-4 with citric acid, extract with n-butanol 200 mL x 3, combine organic layers, concentrate under reduced pressure to give the target product 72.3 g, 92% yield. L-citrulline methyl ester hydrochloride preparation

[0037] To 50 g Boc-L-citrulline in 250 mL DMF, add EDCI (41.8 g, 1.2 eq) / HOBT (14.7 g, 0.5 eq), stir for 30 min, add L-citrulline methyl ester hydrochloride and triethylamine (55.2 g, 3.0 eq), react at room temperature for 8-12 h, TLC shows reaction is complete. Concentrate to remove DMF, load onto a column with DCM:methanol = 20:1-10:1, collect product and concentrate to dryness to give a white solid 44.8 g, 55.0% yield. NMR data: 1 H NMR (400 MHz, DMSO-d6): δ 8.18 (d, 1H), 6.80 (d, 1H), 5.94-5.97 (m, 2H), 5.39 (d, 4H), 4.18-4.24 (m, 1H), 3.95-3.98 (m, 1H), 3.61 (s, 3H), 2.91-2.98 (m, 4H), 1.31-1.71 (m, 17H). Citrulline dipeptide preparation

[0038] To citrulline dipeptide intermediate (10 g, 1.0 eq) in 50 mL water, add sodium hydroxide (0.99 g, 1.1 eq), react at room temperature for 2 h, TLC shows reaction is complete. Add hydrochloric acid (5.7 g, 2.5 eq) and react for 4-6 h. MS shows reaction is complete. Remove salts by nanofiltration, load onto anion resin, elute with 0.5% hydrochloric acid, collect product and concentrate to give a white solid 4.4 g, 59.5% yield. NMR data: 1H NMR (400 MHz, DMSO-d6 + 2d hydrochloric acid): δ 8.90 (d, 1H), 8.33 (d, 3H), 4.14-4.19 (m, 1H), 3.83-3.86 (m, 1H), 3.04-3.08 (m, 4H), 1.46-1.75 (m, 8H). Example 2. Preparation of arginine-citrulline dipeptide Preparation of Boc-L-nitroarginine

[0039] L-nitroarginine (50 g, 1.0 eq) was dissolved in 200 mL water, 100 mL tetrahydrofuran was added, sodium hydroxide (18.2 g, 2.0 eq) was added, the solution was stirred and the temperature was controlled at 0-10 °C, Boc anhydride (59.7 g, 1.2 eq) was added dropwise. After the dropwise addition was completed, the temperature was raised to 20-25 °C and the reaction was carried out for 6-8 h. TLC showed that the reaction was completed. 50 mL of heptane was added, stirred for 15 min, and the layers were separated by standing. The organic layer was discarded. The water layer was adjusted to pH = 3-4 with citric acid, 200 mL of n-butanol was added for extraction, the organic layers were combined, and concentrated under reduced pressure to obtain the target product 66.2 g with a yield of 91%. Preparation of L-citrulline methyl ester hydrochloride

[0040] L-citrulline (50 g, 1.0 eq) was dissolved in 200 mL of methanol, and thionyl chloride (50.9 g, 1.5 eq) was added dropwise while controlling the temperature at 0-10 °C. After the dropwise addition was completed, the temperature was raised to 35-40 °C and the reaction was carried out for 8-12 h. TLC showed that the reaction was completed. Concentrated under reduced pressure to obtain the target product 61.8 g with a yield of 96%. Preparation of Boc-L-nitroarginine-L-citrulline methyl ester

[0041] 50 g of Boc-L-nitroarginine was dissolved in 250 mL of DMF, EDCI (36.0 g, 1.2 eq) / HOBT (10.6 g, 0.5 eq) was added, stirred for 30 min, L-citrulline methyl ester hydrochloride (35.3 g, 1.0 eq) and triethylamine (47.6 g, 3.0 eq) were added, and the reaction was carried out at room temperature for 8-12 h. TLC showed that the reaction was completed. The DMF was removed by concentration, and the product was collected by column chromatography with DCM:methanol = 20:1-10:1. The product was concentrated to dryness to obtain a white solid 51.5 g with a yield of 67.1%. Preparation of L-arginine-L-citrulline

[0042] Boc-L-nitroarginine-L-citrulline methyl ester (20 g, 1.0 eq) in 50 mL water, sodium hydroxide (1.8 g, 1.1 eq) was added, and the reaction was allowed to proceed at room temperature for 2 h, and TLC showed that the reaction was complete. Hydrochloric acid (10.34 g, 2.5 eq) was added, and the reaction was allowed to proceed for 4-6 h. 2 g of 10% palladium on carbon was added to the reaction system, and the system was replaced with hydrogen gas and reacted at 50 °C. After the reaction was completed, the palladium on carbon was filtered off, and the filtrate was concentrated. The product was adsorbed on a suitable resin, washed with purified water to remove inorganic salts, and then eluted with 0.5% hydrochloric acid. The product was collected and concentrated to obtain a white solid (6.0 g, 44.3% yield). NMR data: 1 H NMR (400 MHz, D2O): δ 4.14-4.19 (m, 1H), 3.82-3.85 (m, 1H), 3.02-3.08 (m, 4H), 1.49-1.77 (m, 8H). Preparation of ornithine dipeptide

[0043] L-ornithine (50 g, 1.0 eq) was dissolved in 200 mL water and 100 mL tetrahydrofuran, and sodium hydroxide (37.8 g, 2.5 eq) was added. The solution was stirred to dissolve the solid, and the temperature was controlled at 0-10 °C. Boc anhydride (181.7 g, 2,2 eq) was added dropwise. After the addition was completed, the temperature was increased to 20-25 °C, and the reaction was allowed to proceed for 6-8 h. TLC showed that the reaction was complete. 50 mL of heptane was added, and the mixture was stirred for 15 min. The organic layer was discarded after the layers were separated. The water layer was adjusted to pH = 3-4 with citric acid, and 200 mL of ethyl acetate was added. The organic layers were combined and concentrated under reduced pressure to obtain the target product (110.6 g, 88% yield).

[0044] L-ornithine (50 g, 1.0 eq) was dissolved in 250 mL water and 250 mL tetrahydrofuran, and sodium bicarbonate (47.7 g, 1.5 eq) was added. The temperature was controlled at 0-10 °C, and Cbz-Cl (67.8 g, 1.05 eq) was added dropwise. After the addition was completed, the temperature was increased to 20-25 °C, and the reaction was allowed to proceed for 2 h. TLC showed that the reaction was complete. The pH was adjusted to 5-6 with citric acid, and 250 mL of ethyl acetate was added. The organic layers were combined and concentrated under reduced pressure. The oil was passed through a column with DCM:methanol = 20:1-10:1, and the product was collected and concentrated to obtain a solid (74.4 g, 73.9% yield).

[0045] Cbz-L-Ornithine (50 g, 1.0 eq) in 200 mL of methanol, control temperature 0-10 °C, dropwise addition of thionyl chloride (33.5 g, 1.5 eq). After the end of the dropwise addition, the temperature was raised to 35-40 °C and the reaction was allowed to proceed for 8-12 h. TLC showed that the reaction was complete. Concentration under reduced pressure gave the target product 48.8 g in 82.3% yield.

[0046] Boc-L-Ornithine (50 g, 1.0 eq) in 250 mL of dichloromethane, EDCI (34.6 g, 1.2 eq) / HOBT (10.2 g, 0.5 eq) were added and stirred for 30 min. Cbz-L-Ornithine methyl ester hydrochloride (47.6 g, 1.0 eq) and triethylamine (45.7 g, 3.0 eq) were added and the reaction was allowed to proceed at room temperature for 8-12 h. TLC showed that the reaction was complete. 100 mL of water was added to wash the organic layer, which was concentrated and then column chromatography was performed using DCM:methanol = 20:1-10:1. The product was collected and concentrated to dryness to give a solid 64 g in 71.5% yield.

[0047] Boc-L-Ornithine (50 g, 1.0 eq) in 250 mL of dichloromethane, EDCI (34.6 g, 1.2 eq) / HOBT (10.2 g, 0.5 eq) were added and stirred for 30 min. Cbz-L-Ornithine methyl ester hydrochloride (47.6 g, 1.0 eq) and triethylamine (45.7 g, 3.0 eq) were added and the reaction was allowed to proceed at room temperature for 8-12 h. TLC showed that the reaction was complete. 100 mL of water was added to wash the organic layer, which was concentrated and then column chromatography was performed using DCM:methanol = 20:1-10:1. The product was collected and concentrated to dryness to give a solid 64 g in 71.5% yield. 1 HNMR (400 MHz, D2O): δ 4.53-4.57 (m, 1H), 3.18-3.25 (m, 1H), 2.58-2.67 (m, 4H), 1.73-1.82 (m, 4H), 1.49-1.61 (m, 4H). Example 4. Preparation of hydroxyisoleucine dipeptide

[0048] 4-Hydroxyisoleucine (50 g, 1.0 eq) in 200 mL of hydrochloric acid was heated to 60-65 °C and the reaction was allowed to proceed for 4-6 h. TLC showed that the reaction was complete. Concentration under reduced pressure removed the water and the product was obtained by slurry with ethyl acetate. The target product was obtained in 53.2 g in 96.2% yield.

[0049] To cyclo-4-hydroxyisoleucine hydrochloride (50 g, 1.0 eq) in 200 mL water, sodium bicarbonate (48.1 g, 2.0 eq) was added, and Boc anhydride (79.6 g, 1.2 eq) was added dropwise while controlling the temperature at 0-10 °C. After the dropwise addition was completed, the temperature was increased to 20-25 °C, and the reaction was allowed to proceed for 6-8 h. TLC showed that the reaction was completed. Sodium hydroxide (12.2 g, 1.0 eq) was further added, and the reaction was allowed to proceed at 20-25 °C for 2 h. The pH was adjusted to 3-4 with citric acid, and 200 mL x 2 ethyl acetate was added for extraction. The organic phase was combined and concentrated under reduced pressure until substantially no solvent was left. The target product was obtained in a yield of 61.0 g, 81.3%. Preparation of cyclo-4-hydroxyisoleucine-4-hydroxy-Boc isoleucine

[0050] To cyclo-4-hydroxyisoleucine hydrochloride (50 g, 1.0 eq) and 4-hydroxy-Boc isoleucine (78.8 g, 1.05 eq) in 500 mL dichloromethane, HATU (138.7 g, 1.2 eq) and triethylamine (92.2 g, 3.0 eq) were added, and the reaction was allowed to proceed at room temperature for 4-6 h. TLC showed that the reaction was completed. The product was collected by column chromatography using petroleum ether: ethyl acetate = 10:1-5:1, and concentrated to dryness to obtain a white solid in a yield of 62.4 g, 57.4%. Preparation of 4-hydroxyisoleucine dipeptide

[0051] To cyclo-4-hydroxyisoleucine-4-hydroxy-Boc isoleucine (20 g, 1.0 eq) in 50 mL water, sodium hydroxide (2.3 g, 1.05 eq) was added, and the reaction was allowed to proceed at room temperature for 2 h. TLC showed that the reaction was completed. Hydrochloric acid (14.2 g, 2.5 eq) was added, and the reaction was allowed to proceed for 4-6 h. MS showed that the reaction was completed. The reaction solution was purified by ion resin exchange, and the product was collected and concentrated to obtain a solid in a yield of 8.4 g, 54.5%. NMR data: 1 H NMR (400 MHz, D2O): δ 4.18-4.27 (m, 1H), 3.48-3.53 (m, 2H), 3.33-3.42 (m, 1H), 2.38-2.47 (m, 1H), 1.96-2.01 (m, 1H), 1.11 (dd, 6H), 0.93 (dd, 6H).

[0052] Other configuration or racemic amino acid dipeptides and / or other amino acid dipeptides can also be prepared by a method similar to the above-described examples. Property determination of the amino acid dipeptides of the present application

[0053] The amino acid dipeptides of the present application were respectively subjected to property determination. Odor and taste evaluation

[0054] Sample preparation: 5.4g of citrulline dipeptide was weighed into 240mL of deionized water, numbered, and 10 subjects were randomly recruited to evaluate the odor (fishy, rancid) and taste (fishy, bitter, sweet, sour) of the sample powder and solution, and finally give the total score. The evaluation was carried out in a separate evaluation room, in front of a white wall and white paper background, under white daylight lamp, while only one subject was evaluated and the results were recorded.

[0055] Evaluation criteria: Odor evaluation includes fishy and rancid, subjects in the evaluation room, guided to smell the sample odor for 3s, then scored for fishy (intense fishy 0 points, heavier fishy 1 point, fishy 2 points, moderate fishy 3 points, slightly fishy 4 points, no fishy 5 points) and rancid (intense rancid 0 points, heavier rancid 1 point, rancid 2 points, moderate rancid 3 points, slightly rancid 4 points, no rancid 5 points), the higher the score, the less bad odor. Taste evaluation includes fishy, bitter, sweet, sour, subjects in the evaluation room, guided to taste about 15mL of sample, then scored for fishy (intense fishy 0 points, heavier fishy 1 point, fishy 2 points, moderate fishy 3 points, slightly fishy 4 points, no fishy 5 points), bitter (intense bitter 0 points, stronger bitter 1 point, moderate bitter 2 points, weaker bitter 3 points, weak bitter 4 points, no bitter 5 points), sweet (intense sweet 0 points, stronger sweet 1 point, moderate sweet 2 points, weaker sweet 3 points, weak sweet 4 points, no sweet 5 points), sour (intense sour 0 points, stronger sour 1 point, moderate sour 2 points, weaker sour 3 points, weak sour 4 points, no sour 5 points), the higher the score, the less bad taste. Evaluation results: Table 1. Average score of citrulline dipeptide sample odor evaluation Table 2. Average score of citrulline dipeptide sample taste evaluation

[0056] From the above evaluation average score, it can be seen that the overall bad flavor of citrulline dipeptide is less, there is no special gas flavor, and it is easy to reconstitute later.

[0057] Sample preparation: 5.4g of arginine-citrulline dipeptide and arginine was weighed into 240mL of deionized water, numbered, and 10 subjects were randomly recruited to evaluate the odor (fishy, rancid) and taste (fishy, bitter, sweet, sour) of the sample powder and solution, and finally give the total score. The evaluation was carried out in a separate evaluation room, in front of a white wall and white paper background, under white daylight lamp, while only one subject was evaluated and the results were recorded.

[0058] Evaluation criteria: odor evaluation includes fishy smell and rancid smell. The subjects are guided to smell the sample odor for 3s in the evaluation room, and then score the fishy smell (intense fishy smell 0 points, heavier fishy smell 1 point, fishy smell 2 points, moderate fishy smell 3 points, slightly fishy smell 4 points, no fishy smell 5 points) and rancid smell (intense rancid smell 0 points, heavier rancid smell 1 point, rancid smell 2 points, moderate rancid smell 3 points, slightly rancid smell 4 points, no rancid smell 5 points). The higher the score, the less bad smell. Taste evaluation includes fishy taste, bitter taste, salty taste, aftertaste, sour taste. The subjects are guided to taste the sample about 15mL in the evaluation room, and then score the fishy taste (intense fishy taste 0 points, heavier fishy taste 1 point, fishy taste 2 points, moderate fishy taste 3 points, slightly fishy taste 4 points, no fishy taste 5 points), bitter taste (intense bitter taste 0 points, stronger bitter taste 1 point, moderate bitter taste 2 points, weaker bitter taste 3 points, weak bitter taste 4 points, no bitter taste 5 points), salty taste (intense salty taste 0 points, stronger salty taste 1 point, moderate salty taste 2 points, weaker salty taste 3 points, weak salty taste 4 points, no salty taste 5 points), aftertaste (intense aftertaste 0 points, stronger aftertaste 1 point, moderate aftertaste 2 points, weaker aftertaste 3 points, weak aftertaste 4 points, no aftertaste 5 points). The higher the score, the less bad taste. Evaluation results: Table 3. Average score of arginine-citrulline dipeptide and arginine sample odor evaluation Table 4. Average score of arginine-citrulline dipeptide and arginine sample taste evaluation

[0059] In summary, arginine-citrulline dipeptide has less bad smell and higher overall acceptance than arginine, and its flavor and mouthfeel are superior to arginine in product application. The amino acid dipeptide of the present application has less overall bad flavor, no special smell, and unique advantages in flavor and mouthfeel in product application. In vitro simulation of gastrointestinal stability experiment

[0060] Artificial gastric juice (with enzyme): Take 16.4mL concentrated hydrochloric acid, add about 800mL water and 10g pepsin. Shake well, then dilute with water to a total volume of 1000mL.

[0061] Artificial gastric juice (without enzyme): Take 16.4mL concentrated hydrochloric acid, add about 1000mL water and shake well.

[0062] Artificial intestinal juice (with enzyme): Accurately weigh 6.8g potassium dihydrogen phosphate into 500mL water. Adjust the pH to 6.8 with 0.01mol / L NaOH. Respectively, take 10g trypsin and dissolve in appropriate amount of water. Mix the two solutions, then dilute with water to a total volume of 1000mL.

[0063] Artificial intestinal juice (without enzyme): Accurately weigh 6.8g potassium dihydrogen phosphate into 1000mL water. Adjust the pH to 6.8 with 0.01mol / L NaOH.

[0064] Sample preparation: 25.0 mg of arginine-citrulline dipeptide or arginine or citrulline was accurately weighed and dissolved in 50 mL of simulated gastric fluid or simulated intestinal fluid without enzymes, respectively, after a certain time, sample was taken and injected into high performance liquid chromatography to detect its content.

[0065] Sample preparation: 25.0 mg of arginine-citrulline dipeptide or arginine or citrulline was accurately weighed and dissolved in 50 mL of simulated gastric fluid or simulated intestinal fluid with enzymes, respectively, after a certain time, sample was taken and injected into high performance liquid chromatography to detect its content.

[0066] Chromatographic conditions: Table 5 is the chromatographic conditions used. Table 5 Chromatographic conditions

[0067] Detection results: Table 6 is the stability of the sample after 2h, and Table 7 is the stability of the sample after 4h. As can be seen from Tables 6 and 7, arginine decomposes in artificial intestinal fluid containing enzymes, and after 2h, the content of arginine decreases by about 12%, and then it will be completely degraded in about 4h. Arginine-citrulline dipeptide can be decomposed into 25.12% (w / w) arginine and 31.40% (w / w) citrulline after 2h, and continuously decomposed into arginine and citrulline in 4h, which can stably and continuously provide arginine and citrulline in the intestinal tract. The dipeptide can slowly release amino acids for a long time, thereby prolonging its action time, and has better metabolic properties than amino acids themselves. Table 6 Stability of sample after 2h Table 7 Stability of sample after 4h

[0068] The amino acid dipeptides prepared by the present application have less bad taste, higher overall acceptance, better flavor and taste in product application, and are easy to use in subsequent application. In addition, they can stably and continuously provide amino acid sources in the gastrointestinal tract, are used by the subjects, have good bioavailability, show good comprehensive effect, and are particularly suitable for use in diet or nutritional supplements. In addition, the preparation process is controllable, the cost is low, and the environment is friendly.

[0069] The above only describes the preferred embodiments of the present application, and is not intended to limit the present application. Any person skilled in the art can make various changes, modifications, replacements and variations to these embodiments without departing from the principles and purposes of the present application, the scope of the present application is defined by the claims and their equivalents.

Claims

1. An amino acid dipeptide, characterized in that, The amino acid dipeptide is formed from an amino acid I and an amino acid II, each selected from one of the following: citrulline, arginine, ornithine, hydroxyisoleucine.

2. The amino acid dipeptide according to claim 1, characterized in that, The carboxyl group of the amino acid I is attached to the amino group of the amino acid II, the amino acid I being one of citrulline, arginine, ornithine, hydroxyisoleucine, and the amino acid II being one of citrulline, ornithine, hydroxyisoleucine.

3. The amino acid dipeptide according to claim 1 or 2, characterized in that, The amino acid dipeptide is a citrulline dipeptide, an arginine-citrulline dipeptide, an ornithine dipeptide, a hydroxyisoleucine dipeptide.

4. The amino acid dipeptide according to any one of claims 1 to 3, characterized in that, The amino acid dipeptide has the following structure:

5. The amino acid dipeptide according to any one of claims 1 to 4, characterized in that, The citrulline dipeptide has the following NMR data: 1 H NMR (400 MHz, DMSO-d6 + 2d hydrochloric acid): δ 8.90 (d, 1H), 8.33 (d, 3H), 4.14-4.19 (m, 1H), 3.83-3.86 (m, 1H), 3.04-3.08 (m, 4H), 1.46-1.75 (m, 8H).

6. The amino acid dipeptide according to any one of claims 1 to 4, characterized in that, The arginine-citrulline dipeptide has the following NMR data: 1 H NMR (400 MHz, D20): δ 4.14-4.19 (m, 1H), 3.82-3.85 (m, 1H), 3.02-3.08 (m, 4H), 1.49-1.77 (m, 8H).

7. The amino acid dipeptide according to any one of claims 1 to 4, characterized in that, The ornithine dipeptide has the following NMR data: 1 H NMR (400 MHz, D20): δ 4.53-4.57 (m, 1H), 3.18-3.25 (m, 1H), 2.58-2.67 (m, 4H), 1.73-1.82 (m, 4H), 1.49-1.61 (m, 4H).

8. The amino acid dipeptide according to any one of claims 1 to 4, characterized in that, The hydroxyisoleucine dipeptide has the following NMR data: 1 H NMR (400 MHz, D20): δ 4.18-4.27 (m, 1H), 3.48-3.53 (m, 2H), 3.33-3.42 (m, 1H), 2.38-2.47 (m, 1H), 1.96-2.01 (m, 1H), 1.11 (dd, 6H), 0.93 (dd, 6H).

9. The amino acid dipeptide according to any one of claims 1 to 8, characterized in that, The amino acid dipeptide is prepared as a food, a beverage, a supplement, a nutraceutical.

10. The amino acid dipeptide according to any one of claims 1 to 9, characterized in that, The amino acid dipeptide is prepared as a solid formulation or a liquid formulation.

11. A method of preparing an amino acid dipeptide as claimed in any one of claims 1 to 10, characterized in that, The method comprises the following steps: a. reacting the amino acid I with Boc anhydride under basic conditions to obtain Boc-amino acid I; b. preparing the amino acid II to obtain amino acid II carboxylate hydrochloride; c. reacting the Boc-amino acid I, a condensing agent, the amino acid II carboxylate hydrochloride, a base in a reaction solvent to obtain Boc-amino acid I-amino acid II carboxylate; d. hydrolyzing the Boc-amino acid I-amino acid II carboxylate by reacting with a base, and then removing Boc to obtain the amino acid dipeptide.

12. The method of claim 11, wherein, The condensing agent in step c is selected from one or more of the following: EDCI, DCC, HATU, HOSU, HOBt, DMAP; the base is selected from one or more of the following: triethylamine, DIPEA, N-methylmorpholine, pyridine; the reaction solvent is selected from one or more of the following: dichloromethane, N,N-dimethylformamide, THF, dioxane, NMP.

13. The production method according to claim 11 or 12, characterized by, When the amino acid I is arginine, the arginine is first nitrosylated into nitroarginine, and then step a is performed to react the nitroarginine with Boc anhydride under basic conditions to obtain Boc-nitroarginine as the Boc-amino acid I; and step d is performed after removing Boc to obtain the amino acid dipeptide by reduction.

14. The production method according to any one of claims 11 to 13, characterized by, When the amino acid II is ornithine, the ornithine is first prepared into Cbz-ornithine, and then step b is performed to obtain Cbz-ornithine methyl ester hydrochloride as the amino acid II carboxylate hydrochloride; and step d is performed after removing Boc to obtain the amino acid dipeptide by reduction.

15. The production method according to any one of claims 11 to 14, characterized by, When the amino acid II is hydroxyisoleucine, step b is to react the hydroxyisoleucine with hydrochloric acid to obtain cyclo-hydroxyisoleucine hydrochloride as the amino acid II carboxylate hydrochloride.

16. The production method according to any one of claims 11 to 15, characterized by, When the amino acid I is hydroxyisoleucine, the hydroxyisoleucine is first reacted with hydrochloric acid to obtain cyclo-hydroxyisoleucine hydrochloride, and then step a is performed to react the cyclo-hydroxyisoleucine hydrochloride with Boc anhydride under basic conditions to obtain hydroxy Boc-isoleucine as the Boc-amino acid I.

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