Separation method of single-configuration ring (isoleucine-isoleucine) dipeptide

Abstract

The invention discloses a separation method of a single-configuration cyclic (isoleucine-isoleucine) dipeptide, which is characterized in that one single-configuration (3R, 6S) cyclic (isoleucine-isoleucine) dipeptide can be obtained by using a mixed solvent for thermal pulping. The method specifically comprises the following steps: adding a mixture of diastereoisomers of the cyclo (isoleucine-isoleucine) dipeptide into a mixed solvent, heating to reflux, keeping reflux, directly cooling to crystallize, filtering, washing a filter cake by using at least one of the mixed solvents, and drying the washed filter cake to obtain the single-configuration (3R, 6S) cyclo (isoleucine-isoleucine) dipeptide. The method is simple in process, easy to operate and suitable for large-scale production and preparation of the single-configuration (3R, 6S) ring (isoleucine-isoleucine) dipeptide; the purity and the yield of the product are improved.

Description

Technical Field

[0001] This invention belongs to the field of peptide synthesis technology, and in particular relates to a method for separating a single-configuration ring (isoleucine-isoleucine) dipeptide. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Cyclodipeptides, also known as derivatives of 2,5-piperazine-dione (DKP), are the smallest cyclic peptides. Cyclodipeptides have a stable six-membered ring backbone, formed by two amino acids linked end-to-end, exhibiting excellent rigidity and biological activity. Their stereochemical complexity and biocompatibility make them promising candidates for drug development, agriculture, and materials science. Cyclodipeptides are widely available; they can be extracted and isolated from nature or synthesized through biological or chemical methods. Most chemical synthesis methods for cyclic (isoleucine-isoleucine) dipeptides employ dehydration condensation, a simple and industrially suitable method, but it results in a mixture of three diastereomers. Prior patent CN119799825A, "Preparation Method and Application of Cyclo(Isoleucine-Isoleucine) Dipeptide," discloses a method for preparing cyclic dipeptides through direct amino acid condensation; however, this method still yields a mixture of three diastereomers, failing to address the problem of separating and purifying the stereoisomers.

[0004] Currently available methods for purifying and separating single configurations are adsorption chromatography and preparative liquid chromatography. Both methods are complex, costly, and limited in scale, making them unsuitable for industrial production. Summary of the Invention

[0005] To overcome the shortcomings of existing technologies, this invention provides a method for separating a single-configuration cyclic (isoleucine-isoleucine) dipeptide; one type of single-configuration (3R, 6S) cyclic (isoleucine-isoleucine) dipeptide can be obtained by recrystallization under stirring and reflux with a mixed solvent. The technical solution provided by this invention is as follows: As a first aspect of the present invention, a method for separating a single-configuration cyclic (isoleucine-isoleucine) dipeptide is provided, comprising the following steps: adding a mixture of diastereomers of the cyclic (isoleucine-isoleucine) dipeptide to a mixed solvent, heating to reflux, directly cooling to crystallize, filtering, washing the filter cake with the mixed solvent, and drying the washed filter cake to obtain a single-configuration (3R, 6S) cyclic (isoleucine-isoleucine) dipeptide; The mixed solvent is a mixed solution of haloalkanes and alcohols.

[0006] In some embodiments, the separation method includes the following steps: adding a mixture of diastereomers of the cyclic (isoleucine-isoleucine) dipeptide to a mixed solvent, heating to reflux with stirring, maintaining reflux for 8-10 h, naturally cooling to 0--5℃ for crystallization for 4-6 h, filtering, washing the filter cake with the mixed solvent, and drying the washed filter cake at 50-60℃ to obtain a single configuration (3R, 6S) cyclic (isoleucine-isoleucine) dipeptide.

[0007] In some embodiments of the present invention, the mass-to-volume ratio of the mixture of diastereomers to the mixed solvent is 1:1 to 100 g / mL.

[0008] In some embodiments of the present invention, the volume ratio of haloalkanes to alcohols in the mixed solvent is 10:0.1~100.

[0009] The haloalkane is selected from one or more of dichloromethane, trichloromethane, carbon tetrachloride, 1,2-dichloroethane and chloropropane; preferably dichloromethane.

[0010] The alcohol is selected from one or more of ethanol, isopropanol, butanol, ethylene glycol, glycerol and tert-butanol; preferably ethanol and isopropanol.

[0011] The mixture contains diastereomers with structures represented by formulas I, II, and III as follows: .

[0012] In the mixture, the contents of I, II and III account for approximately 65%, 20% and 12%, respectively.

[0013] The effects of temperature on yield and purity were studied in the embodiments of the present invention: First, the product was completely dissolved by heating and reflux. The different solubility differences between the isomers were utilized in the mixed solvent to crystallize them, thereby improving product purity. The properties of the mixed solvent were found to increase the solubility of non-target products, making the precipitation of the target product smoother. However, the large amount of solvent also meant that some of the target product would not precipitate completely, which would reduce the yield.

[0014] At low temperatures: co-crystallization of different isomers leads to a decrease in purity, but the target product is precipitated more completely. Although the yield is higher, it does not meet the requirements for improving purity.

[0015] It exhibits good yield and purity at reflux temperatures of 50–70 °C, especially when the mixed solvent is dichloromethane and isopropanol in a volume ratio of 10:10.

[0016] More preferably, the optimal balance between purity and yield is achieved at 50°C.

[0017] The mixture of diastereomers was prepared by the following method: Add the required amount of ethylene glycol and L-isoleucine to the reactor. Start stirring and heat to reflux at high speed. Maintain the temperature and time the reaction. After the reaction is complete, turn off the heating and allow it to cool naturally. Add the required amount of isopropanol dropwise to the reactor. After the addition is complete, cool and continue stirring. Centrifuge, and wash the filter cake with isopropanol. Discharge, dry, and obtain a solid cyclic dipeptide.

[0018] As a second aspect of the invention, it is provided that a single-configuration (3R,6S)-cyclic (isoleucine-isoleucine) dipeptide is used to promote plant growth.

[0019] Furthermore, the plant in question is lettuce.

[0020] Compared with the prior art, the beneficial effects of the present invention are: 1. This invention provides a method for separating three diastereomers of cyclic (isoleucine-isoleucine) dipeptide. The method can obtain one of the single configurations (3R, 6S) cyclic (isoleucine-isoleucine) dipeptide with better bioassay results by purification by recrystallization in a mixed solvent, thereby improving the purity and yield of the product.

[0021] 2. This method is simple and easy to operate, and is suitable for large-scale production of single-configuration (3R, 6S) cyclic (isoleucine-isoleucine) dipeptides.

[0022] 3. Experiments of this invention have demonstrated that the single-configuration (3R, 6S) ring (isoleucine-isoleucine) dipeptide is significantly more effective than the mixture of three diastereomers in promoting plant growth. This invention provides a new plant growth promoting component by purifying this single-configuration isomer. Attached Figure Description

[0023] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0024] Figure 1 MS chromatogram of the cyclic (isoleucine-isoleucine) dipeptide mixture obtained in the preparation example.

[0025] Figure 2 To prepare the cyclic (isoleucine-isoleucine) dipeptide mixture obtained in the example 1 HNMR image.

[0026] Figure 3The image shows the HPLC chromatogram of the cyclic (isoleucine-isoleucine) dipeptide mixture obtained in the preparation example.

[0027] Figure 4 The image shows the HPLC chromatogram of the single-configuration (3R, 6S) cyclic (isoleucine-isoleucine) dipeptide prepared in Example 2. Detailed Implementation

[0028] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0029] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of exemplary embodiments according to the invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0030] Preparation example, Preparation of a mixture of diastereomers of cyclic (isoleucine-isoleucine) dipeptides Add 10 mL of ethylene glycol and 5.0 g of L-isoleucine (the amount required for the process) to a 50 mL reaction flask. Start stirring and heat to reflux at high speed. Maintain the temperature and time the reaction for 10 h. After the reaction is complete, turn off the heating and allow it to cool naturally. Add 10 mL of isopropanol dropwise to the reaction flask. After the addition is complete, cool and continue stirring. Filter, and wash the filter cake with isopropanol. Dry the filter cake at 50–60 °C using forced air to obtain 3.0 g of a mixture of diastereomers of the cyclic (isoleucine-isoleucine) dipeptide, with a yield of 60%. The isomers include those with the following structures:

[0031] This mixture was used for the purification of Examples 1 to 3.

[0032] Detection method: 1. Mass spectrometry detection: Instrument model: Waters SQ-D-2; Testing conditions: Liquid chromatography: Injection volume: 2 μL; Column temperature: 30℃; Mobile phase: A - 0.1% formic acid aqueous solution, B - acetonitrile, 0 min A: 90%, 10 min A: 10%, 12-15 min A: 90%; Run time: 15 min; Detection wavelength: 210 nm; Flow rate: 1 mL / min; Column: ZORBAX SB-C18 4.6 × 150 mm 3.5 μm; Mass spectrometry section: Ion source: API-ES, positive and negative ion detection mode; capillary voltage: 3.0kV; cone voltage: 40V; desolventizing temperature: 450℃; flow rate: 650L / Hr.

[0033] The product prepared in Preparation Example 1 was analyzed by mass spectrometry, and the mass spectrum of the synthesized compound cyclic (isoleucine-isoleucine) dipeptide was determined. The positive ion chromatogram is shown below. Figure 1 As shown.

[0034] 2. Hydrogen spectrum detection HNMR testing conditions: Nuclear magnetic resonance spectrometer model: Bruker 400M Detection conditions: Dissolve 5 mg of sample in 0.6 mL of DMSO and detect the proton NMR spectrum using 400 M NMR.

[0035] The product prepared in Preparation Example 1 was analyzed by mass spectrometry, and the cyclic (isoleucine-isoleucine) dipeptide spectrum was obtained as follows: Figure 2 As shown.

[0036] 3. Detection methods for cyclic (isoleucine-isoleucine) dipeptides and their single configurations Sample pretreatment: Weigh 5 mg of the compound sample prepared in Preparation Example 1, dissolve it in ethanol and bring the volume to 5 mL. After mixing, take 1 mL and filter it through a 0.22 μm organic filter membrane. Perform the analysis according to the following liquid chromatography reference conditions.

[0037] Reference conditions for liquid chromatography: Column: Inertsil ODS-3, 5 μm 4.6×250 mm; Mobile phase: water:acetonitrile = 80:20; UV detector wavelength: 200 nm; Column temperature: 40℃; Flow rate: 1 mL / min.

[0038] The synthesized sample was analyzed using the area normalization method, and the content of the cyclic (isoleucine-isoleucine) dipeptide was in the range of 90-95%. By comparing the chromatographic behavior with that reported in the literature (DOI:10.1039 / P19920001199), the diastereomers corresponding to each chromatographic peak were clearly assigned. Figure 3 .

[0039] Example 1: Separation of mixtures of diastereomers of cyclic (isoleucine-isoleucine) dipeptides 5.0 g of the mixture of diastereomers of the cyclic (isoleucine-isoleucine) dipeptide prepared in Preparation Example 1 was added to a 250 mL single-necked round-bottom flask. 50 mL of mixed solvents in different proportions (dichloromethane:isopropanol, v:v = 10:0.1, 10:10, 10:100) were added respectively. The reaction system was heated (30℃, 50℃, 70℃) and refluxed for 10 h. After reflux, the mixture was directly cooled to -5℃ for 5 h to crystallize. The crystals were filtered, and the filter cake was washed with 10 mL of the mixed solvent. After drying, the single-configuration cyclic (isoleucine-isoleucine) dipeptide was obtained. The yield was calculated, and the purity of the product was determined by high-performance liquid chromatography (HPLC). The results are shown in Table 1.

[0040] Table 1. Yields and purity of monomorphic cyclic (isoleucine-isoleucine) dipeptides

[0041] Table 2. Yields and purities of the cyclic (isoleucine-isoleucine) dipeptide in other solvents.

[0042] It is evident that the cyclic (isoleucine-isoleucine) dipeptide cannot be completely dissolved in other non-alkanol systems, making recrystallization impossible.

[0043] Example 2: Add 5.0 g of the mixture of diastereomers of the cyclic (isoleucine-isoleucine) dipeptide prepared in Preparation Example 1 to a 250 mL single-necked flask, add 50 mL of mixed solvent (dichloromethane:isopropanol = 10:10), heat to 45-50 °C, and reflux for 10 h. After reflux, cool directly to -5 °C for 5 h to crystallize. Filter, wash the filter cake with 10 mL of mixed solvent, and dry the washed filter cake to obtain 2.90 g of a single-configuration cyclic (isoleucine-isoleucine) dipeptide (yield and purity are shown in Table 2). HPLC chromatogram is shown in [Figure 2]. Figure 4 .

[0044] Example 3: Add 5.0 g of the mixture of diastereomers of the cyclic (isoleucine-isoleucine) dipeptide prepared in Preparation Example 1 to a 250 mL single-necked flask, add 50 mL of different mixed solvents, heat to 45-50 °C, and reflux for 10 h. After reflux, cool directly to -5 °C to crystallize for 5 h, filter, and wash the filter cake with 10 mL of mixed solvent or 10 mL of single solvent (ethanol, isopropanol, dichloromethane). After washing, dry the filter cake to obtain a single configuration of cyclic (isoleucine-isoleucine) dipeptide. The yield and purity are shown in Table 2.

[0045] Table 3. Yields and purity of single-configuration cyclic (isoleucine-isoleucine) dipeptides

[0046] A comparison of the results from Examples 1 and 4 shows that, in the mixed solvent recrystallization step, the solvent ratio and system temperature have a significant impact on the product yield and purity. The optimal conditions are a mixed solvent of dichloromethane:isopropanol = 10:10 and a system temperature of 50°C.

[0047] Example 4: The effects of the (3R, 6S) cyclic (isolus-isolus) dipeptide single configuration (single crystal) prepared in Example 2 and the mixture of cyclic (isolus-isolus) dipeptide isomers (cyclic H) prepared in the preparation example on plant growth were compared.

[0048] 4.1 Lettuce seedling raising Thoroughly mix the sterilized vermiculite, fill the seedling trays, level the surface, and sow lettuce seeds at a depth of 0.5cm. Cultivate in an indoor environment at a temperature of 23℃, humidity of 50%-60%, and light intensity of 6000-7000 lux. When the seedlings reach the two-leaf-one-heart stage, select seedlings with uniform growth for transplanting.

[0049] 4.2 Preparation of the application concentration of the cyclic dipeptide solution Preparation of the cyclic dipeptide stock solution: Weigh 10 mg of cyclic dipeptide powder accurately. Dissolve 10 mg of cyclic dipeptide in 5 mL of dimethyl sulfoxide (DMSO) to prepare a stock solution with a concentration of 2 mg / mL.

[0050] Preparation of a 20 ng / mL cyclic dipeptide solution: Take 5 µl (0.005 mL) of a 2 mg / mL solution from the stock solution. Add 500 mL of pure water and mix well to obtain a solution with a concentration of 20 ng / mL.

[0051] 4.3 Lettuce Cultivation and Processing Nutrient soil was filled into flowerpots, the surface was leveled, and lettuce seedlings were transplanted into them. Five pots were used for each treatment, with replicates. The specific experimental design is shown in Table 3.

[0052] Table 3 Experimental Design

[0053] 4.4 Indicator Measurement and Methods The fresh weight of the above-ground parts and the number of leaves were measured, and photographs were taken when the effects were significant throughout the entire growth period. The experimental results are shown in Table 4.

[0054] Table 4 Growth indicators of lettuce under different treatments

[0055] Based on the results of the bioassay on the effects of different treatments on the fresh weight of lettuce, it can be seen that the single-configuration (3R,6S) cyclic (isolus-isolus) dipeptide has a significantly better growth-promoting effect on plants than the mixture. The aboveground fresh weight of the single crystal treatment group is significantly different from that of the control group, with the aboveground fresh weight of the single crystal group being higher, increasing by 24.17% compared to the control group.

[0056] It is evident that the (3R,6S)-cyclic (isoluene-isoluene) dipeptide has a better plant growth-promoting effect, making it necessary to isolate this single configuration.

[0057] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for isolating a single-configurational-cyclic (isoleucine-isoleucine) dipeptide, characterized in that, Includes the following steps: A mixture of diastereomers of the cyclic (isoleucine-isoleucine) dipeptide was added to a mixed solvent, heated to reflux, directly cooled to crystallize, filtered, and the filter cake was washed with at least one of the mixed solvents. After drying, a single configuration of (3R,6S) cyclic (isoleucine-isoleucine) dipeptide was obtained. Diastereomers include structures represented by formulas I, II, and III as follows: ； The mixed solvent is a mixed solution of haloalkanes and alcohols.

2. The method for separating a single-configurational-cyclic (isoleucine-isoleucine) dipeptide according to claim 1, characterized in that, The mass-to-volume ratio of the mixture of diastereomers to the mixed solvent is 1:1~100 g / mL.

3. The method for separating a single-configurational-cyclic (isoleucine-isoleucine) dipeptide according to claim 1, characterized in that, The volume ratio of haloalkanes to alcohols is 10:0.1~100.

4. The method for separating a single-configurational-cyclic (isoleucine-isoleucine) dipeptide according to claim 1, characterized in that, The haloalkane is selected from one or more of dichloromethane, trichloromethane, carbon tetrachloride, 1,2-dichloroethane and chloropropane; preferably dichloromethane.

5. The method for separating a single-configurational-cyclic (isoleucine-isoleucine) dipeptide according to claim 1, characterized in that, The alcohol is selected from one or more of ethanol, isopropanol, butanol, ethylene glycol, glycerol and tert-butanol; preferably ethanol and isopropanol.

6. The method for separating a single-configurational-cyclic (isoleucine-isoleucine) dipeptide according to claim 1, characterized in that, The reflux temperature is 50~70℃.

7. The method for separating a single-configurational-cyclic (isoleucine-isoleucine) dipeptide according to claim 1, characterized in that, The mixed solvent is dichloromethane and isopropanol in a volume ratio of 10:

10.

8. The method for separating a single-configurational-cyclic (isoleucine-isoleucine) dipeptide according to claim 7, characterized in that, The diastereomer mixture was prepared as follows: Add the required amount of ethylene glycol and L-isoleucine to the reactor; turn on the stirrer and heat to reflux at high speed; maintain the temperature and start the reaction; after the reaction is complete, turn off the heating and allow it to cool naturally; add the required amount of isopropanol dropwise to the reactor; after the dropwise addition is complete, cool down and continue stirring. Centrifuge, wash the filter cake with isopropanol; discharge, dry, and obtain solid cyclic dipeptide.

9. Application of a single-configuration (3R,6S)-cyclic (isoleucine-isoleucine) dipeptide in promoting plant growth.

10. The application according to claim 9, characterized in that, The plant in question is lettuce.

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