Synthesis method of Kyotrophin
By optimizing the protection and simplifying the purification steps of Kyotrophin starting materials, the problems of low yield and high cost in the prior art are solved, and high purity, high yield and low cost Kyotrophin production is achieved, which is suitable for industrial production.
Patent Information
- Application Number
- CN202411182910.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-08-27
- Publication Date
- 2024-12-13
AI Technical Summary
The existing chemical synthesis methods of Kyotrophin have problems such as low yield, high cost and are not conducive to large-scale industrial production. They are mainly due to the low yield and high lyophilization post-treatment method caused by intermediate purification and final product preparation liquid chromatography purification. cost.
Adopt a relatively better protection strategy for starting raw materials to reduce the generation of by-products, and directly perform simple removal of impurities. Use the differences in solubility between by-products and final products to remove impurities in the final product post-treatment, and use pH adjustment and crystallization purification. High purity Kyotrophin is obtained, which simplifies operation and reduces production costs.
It realizes high yield (over 70%) and low-cost production of high-purity Kyotrophin, simplifies the process flow, is suitable for industrial production, and is simple in purification, avoiding the problems of early product precipitation and impurity wrapping.
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Abstract
Description
Technical Field
[0001] The invention relates to the technical field of organic synthesis, and in particular to a method for synthesizing Kyotrophin. Background Art
[0002] Kyotrophin (L-tyrosyl-L-arginine) is a neuroactive dipeptide that plays a role in pain regulation in the brain. It was first isolated by Japanese scientists from the brain of cattle in 1979. Kyotrophin is named for Kyoto, Japan, where it was discovered, and for its analgesic activity similar to morphine (or endorphins). Kyotrophin has analgesic effects but does not interact with opioid receptors. Instead, it acts to release alpha-fetoenkephalin and stabilize it for degradation. It may also have properties as a neurotransmitter / neuromodulator. Studies have shown that carotid peptide is present in human cerebrospinal fluid and that patients with persistent pain have lower concentrations of carotid peptide.
[0003] Kyotrophin is an endogenous neuroactive dipeptide with analgesic properties. Kyotrophin has anti-inflammatory and antimicrobial activities. Kyotrophin levels in cerebrospinal fluid are negatively correlated with the progression of neurodegeneration in patients with Alzheimer's disease.
[0004] The synthesis methods of kyotrophin are mainly divided into biosynthesis and chemical synthesis. Biosynthesis has the following disadvantages: the enzyme is not specific enough, the generated tripeptide impurities (such as H-Tyr-Tyr-Arg-OH) are large, and the post-treatment process will cause the enzyme to denature and inactivate, making it difficult to recycle the enzyme, resulting in high costs.
[0005] The reported chemical synthesis method of Kyotrophin is as follows: Z(OMe)-Tyr-NHNH 2 and H-Arg(Mts)-OH as raw materials, condensed at room temperature to obtain an intermediate, the intermediate was purified by recrystallization with n-butanol and ether, then deprotected, post-processed and purified by ion exchange resin, then purified by preparative liquid chromatography, and finally freeze-dried to obtain Kyotrophin with a total yield of 48.23%. In this method, the amino group of Z(OMe)-Tyr-NHNH2 is not protected, and the self-reactive dipeptide impurity H-Tyr-Tyr-OH is easily generated during the reaction. At the same time, the intermediate purification and the final product preparative liquid chromatography purification result in a low yield, and the post-processing method of freeze-drying is costly and not conducive to large-scale industrial production.
[0006]
[0007] Using Boc-Tyr(Bn)-OSu and H-Arg(Mtr)-OH as raw materials, condensation at room temperature obtains an intermediate, which is then purified by column, deprotected, and post-processed by preparative liquid chromatography and ion exchange resin to obtain Kyotrophin, with a total yield of about 40.04%. Similarly, the intermediate purification and final product preparative liquid chromatography purification in this method result in a low yield, and the post-processing method of freeze-drying is costly and not conducive to large-scale industrial production.
[0008] Boc-Tyr-OSu or Boc-Tyr-ONB and H-Arg (Mtr or Pme) -OH are used as raw materials, and condensation is carried out at room temperature to obtain an intermediate, and then the intermediate is purified by preparative liquid chromatography, and then deprotected, and the post-treatment is also treated by ion exchange resin, and then a preparative liquid chromatography method is used, and finally lyophilization is used to obtain Kyotrophin, and the total yield is about 34.48%. In this method, the hydroxyl group of Boc-Tyr-OSu or Boc-Tyr-ONB is not protected, and self-condensation impurities will be generated during the synthesis process of the activated ester. At the same time, the intermediate purification and the final product preparative liquid chromatography purification result in a low yield, and the post-treatment method of lyophilization is costly and not conducive to large-scale industrial production.
[0009]
[0010] The yields of the above-mentioned chemical synthesis methods are generally low. Firstly, in the first step of the synthesis process, the intermediate products are separated and purified by purification methods such as recrystallization or column chromatography; secondly, the final products are converted into salt forms using ion exchange resins and purified by methods such as preparative liquid chromatography, resulting in low yields and high costs. Summary of the invention
[0011] The starting materials used in the present invention adopt relatively better protection strategies for the corresponding active sites. Therefore, there are few by-products generated by the starting materials during the reaction, and the intermediate products can be directly treated without separation and purification, and only simple impurities are removed to ensure the total yield. The by-product DIU generated by the condensing agent, which is difficult to remove in the first step intermediate product, is removed in the post-treatment process of the final product by utilizing the solubility difference between the by-product and the final product. At the same time, desalting is performed according to the isoelectric point of Kyotrophin, and purification can effectively remove impurities in the final product by adjusting the pH to obtain high-purity Kyotrophin. The method is simple to operate and easy to industrialize, while reducing production costs. The reaction formula of its synthesis is as follows: .
[0012] The synthesis method of Kyotrophin provided by the present invention comprises: Boc-Tyr(tBu)-OH and HOSu are reacted with a condensing agent to obtain Boc-Tyr(tBu)-OSu, Boc-Tyr(tBu)-OSu is reacted with protected arginine, and Kyotrophin is obtained by deprotection; wherein the condensing agent is DIC, and a Boc-Tyr(tBu)-OSu solution is obtained without any treatment after the reaction is completed.
[0013] The protected arginine is H-Arg(Mtr)-OH. After the reaction is completed, acid is added to adjust the pH value to 2-3, extraction is performed, and the organic phase is concentrated to dryness to obtain Boc-Tyr(tBu)-Arg(Mtr)-OH. Deprotection is performed to obtain the reaction solution of Kyotrophin, which is concentrated under reduced pressure to remove the solvent and excess deprotection reagent, water and an extractant are added, and the aqueous phase is concentrated under reduced pressure until the system becomes viscous. Alkali is added while hot to adjust the pH value to 10.5-11.2, and methanol is added. Solid-liquid separation is performed to obtain a crude Kyotrophin product.
[0014] The crude Kyotrophin is dissolved in water, heated to 50-60°C to dissolve, alkali is added to adjust the pH value to 11.5-12.0, a crystallization solvent is added, and after the Kyotrophin is completely crystallized, the temperature is lowered to 0-10°C, and solid-liquid separation is performed to obtain Kyotrophin. The crystallization solvent is selected from methanol, ethanol or isopropanol, preferably isopropanol. The amount of water used is 1-3 times the weight of the crude Kyotrophin, and the amount of the crystallization solvent used is 3-5 times the weight of the crude Kyotrophin.
[0015] Specifically, the synthesis method of Kyotrophin provided by the present invention comprises: (1) Synthesis of Boc-Tyr(tBu)-OSu In an aprotic solvent, Boc-Tyr(tBu)-OH and HOSu react at room temperature under the action of DIC to obtain Boc-Tyr(tBu)-OSu, wherein the molar ratio of Boc-Tyr(tBu)-OH, HOSu and DIC is 1:1.0-1.1:1.0-1.1.
[0016] (2) Synthesis of Boc-Tyr(tBu)-Arg(Mtr)-OH In solvent A, under the action of an acid-binding agent, Boc-Tyr(tBu)-OSu and H-Arg(Mtr)-OH are condensed at room temperature to obtain Boc-Tyr(tBu)-Arg(Mtr)-OH, wherein the molar ratio of Boc-Tyr(tBu)-OSu, H-Arg(Mtr)-OH and the acid-binding agent is 1:1.0-1.2:1.0-1.5.
[0017] (3) Synthesis of Kyotrophin In solvent B, at room temperature, Boc-Tyr(tBu)-Arg(Mtr)-OH reacts with a deprotection agent, and Kyotrophin is obtained after post-treatment and purification. The molar ratio of Boc-Tyr(tBu)-Arg(Mtr)-OH to the deprotection agent is 1:3-20.
[0018] Wherein, in step (1), the aprotic solvent is selected from dichloromethane, tetrahydrofuran, acetone, 1,4-dioxane or acetonitrile, and is preferably tetrahydrofuran.
[0019] Wherein, in step (2), solvent A is selected from dichloromethane, tetrahydrofuran, acetone, 1,4-dioxane, acetonitrile, a mixed solvent of tetrahydrofuran and water, a mixed solvent of acetone and water, a mixed solvent of 1,4-dioxane and water, or a mixed solvent of acetonitrile and water, and is preferably a mixed solvent of tetrahydrofuran and water. The acid-binding agent is selected from triethylamine, DIEA, sodium carbonate, potassium carbonate or sodium bicarbonate, and is preferably sodium bicarbonate.
[0020] Wherein, in step (3), solvent B is selected from water, dichloromethane, ethyl acetate or tetrahydrofuran, preferably dichloromethane. The deprotection reagent is selected from trifluoroacetic acid, hydrogen chloride, a dichloromethane solution of hydrogen chloride, a tetrahydrofuran solution of hydrogen chloride, an ethyl acetate solution of hydrogen chloride or hydrochloric acid, preferably trifluoroacetic acid.
[0021] After the deprotection reaction is completed, the solvent and excess deprotection reagent are removed by concentration under reduced pressure, water (specifically 1.5-4.0 times the weight of the crude Kyotrophin product) and ethyl acetate (specifically 1.5-4.0 times the weight of the crude Kyotrophin product) are added, the aqueous phase is concentrated under reduced pressure until the system becomes viscous, ammonia water is added while hot to adjust the pH value to 10.5-11.2, methanol is added, and solid-liquid separation is performed to obtain the crude Kyotrophin product. The amount of methanol used is 6-9 times the weight of the crude Kyotrophin product.
[0022] The base is selected from aqueous sodium hydroxide solution, aqueous lithium hydroxide solution, aqueous potassium hydroxide solution or aqueous ammonia, and is preferably aqueous ammonia.
[0023] Preferably, the crude Kyotrophin is dissolved in water, heated to 50-60°C to dissolve, ammonia water is added to adjust the pH value to 11.5-12.0, and then isopropanol is added dropwise. After the Kyotrophin is completely crystallized, the temperature is lowered to 0-10°C and filtered to obtain the Kyotrophin. The amount of water used is 1-3 times the weight of the crude Kyotrophin, and the amount of isopropanol used is 3-5 times the weight of the crude Kyotrophin.
[0024] Preferably, the synthesis method of Kyotrophin provided by the present invention comprises: (1) Synthesis of Boc-Tyr(tBu)-OSu In an aprotic solvent, Boc-Tyr(tBu)-OH and HOSu react at room temperature under the action of DIC to obtain a Boc-Tyr(tBu)-OSu solution, wherein the molar ratio of Boc-Tyr(tBu)-OH, HOSu and DIC is 1:1.0-1.1:1.0-1.1; the aprotic solvent is selected from dichloromethane, tetrahydrofuran, acetone, 1,4-dioxane or acetonitrile.
[0025] (2) Synthesis of Boc-Tyr(tBu)-Arg(Mtr)-OH In solvent A, under the action of an acid-binding agent, Boc-Tyr(tBu)-OSu and H-Arg(Mtr)-OH undergo condensation reaction at room temperature. After the reaction is completed, acid is added to adjust the pH value to 2-3, extraction is performed, the organic phase is washed with water, and the organic phase is concentrated to dryness to obtain Boc-Tyr(tBu)-Arg(Mtr)-OH. The molar ratio of Boc-Tyr(tBu)-OSu, H-Arg(Mtr)-OH and the acid-binding agent is 1:1.0-1.2:1.0-1.5; solvent A is selected from dichloromethane, tetrahydrofuran, acetone, 1.4-dioxane, acetonitrile, a mixed solvent of tetrahydrofuran and water, a mixed solvent of acetone and water, a mixed solvent of 1.4-dioxane and water, or a mixed solvent of acetonitrile and water; and the acid-binding agent is selected from triethylamine, DIEA, sodium carbonate, potassium carbonate or sodium bicarbonate.
[0026] (3) Synthesis of Kyotrophin In solvent B, at room temperature, Boc-Tyr(tBu)-Arg(Mtr)-OH reacts under the action of a deprotecting agent. After the reaction is completed, the solvent and excess deprotecting agent are removed by vacuum concentration, and water and an extractant are added. The aqueous phase is vacuum concentrated until the system becomes viscous, and a base is added while hot to adjust the pH value to 10.5-11.2, and methanol is added to separate the solid and liquid to obtain a crude Kyotrophin product. The molar ratio of Boc-Tyr(tBu)-Arg(Mtr)-OH to the deprotecting agent is 1:3-20, and the amount of methanol used is 6-9 times the weight of the crude Kyotrophin product; the solvent B is selected from water, dichloromethane, ethyl acetate or tetrahydrofuran, and the deprotecting agent is selected from trifluoroacetic acid, hydrogen chloride, a dichloromethane solution of hydrogen chloride, a tetrahydrofuran solution of hydrogen chloride, an ethyl acetate solution of hydrogen chloride or hydrochloric acid.
[0027] (4) Purification of Kyotrophin The crude Kyotrophin is dissolved in water, heated to 50-60°C to dissolve, alkali is added to adjust the pH value to 11.5-12.0, and then a crystallization solvent is added dropwise. After the Kyotrophin is completely crystallized, the temperature is lowered to 0-10°C, and solid-liquid separation is performed to obtain Kyotrophin. The crystallization solvent is selected from methanol, ethanol or isopropanol, etc., the amount of water used is 1-3 times the weight of the crude Kyotrophin, and the amount of the crystallization solvent used is 3-5 times the weight of the crude Kyotrophin; the alkali is selected from sodium hydroxide aqueous solution, lithium hydroxide aqueous solution, potassium hydroxide aqueous solution or ammonia water, etc.
[0028] More preferably, the synthesis method of Kyotrophin provided by the present invention comprises: (1) Synthesis of Boc-Tyr(tBu)-OSu In tetrahydrofuran, Boc-Tyr(tBu)-OH and HOSu react under the action of DIC at room temperature to obtain a tetrahydrofuran solution of Boc-Tyr(tBu)-OSu, wherein the molar ratio of Boc-Tyr(tBu)-OH, HOSu and DIC is 1:1.0-1.1:1.0-1.1.
[0029] (2) Synthesis of Boc-Tyr(tBu)-Arg(Mtr)-OH Add H-Arg(Mtr)-OH to water, add sodium bicarbonate solution dropwise, then add tetrahydrofuran solution of Boc-Tyr(tBu)-OSu dropwise, react at room temperature, add hydrochloric acid to adjust pH value to 2-3 after the reaction is completed, extract with ethyl acetate, wash the organic phase with water, and concentrate the organic phase to obtain Boc-Tyr(tBu)-Arg(Mtr)-OH. The molar ratio of Boc-Tyr(tBu)-OSu, H-Arg(Mtr)-OH and sodium bicarbonate is 1:1.0-1.2:1.0-1.5.
[0030] (3) Synthesis of Kyotrophin Boc-Tyr(tBu)-Arg(Mtr)-OH is added to dichloromethane, and trifluoroacetic acid is added dropwise at room temperature. After the reaction is completed, the dichloromethane and excess trifluoroacetic acid are removed by vacuum concentration, and water and ethyl acetate are added to obtain an aqueous phase. The aqueous phase is vacuum concentrated until the system becomes viscous, and ammonia water is added while hot to adjust the pH value to 10.5-11.2, and methanol is added and filtered to obtain a crude Kyotrophin product. The molar ratio of Boc-Tyr(tBu)-Arg(Mtr)-OH to trifluoroacetic acid is 1:3-20, and the amount of methanol used is 6-9 times the weight of the crude Kyotrophin product. (4) Purification of Kyotrophin Dissolve the crude Kyotrophin in water, heat it to 50-60°C to dissolve it, add ammonia water to adjust the pH value to 11.5-12.0, then add isopropanol dropwise, wait for the Kyotrophin to crystallize completely, cool it to 0-10°C and filter it to obtain Kyotrophin. The amount of water used is 1-3 times the weight of the crude Kyotrophin, and the amount of isopropanol used is 3-5 times the weight of the crude Kyotrophin.
[0031] This patent has the following advantages: (1) The purification process is simple. Step 1 does not require any treatment, step 2 only requires extraction, step 3 involves extraction and pH adjustment (the isoelectric point of Kyotrophin is about 10.8), and step 4 involves recrystallization at a relatively high temperature (at a specific pH value). Compared with the column treatment in the prior art, this process is much simpler and suitable for industrial production. (2) The Kyotrophin obtained in this patent has high purity (more than 99%) and high yield (more than 70%, more than 75% under preferred conditions); (3) During the purification process, alkali is added first, and then the solvent is added dropwise to avoid premature precipitation of the product, which will cause impurities to be wrapped in the product and cannot be removed; (4) This patent uses HOSu to activate Boc-Tyr(tBu)-OH and adopts arginine with a specific protective group (specifically H-Arg(Mtr)-OH) to perform acid-amine condensation through the activated ester method. There is no need to further protect the carboxyl group of arginine, which not only improves the yield and reduces the cost, but also simplifies the subsequent processing process; the three protective groups are removed simultaneously by a deprotection reagent, which simplifies the process. DETAILED DESCRIPTION
[0032] The invention is further described below through specific examples, but it should be noted that these examples are merely illustrative and do not limit the scope of the invention.
[0033] Example 1: Synthesis of Boc-Tyr(tBu)-OSu 200.00 g of tetrahydrofuran, 100.00 g of Boc-Tyr(tBu)-OH and 35.81 g of HOSu were added to a four-necked flask in sequence, and then 39.27 g of DIC was dissolved in 100 g of tetrahydrofuran, and the tetrahydrofuran solution of DIC was slowly added dropwise at room temperature. After the addition, the mixture was stirred at room temperature for 6-8 hours to obtain a Boc-Tyr(tBu)-OSu solution, and the yield was 100%.
[0034] Example 2: Synthesis of Boc-Tyr(tBu)-Arg(Mtr)-OH Then, 126.00 g of H-Arg(Mtr)-OH and 500 g of water were added to another four-necked flask, 37.35 g of sodium bicarbonate was added dropwise, and then the whole batch of Boc-Tyr(tBu)-OSu solution in Example 1 was added dropwise at room temperature. After the addition was completed, the reaction was carried out for 10-12 hours. After the reaction was completed, the pH value was adjusted to 2-3 with dilute hydrochloric acid, 500 g of ethyl acetate was added for extraction, and then the organic phase was washed twice with 200 g*2 water, dried over anhydrous sodium sulfate to obtain an organic phase, and concentrated to obtain Boc-Tyr(tBu)-Arg(Mtr)-OH, which was directly used in the next step. The yield was 100%.
[0035] Example 3: Synthesis of Kyotrophin To the whole batch of Boc-Tyr(tBu)-Arg(Mtr)-OH in Example 2, add 600.0g of dichloromethane, add 168.96g of trifluoroacetic acid dropwise at room temperature, react at room temperature for 4-6 hours, and after the reaction, concentrate under reduced pressure to remove dichloromethane and excess trifluoroacetic acid, add 200g of water and 200g of ethyl acetate, stir and extract to obtain an aqueous phase, and then extract the aqueous phase twice with 200g*2 ethyl acetate to obtain an aqueous phase. Concentrate under reduced pressure until the system is viscous, add ammonia water dropwise, adjust the pH value to 10.5-11.2, add 600g of methanol dropwise, precipitate a large amount of solids, and filter to obtain 82.15g of crude Kyotrophin, with a crude yield of 82.16%.
[0036] Example 4: Purification of Kyotrophin 20.00 g of crude Kyotrophin and 40.00 g of water were added to a four-necked flask in sequence, and then the temperature was raised to 50-60°C to dissolve. A small amount of ammonia water was used to adjust the pH value to 11.5-12.0, and 80.00 g of methanol was added dropwise. A large amount of solid precipitated from the system. The temperature was lowered to 0-10°C, filtered, and dried to obtain 17.1 g of Kyotrophin with a purity of 99.53% and a yield of 85.50%.
[0037] Example 5: Purification of Kyotrophin Add 20.00 g of crude Kyotrophin and 40.00 g of water to a four-necked flask in sequence, then heat to 50-60°C to dissolve, adjust the pH to 11.5-12.0 with a small amount of 10% aqueous sodium hydroxide solution, add 80.00 g of ethanol dropwise, a large amount of solid precipitates in the system, cool to 0-10°C, filter and dry to obtain 17.5 g of Kyotrophin with a purity of 99.33% and a yield of 87.5%.
[0038] Example 6: Purification of Kyotrophin 20.00 g of crude Kyotrophin and 40.00 g of water were added to a four-necked flask in sequence, and then the temperature was raised to 50-60°C to dissolve. A small amount of ammonia water was used to adjust the pH to 11.5-12.0, and 80.00 g of isopropanol was added dropwise. A large amount of solid precipitated from the system. The temperature was lowered to 0-10°C, filtered, and dried to obtain 18.30 g of Kyotrophin with a purity of 99.20% and a yield of 91.50%.
[0039] Example 7: Synthesis of Kyotrophin 400.00 g of tetrahydrofuran, 200 g of Boc-Tyr(tBu)-OH and 71.62 g of HOSu were added to a four-necked flask in sequence, and then 78.54 g of DIC was dissolved in 200 g of tetrahydrofuran, and the tetrahydrofuran solution of DIC was slowly added dropwise at room temperature. After the addition was completed, the reaction was stirred at room temperature for 6-8 hours to obtain a Boc-Tyr(tBu)-OSu solution. Then 1000 g of water and 74.70 g of sodium bicarbonate were added to another four-necked flask, and 252.00 g of H-Arg(Mtr)-OH, then add the whole batch of Boc-Tyr(tBu)-OSu solution dropwise at room temperature, react for 10-12 hours, adjust the pH to 2-3 with dilute hydrochloric acid, extract with 1000g ethyl acetate, then wash the organic phase twice with 400g*2 water, dry with anhydrous sodium sulfate to obtain the organic phase, and concentrate under reduced pressure to dryness to obtain Boc-Tyr(tBu)-Arg(Mtr)-OH, then add 1200g dichloromethane to Boc-Tyr(tBu)-Arg(Mtr)-OH, add 337.92g trifluoroacetic acid dropwise at room temperature, react at room temperature for 4-6 hours, concentrate under reduced pressure to dryness, add 400g water and 400g ethyl acetate to extract to obtain the aqueous phase, and then extract the aqueous phase twice with 400g*2 ethyl acetate to obtain the aqueous phase. The aqueous phase was concentrated under reduced pressure until the system was viscous, and ammonia water was added dropwise to adjust the pH value to 10.5-11.2, and 1200g of methanol was added dropwise to precipitate a large amount of solids, which were filtered to obtain crude Kyotrophin. Then, the whole batch of crude Kyotrophin and 340g of water were added to a four-necked flask, and the temperature was raised to 50-60°C, and then the pH value was adjusted to 11.5-12.0 with ammonia water, and 680.0g of methanol was added dropwise to precipitate a large amount of solids, and the temperature was lowered to 0-10°C, filtered, and dried to obtain 140.83g of Kyotrophin with a purity of 99.46% and a total yield of 70.42%.
[0040] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims
1. A method for synthesizing Kyotrophin, the method comprising: Boc-Tyr(tBu)-OH and HOSu are reacted with a condensing agent to obtain Boc-Tyr(tBu)-OSu, and Boc-Tyr(tBu)-OSu is reacted with protected arginine and deprotected to obtain Kyotrophin; characterized in that: The condensation agent is DIC, and after the reaction is completed, no treatment is performed to obtain a Boc-Tyr(tBu)-OSu solution; The protected arginine is H-Arg(Mtr)-OH. After the reaction is completed, an acid is added to adjust the pH value to 2-3, extraction is performed, and the organic phase is concentrated to dryness to obtain Boc-Tyr(tBu)-Arg(Mtr)-OH; the reaction solution of Kyotrophin is deprotected, and the solvent and excess deprotection reagent are removed by vacuum concentration, water and an extractant are added, and the aqueous phase is concentrated under reduced pressure until the system becomes viscous, and a base is added while hot to adjust the pH value to 10.5-11.2, and methanol is added, and solid-liquid separation is performed to obtain a crude Kyotrophin product; The crude Kyotrophin is dissolved in water, heated to 50-60°C to dissolve, alkali is added to adjust the pH value to 11.5-12.0, a crystallization solvent is added, and after the Kyotrophin is completely crystallized, the temperature is lowered to 0-10°C, and the solid-liquid separation is performed to obtain Kyotrophin; the crystallization solvent is selected from methanol, ethanol or isopropanol, the amount of water used is 1-3 times the weight of the crude Kyotrophin, and the amount of the crystallization solvent used is 3-5 times the weight of the crude Kyotrophin.
2. The method for synthesizing Kyotrophin according to claim 1, characterized in that: The method comprises: (1) Synthesis of Boc-Tyr(tBu)-OSu In an aprotic solvent, Boc-Tyr(tBu)-OH and HOSu react at room temperature under the action of DIC to obtain Boc-Tyr(tBu)-OSu; the molar ratio of Boc-Tyr(tBu)-OH, HOSu and DIC is 1:1.0-1.1:1.0-1.1; (2) Synthesis of Boc-Tyr(tBu)-Arg(Mtr)-OH In solvent A, under the action of an acid-binding agent, Boc-Tyr(tBu)-OSu and H-Arg(Mtr)-OH are condensed at room temperature to obtain Boc-Tyr(tBu)-Arg(Mtr)-OH; the molar ratio of Boc-Tyr(tBu)-OSu, H-Arg(Mtr)-OH and the acid-binding agent is 1:1.0-1.2:1.0-1.5; (3) Synthesis of Kyotrophin In solvent B, at room temperature, Boc-Tyr(tBu)-Arg(Mtr)-OH reacts with a deprotection agent, and Kyotrophin is obtained after post-treatment and purification. The molar ratio of Boc-Tyr(tBu)-Arg(Mtr)-OH to the deprotection agent is 1:3-20.
3. The method for synthesizing Kyotrophin according to claim 2, characterized in that: In step (1), the aprotic solvent is selected from dichloromethane, tetrahydrofuran, acetone, 1,4-dioxane or acetonitrile.
4. The method for synthesizing Kyotrophin according to claim 2, characterized in that: In step (2), the solvent A is selected from dichloromethane, tetrahydrofuran, acetone, 1,4-dioxane, acetonitrile, a mixed solvent of tetrahydrofuran and water, a mixed solvent of acetone and water, a mixed solvent of 1,4-dioxane and water, or a mixed solvent of acetonitrile and water; and the acid binding agent is selected from triethylamine, DIEA, sodium carbonate, potassium carbonate, or sodium bicarbonate.
5. The method for synthesizing Kyotrophin according to claim 2, characterized in that: In step (3), the solvent B is selected from water, dichloromethane, ethyl acetate or tetrahydrofuran, and the deprotection reagent is selected from trifluoroacetic acid, hydrogen chloride, a dichloromethane solution of hydrogen chloride, a tetrahydrofuran solution of hydrogen chloride, an ethyl acetate solution of hydrogen chloride or hydrochloric acid.
6. The method for synthesizing Kyotrophin according to claim 1, characterized in that: After the deprotection reaction is completed, the solvent and excess deprotection reagent are removed by concentration under reduced pressure, water and ethyl acetate are added, and the aqueous phase is concentrated under reduced pressure until the system becomes viscous. Ammonia water is added while hot to adjust the pH value to 10.5-11.2, and methanol is added to separate the solid and liquid to obtain the crude Kyotrophin; the amount of methanol used is 6-9 times the weight of the crude Kyotrophin.
7. The method for synthesizing Kyotrophin according to claim 1, characterized in that: The alkali is selected from aqueous sodium hydroxide solution, aqueous lithium hydroxide solution, aqueous potassium hydroxide solution or aqueous ammonia.
8. The method for synthesizing Kyotrophin according to claim 1, characterized in that: The crude Kyotrophin is dissolved in water, heated to 50-60°C to dissolve, ammonia water is added to adjust the pH value to 11.5-12.0, and then isopropanol is added dropwise. After the Kyotrophin is completely crystallized, the temperature is lowered to 0-10°C and then filtered to obtain the Kyotrophin; the amount of water used is 1-3 times the weight of the crude Kyotrophin, and the amount of isopropanol used is 3-5 times the weight of the crude Kyotrophin.
9. The method for synthesizing Kyotrophin according to claim 1, characterized in that: The method comprises: (1) Synthesis of Boc-Tyr(tBu)-OSu In an aprotic solvent, Boc-Tyr(tBu)-OH and HOSu react at room temperature under the action of DIC to obtain a Boc-Tyr(tBu)-OSu solution; the molar ratio of Boc-Tyr(tBu)-OH, HOSu and DIC is 1:1.0-1.1:1.0-1.1; the aprotic solvent is selected from dichloromethane, tetrahydrofuran, acetone, 1,4-dioxane or acetonitrile; (2) Synthesis of Boc-Tyr(tBu)-Arg(Mtr)-OH In solvent A, under the action of an acid-binding agent, Boc-Tyr(tBu)-OSu and H-Arg(Mtr)-OH undergo a condensation reaction at room temperature. After the reaction is completed, acid is added to adjust the pH value to 2-3, extraction is performed, the organic phase is washed with water, and the organic phase is concentrated to obtain Boc-Tyr(tBu)-Arg(Mtr)-OH; the molar ratio of Boc-Tyr(tBu)-OSu, H-Arg(Mtr)-OH and the acid-binding agent is 1:1.0-1.2:1.0-1.5; the solvent A is selected from dichloromethane, tetrahydrofuran, acetone, 1.4-dioxane, acetonitrile, a mixed solvent of tetrahydrofuran and water, a mixed solvent of acetone and water, a mixed solvent of 1.4-dioxane and water, or a mixed solvent of acetonitrile and water; the acid-binding agent is selected from triethylamine, DIEA, sodium carbonate, potassium carbonate or sodium bicarbonate; (3) Synthesis of Kyotrophin In solvent B, at room temperature, Boc-Tyr(tBu)-Arg(Mtr)-OH reacts under the action of a deprotecting agent. After the reaction is completed, the solvent and excess deprotecting agent are removed by concentration under reduced pressure, water and an extractant are added, the aqueous phase is concentrated under reduced pressure until the system becomes viscous, alkali is added while hot to adjust the pH value to 10.5-11.2, methanol is added, and solid-liquid separation is performed to obtain a crude Kyotrophin product; the molar ratio of the Boc-Tyr(tBu)-Arg(Mtr)-OH to the deprotecting agent is 1:3-20, and the amount of the methanol is 6-9 times the weight of the crude Kyotrophin product; the solvent B is selected from water, dichloromethane, ethyl acetate or tetrahydrofuran, and the deprotecting agent is selected from trifluoroacetic acid, hydrogen chloride, a dichloromethane solution of hydrogen chloride, a tetrahydrofuran solution of hydrogen chloride, an ethyl acetate solution of hydrogen chloride or hydrochloric acid; (4) Purification of Kyotrophin The crude Kyotrophin is dissolved in water, heated to 50-60°C to dissolve, alkali is added to adjust the pH value to 11.5-12.0, and then a crystallization solvent is added dropwise. After the Kyotrophin is completely crystallized, the temperature is lowered to 0-10°C, and the Kyotrophin is separated from the solid and liquid to obtain the Kyotrophin; the crystallization solvent is selected from methanol, ethanol or isopropanol, the amount of water is 1-3 times the weight of the crude Kyotrophin, and the amount of the crystallization solvent is 3-5 times the weight of the crude Kyotrophin; the alkali is selected from sodium hydroxide aqueous solution, lithium hydroxide aqueous solution, potassium hydroxide aqueous solution or ammonia water.
10. The method for synthesizing Kyotrophin according to claim 1, characterized in that: The method comprises: (1) Synthesis of Boc-Tyr(tBu)-OSu In tetrahydrofuran, Boc-Tyr(tBu)-OH and HOSu react at room temperature under the action of DIC to obtain a tetrahydrofuran solution of Boc-Tyr(tBu)-OSu; the molar ratio of Boc-Tyr(tBu)-OH, HOSu and DIC is 1:1.0-1.1:1.0-1.1; (2) Synthesis of Boc-Tyr(tBu)-Arg(Mtr)-OH Add H-Arg(Mtr)-OH to water, add sodium bicarbonate solution dropwise, then add tetrahydrofuran solution of Boc-Tyr(tBu)-OSu dropwise, react at room temperature, add hydrochloric acid to adjust pH value to 2-3 after the reaction is completed, extract with ethyl acetate, wash the organic phase with water, and concentrate the organic phase to obtain Boc-Tyr(tBu)-Arg(Mtr)-OH; the molar ratio of Boc-Tyr(tBu)-OSu, H-Arg(Mtr)-OH and sodium bicarbonate is 1:1.0-1.2:1.0-1.5; (3) Synthesis of Kyotrophin Boc-Tyr(tBu)-Arg(Mtr)-OH is added to dichloromethane, and trifluoroacetic acid is added dropwise at room temperature. After the reaction is completed, the dichloromethane and excess trifluoroacetic acid are removed by vacuum concentration, and water and ethyl acetate are added to obtain an aqueous phase. The aqueous phase is concentrated under reduced pressure until the system becomes viscous, and ammonia water is added while hot to adjust the pH value to 10.5-11.2, and methanol is added. The crude Kyotrophin is filtered to obtain the crude Kyotrophin product; the molar ratio of the Boc-Tyr(tBu)-Arg(Mtr)-OH to the trifluoroacetic acid is 1:3-20, and the amount of the methanol used is 6-9 times the weight of the crude Kyotrophin product; (4) Purification of Kyotrophin Dissolve the crude Kyotrophin in water, heat it to 50-60°C to dissolve it, add ammonia water to adjust the pH value to 11.5-12.0, then add isopropanol dropwise, wait for the Kyotrophin to crystallize completely, cool it to 0-10°C and filter it to obtain Kyotrophin, the amount of water used is 1-3 times the weight of the crude Kyotrophin, and the amount of isopropanol used is 3-5 times the weight of the crude Kyotrophin.