Synthesis method of N, N-di-L-lysyl-L-cystine dihydrochloride
Through the protection and peptide connection reaction of L-lysine and L-cysteine, combined with hydrochloric acid acidification, the scarcity of N,N-di-L-lysine-L-cysteine dihydrochloride synthesis scheme was solved. The problem is that high purity and efficient production are achieved, and the quality of cell culture medium and biopharmaceutical production efficiency are improved.
Patent Information
- Application Number
- CN202411179198.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-08-27
- Publication Date
- 2024-12-10
AI Technical Summary
In the prior art, the synthesis scheme of N,N-di-L-lysine-L-cystinine dihydrochloride is fewer, and the effective industrial synthesis route is lacking, which affects the efficient production of cell culture medium and the quality of biopharmaceuticals. .
Through the protection of L-lysine and acid chloride, the reaction of L-cysteine and trimethylchlorosilane is combined, and then the peptide connection and deprotection of bispheryl anhydride-L-lysyl chloride is obtained by hydrochloric acid acidification. The purity of N,N-di-L-lysyl-L-cystinine dihydrochloride is achieved by simplified process flow to achieve high yield and high purity product acquisition.
Achieve high purity (up to 99%) of N,N-di-L-lysyl-L-cystinine dihydrochloride, simplify the process flow, improve the production efficiency and the market prospects of the product, and provide A feasible industrial synthesis route was established.
Smart Images
Figure QLYQS_1
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of organic synthesis, and in particular to a method for synthesizing N,N-di-L-lysyl-L-cystine dihydrochloride. Background Art
[0002] Efficient cell culture process is the cornerstone of biopharmaceutical production. N,N-di-L-lysine-L-cystine dihydrochloride is a high-purity peptide product portfolio with clear chemical composition, which helps to prepare more concentrated and stable culture medium, simplify cell culture process and improve productivity.
[0003] As a highly soluble peptide, N,N-di-L-lysine-L-cystine dihydrochloride is designed to address the challenges of L-cystine supply in cell culture, and can be configured with high-concentration, neutral pH basal medium, feed medium and perfusion medium, thereby strengthening and simplifying complex production processes. By improving cell proliferation, survival rate and cell-specific productivity, N,N-di-L-lysine-L-cystine dihydrochloride also helps to improve antibody titer and the overall quality of biopharmaceuticals.
[0004] N,N-Di-L-Lysine-L-Cystine dihydrochloride is a precursor of the key amino acids L-cystine and L-lysine for high-concentration L-cystine-optimized cell culture media. It is highly soluble and acts on cells sensitive to L-alanine accumulation, performing better than alanine-based molecules.
[0005] N,N-di-L-lysine-L-cystine dihydrochloride is an emerging raw material for cell culture medium with good market prospects. Currently, there are relatively few synthetic schemes for N,N-di-L-lysine-L-cystine dihydrochloride reported in relevant materials. Therefore, it is of great significance to independently develop an industrial synthetic route for N,N-di-L-lysine-L-cystine dihydrochloride. Summary of the invention
[0006] The present invention provides a method for synthesizing N,N-di-L-lysyl-L-cystine dihydrochloride, and the reaction scheme is as follows: ; Among them, L-lysine is protected by phthalic anhydride and chlorinated to obtain compound I (diphthalic anhydride-L-lysyl chloride). In a solvent, L-cystine, base B and trimethylsilyl chloride react at 0-50°C to obtain compound II, and the molar ratio of L-cystine, base B and trimethylsilyl chloride is 1:4-6:4-6. In a solvent, compound I and compound II react at 0-40°C. After the reaction is completed, water is added to quench, and a solid is precipitated at room temperature to obtain compound III (N,N-di-diphthalic anhydride-L-lysyl-L-cystine), and the molar ratio of compound I, compound II and water is 2-2.5:1:4-8. Compound III is deprotected by hydrazine removal, and N,N-di-L-lysyl-L-cystine dihydrochloride is obtained after acidification with hydrochloric acid.
[0007] Specifically, the synthesis method of N,N-di-L-lysyl-L-cystine dihydrochloride provided in an embodiment of the present invention comprises: (1) In solvent A, L-lysine, phthalic anhydride and base A react at 80-120° C. to obtain diphthalic anhydride-L-lysine, and the molar ratio of L-lysine, base A and phthalic anhydride is 1:0.01-0.5:2-4. Wherein, solvent A is selected from toluene, xylene, dioxane or no solvent, preferably toluene; base A is selected from triethylamine, pyridine, DBU, potassium carbonate, sodium carbonate, sodium hydroxide or potassium hydroxide, preferably triethylamine.
[0008] (2) Dispersing bisphthalic anhydride-L-lysine in solvent B, adding dichlorothionyl dropwise, and reacting at 40-80° C. to obtain compound I, wherein the molar ratio of bisphthalic anhydride-L-lysine to dichlorothionyl is 1:1-3; wherein solvent B is selected from toluene, dichloromethane, chloroform, acetone or tetrahydrofuran, and preferably toluene or chloroform.
[0009] (3) In solvent C, L-cystine, base B and trimethylsilyl chloride react at 0-70° C. to obtain a solution of compound II, wherein the molar ratio of L-cystine, base B and trimethylsilyl chloride is 1:4-6:4-6. Solvent C is selected from dichloromethane, chloroform, acetone, tetrahydrofuran, acetonitrile or dioxane, preferably dichloromethane or chloroform; base B is selected from triethylamine, pyridine, DBU, potassium carbonate, sodium carbonate, sodium bicarbonate or sodium hydroxide, preferably triethylamine.
[0010] (4) Compound I is dissolved in solvent C and added dropwise to the solution of compound II, and reacted at 0-40° C. After the reaction is completed, water is added to quench the reaction, and solid-liquid separation is performed to obtain compound III. The molar ratio of compound I, compound II and water is 2-2.5:1:4-8.
[0011] (5) In solvent D, compound III reacts with hydrazine hydrate at 40-80° C. After the reaction is completed, hydrochloric acid is added to adjust the pH value to 5-6 to obtain N,N-di-L-lysyl-L-cystine dihydrochloride, and the molar ratio of compound III to hydrazine hydrate is 1:4-6. Wherein, solvent D is selected from water, methanol, ethanol, acetone, tetrahydrofuran, acetonitrile or dioxane, and is preferably water.
[0012] In this patent, steps (1)-(3) do not require further purification and can be directly subjected to subsequent reactions. The purification processes of steps (4) (product precipitation and separation from the reaction system) and (5) (purification by recrystallization) can obtain high-purity products, thus simplifying the process.
[0013] In step (3), TMS protection is used because it can protect both the amino group and the carboxyl group, enhance the selectivity of the reaction, and increase the solubility and stability of cystine in the solvent. Cystine has very poor solubility in common solvents and is not resistant to strong alkali and high temperature.
[0014] In step (4), the peptide grafting process, the deprotection process and the product precipitation process are realized, and the product will slowly precipitate in the solution after the reaction (a mixed system of solvent C and water).
[0015] Further, in step (5), after adding hydrochloric acid to adjust the pH value to 5-6, solid-liquid separation is performed, the filtrate is concentrated under reduced pressure to remove part of the water, and anhydrous ethanol is added dropwise at 50-60° C. After the addition is complete, the temperature is gradually lowered to 10-20° C., crystallization is performed, and solid-liquid separation is performed to obtain N,N-di-L-lysyl-L-cystine dihydrochloride. The product is concentrated under reduced pressure to 2.5-4.0 times the weight of the product, and the amount of anhydrous ethanol used is 5-8 times the weight of the product.
[0016] More specifically, the synthesis method of N,N-di-L-lysyl-L-cystine dihydrochloride provided by the embodiment of the present invention comprises: (1) In toluene, L-lysine, phthalic anhydride and triethylamine react at 80-120° C. to obtain diphthalic anhydride-L-lysine, wherein the molar ratio of L-lysine, triethylamine and phthalic anhydride is 1:0.01-0.5:2-4.
[0017] (2) Disperse bisphthalic anhydride-L-lysine in chloroform or toluene, add thionyl chloride dropwise, react at 40-80°C, and after the reaction is completed, concentrate under reduced pressure to obtain compound I. The molar ratio of bisphthalic anhydride-L-lysine to thionyl chloride is 1:1-3.
[0018] (3) In dichloromethane or chloroform, L-cystine, triethylamine and trimethylsilyl chloride react at 0-70° C. to obtain a solution of compound II, wherein the molar ratio of L-cystine, triethylamine and trimethylsilyl chloride is 1:4-6:4-6.
[0019] (4) Compound I is dissolved in dichloromethane or chloroform and added dropwise to the solution of Compound II, and reacted at 0-40°C. After the reaction is completed, water is added to quench the reaction, and solid-liquid separation is performed to obtain Compound III. The molar ratio of Compound I, Compound II and water is 2-2.5:1:4-8.
[0020] (5) Compound III reacts with hydrazine hydrate in water at 40-80°C. After the reaction is completed, hydrochloric acid is added to adjust the pH value to 5-6, solid-liquid separation is performed, and the filtrate is concentrated under reduced pressure to 2.5-4.0 times the weight of the product. Anhydrous ethanol is added dropwise at 50-60°C. After the addition is completed, the temperature is gradually lowered to 10-20°C, crystallization is performed, and solid-liquid separation is performed to obtain N,N-di-L-lysyl-L-cystine dihydrochloride. The molar ratio of compound III to hydrazine hydrate is 1:4-6, and the amount of anhydrous ethanol used is 5-8 times the weight of the product.
[0021] The invention has the following advantages: the method uses L-lysine and L-cystine as starting materials, and finally obtains high-purity (purity up to 99%) N, N-di-L-lysyl-L-cystine dihydrochloride through L-lysine protection and L-cystine protection, peptide grafting, deprotection, acidification and purification. The method has the advantages of cheap and easy-to-obtain starting materials, high yield (molar yield up to 60%), etc., and provides a feasible scheme for the process research of N, N-di-L-lysyl-L-cystine dihydrochloride. DETAILED DESCRIPTION
[0022] 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.
[0023] (I) Synthesis of bis(phthalic anhydride)-L-lysine 1.1 Add 149.19g of L-lysine (1.0mol), 450g of toluene, 10.12g of triethylamine (0.1mol) and 325.86g of phthalic anhydride (2.2mol) to a 2L reaction bottle with a water separator, stir evenly and gradually heat up to 110-115℃, reflux with water until no water is separated, cool to 50-80℃, and concentrate under reduced pressure until no liquid drops out to obtain colorless and transparent oily phthalic anhydride-L-lysine, with a yield of 100%.
[0024] 1.2 Add 149.19 g of L-lysine (1.0 mol), 300 g of toluene, 15.18 g of triethylamine (0.15 mol) and 370.30 g of phthalic anhydride (2.5 mol) into a 2 L reaction bottle with a water separator, stir evenly and gradually heat to 115-120 ° C, reflux with water until no water is removed, cool to room temperature to obtain a toluene solution of phthalic anhydride-L-lysine, the yield is calculated as 100%, and it is directly used in the next step.
[0025] (II) Synthesis of bis(phthalic anhydride)-L-lysine chloride 2.1 Add the colorless and transparent oily bisphthalanhydride-L-lysine (1.0 mol) obtained in Example 1.1 and 300 g of chloroform to a 2 L reaction bottle, and add 142.76 g of thionyl chloride (1.2 mol) dropwise at a temperature below 50 °C. After the addition is complete, gradually increase the temperature to 55-65 °C and keep the temperature for 4-5 hours. After the reaction is complete, concentrate under reduced pressure at below 60 °C until no liquid drips out, and continue to concentrate for 2 hours. Cool down to room temperature to obtain slightly yellow oily bisphthalanhydride-L-lysine chloride, and the yield is calculated as 100%.
[0026] 2.2 Add the toluene solution (1.0 mol) of bis(phthalic anhydride)-L-lysine obtained in Example 1.2 to a 2L reaction bottle, and add 178.45 g of thionyl chloride (1.5 mol) dropwise at a temperature below 50°C. After the addition is complete, gradually increase the temperature to 65-75°C and keep the temperature for 4-5 hours. After the reaction is complete, concentrate under reduced pressure at below 60°C until no liquid drips out, and continue to concentrate for 2 hours. Cool down to room temperature to obtain slightly yellow oily bis(phthalic anhydride)-L-lysine chloride, and the yield is calculated as 100%.
[0027] (III) Synthesis of L-cystine protective solution 3.1 In a 2L reaction bottle, add 120.15g of L-cystine (0.5mol), 400g of dichloromethane, and 212.52g of triethylamine (2.1mol), cool to 0-10℃, gradually add 228.14g of trimethylsilyl chloride (2.1mol), gradually raise the temperature to 35-40℃ after the addition is complete, keep the temperature for reaction for 8-10h, and after the reaction is complete, cool to room temperature to obtain L-cystine protective solution, protect with nitrogen, and set aside. The yield is calculated as 100%.
[0028] 3.2 In a 2L reaction bottle, add 114.43g of L-cystine (0.48mol), 400g of chloroform, and 252.98g of triethylamine (2.5mol), cool to 0-10℃, gradually add 217.28g of trimethylsilyl chloride (2.0mol), gradually increase the temperature to 50-60℃ after the addition is complete, keep the temperature for reaction for 6-8h, and after the reaction is complete, cool to room temperature to obtain L-cystine protective solution, protect with nitrogen, and set aside. The yield is calculated as 100%.
[0029] (IV) Synthesis of N,N-di-di-phthalic anhydride-L-lysyl-L-cystine 4.1 The bis-phthalic anhydride-L-lysyl chloride obtained in Example 2.1 was added with 300 g of dichloromethane, stirred to dissolve, and added dropwise to the L-cystine protective solution obtained in Example 3.1 at a temperature below 40°C. After the addition was completed, the reaction was maintained at 15-25°C for 5-8 hours. After the reaction was completed, 72 g of water (4.0 mol) gradually precipitated a solid, which was crystallized at room temperature for 2-4 hours and filtered to obtain a brown-yellow powdery solid. The solid was dried in vacuo at 40-50°C to obtain 445 g of solid N,N-bis-phthalic anhydride-L-lysyl-L-cystine, with a molar yield (calculated as cystine) of 82.1%.
[0030] 4.2 Add 300g of chloroform to the bis-phthalic anhydride-L-lysyl chloride obtained in Example 2.2, stir to dissolve, and add dropwise to the L-cystine protective solution obtained in Example 3.2 at a temperature below 40°C. After the addition is complete, keep the temperature at 15-25°C for 5-8h. After the reaction is complete, 51.8g of water (2.88mol) gradually precipitates a solid. Crystallize at room temperature for 2-4h, filter to obtain a brown-yellow powdery solid, and dry in vacuo at 40-50°C to obtain 408.5g of solid N,N-bis-phthalic anhydride-L-lysyl-L-cystine, with a molar yield (calculated as cystine) of 78.5%.
[0031] (V) Synthesis of N,N-di-L-lysyl-L-cystine dihydrochloride 5.1 Add 445g of N,N-di-diphthalic anhydride-L-lysyl-L-cystine (0.41mol) and 1000g of water to a 5L reaction bottle, gradually add 115.89g of 80% hydrazine hydrate (1.85mol) under stirring, gradually heat to 50-60℃ after addition, and keep warm for 8-10h. After the reaction is completed, adjust the pH to 5-6 with concentrated hydrochloric acid, and continue stirring for 2h. Filter, and concentrate the filtrate under reduced pressure below 60℃ to a total weight of 500-600g of the system, stop concentrating, and add 1000g of anhydrous ethanol at 50-60℃. After the addition is complete, gradually cool to 15-25℃, keep warm for crystallization for 2h, filter, and dry under reduced pressure at 50℃ to obtain 182.1g of white powder, with a yield of 64% (calculated as cystine), HPLC purity of 99.18%, and chloride ion content of 12.6%.
[0032] 5.2 Add 408.5g of N,N-di-diphthalic anhydride-L-lysyl-L-cystine (0.38mol) and 900g of water to a 5L reaction bottle, and gradually add 117.76g of 80% hydrazine hydrate (1.88mol) under stirring. After adding, gradually heat to 65-75℃ and keep warm for 6-8h. After the reaction is completed, adjust the pH to 5-6 with concentrated hydrochloric acid and continue stirring for 2h. Filter, and concentrate the filtrate under reduced pressure below 60℃ to a total weight of 500-600g. Stop concentrating, and add 1200g of anhydrous ethanol at 50-60℃. After the addition is complete, gradually cool to 10-20℃, keep warm for crystallization for 2h, filter, and dry under reduced pressure at 50℃ to obtain 163.9g of white powder, with a yield of 60% (calculated as cystine), HPLC purity of 99.08%, and chloride ion content of 12.8%.
[0033] 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 N,N-di-L-lysyl-L-cystine dihydrochloride, characterized in that: The reaction route is as follows: ； The method comprises the following steps: L-lysine is protected by phthalic anhydride and chlorinated to obtain compound I; L-cystine, base B and trimethylsilyl chloride are reacted at 0-50°C in a solvent to obtain compound II, wherein the molar ratio of L-cystine, base B and trimethylsilyl chloride is 1:4-6:4-6; compound I and compound II are reacted at 0-40°C in a solvent, and after the reaction is completed, water is added to quench the reaction, and a solid is precipitated at room temperature to obtain compound III, wherein the molar ratio of compound I, compound II and water is 2-2.5:1:4-8; compound III is subjected to hydrazine deprotection to remove the protecting group, and N,N-di-L-lysyl-L-cystine dihydrochloride is obtained after acidification with hydrochloric acid.
2. The synthetic method of N,N-di-L-lysyl-L-cystine dihydrochloride according to claim 1, characterized in that: The method comprises: (1) In solvent A, L-lysine, phthalic anhydride and base A react at 80-120° C. to obtain diphthalic anhydride-L-lysine, wherein the molar ratio of L-lysine, base A and phthalic anhydride is 1:0.01-0.5:2-4; (2) Dispersing bisphthalic anhydride-L-lysine in solvent B, adding dithionyl chloride dropwise, and reacting at 40-80° C. to obtain compound I, wherein the molar ratio of bisphthalic anhydride-L-lysine to dithionyl chloride is 1:1-3; (3) In solvent C, L-cystine, base B and trimethylsilyl chloride react at 0-70° C. to obtain a solution of compound II, wherein the molar ratio of L-cystine, base B and trimethylsilyl chloride is 1:4-6:4-6; (4) dissolving compound I in solvent C and adding dropwise to the solution of compound II, reacting at 0-40° C., adding water to quench the reaction after completion, and performing solid-liquid separation to obtain compound III, wherein the molar ratio of compound I, compound II and water is 2-2.5:1:4-8; (5) In solvent D, compound III is reacted with hydrazine hydrate at 40-80° C. After the reaction is completed, hydrochloric acid is added to adjust the pH value to 5-6 to obtain N,N-di-L-lysyl-L-cystine dihydrochloride, wherein the molar ratio of compound III to hydrazine hydrate is 1:4-6.
3. The synthetic method of N,N-di-L-lysyl-L-cystine dihydrochloride according to claim 2, characterized in that, In step (1), the solvent A is selected from toluene, xylene, dioxane or no solvent, and the base A is selected from triethylamine, pyridine, DBU, potassium carbonate, sodium carbonate, sodium hydroxide or potassium hydroxide.
4. The synthetic method of N,N-di-L-lysyl-L-cystine dihydrochloride according to claim 2, characterized in that, In step (2), the solvent B is selected from toluene, dichloromethane, chloroform, acetone or tetrahydrofuran.
5. The method for synthesizing N,N-di-L-lysyl-L-cystine dihydrochloride according to claim 2, characterized in that: In step (3), the solvent C is selected from dichloromethane, chloroform, acetone, tetrahydrofuran, acetonitrile or dioxane, and the base B is selected from triethylamine, pyridine, DBU, potassium carbonate, sodium carbonate, sodium bicarbonate or sodium hydroxide.
6. The method for synthesizing N,N-di-L-lysyl-L-cystine dihydrochloride according to claim 2, characterized in that: In step (5), the solvent D is selected from water, methanol, ethanol, acetone, tetrahydrofuran, acetonitrile or dioxane.
7. The method for synthesizing N,N-di-L-lysyl-L-cystine dihydrochloride according to claim 2, characterized in that: In step (5), hydrochloric acid is added to adjust the pH value to 5-6, and then the solid-liquid is separated. The filtrate is concentrated under reduced pressure to remove part of the water, and then anhydrous ethanol is added dropwise at 50-60° C. After the addition is complete, the temperature is gradually lowered to 10-20° C., crystallization is performed, and solid-liquid separation is performed to obtain N,N-di-L-lysyl-L-cystine dihydrochloride.
8. The method for synthesizing N,N-di-L-lysyl-L-cystine dihydrochloride according to claim 7, characterized in that: The mixture is concentrated under reduced pressure to 2.5-4.0 times the weight of the product, and the amount of anhydrous ethanol used is 5-8 times the weight of the product.
9. The method for synthesizing N,N-di-L-lysyl-L-cystine dihydrochloride according to claim 1, characterized in that: The method comprises: (1) In toluene, L-lysine, phthalic anhydride and triethylamine react at 80-120° C. to obtain diphthalic anhydride-L-lysine, wherein the molar ratio of L-lysine, triethylamine and phthalic anhydride is 1:0.01-0.5:2-4; (2) dispersing bisphthalic anhydride-L-lysine in chloroform or toluene, adding dithionyl chloride dropwise, reacting at 40-80° C., and after the reaction is completed, concentrating under reduced pressure to obtain compound I, wherein the molar ratio of bisphthalic anhydride-L-lysine to dithionyl chloride is 1:1-3; (3) In dichloromethane or chloroform, L-cystine, triethylamine and trimethylsilyl chloride react at 0-70° C. to obtain a solution of compound II, wherein the molar ratio of L-cystine, triethylamine and trimethylsilyl chloride is 1:4-6:4-6; (4) dissolving compound I in dichloromethane or chloroform and adding dropwise to the solution of compound II, reacting at 0-40° C., adding water to quench the reaction after completion, and performing solid-liquid separation to obtain compound III, wherein the molar ratio of compound I, compound II and water is 2-2.5:1:4-8; (5) Compound III and hydrazine hydrate are reacted in water at 40-80° C. After the reaction is completed, hydrochloric acid is added to adjust the pH value to 5-6, solid-liquid separation is performed, and the filtrate is concentrated under reduced pressure to 2.5-4.0 times the weight of the product. Anhydrous ethanol is added dropwise at 50-60° C. After the addition is completed, the temperature is gradually lowered to 10-20° C., crystallization is performed, and solid-liquid separation is performed to obtain N,N-di-L-lysyl-L-cystine dihydrochloride. The molar ratio of compound III to hydrazine hydrate is 1:4-6, and the amount of anhydrous ethanol used is 5-8 times the weight of the product.