A method for separating and purifying small molecule peptides by coupling beating-extraction-crystallization
Patent Information
- Application Number
- CN202610536305.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-04-22
- Publication Date
- 2026-08-21
AI Technical Summary
①尽管这种方法成本较低、操作相对简便，但产品析出不完全，回收率低，通常在80%以下；②杂质与肽同时析出，产品分离效果不理想，纯度低
本发明提供的打浆-萃取-结晶耦合技术分离纯化小分子肽的方法，其技术原理是将含肽混合物置于水和有机溶剂中，在强烈搅拌、打浆分散的同时，依据不同成份在水和有机溶剂中分配系数的差异，分别被萃取转移，水溶性杂质转移至水相，有机杂质分散溶解于有机溶剂，小子肽在两相界面之间不断积累，达到过饱和后以固体的形式析出，脱离于溶剂，实现分离的效果；而传统的结晶或重结晶是基于被纯化物在良性溶剂和不良溶剂中的溶解性差异，从混合溶剂的过饱和真溶液溶液中析出固体的过程；其技术特征是不同溶剂必须是互溶的，且结晶母液中含有相当量的被纯化物；与传统的结晶或重结晶的原理和方法相比，本发明的打浆-萃取-结晶分离纯化耦合技术，是在相互不溶解的两类溶剂中实现的，被纯化物是在极性差异极大的、互不相溶的溶剂的界面上析出来的；本发明的打浆过程是为了强化传质，加快水溶性杂质和有机杂质在两相的分散速度，起到萃取的效果，随着乳体浆液中杂质不断地转移，肽从两相界面不断析出。打浆-萃取-结晶技术是将后处理与分离工艺过程耦合在一起，一次完成；而传统的结晶是在完成后处理后，进一步实施的分离和纯化过程，两种技术原理和分离机制完全不同。本发明与传统的结晶或重结晶相比，技术优势是被纯化物回收率能达到90%以上，母液中极少含有被纯化物残留，分离效率高，有机溶剂便于回收再利用。
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Abstract
Description
Technical Field
[0001] This invention relates to the field of small molecule peptide intermediates and drug purification technology, and in particular to a method for separating and purifying small molecule peptides using a pulping-extraction-crystallization coupling technique. Background Technology
[0002] Small molecule peptides, as an important class of bioactive molecules, play a crucial role in drug development and disease treatment. The chemical synthesis of small molecule peptides is generally divided into two main methods: solid-phase and liquid-phase synthesis. Liquid-phase synthesis has unique advantages in preparing peptides of small to medium length, complexly modified peptides, and large-scale applications, such as easy scale-up of reactions, easy separation and identification of intermediates, and relatively low overall cost. However, liquid-phase synthesis still faces many challenges in the post-processing of reaction products and the separation and purification of target products. These challenges include cumbersome and lengthy processes, low efficiency, high solvent consumption, and, in particular, low recovery rates, leading to increased costs and environmental pollution, thus limiting its industrial application efficiency and economic benefits.
[0003] Currently, the main methods for separating and purifying small molecule peptides synthesized by liquid phase include neutralization, washing, extraction, concentration, column chromatography, and precipitation crystallization. The first few operations can be referred to as post-processing, while the latter two belong to purification. Although column chromatography can obtain products with high purity, it usually requires a large amount of stationary phase and organic solvent as the mobile phase. The operation is cumbersome and time-consuming, suitable for small-scale purification in the laboratory, but difficult to directly scale up for industrial production. Precipitation method, on the other hand, utilizes the difference in solubility of small molecule peptides in different solvents, forcing them to precipitate from the solution by using a poor solvent. The common operation is to directly add the condensed reaction solution to water or cold ether, and stir to precipitate the peptides. ① Although this method is low-cost and relatively simple to operate, the product precipitation is incomplete, and the recovery rate is low, usually below 80%; ② Impurities and peptides precipitate simultaneously, resulting in unsatisfactory product separation and low purity; ③ If cold ether is used as a precipitant, due to its low boiling point, low flash point, volatility, flammability, explosiveness, and anesthetic toxicity, it may pose production safety risks and environmental hazards.
[0004] Therefore, proposing a new, efficient, and easy-to-operate technique to meet the needs of small molecule peptide separation and purification has important guiding significance and significant economic value for the industrial production of peptide drugs. Summary of the Invention
[0005] This invention provides a method for post-processing, separation, and purification of small molecule peptides using a pulping-extraction-crystallization coupling technology. This method addresses key technical issues in the current post-processing and separation / purification processes of peptide synthesis, providing an industrially valuable technical approach for the large-scale production of small molecule peptides.
[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A method for separating and purifying small molecule peptides using a pulping-extraction-crystallization coupling technique is disclosed. The method involves placing a mixture containing small molecule peptides in water and a low-polarity organic solvent, dispersing them by pulping, and extracting and transferring them according to the differences in partition coefficients of different components in different solvents. The small peptides continuously accumulate at the interface between the two phases, precipitating out in solid form after reaching supersaturation, thus obtaining the small molecule peptides.
[0007] Furthermore, the small molecule peptides are small molecule chain peptides or small molecule cyclic peptides; Small peptides have a molecular weight of less than or equal to 1200 Daltons and are peptides containing 2 to 12 amino acids, especially peptides containing 2 to 10 amino acids.
[0008] Furthermore, the amino acids contained in the peptide chains of small molecule peptides include natural amino acids and / or non-natural amino acids.
[0009] Furthermore, the natural amino acid is one or more of the 20 L-amino acids that make up the protein; the non-natural amino acid is any one or more of the following: D-configuration amino acid, L-arylglycine, D-arylglycine, L-aryl-alanine, D-aryl-alanine, and N-methyl amino acid.
[0010] Furthermore, the mixture containing small molecule peptides is a concentrate obtained by directly concentrating the reaction solution used to synthesize the small molecule peptides.
[0011] Furthermore, the specific process of the method involves slowly adding water to a mixture containing small molecule peptides under stirring conditions, continuing to stir, then slowly adding an organic solvent, continuing to stir, precipitating a solid, filtering and washing to obtain the small molecule peptides.
[0012] Furthermore, the stirring speed is 1000~3000 rpm.
[0013] Furthermore, the filtrate obtained by filtration is subjected to phase separation, the organic phase is collected, dried, and the organic solvent is recovered by distillation; the recovery rate of small molecule peptides is ≥90%, and the purity is ≥92%.
[0014] Furthermore, the organic solvent is a nonpolar or slightly polar organic compound, including any one or more of n-pentane, cyclohexane, hexane, heptane, petroleum ether, cyclopentyl methyl ether, tetrahydrofuran, methyl tert-butyl ether, diisopropyl ether, ethylene glycol dimethyl ether, and ethylene glycol diethyl ether.
[0015] Furthermore, the amount of organic solvent used is 0.3 to 1.0 times the volume of water.
[0016] The beneficial effects of the method for separating and purifying small molecule peptides using a pulping-extraction-crystallization coupling technique of the present invention are as follows: The present invention provides a method for separating and purifying small molecule peptides using a pulping-extraction-crystallization coupling technology. The technical principle involves placing a peptide-containing mixture in water and an organic solvent. During vigorous stirring and pulping dispersion, the components are extracted and transferred based on their different partition coefficients in the water and organic solvents. Water-soluble impurities are transferred to the aqueous phase, while organic impurities are dispersed and dissolved in the organic solvent. Small peptides accumulate between the two phases, precipitating as solids upon reaching supersaturation, thus achieving separation. Traditional crystallization or recrystallization, on the other hand, relies on the difference in solubility of the purified substance in good and bad solvents, relying on the difference in partition coefficients between the components in the mixed solvent. This invention describes the process of precipitating a solid from a saturated true solution. Its key technical feature is that the different solvents must be miscible, and the mother liquor contains a significant amount of the purified substance. Compared to traditional crystallization or recrystallization principles and methods, the pulping-extraction-crystallization separation and purification coupling technology of this invention is implemented in two immiscible solvents with vastly different polarities. The purified substance precipitates at the interface of these immiscible solvents. The pulping process of this invention aims to enhance mass transfer and accelerate the dispersion of water-soluble and organic impurities in both phases, achieving an extraction effect. As impurities continuously transfer within the emulsion, peptides continuously precipitate from the two-phase interface. The pulping-extraction-crystallization technology couples the post-treatment and separation processes together, completing them in one step. Traditional crystallization, on the other hand, is a further separation and purification process implemented after post-treatment; the two technologies have completely different principles and separation mechanisms. Compared to traditional crystallization or recrystallization, the technical advantages of this invention are that the purified substance recovery rate can reach over 90%, the mother liquor contains very little purified substance residue, the separation efficiency is high, and the organic solvent is easy to recover and reuse.
[0017] This invention provides a method for the separation and purification of small molecule peptides synthesized in liquid phase. After the small molecule chain peptide or cyclic peptide completes its condensation reaction, a certain amount of water is directly added to the concentrated reaction solution under rapid stirring to form a flocculent emulsion. Then, a certain amount of organic solvent is added to cause the small molecule chain peptide or cyclic peptide to precipitate out in large quantities from the solvent in solid form, thus achieving separation. The extraction-washing-pulping coupling technology of this invention combines the post-processing and separation of the target peptide synthesis concentrate into one process. It is simple to operate, has high separation efficiency, and can be used for the post-processing and purification of various amino acids or target small peptides. It avoids the technical problems and risks of low efficiency, limited scalability, high organic solvent consumption, and environmental pollution associated with column chromatography separation technology. It is a highly efficient, green, and clean peptide separation and purification technology with extremely high industrial-scale application value. The alkane solvents in this invention can be reused through distillation and other recovery methods, which has certain environmental protection characteristics. Attached Figure Description
[0018] Figure 1This is the HPLC chromatogram of N-Boc-L-leucine-L-leucine dipeptide methyl ester in Example 1 of the present invention; Figure 2 This is the HPLC chromatogram of N-Boc-L-leucine-L-phenylalanine dipeptide methyl ester in Example 2 of the present invention; Figure 3 This is the HPLC chromatogram of N-Boc-L-leucine-3-(2-naphthalene)-L-alanine dipeptide methyl ester in Example 4 of this invention; Figure 4 This is the HPLC chromatogram of N-Boc-L-valine-L-phenylalanine dipeptide methyl ester in Example 7 of the present invention; Figure 5 This is the HPLC chromatogram of N-Boc-L-valine-L-leucine-3-(2-naphthalene)-L-alanine tripeptide methyl ester in Example 8 of the present invention; Figure 6 This is the HPLC chromatogram of N-Boc-L-leuc-L-leuc-L-valine-LN-Me-leuc-3-(2-naphthyl)-L-alanine pentapeptide methyl ester in Example 9 of the present invention; Figure 7 This is the HPLC chromatogram of N-Boc-propionic-D-leucine-leucine-valine-proline-tyrosine-phenylalanine octapeptide methyl ester in Example 11 of this invention; Figure 8 This is the HPLC chromatogram of N-Boc-glyco-propanol-valine-leucine-phenylpropanol-glyco-glyco-tyrosine-alanine decapeptide methyl ester in Example 12 of the present invention; Figure 9 This is the HPLC chromatogram of N-(4-BnO)-Boc-aspartic acid-glyco-propan-leucine-leucine-valine-pro-tyrosine-glyco-phenylalanine decapeptide methyl ester in Example 14 of the present invention. Figure 10 This is the HPLC chromatogram of the cyclic pentapeptide ring (leucine-leucine-valine-N-Me-leucine-3-(2-naphthalene)-alanine) in Example 15 of the present invention. Detailed Implementation
[0019] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Many specific details are set forth in the following description to provide a thorough understanding of the present invention. However, the present invention may also be implemented in other ways different from those described herein. Those skilled in the art can make similar extensions without departing from the spirit of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.
[0020] The method of this invention is to directly concentrate the reaction solution for synthesizing small molecule peptides to obtain a concentrated solution. The concentrated solution is placed in water and organic solvent with a volume ratio of 1:0.3~1.0 and dispersed by stirring at a speed of 1000~3000 rpm. According to the difference in the partition coefficient of different components in different solvents, they are extracted and transferred respectively. The small peptides accumulate continuously between the two phase interfaces. After reaching supersaturation, they precipitate in solid form to obtain small molecule peptides with a recovery rate of ≥90% and a purity of ≥92%.
[0021] Among them, small molecule peptides are small molecule chain peptides or small molecule cyclic peptides; the molecular weight of small molecule peptides is less than or equal to 1200 Daltons, and they are peptides containing 2 to 12 amino acids, especially peptides containing 2 to 10 amino acids. The amino acids contained in the peptide chain of small molecule peptides include natural amino acids and / or non-natural amino acids. Natural amino acids are one or more of the 20 L-amino acids that make up proteins; non-natural amino acids are any one or more of D-configuration amino acids, L-arylglycine, D-arylglycine, L-aryl-alanine, D-aryl-alanine, and N-methyl amino acids.
[0022] The organic solvent is a nonpolar or slightly polar organic compound, including any one or more of n-pentane, cyclohexane, hexane, heptane, petroleum ether, cyclopentyl methyl ether, tetrahydrofuran, methyl tert-butyl ether, diisopropyl ether, ethylene glycol dimethyl ether, and ethylene glycol diethyl ether.
[0023] Example 1: Preparation method of N-Boc-L-leucine-L-leucine dipeptide methyl ester This embodiment describes the preparation method of N-Boc-L-leucine-L-leucine dipeptide methyl ester, as detailed below: S1, Synthesis: In a mixed solvent of 640 mL dichloromethane and 20 mL N,N-dimethylformamide, 16.69 g of condensing agent HBTU, 26 mL of N,N-diisopropylethylamine, 10.18 g of Boc-L-leucine, and 7.27 g of L-leucine methyl ester hydrochloride were added to synthesize N-Boc-L-leucine-L-leucine dipeptide methyl ester. After the reaction was completed, the solution was concentrated under reduced pressure to obtain a concentrated solution.
[0024] S2. Separation and purification: The concentrate was slowly added to 240 mL of purified water under stirring at room temperature to form a flocculent emulsion. Stirring was continued for 30 min, followed by the addition of 120 mL of petroleum ether. Stirring was maintained at 1000 rpm for 3 h, during which a large amount of solid precipitated. The solid was filtered, washed, and the filter cake and filtrate were collected. The filter cake was N-Boc-L-leucine-L-leucine dipeptide methyl ester, with a yield of 83%, a product recovery rate of 92%, and an HPLC relative peak area of 95.2% (see appendix). Figure 1 ).
[0025] S3, Solvent Recovery: The filtrate is subjected to phase separation, the organic phase is collected, dried, and the organic solvent is recovered by distillation.
[0026] The chemical formula for synthesizing N-Boc-L-leucine-L-leucine dipeptide methyl ester is as follows: .
[0027] Example 2: Preparation method of N-Boc-L-leucine-L-phenylalanine dipeptide methyl ester This embodiment describes the preparation method of N-Boc-L-leucine-L-phenylalanine dipeptide methyl ester, as detailed below: S1, Synthesis: In a reactor, 660 mL of dichloromethane, 10.18 g of Boc-L-leucine, 16.69 g of HBTU, 8.63 g of L-phenylalanine methyl ester hydrochloride, and 27 mL of N,N-diisopropylethylamine were added sequentially. The mixture was stirred at room temperature for 16 h, and the reaction solution was concentrated to obtain a concentrated solution.
[0028] S2. Separation and purification: While stirring, 300 mL of purified water was slowly added to the concentrate. The mixture was stirred at 3000 rpm for 30 min at room temperature. Then, 180 mL of n-hexane was slowly added, and stirring continued for 3 h. During this time, a large amount of solid precipitated. The solid was filtered, washed, and the filter cake and filtrate were collected. The filter cake was N-Boc-L-leucine-L-phenylalanine dipeptide methyl ester, with a yield of 90%, a recovery rate of 96%, and an HPLC relative peak area of 95.33% (see Appendix). Figure 2 ).
[0029] S3, Solvent Recovery: The filtrate is separated, the organic phase is collected, dried, and the organic solvent is recovered by distillation.
[0030] The chemical formula for synthesizing N-Boc-L-leucine-L-phenylalanine dipeptide methyl ester is as follows: .
[0031] Example 3: Preparation method of N-Boc-L-valine-L-leucine-L-phenylalanine tripeptide methyl ester This embodiment describes the preparation method of N-Boc-L-valine-L-leucine-L-phenylalanine tripeptide methyl ester, as detailed below: S1, Synthesis: In a reactor, 620 mL of dichloromethane, 40 mL of N,N-dimethylformamide, 9.56 g of Boc-L-valine, 18.20 g of HBTU, 11.68 g of L-leucine-L-phenylalanine dipeptide methyl ester, and 10 mL of N,N-diisopropylethylamine were added sequentially. The mixture was stirred at room temperature for 16 h, and the reaction solution was concentrated under reduced pressure to obtain a concentrated solution.
[0032] S2. Separation and purification: The concentrate was slowly added to 400 mL of purified water at room temperature with stirring, and stirring continued for 30 min. Then, 250 mL of cyclohexane was slowly added while stirring continued for 3 h. During this period, a large amount of solid precipitated. The solid was filtered, washed, and the filter cake and filtrate were collected. The filter cake was N-Boc-L-valine-L-leucine-L-phenylalanine tripeptide methyl ester, with a yield of 84%, a recovery rate of 92%, and a relative peak area of 95.63% on HPLC.
[0033] S3, Solvent Recovery: The filtrate is separated, the organic phase is collected, dried, and the organic solvent is recovered by distillation.
[0034] The chemical formula for synthesizing N-Boc-L-valine-L-leucine-L-phenylalanine tripeptide methyl ester is as follows: .
[0035] Example 4: Preparation method of N-Boc-L-leucine-3-(2-naphthalene)-L-alanine dipeptide methyl ester This embodiment describes the preparation method of N-Boc-L-leucine-3-(2-naphthalene)-L-alanine dipeptide methyl ester. The synthesis method is basically the same as that in step S1 of Example 1, except that dichloromethane in Example 1 is replaced with an equal volume of ethyl acetate, and L-leucine methyl ester hydrochloride is replaced with an equal molar amount of 3-(2-naphthalene)-L-alanine methyl ester hydrochloride, to obtain a concentrated solution. The separation and purification method of the obtained concentrated solution is exactly the same as that in step S2 of Example 1, yielding N-Boc-L-leucine-3-(2-naphthalene)-L-alanine dipeptide methyl ester with a yield of 84%, a recovery rate of 93%, and an HPLC relative peak area of 96.11% (see Appendix). Figure 3 Furthermore, the solvent recovery method in this embodiment is exactly the same as the solvent recovery method in step S3 of Embodiment 1.
[0036] The chemical formula for synthesizing N-Boc-L-leucine-3-(2-naphthalene)-L-alanine dipeptide methyl ester is as follows: .
[0037] Example 5: Preparation method of N-Boc-L-alanine-L-phenylalanine dipeptide methyl ester This embodiment describes the preparation method of N-Boc-L-alanine-L-phenylalanine dipeptide methyl ester. The synthesis method is basically the same as that in step S1 of Example 1, except that Boc-L-leucine is replaced with an equimolar amount of Boc-L-alanine, L-leucine methyl ester hydrochloride is replaced with an equimolar amount of L-phenylalanine methyl ester hydrochloride, and petroleum ether is replaced with an equal volume of n-hexane to obtain a concentrated solution. The separation and purification method of the obtained concentrated solution is exactly the same as that in step S2 of Example 1, yielding N-Boc-L-alanine-L-phenylalanine dipeptide methyl ester with a yield of 89%, a recovery rate of 91%, and an HPLC relative peak area of 96.45%. Furthermore, the solvent recovery method in this embodiment is also exactly the same as that in step S3 of Example 1.
[0038] The chemical formula for synthesizing N-Boc-L-alanine-L-phenylalanine dipeptide methyl ester is as follows: .
[0039] Example 6: Preparation method of N-Boc-L-phenylalanine-L-leucine dipeptide methyl ester This embodiment describes the preparation method of N-Boc-L-phenylalanine-L-leucine dipeptide methyl ester. The synthesis method is essentially the same as that in step S1 of Example 1, except that Boc-L-leucine in Example 1 is replaced with an equimolar amount of Boc-L-phenylalanine, and petroleum ether is replaced with an equal volume of cyclohexane to obtain a concentrated solution. The separation and purification method of the obtained concentrated solution is exactly the same as that in step S2 of Example 1, yielding N-Boc-L-phenylalanine-L-leucine dipeptide methyl ester with a yield of 83%, a recovery rate of 91%, and an HPLC relative peak area of 95.44%. Furthermore, the solvent recovery method in this embodiment is also exactly the same as that in step S3 of Example 1.
[0040] The chemical formula for synthesizing N-Boc-L-phenylalanine-L-leucine dipeptide methyl ester is as follows: .
[0041] Example 7: Preparation method of N-Boc-L-valine-L-phenylalanine dipeptide methyl ester This embodiment describes the preparation method of N-Boc-L-valine-L-phenylalanine dipeptide methyl ester. The synthesis method is basically the same as that in step S1 of Example 2, except that Boc-L-leucine in Example 2 is replaced with an equimolar amount of Boc-L-valine, and 27 mL of N,N-diisopropylethylamine is replaced with 10 mL of triethylamine to obtain a concentrated solution. The separation and purification method of the obtained concentrated solution is exactly the same as that in step S2 of Example 2, yielding N-Boc-L-valine-L-phenylalanine dipeptide methyl ester with a yield of 90%, a recovery rate of 91%, and an HPLC relative peak area of 98.08% (see Appendix). Figure 4 Furthermore, the solvent recovery method in this embodiment is exactly the same as the solvent recovery method in step S3 of embodiment 2.
[0042] The chemical formula for synthesizing N-Boc-L-valine-L-phenylalanine dipeptide methyl ester is as follows: .
[0043] Example 8: Preparation method of N-Boc-L-valine-L-leucine-3-(2-naphthalene)-L-alanine tripeptide methyl ester This embodiment describes the preparation method of N-Boc-L-valine-L-leucine-3-(2-naphthalene)-L-alanine tripeptide methyl ester. The synthesis method is basically the same as that in step S1 of Example 3, except that the L-leucine-L-phenylalanine dipeptide methyl ester in Example 3 is replaced with an equimolar amount of L-leucine-3-(2-naphthalene)-L-alanine dipeptide methyl ester to obtain a concentrated solution. The separation and purification method of the obtained concentrated solution is exactly the same as that in step S2 of Example 1, yielding N-Boc-L-valine-L-leucine-3-(2-naphthalene)-L-alanine tripeptide methyl ester with a yield of 83%, a recovery rate of 90%, and an HPLC relative peak area of 87.00% (see Appendix). Figure 5 Furthermore, the solvent recovery method in this embodiment is exactly the same as the solvent recovery method in step S3 of embodiment 3.
[0044] The chemical formula for synthesizing N-Boc-L-valine-L-leucine-3-(2-naphthalene)-L-alanine tripeptide methyl ester is as follows: .
[0045] Example 9: Preparation method of N-Boc-L-Lewis-L-Lewis-L-Val-LN-Me-Lewis-3-(2-Naphthyl)-L-Alanine Pentapeptide Methyl Ester This embodiment describes the preparation method of N-Boc-L-Leuc-L-Leuc-L-Val-LN-Me-Leuc-3-(2-naphthyl)-L-alanine pentapeptide methyl ester, as detailed below: S1, Synthesis: In a reactor, 610 mL of dichloromethane, 50 mL of N,N-dimethylformamide, 15.32 g of dipeptide Boc-L-leucine-L-leucine, 18.20 g of HBTU, 18.20 g of L-valine-LN-Me-leucine-3-(2-naphthyl)-L-alanine tripeptide methyl ester, and 18 mL of N,N-diisopropylethylamine were added sequentially. The mixture was stirred at room temperature for 18 h, and the reaction mixture was concentrated under reduced pressure to obtain a concentrated solution.
[0046] S2. Separation and purification: While stirring at room temperature, slowly add 500 mL of purified water to the concentrate and continue stirring for 30 min. Then, while stirring at approximately 2000 rpm, slowly add 250 mL of methyl tert-butyl ether and continue stirring for 3 h. A large amount of solid will precipitate during this process. Filter, wash, and collect the filter cake and filtrate. The filter cake is N-Boc-L-leuc-L-leuc-L-valine-LN-Me-leuc-3-(2-naphthyl)-L-alanine pentapeptide methyl ester, with a yield of 80%, a recovery rate of 91%, and an HPLC relative peak area of 95.04% (see Appendix). Figure 6 ).
[0047] S3, Solvent Recovery: The filtrate is separated, the organic phase is collected, dried, and the organic solvent is recovered by distillation.
[0048] The chemical formula of N-Boc-L-Lewis-L-Lewis-L-Val-LN-Me-Lewis-3-(2-naphthyl)-L-alanine pentapeptide methyl ester is as follows: .
[0049] Example 10: Preparation method of N-Boc-L-va-L-prop-L-prop-gly-L-phenylprop-L-(N'-Cbz)lys-glycine heptapeptide methyl ester This embodiment describes the preparation method of N-Boc-L-va-L-prop-L-prop-gly-L-phenylprop-L-(N'-Cbz)lys-glycine heptapeptide methyl ester, as follows: S1, Synthesis: In a reactor, 600 mL of dichloromethane, 60 mL of N,N-dimethylformamide, 21.32 g of Boc-va-propan-propan-glycine tetrapeptide, 18.20 g of HBTU, 22.91 g of phenylalanine-(N'-Cbz)lys-glycine tripeptide methyl ester, and 15 mL of N,N-diisopropylethylamine were added sequentially. The mixture was stirred at room temperature for 18 h, and the reaction solution was concentrated under reduced pressure to obtain a concentrated solution.
[0050] S2. Separation and purification: Under room temperature and stirring conditions, 600 mL of purified water was slowly added to the concentrate, and stirring continued for 30 min. Then, controlling the stirring speed at approximately 2500 rpm, 300 mL of cyclopentyl methyl ether was slowly added, and stirring continued for 3 h, during which a large amount of solid precipitated. The mixture was filtered, washed, and the filter cake and filtrate were collected. The filter cake was N-Boc-L-valine-L-propane-glycine-L-phenylpropane-L-(N'-Cbz)lysine heptapeptide methyl ester, with a yield of 74% and a recovery rate of 90.0%.
[0051] S3, Solvent Recovery: The filtrate was separated, the organic phase was collected, dried, and the organic solvent was recovered by distillation. The chemical formula of N-Boc-L-va-L-prop-L-prop-gly-L-phenylprop-L-(N`-Cbz)lys-glycine heptapeptide methyl ester is as follows: .
[0052] Example 11: Preparation method of N-Boc-L-propane-D-leucine-L-leucine-L-val-L-pro-L-tyrosine-L-phenylalanine octapeptide methyl ester This embodiment describes the preparation method of N-Boc-L-propan-D-leucine-L-leucine-L-valine-L-proline-L-tyrosine-L-phenylalanine octapeptide methyl ester, as detailed below: S1, Synthesis: Preparation of S1-1, tetrapeptide Boc-propyl-D-leuc-leuc-valine The method for obtaining the tetrapeptide Boc-propan-D-leucine-leucine methyl ester in this embodiment is basically the same as the method for obtaining N-Boc-L-leucine-L-leucine dipeptide methyl ester in Example 1. The synthesis method is basically the same as the synthesis method in step S1 of Example 1, except that Boc-L-leucine in Example 1 is replaced with an equimolar amount of dipeptide Boc-alanine-D-alanine, and L-leucine methyl ester hydrochloride is replaced with an equimolar amount of dipeptide L-leucine-valine methyl ester to obtain a concentrated solution. The separation and purification method of the obtained concentrated solution is exactly the same as the purification method in step S2 of Example 1 to obtain tetrapeptide Boc-propan-D-leucine-leucine methyl ester, which is then hydrolyzed and deesterified under alkaline conditions to obtain tetrapeptide Boc-propan-D-leucine-leucine.
[0053] Preparation of S1-2, proline-tyrosine-phenylalanine tetrapeptide methyl ester The method for obtaining the tetrapeptide Boc-proline-tyrosine-glycopeptide methyl ester in this embodiment is basically the same as the method for obtaining N-Boc-L-leucine-L-leucine dipeptide methyl ester in Example 1. The synthesis method is basically the same as the synthesis method in step S1 of Example 1, except that the L-leucine methyl ester hydrochloride in Example 1 is replaced with an equimolar amount of dipeptide Boc-proline-tyrosine, and the L-leucine methyl ester hydrochloride is replaced with an equimolar amount of dipeptide glycine-phenylalanine methyl ester to obtain a concentrated solution. The separation and purification method of the obtained concentrated solution is exactly the same as the purification method in step S2 of Example 1 to obtain the tetrapeptide Boc-proline-tyrosine-glycopeptide methyl ester. Then, the Boc is removed under acidic conditions to obtain proline-tyrosine-glycopeptide methyl ester.
[0054] S1-3, Preparation of Concentrate In a reactor, 600 mL of dichloromethane, 60 mL of N,N-dimethylformamide, 22.56 g of tetrapeptide Boc-propyl-D-leucine-leucine, 18.20 g of HBTU, 20.10 g of proline-tyrosine-phenylalanine tetrapeptide methyl ester, and 15 mL of N,N-diisopropylethylamine were added sequentially. The mixture was stirred at room temperature for 30 h, and the reaction mixture was concentrated under reduced pressure to obtain a concentrated solution.
[0055] S2. Separation and purification: Under room temperature and stirring conditions, 600 mL of purified water was slowly added to the concentrate, and stirring continued for 30 min. Then, while maintaining a stirring speed of 3000 rpm, 200 mL of a 1:1 mixture of ethylene glycol dimethyl ether and tetrahydrofuran was slowly added, and stirring was maintained for 3 h. During this period, a large amount of solid precipitated. The mixture was filtered, washed, and the filter cake and filtrate were collected. The filter cake was N-Boc-propyl-D-leucine-leucine-valine-proline-tyrosine-phenylalanine octapeptide methyl ester, with a yield of 65%, a recovery rate of 90.02%, and an HPLC relative peak area of 91.97% (see Appendix). Figure 7 ); S3, Solvent Recovery: The filtrate is separated, the organic phase is collected, dried, and the organic solvent is recovered by distillation.
[0056] The chemical formula of N-Boc-L-propionic-D-leucine-L-leucine-L-va-L-pro-L-tyrosine-L-phenylalanine octapeptide methyl ester is as follows: .
[0057] Example 12: Preparation method of N-Boc-glycine-L-propanol-L-valine-L-leucine-L-phenylpropanol-L-proline-glycine-L-tyrosine-L-alanine decapeptide methyl ester This embodiment describes the preparation method of N-Boc-glycine-L-propanol-L-valine-L-leucine-L-phenylpropanol-L-proline-glycine-L-tyrosine-L-alanine decapeptide methyl ester, as follows: S1, Synthesis: Preparation of S1-1, Boc-glyco-alanine-valine-leucine pentapeptide The method for obtaining the pentapeptide Boc-glycine-valyl-leucine-phenylalanine methyl ester in this embodiment is basically the same as the method for obtaining N-Boc-L-leucine-L-leucine dipeptide methyl ester in Example 1. The synthesis method is basically the same as the synthesis method in step S1 of Example 1, except that Boc-L-leucine in Example 1 is replaced with an equimolar amount of dipeptide Boc-glycine-alanine, and L-leucine methyl ester hydrochloride is replaced with an equimolar amount of tripeptide L-valine-leucine-phenylalanine methyl ester to obtain a concentrated solution. The separation and purification method of the obtained concentrated solution is exactly the same as the purification method in step S2 of Example 1 to obtain the pentapeptide Boc-glycine-valyl-leucine-phenylalanine methyl ester, which is then hydrolyzed and deesterified under alkaline conditions to obtain Boc-glycine-valyl-leucine-phenylalanine pentapeptide.
[0058] Preparation of S1-2, proline-glycine-tyrosine-alanine pentapeptide methyl ester In this embodiment, the method for obtaining the pentapeptide Boc-proline-glycine-tyrosine-alanine methyl ester is basically the same as the method for obtaining N-Boc-L-leucine-L-leucine dipeptide methyl ester in Example 1. The synthesis method is basically the same as the synthesis method in step S1 of Example 1, except that the L-leucine methyl ester hydrochloride in Example 1 is replaced with an equimolar amount of dipeptide Boc-proline-glycine, and the L-leucine methyl ester hydrochloride is replaced with an equimolar amount of tripeptide glycine-tyrosine-phenylalanine methyl ester to obtain a concentrated solution. The separation and purification method of the obtained concentrated solution is exactly the same as the purification method in step S2 of Example 1 to obtain the pentapeptide Boc-proline-glycine-tyrosine-alanine methyl ester. Then, the Boc is removed under acidic conditions to obtain proline-glycine-tyrosine-alanine pentapeptide methyl ester.
[0059] S1-3, Preparation of Concentrate In a reactor, 600 mL of dichloromethane, 60 mL of N,N-dimethylformamide, 26.65 g of Boc-glycine-valine-leucine-phenylalanine pentapeptide, 18.20 g of HBTU, 18.98 g of proline-glycine-tyrosine-alanine pentapeptide methyl ester, and 15 mL of N,N-diisopropylethylamine were added sequentially. The mixture was stirred at room temperature for 36 h, and the reaction solution was concentrated under reduced pressure to obtain a concentrated solution.
[0060] S2. Separation and purification: Under room temperature and stirring conditions, 600 mL of purified water was slowly added to the obtained concentrate, and stirring was continued for 30 min at approximately 3000 rpm. Then, while maintaining the stirring speed, 250 mL of a 1:1 mixture of ethylene glycol dimethyl ether and diisopropyl ether was slowly added and stirred for 3 h, during which a large amount of solid precipitated. The mixture was filtered, washed, and the filter cake and filtrate were collected. The filter cake was N-Boc-glycine-propane-valine-leucine-phenylpropane-proline-glycine-tyrosine-alanine decapeptide methyl ester, with a yield of 58%, a recovery rate of 90%, and an HPLC relative peak area of 93.86% (see Appendix). Figure 8 ).
[0061] S3, Solvent Recovery: The filtrate is separated, the organic phase is collected, dried, and the organic solvent is recovered by distillation.
[0062] The chemical formula of N-Boc-glycolic acid-valyl-leucine-phenylpropanoid-glycolic acid-tyrosine-alanine decapeptide methyl ester is as follows: .
[0063] Example 13: Preparation method of N-Boc-L-propan-glycine-L-propan-L-val-L-leucine-L-hizo-L-pro-glycine-D-propan-L-(N`-Cbz)-lysine decapeptide methyl ester This embodiment describes the preparation method of N-Boc-L-propan-glycine-L-propan-L-val-L-leucine-L-hist-L-pro-glycine-D-propan-L-(N`-Cbz)-lysine decapeptide methyl ester, as follows: S1, Synthesis: Preparation of S1-1, Boc-alanyl-galanyl-valine pentapeptide The method for obtaining Boc-prop-glycolic acid-prop-valine-leucine pentapeptide methyl ester in this embodiment is basically the same as the method for obtaining N-Boc-L-leucine-L-leucine dipeptide methyl ester in Example 1. The synthesis method is basically the same as the synthesis method in step S1 of Example 1, except that Boc-L-leucine in Example 1 is replaced with an equimolar amount of Boc-prop-glycolic dipeptide, and L-leucine methyl ester hydrochloride is replaced with an equimolar amount of L-prop-valine-leucine tripeptide methyl ester to obtain a concentrated solution. The separation and purification method of the obtained concentrated solution is exactly the same as the purification method in step S2 of Example 1 to obtain Boc-prop-glycolic acid-prop-valine-leucine pentapeptide methyl ester, which is then hydrolyzed and deesterified under alkaline conditions to obtain Boc-prop-glycolic acid-prop-valine-leucine pentapeptide.
[0064] Preparation of S1-2, histidine-pro-glycine-D-propan-L-(N'-Cbz)-lysine pentapeptide methyl ester In this embodiment, the method for obtaining the pentapeptide Fmoc-histidine-proline-D-propanol-L-(N'-Cbz)-lysine methyl ester is basically the same as the method for obtaining N-Boc-L-leucine-L-leucine dipeptide methyl ester in Example 1. The synthesis method is essentially the same as the synthesis method in step S1 of Example 1, except that the L-leucine methyl ester hydrochloride in Example 1 is replaced with an equimolar amount of Fmoc-histidine-proline-D-propanol-L-(N'-Cbz)-lysine methyl ester. The amino acid methyl ester hydrochloride was replaced with an equimolar amount of D-propane-(N'-Cbz)-lysine dipeptide methyl ester to prepare a concentrated solution. The separation and purification method of the obtained concentrated solution was exactly the same as the purification method in step S2 of Example 1, to obtain the pentapeptide Fmoc-histidine-pro-gly-D-propane-L-(N'-Cbz)-lysine methyl ester. Then, Fmoc was removed under alkaline conditions to obtain histidine-pro-gly-D-propane-L-(N'-Cbz)-lysine pentapeptide methyl ester.
[0065] S1-3, Preparation of Concentrate In a reactor, 600 mL of dichloromethane, 60 mL of N,N-dimethylformamide, 23.26 g of Boc-propane-propane-valine-leucine pentapeptide, 18.20 g of HBTU, 25.79 g of histidine-proline-D-propane-L-(N'-Cbz)-lysine pentapeptide methyl ester, and 15 mL of N,N-diisopropylethylamine were added sequentially. The resulting mixture was stirred at room temperature for 36 h, and then concentrated under reduced pressure to obtain a concentrated solution.
[0066] S2. Separation and purification: Under room temperature and stirring conditions, 600 mL of purified water was slowly added to the obtained concentrate, and stirring was maintained for 30 min. The stirring speed was controlled at approximately 3000 rpm, and then 250 mL of a 1:1 mixture of diisopropyl ether and tetrahydrofuran was slowly added, maintaining the mixture for 3 h. During this period, a large amount of solid precipitated. The mixture was filtered, washed, and the filter cake and filtrate were collected. The filter cake was N-Boc-propan-glycopeptide-val-leucine-histyl-proline-glycopeptide-D-propan-(N'-Cbz)-lysine decapeptide methyl ester, with a yield of 75%, a recovery rate of 90%, and a relative peak area of 91.81% on HPLC.
[0067] S3, Solvent Recovery: The filtrate is separated, the organic phase is collected, dried, and the organic solvent is recovered by distillation.
[0068] The chemical formula of N-Boc-L-propan-glycine-L-propan-L-va-L-leucine-L-hist-L-pro-glycine-D-propan-L-(N`-Cbz)-lysine decapeptide methyl ester is as follows: .
[0069] Example 14: Preparation method of N-(4-BnO)-Boc-L-aspartic-glycine-L-propanic-L-leucine-L-leucine-L-val-L-pro-L-tyrosine-L-phenylalanine decapeptide methyl ester This embodiment describes the preparation method of N-(4-BnO)-Boc-L-aspartic-glycine-L-propanic-L-leucine-L-leucine-L-val-L-pro-L-tyrosine-L-phenylalanine decapeptide methyl ester, as follows: S1, Synthesis: Preparation of S1-1, (4-BnO)-Boc-aspartic-glycine-leucine pentapeptide The method for obtaining (4-BnO)-Boc-aspartic-glycolic-leucine-leucine pentapeptide methyl ester in this embodiment is basically the same as the method for obtaining N-Boc-L-leucine-L-leucine dipeptide methyl ester in Example 1. The synthesis method is essentially the same as the synthesis method in step S1 of Example 1, except that Boc-L-leucine in Example 1 is replaced with an equimolar amount of (4-BnO)-Boc-aspartic-glycolic-leucine tripeptide, and an equimolar amount of L-leucine-leucine dipeptide methyl ester is replaced with L-leucine methyl ester hydrochloride to obtain a concentrated solution. The separation and purification method of the obtained concentrated solution is exactly the same as the purification method in step S2 of Example 1, yielding the pentapeptide (4-BnO)-Boc-aspartic-glycolic-leucine methyl ester. Then, it is hydrolyzed and deesterified under alkaline conditions to obtain (4-BnO)-Boc-aspartic-glycolic-leucine pentapeptide.
[0070] Preparation of S1-2, valine-proline-tyrosine-phenylalanine pentapeptide methyl ester The method for obtaining Boc-valine-proline-tyrosine-phenylalanine methyl ester in this embodiment is basically the same as the method for obtaining N-Boc-L-leucine-L-leucine dipeptide methyl ester in Example 1. The synthesis method is essentially the same as the synthesis method in step S1 of Example 1, except that L-leucine methyl ester hydrochloride in Example 1 is replaced with an equimolar amount of Boc-valine-proline-tyrosine tripeptide, and L-leucine methyl ester hydrochloride is replaced with an equimolar amount of phenylalanine dipeptide methyl ester, to obtain a concentrated solution. The separation and purification method of the obtained concentrated solution is exactly the same as the purification method in step S2 of Example 1, yielding the pentapeptide Boc-valine-proline-tyrosine-phenylalanine methyl ester. Then, Boc is removed under acidic conditions to obtain valine-proline-tyrosine-phenylalanine pentapeptide methyl ester.
[0071] S1-3, Preparation of Concentrate In a reactor, 600 mL of dichloromethane, 60 mL of N,N-dimethylformamide, 29.38 g of (4-BnO)-Boc-aspartic-glycolic-leucine pentapeptide, 18.20 g of HBTU, 23.75 g of valine-proline-tyrosine-phenylalanine pentapeptide methyl ester, and 15 mL of N,N-diisopropylethylamine were added sequentially. The resulting mixture was stirred at room temperature for 36 h. After the reaction was completed, the mixture was concentrated under reduced pressure to obtain the concentrated reaction solution.
[0072] S2. Separation and purification: Under room temperature and stirring conditions, 600 mL of purified water was slowly added to the obtained concentrate, and stirring was continued for 30 min. Then, while controlling the stirring speed at approximately 3000 rpm, 300 mL of a 1:1 mixture of ethylene glycol diethyl ether and diisopropyl ether was slowly added, and stirring was maintained for 3 h. During this period, a large amount of solid precipitated. The mixture was filtered, washed, and the filter cake and filtrate were collected. The filter cake was N-(4-BnO)-Boc-aspartic acid-glycolic acid-propanol-leucine-leucine-valine-proline-tyrosine-phenylalanine decapeptide methyl ester, with a yield of 75%, a recovery rate of 89%, and an HPLC relative peak area of 91.16% (see Appendix). Figure 9 ).
[0073] S3, Solvent Recovery: The filtrate is separated, the organic phase is collected, dried, and the organic solvent is recovered by distillation.
[0074] The chemical formula of N-(4-BnO)-Boc-L-aspartic-glycine-L-propanic-L-leucine-L-leucine-L-val-L-pro-L-tyrosine-L-phenylalanine decapeptide methyl ester is as follows: .
[0075] Example 15: Preparation method of cyclic pentapeptide (L-leucine-L-leucine-L-valine-LN-Me-leucine-3-(2-naphthyl)-L-alanine) This embodiment describes the preparation method of a cyclic pentapeptide (L-leucine-L-leucine-L-valine-LN-Me-leucine-3-(2-naphthyl)-L-alanine), as detailed below: S1, Synthesis: Dissolve 0.65g of the chain pentapeptide L-leucine-L-leucine-L-valine-LN-Me-leucine-3-(2-naphthalene)-L-alanine in a mixed solution of 10mL dichloromethane and 10mL N,N-dimethylformamide to obtain a peptide solution. In a reactor, 1180 mL of dichloromethane, 1.24 g of HCTU, and 5 mL of N,N-diisopropylethylamine were added sequentially. While stirring, the peptide solution was slowly added dropwise to the reactor using a syringe pump, and the reactor was placed at room temperature for 36 h. After the reaction was completed, the reaction solution was concentrated under reduced pressure to obtain a concentrated solution.
[0076] S2. Separation and purification: Under room temperature and stirring conditions, 120 mL of purified water was slowly added to the obtained concentrate, and stirring was continued for 30 min. Then, controlling the stirring speed at approximately 2500 rpm, 60 mL of a 1:1 mixture of diisopropyl ether and hexane was added, and stirring was continued for 3 h, during which solids continuously precipitated. The mixture was filtered, washed, and the filter cake and filtrate were collected. The filter cake was the cyclic pentapeptide ring (leucine-leucine-valine-N-Me-leucine-3-(2-naphthalene)-alanine), with a yield of 53.3%, a recovery rate of 91%, and an HPLC relative peak area of 92.07% (see Appendix). Figure 10 ).
[0077] S3, Solvent Recovery: The filtrate is separated, the organic phase is collected, dried, and the organic solvent is recovered by distillation.
[0078] The chemical formula of the synthetic cyclic pentapeptide (L-leucine-L-leucine-L-valine-LN-Me-leucine-3-(2-naphthyl)-L-alanine) is as follows: .
[0079] Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.
Claims
1. A method for separating and purifying small molecule peptides using a pulping-extraction-crystallization coupling technique, characterized in that, The method involves placing a mixture containing small molecule peptides in water and an organic solvent, dispersing it by slurry, and extracting and transferring the components according to the differences in their partition coefficients in different solvents. The small peptides accumulate continuously between the two phase interfaces, and after reaching supersaturation, they precipitate out in solid form to obtain the small molecule peptides.
2. The method for separating and purifying small molecule peptides using the pulping-extraction-crystallization coupling technology according to claim 1, characterized in that, Small molecule peptides are either small molecule chain peptides or small molecule cyclic peptides; Small peptides have a molecular weight of less than or equal to 1200 Daltons and consist of 2 to 12 amino acids.
3. The method for separating and purifying small molecule peptides using the pulping-extraction-crystallization coupling technology according to claim 1 or 2, characterized in that, The amino acids contained in the peptide chains of small molecule peptides include natural amino acids and / or non-natural amino acids.
4. The method for separating and purifying small molecule peptides using the pulping-extraction-crystallization coupling technology according to claim 3, characterized in that, Natural amino acids are one or more of the 20 L-amino acids that make up proteins; non-natural amino acids are any one or more of D-configuration amino acids, L-arylglycine, D-arylglycine, L-aryl-alanine, D-aryl-alanine, and N-methyl amino acids.
5. The method for separating and purifying small molecule peptides using the pulping-extraction-crystallization coupling technique according to claim 1, 2, or 4, characterized in that, The mixture containing small molecule peptides is a concentrated solution obtained by directly concentrating the reaction solution used to synthesize small molecule peptides.
6. The method for separating and purifying small molecule peptides using the pulping-extraction-crystallization coupling technique according to claim 1, 2, or 4, characterized in that, The specific process of the method is as follows: under stirring conditions, water is slowly added to a mixture containing small molecule peptides, stirring is continued, then an organic solvent is slowly added, stirring is continued, a solid is precipitated, and the solid is filtered and washed to obtain the small molecule peptides.
7. The method for separating and purifying small molecule peptides using the pulping-extraction-crystallization coupling technology according to claim 6, characterized in that, The stirring speed is 1000~3000 rpm.
8. The method for separating and purifying small molecule peptides using the pulping-extraction-crystallization coupling technology according to claim 6, characterized in that, The filtrate obtained by filtration is subjected to phase separation, the organic phase is collected, dried, and the organic solvent is recovered by distillation.
9. The method for separating and purifying small molecule peptides using a pulping-extraction-crystallization coupling technique according to claim 1, 2, 4, 7, or 8, characterized in that, The organic solvent is a nonpolar or slightly polar organic compound, including any one or more of n-pentane, cyclohexane, hexane, heptane, petroleum ether, cyclopentyl methyl ether, tetrahydrofuran, methyl tert-butyl ether, diisopropyl ether, ethylene glycol dimethyl ether, and ethylene glycol diethyl ether.
10. The method for separating and purifying small molecule peptides using a pulping-extraction-crystallization coupling technique according to claim 1, 2, 4, 7, or 8, characterized in that, The amount of organic solvent used is 0.3 to 1.0 times the volume of water.