WO2023221978A1 - 一种多赖氨酸盐及其制备方法和纯化方法 - Google Patents

一种多赖氨酸盐及其制备方法和纯化方法 Download PDF

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Publication number
WO2023221978A1
WO2023221978A1 PCT/CN2023/094530 CN2023094530W WO2023221978A1 WO 2023221978 A1 WO2023221978 A1 WO 2023221978A1 CN 2023094530 W CN2023094530 W CN 2023094530W WO 2023221978 A1 WO2023221978 A1 WO 2023221978A1
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Prior art keywords
trilysine
acetate
add
polylysine
stir
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PCT/CN2023/094530
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English (en)
French (fr)
Inventor
陶秀梅
高宏伟
秦元满
高启福
马建佳
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北京诺康达医药科技股份有限公司
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Priority claimed from CN202210542271.4A external-priority patent/CN117106014A/zh
Priority claimed from CN202210542240.9A external-priority patent/CN117106013A/zh
Application filed by 北京诺康达医药科技股份有限公司 filed Critical 北京诺康达医药科技股份有限公司
Publication of WO2023221978A1 publication Critical patent/WO2023221978A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
    • A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/34—Macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyesters, polyamino acids, polysiloxanes, polyphosphazines, copolymers of polyalkylene glycol or poloxamers
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J3/00—Processes of treating or compounding macromolecular substances
    • C08J3/02—Making solutions, dispersions, lattices or gels by other methods than by solution, emulsion or suspension polymerisation techniques
    • C08J3/03—Making solutions, dispersions, lattices or gels by other methods than by solution, emulsion or suspension polymerisation techniques in aqueous media
    • C08J3/075—Macromolecular gels

Definitions

  • the present invention relates to chemical synthesis, specifically to polylysine salts and their preparation and purification methods.
  • Trilysine acetate is a tripeptide of lysine that serves as a cross-linking agent for hydrogels. Trilysine acetate is a tripeptide, and the synthesis of peptides mainly uses solid-phase synthesis.
  • Fmoc solid-phase synthesis method has been widely used at home and abroad for the synthesis of various performance peptides, with the advantages of convenience, speed, simple operation, and high yield.
  • peptides are synthesized by solid-phase synthesis, and chromatography is often used for purification, and freeze-drying is used when the product is dried, which increases production costs.
  • the existing preparation method is to prepare trilysine acetate through HPLC salt transfer method after purification by column chromatography.
  • the prepared trilysine acetate is trilysine triacetate
  • trilysine triacetate is Lysine triacetate is highly hygroscopic and has unstable physical properties, making it inconvenient for later use and storage.
  • the present invention aims to solve at least one of the above technical problems.
  • the present invention first provides a trilysine acetate represented by the following formula (I),
  • Trilysine acetate represented by the following formula TLYS is also provided.
  • the molecular formula of the trilysine acetate is C 18 H 38 N 6 O 4 ⁇ CH 3 CO 2 H.
  • the existing trilysine acetate is trilysine triacetate combined with three acetic acids. It has strong hygroscopicity and is difficult to obtain a good solid through concentration and drying, so its application is limited.
  • the trilysine acetate prepared by the present invention only combines one acetic acid, and can obtain a better solid through concentration and drying, which is convenient for weighing and use, and has a higher trilysine content.
  • the present invention also provides crystal form A of trilysine acetate represented by the above formula TLYS.
  • the crystal form A of the trilysine acetate uses Cu-K ⁇ radiation, and the X-ray powder diffraction expressed by the diffraction angle 2 ⁇ has characteristic peaks at 20.227, 23.194, and 23.686, wherein, The error range of the 2 ⁇ angle of each characteristic peak is ⁇ 0.2.
  • the crystal form A of the trilysine acetate uses Cu-K ⁇ radiation, and the X-ray powder diffraction expressed by the diffraction angle 2 ⁇ angle is at 4.632, 19.695, 20.227, 22.172, 23.194, 23.686, There is a characteristic peak at 28.079, and the error range of the 2 ⁇ angle of each characteristic peak is ⁇ 0.2.
  • the crystal form A of the trilysine acetate uses Cu-K ⁇ radiation, and the X-ray powder diffraction expressed by the diffraction angle 2 ⁇ angle is at 4.632, 7.951, 11.825, 13.980, 17.667, 18.307, There are characteristic peaks at 19.695, 20.227, 21.686, 22.172, 23.194, 23.686, and 28.079.
  • the error range of the 2 ⁇ angle of each characteristic peak is ⁇ 0.2.
  • the crystalline form A of trilysine acetate uses Cu-K ⁇ radiation, and the X-ray powder diffraction expressed by the diffraction angle 2 ⁇ angle is at 4.632, 7.951, 9.272, 11.825, 13.980, 16.544, There are characteristic peaks at 17.343, 17.667, 18.307, 19.695, 20.227, 21.686, 22.172, 23.194, 23.686, 27.327, 28.079, 29.270, and 32.239.
  • the error range of the 2 ⁇ angle of each characteristic peak is ⁇ 0.2.
  • the crystal form A of the trilysine acetate uses Cu-K ⁇ radiation, and the X-ray powder diffraction expressed by the diffraction angle 2 ⁇ angle is at 4.632, 5.478, 7.951, 9.272, 9.742, 11.825, 13.980, 16.192, 16.544, 17.343, 17.667, 18.307, 18.716, 19.695, 20.227, 21.686, 22.172, 23.194, 23.686, 24.672, 27.327, 28.079, 29.270, 32 There is a characteristic peak at .239, among which, the error of 2 ⁇ angle of each characteristic peak The range is ⁇ 0.2.
  • the crystal form A of the trilysine acetate uses Cu-K ⁇ radiation, and the X-ray powder diffraction peak expressed in the diffraction angle 2 ⁇ is as shown in Table 1, with an error range of ⁇ 0.2:
  • the crystal form A of trilysine acetate has a powder X-ray diffraction pattern basically as shown in Figure 1 .
  • the crystal form A of the trilysine acetate was measured by thermogravimetric analysis, and the heat loss at 176.10°C was 8.1wt% ⁇ 2wt%, optionally 8.1wt% ⁇ 1wt%.
  • the DSC curve of the differential scanning calorimetry analysis spectrum of the crystal form A of trilysine acetate has an endothermic peak at 147.81°C.
  • the crystal form A of trilysine acetate has a DSC chart basically as shown in Figure 7.
  • the trilysine acetate (trilysine monoacetate) and the above-mentioned crystal form A provided by the invention have good physical stability, low hygroscopicity, convenient storage, and can avoid crystallization during the development and production process of drugs. risks of.
  • the present invention also provides a method for preparing trilysine acetate represented by the above formula (I) or formula TLYS, which includes: using a strong alkaline method to prepare the compound represented by formula TLYS-4 (trilysine trifluoroacetate).
  • the anion exchange resin removes trifluoroacetic acid and then reacts with acetic acid to generate the compound shown as TLYS;
  • the strong basic anion exchange resin is selected from type 201 ⁇ 4 or type 201 ⁇ 7. Studies have found that if other resins are used, the obtained trilysine acetate is a mixture of trilysine acetate and trilysine trifluoroacetate, and the product is highly hygroscopic.
  • This salt conversion method is cumbersome to operate, requires column separation, and can only prepare trilysine triacetate, but not trilysine monoacetate.
  • the inventor unexpectedly discovered that by adding a strong alkaline anion exchange resin to an aqueous solution of the compound represented by TLYS-4 (trilysine trifluoroacetate) and stirring for a certain period of time, the trifluoroacetic acid can be removed, and then removed by filtration. Resin, add glacial acetic acid to the filtrate to form a salt, forming trilysine acetate (shown in the above formula I or TLYS). This method is simple to operate and easy to control, and does not require column separation.
  • TLYS-4 trilysine trifluoroacetate
  • the above preparation method further includes the steps of using activated carbon to decolorize and filter the solution, and then adding glacial acetic acid to the filtrate to form a salt to form trilysine monoacetate (formula I above). or TLYS shown).
  • the step of preparing the compound represented by formula TLYS-4 is also included.
  • the reaction route is as follows:
  • the preparation method of trilysine acetate represented by the above formula (I) or formula TLYS includes: immobilization of the first amino acid, de-Fmoc group, coupling of the second amino acid, and de-Fmoc group. , third amino acid coupling, de-Fmoc group, cleavage, and salt transfer. This method does not require the use of chromatographic purification and lyophilization.
  • the trilysine acetate and crystal form represented by formula (I) and formula TLYS prepared by this method have good physical stability, low hygroscopicity, convenient storage, and can avoid the risk of crystallization during drug development and production.
  • Fmoc is 9-fluorenylmethoxycarbonyl. Boc is tert-butyloxycarbonyl. DIPEA is diisopropylethylamine. TFA is trifluoroacetic acid. DMF is N,N-dimethylformamide. HBTU is O-benzotriazole-N,N,N,N-tetramethylurea hexafluorophosphate. HOBt is 1-hydroxybenzotriazole. 2-CTC is 2-chlorotrityl chloride. TLYS-A is N-fluorenemethoxycarbonyl-N'-tert-butyloxycarbonyl-L-lysine.
  • the degree of substitution of the 2-CTC resin used is 1.0-1.2 mmol/g, for example, 1.10 mmol/g.
  • the 2-CTC resin used in the step of immobilizing the first amino acid, needs to be soaked and swollen in N,N-dimethylformamide at 20-30°C for more than 12 hours, so that the resin can be fully swollen. Conducive to the immobilization of the first amino acid.
  • the reaction temperature during the immobilization of the first amino acid is 25-35°C, preferably 28-30°C; the preferred reaction time is 15-18 hours. If the reaction temperature is lower than 25°C, the reaction will not proceed completely and the The efficiency is low; if the temperature is higher than 35°C, the purity of the product will be reduced. In addition, if the reaction temperature is lower than 25°C, the trilysine acetate obtained will be a mixture of trilysine diacetate and trilysine triacetate, and the product will be highly hygroscopic. If the reaction time is less than 15h, the reaction will not proceed completely and the yield will be low. If the reaction time is more than 18h, it will have no effect on product quality and yield, but will increase production costs.
  • the step of immobilizing the first amino acid also includes using anhydrous methanol to cap the 2-CTC resin, for example, using anhydrous methanol and stirring at 20-30°C for 1 hour to cap the resin. If end-capping is not performed, the purity of the product will be reduced.
  • the method for immobilizing the first amino acid in the above preparation method is as follows:
  • Resin pretreatment Add 2-CTC resin with a substitution degree of 1.10mmol/g into a stainless steel barrel, continue to add N,N-dimethylformamide, and soak at 20 to 30°C to fully swell the resin. The swelling time ⁇ 12h, filter, and wash the filter cake with N,N-dimethylformamide.
  • the preparation method of the crystal form A of the above-mentioned trilysine acetate includes: adding the above-mentioned trilysine acetate (shown in the above formula I or TLYS) into ethanol, and isolating the trilysine ester.
  • Form A of the acid salt it is preferable to stir and beat at a temperature of 20 to 30°C after adding ethanol, and then separate the crystal form A of trilysine acetate. This temperature condition is beneficial to better removal of impurities. After filtration, the filter cake can be dried, for example, under vacuum, to obtain crystal form A of trilysine acetate.
  • the salt conversion and refining are completed at the same time, that is, the compound represented by the formula TLYS-4 (trilysine trifluoroacetate) is removed from the trifluoroacetic acid using a strong basic anion exchange resin, and is decolorized and filtered using activated carbon. Then add acetic acid (glacial acetic acid) to form a salt, and concentrate under vacuum (35 ⁇ 40°C). Add ethanol to the concentrate and continue vacuum concentration until no droplets drip out. Add absolute ethanol to the concentrated residue, and stir and beat at 20 ⁇ 30°C. (for example, 2h), filter, and dry the filter cake in vacuum (drying temperature is 30 ⁇ 35°C, vacuum degree ⁇ -0.095Mpa).
  • TLYS-4 trilysine trifluoroacetate
  • the above preparation method can complete salt conversion and refining at the same time, and can obtain the crystalline form A of trilysine acetate with good physical stability and low hygroscopicity.
  • This crystalline form A is convenient for storage and can avoid the use of drugs. Risk of transcrystallization during development and production.
  • the present invention also includes trilysine acetate prepared by the above method and its crystal form A.
  • the present invention also includes the use of the above-mentioned trilysine acetate or its crystal form A in the preparation of medicines, for example, as a cross-linking agent for hydrogels.
  • the present invention also provides polylysine salts and purification methods thereof.
  • the polylysine salt is represented by the following formula (II),
  • Lys represents lysine
  • A represents acid, including acetic acid, hydrochloric acid, etc.
  • n is a natural number ⁇ 3, for example, n is 3-20, or 3-11, or 3-5.
  • polylysine salts include polylysine acetate, polylysine hydrochloride.
  • the polylysine acetate can be selected from three lysine acetates, four lysine acetates, five lysine acetates, six lysine acetates, and seven lysine acetates.
  • the polylysine salt combines only one acetic acid or one hydrochloric acid, compared to a polylysine salt that combines three (or two) acetic acids or three (or two) hydrochloric acids. It is easier to obtain better solids through concentration and drying, which is convenient for weighing and use, and the content of trilysine is higher.
  • the above-mentioned polylysine salt has no obvious moisture absorption when it is packaged in a pharmaceutical low-density polyethylene bag as the inner packaging and a polyester/aluminum/polyethylene pharmaceutical packaging composite film bag as the outer layer.
  • the purification method of the above-mentioned polylysine salt includes: providing a crude product to be purified; mixing a mixed solution of the crude product to be purified and a solvent with a strong basic anion exchange resin to separate the filtrate; adding an acidic solvent to the filtrate to convert the salt
  • the polylysine salt can be prepared by removing the solvent.
  • the solvent is water.
  • the crude product to be purified includes the crude product of the polylysine salt, polylysine trifluoroacetate, polylysine salt combined with three or two acetic acids, polylysine salt combined with three At least one of two or two polylysine salts of hydrochloric acid.
  • polylysine trifluoroacetate structure is represented by the formula PLYS-N:
  • Lys represents lysine
  • n is a natural number ⁇ 3, for example, n is 3-20, or 3-11, or 3-5.
  • the crude product to be purified includes trilysine triacetate, trilysine diacetate, tetralysine triacetate, tetralysine diacetate, trilysine At least one of amino acid trihydrochloride, trilysine dihydrochloride, tetralysine trihydrochloride, and tetralysine dihydrochloride.
  • the crude polylysine salt is commercially available or prepared according to existing methods or the method of the present invention.
  • the crude product to be purified also contains strongly acidic impurities.
  • the strongly acidic impurity is trifluoroacetic acid.
  • methods for removing solvent include lyophilization and concentration.
  • the acidic solvent is selected from acetic acid and hydrochloric acid.
  • the above-mentioned purification method of polylysine salt avoids the use of chromatographic columns, is simple and low-cost, and is conducive to industrial production.
  • LYS, n, and A have the same meanings as above.
  • the strong basic anion exchange resin is selected from type 201 ⁇ 4 or type 201 ⁇ 7. If other resins are used, the obtained trilysine acetate is a mixture of trilysine acetate and trilysine trifluoroacetate, and the product is highly hygroscopic.
  • the compound represented by the formula PLYS-N can be prepared by referring to the method of the compound represented by PLYS-4 above, that is, continuing to couple lysine on the main chain.
  • the polylysine salt represented by the above formula (II) After preparing the polylysine salt represented by the above formula (II), it also includes the step of adding it to ethanol to separate and obtain its crystal form. Among them, it is preferable to stir and beat while maintaining the temperature at 20 to 30°C after adding ethanol, and then separate and obtain the crystal form. Specific operations can be found above.
  • Another advantage of the polylysine salt preparation method provided by the present invention is that the method is simple and low-cost, and is conducive to industrial production.
  • the present invention also provides the use of the polylysine salt represented by formula (II) or its crystal form in the preparation of drugs; for example, as a cross-linking agent for hydrogels.
  • Figure 1 and Figure 2 Crystal Form A powder X-ray diffraction pattern and peak data of trilysine acetate prepared in Example 1 of the present invention.
  • FIG. 3 and Figure 4 High-resolution mass spectrometry spectrum of crystal form A of trilysine acetate prepared in Example 1 of the present invention.
  • Figure 5 Infrared spectrum of crystal form A of trilysine acetate prepared in Example 1 of the present invention.
  • Figure 6 TGA thermal analysis spectrum of crystal form A of trilysine acetate prepared in Example 1 of the present invention.
  • Figure 7 DSC thermal analysis pattern of crystal form A of trilysine acetate prepared in Example 1 of the present invention.
  • the preparation method of trilysine acetate and its crystal form A is as follows:
  • Resin pretreatment Add 2-CTC resin with a substitution degree of 1.10mmol/g into a stainless steel barrel, continue to add N,N-dimethylformamide, and soak at 20 to 30°C to fully swell the resin. The swelling time ⁇ 12h, filter, and wash the filter cake with N,N-dimethylformamide.
  • the preferred temperature for the above reaction is 25 to 35°C.
  • the preferred temperature for the above reaction is 20 to 30°C.
  • N,N-dimethylformamide to the reaction kettle, stir and add N-fluorenemethoxycarbonyl-N'-tert-butyloxycarbonyl-L-lysine, HBTU, and DIPEA at 20 to 30°C, stir for 0.5 h, add the resin obtained in step 2, stir and react for 12 to 15 hours at 25-35°C, filter, wash the filter cake with N,N-dimethylformamide, no droplets will drip out, collect the filter cake, and use the obtained resin directly to the next step.
  • the preferred temperature for the above reaction is 25 to 35°C.
  • the preferred temperature for the above reaction is 20 to 30°C.
  • N,N-dimethylformamide to the reaction kettle, stir and add N-fluorenemethoxycarbonyl-N'-tert-butyloxycarbonyl-L-lysine, HBTU, and DIPEA at 20 to 30°C, stir for 0.5 h, add the resin obtained in step 4, stir and react for 12 to 15 hours at 25-35°C, filter, wash the filter cake with N,N-dimethylformamide, no droplets will drip out, collect the filter cake, and use the obtained resin directly to the next step.
  • the preferred temperature for the above reaction is 25 to 35°C.
  • the preferred temperature for the above reaction is 20 to 30°C.
  • the preferred temperature for the above reaction is 20 to 30°C.
  • a strong basic anion exchange resin (type 201 ⁇ 4 or type 201 ⁇ 7) is used to adjust the pH to 9-10, preferably 9.5 to 9.8, and glacial acetic acid is used to adjust the pH to 2-4, preferably 2.5 to 3.5.
  • Vacuum drying is used in the above-mentioned salt conversion process, the temperature is 30 ⁇ 35°C, and the vacuum degree is ⁇ -0.095Mpa.
  • the sample is passed through a 45-mesh sieve (0.4mm).
  • the main purpose is to crush the particles to facilitate complete drying.
  • This embodiment provides trilysine acetate and its crystal form A, and its preparation method is as follows:
  • Resin pretreatment Add 2-CTC resin (350.00g) with a substitution degree of 1.10mmol/g into a 10L stainless steel bucket, continue to add N,N-dimethylformamide (3.5L), and soak at 25°C. The resin was fully swollen for 15 hours, filtered, and the filter cake was washed with N,N-dimethylformamide (3.5L).
  • N,N-dimethylformamide (3.5L) to a 10L glass reaction kettle, add TLYS-A (270.59g) and DIPEA (199.05g) with stirring, stir to dissolve, add the above-mentioned swollen 2-CTC resin, and Stir the reaction at 35°C for 16 hours.
  • Filter wash the filter cake with N,N-dimethylformamide (700ml ⁇ 3), collect the filter cake, transfer it to a 10L glass reaction kettle, add 3.5L anhydrous methanol, and stir at 25°C for 1 hour to seal the resin. Suction filtration, the filter cake was washed with N,N-dimethylformamide (700ml ⁇ 5), no droplets dripped out, the filter cake was collected, and the resin obtained was directly used in the next step.
  • N,N-dimethylformamide (3.5L) to a 10L glass reaction kettle, stir and add TLYS-A (270.59g), HBTU (240.91g), and DIPEA (82.11g), and stir for 0.5h at 25°C.
  • TLYS-A 270.59g
  • HBTU 240.91g
  • DIPEA 82.11g
  • Add the resin obtained in step 2 stir and react for 12 hours at 25°C, filter, wash the filter cake with N,N-dimethylformamide (700ml ⁇ 5), no droplets will drip out, collect the filter cake, and use the resin directly Next step.
  • N,N-dimethylformamide (3.5L) to a 10L glass reaction kettle, stir and add TLYS-A (270.59g), HBTU (240.91g), and DIPEA (82.11g), and stir for 0.5h at 25°C.
  • TLYS-A 270.59g
  • HBTU 240.91g
  • DIPEA 82.11g
  • Add the resin obtained in step 2 stir and react for 12 hours at 25°C, filter, wash the filter cake with N,N-dimethylformamide (700ml ⁇ 5), no droplets will drip out, collect the filter cake, and use the resin directly Next step.
  • the crystal form A of trilysine acetate prepared in Example 1 is shown by the following formula TLYS, and the molecular formula is C 18 H 38 N 6 O 4 ⁇ CH 3 CO 2 H.
  • step 1 This embodiment provides trilysine acetate and its crystal form A.
  • step 1 is as follows:
  • Resin pretreatment Add 2-CTC resin (350.00g) with a substitution degree of 1.10mmol/g into a 10L stainless steel bucket, continue to add N,N-dimethylformamide (3.5L), and soak at 20°C. Fully swell the resin for 20 hours, filter, and wash the filter cake with N,N-dimethylformamide (3.5L).
  • N,N-dimethylformamide (3.5L) to a 10L glass reaction kettle, add TLYS-A (265.0g) and DIPEA (195.0g) with stirring, stir to dissolve, add the above-mentioned swollen 2-CTC resin, and add Stir the reaction for 15 hours at 25°C.
  • Filter wash the filter cake with N,N-dimethylformamide (700ml ⁇ 3), collect the filter cake, transfer it to a 10L glass reaction kettle, add 3.5L anhydrous methanol, and stir at 20°C for 1 hour to seal the resin. Suction filtration, the filter cake was washed with N,N-dimethylformamide (700ml ⁇ 5), no droplets dripped out, the filter cake was collected, and the resin obtained was directly used in the next step.
  • step 1 This embodiment provides trilysine acetate and its crystal form A.
  • step 1 is as follows:
  • Resin pretreatment Add 2-CTC resin (350.00g) with a substitution degree of 1.10mmol/g into a 10L stainless steel bucket, continue to add N,N-dimethylformamide (3.5L), and soak at 30°C. Fully swell the resin for 20 hours, filter, and wash the filter cake with N,N-dimethylformamide (3.5L).
  • N,N-dimethylformamide (3.5L) to a 10L glass reaction kettle, add TLYS-A (280.0g) and DIPEA (205.0g) with stirring, stir to dissolve, add the above-mentioned swollen 2-CTC resin, and Stir the reaction at 35°C for 18 hours.
  • Filter wash the filter cake with N,N-dimethylformamide (700ml ⁇ 3), collect the filter cake, transfer it to a 10L glass reaction kettle, add 3.5L anhydrous methanol, and stir at 30°C for 1 hour to seal the resin. Suction filtration, the filter cake was washed with N,N-dimethylformamide (700ml ⁇ 5), no droplets dripped out, the filter cake was collected, and the resin obtained was directly used in the next step.
  • 99.2 g of crystal form A of trilysine acetate has a product purity of 99.45%, an acetic acid content of 12.9%, and a yield of 55.7%.
  • This embodiment provides tetralysine acetate, and its preparation method is as follows:
  • Resin pretreatment Add 2-CTC resin (350.00g) with a substitution degree of 1.10mmol/g into a 10L stainless steel bucket, continue to add N,N-dimethylformamide (3.5L), and soak at 20°C. Fully swell the resin for 20 hours, filter, and wash the filter cake with N,N-dimethylformamide (3.5L).
  • N,N-dimethylformamide (3.5L) to a 10L glass reaction kettle, add TLYS-A (265.0g) and DIPEA (195.0g) with stirring, stir to dissolve, add the above-mentioned swollen 2-CTC resin, and add Stir the reaction for 15 hours at 25°C.
  • Filter wash the filter cake with N,N-dimethylformamide (700ml ⁇ 3), collect the filter cake, transfer it to a 10L glass reaction kettle, add 3.5L anhydrous methanol, and stir at 20°C for 1 hour to seal the resin. Suction filtration, the filter cake was washed with N,N-dimethylformamide (700ml ⁇ 5), no droplets dripped out, the filter cake was collected, and the resin obtained was directly used in the next step.
  • N,N-dimethylformamide (3.5L) to a 10L glass reaction kettle, stir and add TLYS-A (265.0g), HBTU (231.0g), and DIPEA (79.0g), and stir for 0.5h at 20°C.
  • TLYS-A 265.0g
  • HBTU 231.0g
  • DIPEA 79.0g
  • Add the resin obtained in step 2 stir and react for 12 hours at 20°C, filter, wash the filter cake with N,N-dimethylformamide (700ml ⁇ 5), no droplets will drip out, collect the filter cake, and use the resin directly Next step.
  • N,N-dimethylformamide (2.8L) and piperidine (700g) to a 10L glass reaction kettle, stir evenly, add the above filter cake resin, stir at 20 ⁇ 30°C for 0.5h, filter, use N for the filter cake , wash with N-dimethylformamide (700ml ⁇ 5), no droplets drip out, collect the filter cake, and the resin obtained is directly used in the next step.
  • N,N-dimethylformamide (3.5L) to a 10L glass reaction kettle, stir and add TLYS-A (265.0g), HBTU (231.0g), and DIPEA (79.0g), and stir for 0.5h at 20°C.
  • TLYS-A 265.0g
  • HBTU 231.0g
  • DIPEA 79.0g
  • Add the resin obtained in step 2 stir and react for 15 hours at 20°C, filter, wash the filter cake with N,N-dimethylformamide (700ml ⁇ 5), no droplets will drip out, collect the filter cake, and the resin obtained is used directly Next step.
  • N,N-dimethylformamide (2.8L) and piperidine (700g) to a 10L glass reaction kettle, stir evenly, add the above filter cake resin, stir at 20 ⁇ 30°C for 0.5h, filter, use N for the filter cake , wash with N-dimethylformamide (700ml ⁇ 5), no droplets drip out, collect the filter cake, and the resin obtained is directly used in the next step.
  • N,N-dimethylformamide (3.5L) to a 10L glass reaction kettle, stir and add TLYS-A (265.0g), HBTU (231.0g), and DIPEA (79.0g), and stir for 0.5h at 20°C.
  • TLYS-A 265.0g
  • HBTU 231.0g
  • DIPEA 79.0g
  • Add the resin obtained in step 2 stir and react for 15 hours at 20°C, filter, wash the filter cake with N,N-dimethylformamide (700ml ⁇ 5), no droplets will drip out, collect the filter cake, and the resin obtained is used directly Next step.
  • the obtained tetralysine acetate is a white powder.
  • a low-density polyethylene bag for inner packaging and a polyester/aluminum/polyethylene composite film bag for pharmaceutical packaging there is no obvious moisture absorption.
  • This embodiment provides pentalysine acetate, and its preparation method is as follows:
  • Resin pretreatment Add 2-CTC resin (350.00g) with a substitution degree of 1.10mmol/g into a 10L stainless steel barrel, and continue Add N,N-dimethylformamide (3.5L), soak at 20°C to fully swell the resin, swell for 20 hours, filter, and wash the filter cake with N,N-dimethylformamide (3.5L).
  • N,N-dimethylformamide (3.5L) to a 10L glass reaction kettle, add TLYS-A (265.0g) and DIPEA (195.0g) with stirring, stir to dissolve, add the above-mentioned swollen 2-CTC resin, and add Stir the reaction for 15 hours at 25°C.
  • Filter wash the filter cake with N,N-dimethylformamide (700ml ⁇ 3), collect the filter cake, transfer it to a 10L glass reaction kettle, add 3.5L anhydrous methanol, and stir at 20°C for 1 hour to seal the resin. Suction filtration, the filter cake was washed with N,N-dimethylformamide (700ml ⁇ 5), no droplets dripped out, the filter cake was collected, and the resin obtained was directly used in the next step.
  • N,N-dimethylformamide (3.5L) to a 10L glass reaction kettle, stir and add TLYS-A (265.0g), HBTU (231.0g), and DIPEA (79.0g), and stir for 0.5h at 20°C.
  • TLYS-A 265.0g
  • HBTU 231.0g
  • DIPEA 79.0g
  • Add the resin obtained in step 2 stir and react for 12 hours at 20°C, filter, wash the filter cake with N,N-dimethylformamide (700ml ⁇ 5), no droplets will drip out, collect the filter cake, and use the resin directly Next step.
  • N,N-dimethylformamide (2.8L) and piperidine (700g) to a 10L glass reaction kettle, stir evenly, add the above filter cake resin, stir at 20 ⁇ 30°C for 0.5h, filter, use N for the filter cake , wash with N-dimethylformamide (700ml ⁇ 5), no droplets drip out, collect the filter cake, and the resin obtained is directly used in the next step.
  • N,N-dimethylformamide (3.5L) to a 10L glass reaction kettle, stir and add TLYS-A (265.0g), HBTU (231.0g), and DIPEA (79.0g), and stir for 0.5h at 20°C.
  • TLYS-A 265.0g
  • HBTU 231.0g
  • DIPEA 79.0g
  • Add the resin obtained in step 2 stir and react for 15 hours at 20°C, filter, wash the filter cake with N,N-dimethylformamide (700ml ⁇ 5), no droplets will drip out, collect the filter cake, and the resin obtained is used directly Next step.
  • N,N-dimethylformamide (2.8L) and piperidine (700g) to a 10L glass reaction kettle, stir evenly, add the above filter cake resin, stir at 20 ⁇ 30°C for 0.5h, filter, use N for the filter cake , wash with N-dimethylformamide (700ml ⁇ 5), no droplets drip out, collect the filter cake, and the resin obtained is directly used in the next step.
  • N,N-dimethylformamide (3.5L) to a 10L glass reaction kettle, stir and add TLYS-A (265.0g), HBTU (231.0g), and DIPEA (79.0g), and stir for 0.5h at 20°C.
  • TLYS-A 265.0g
  • HBTU 231.0g
  • DIPEA 79.0g
  • Add the resin obtained in step 2 stir and react for 15 hours at 20°C, filter, wash the filter cake with N,N-dimethylformamide (700ml ⁇ 5), no droplets will drip out, collect the filter cake, and the resin obtained is used directly Next step.
  • N,N-dimethylformamide (2.8L) and piperidine (700g) to a 10L glass reaction kettle, stir evenly, add the above filter cake resin, stir at 20 ⁇ 30°C for 0.5h, filter, use N for the filter cake , wash with N-dimethylformamide (700ml ⁇ 5), no droplets drip out, collect the filter cake, and the resin obtained is directly used in the next step.
  • N,N-dimethylformamide (3.5L) to a 10L glass reaction kettle, stir and add TLYS-A (265.0g), HBTU (231.0g), and DIPEA (79.0g), and stir for 0.5h at 20°C.
  • TLYS-A 265.0g
  • HBTU 231.0g
  • DIPEA 79.0g
  • Add the resin obtained in step 2 stir and react for 15 hours at 20°C, filter, wash the filter cake with N,N-dimethylformamide (700ml ⁇ 5), no droplets will drip out, collect the filter cake, and the resin obtained is used directly Next step.
  • N,N-dimethylformamide (2.8L) and piperidine (600g) to a 10L glass reaction kettle, stir evenly, and add the above filter cake tree fat, stir at 20°C for 0.5h, filter, and wash the filter cake with N,N-dimethylformamide (700ml ⁇ 5). If no droplets drip out, collect the filter cake.
  • the obtained pentalysine acetate is a white powder.
  • a low-density polyethylene bag for inner packaging and a polyester/aluminum/polyethylene composite film bag for pharmaceutical packaging there is no obvious moisture absorption.
  • This embodiment provides tetralysine hydrochloride, the preparation method of which is different from that of Example 4 only in step 10, which is as follows: Step 10: salt transfer
  • the obtained tetralysine hydrochloride is a white powder.
  • a low-density polyethylene bag for inner packaging and a polyester/aluminum/polyethylene composite film bag for pharmaceutical packaging there is no obvious moisture absorption.
  • This embodiment provides pentalysine hydrochloride, the preparation method of which is different from that of Example 5 only in step 12, which is as follows: Step 12: salt transfer
  • the obtained pentalysine hydrochloride is a white powder.
  • a pharmaceutical low-density polyethylene bag as an inner packaging
  • a polyester/aluminum/polyethylene pharmaceutical packaging composite film bag as an outer layer
  • High-resolution test conditions LCMS-IT/TOF, ion source ESI, scanning range 100 ⁇ 1000m/z.
  • Infrared test conditions Fourier transform infrared spectrometer, using KBr tableting method to record spectra.
  • TGA thermal analysis test conditions TG/DTA6200 thermogravimetric analyzer, heating rate 10°C/min.
  • DSC thermal analysis test conditions DSC6220 differential scanning calorimeter, heating rate: 10°C/min.
  • the trilysine acetate sample prepared in Examples 1-3 was packaged in a pharmaceutical low-density polyethylene bag as an inner package and a polyester/aluminum/polyethylene pharmaceutical packaging composite film bag as the outer layer, and was placed out at a temperature of 6 ⁇ 2°C. , placed for 6 months, and samples were taken for measurement at 0, 1, 2, 3, 6, 12, and 18 months.
  • the result is as follows:
  • This product was placed at a temperature of 6 ⁇ 2°C for 18 months. There was no significant change in various test indicators compared with 0 days, indicating that this product has good stability under the condition of 6 ⁇ 2°C.
  • Preparation method of trilysine triacetate Repeat steps 1 to 7 of Example 1, use HPLC to convert the obtained filter cake into salt (acetic acid), concentrate the obtained solution, and freeze-dry to obtain trilysine triacetate. acetate. After testing, its tripeptide content is 69.79%, indicating that its acetic acid content is about 30%, which is triacetate.
  • This comparative example provides trilysine acetate, the preparation method of which is different from Example 1 only in step 1, which is as follows: Step 1: Immobilization of the first amino acid
  • Resin pretreatment Add 2-CTC resin (350.00g) with a substitution degree of 1.10mmol/g into a 10L stainless steel bucket, continue to add N,N-dimethylformamide (3.5L), and soak at 20°C. Fully swell the resin for 20 hours, filter, and wash the filter cake with N,N-dimethylformamide (3.5L).
  • N,N-dimethylformamide (3.5L) to a 10L glass reaction kettle, add TLYS-A (265.0g) and DIPEA (195.0g) with stirring, stir to dissolve, add the above-mentioned swollen 2-CTC resin, and add 18°C, stirring reaction for 15h.
  • Filter wash the filter cake with N,N-dimethylformamide (700ml ⁇ 3), collect the filter cake, transfer it to a 10L glass reaction kettle, add 3.5L anhydrous methanol, and stir at 18°C for 1 hour to seal the resin. Suction filtration, the filter cake was washed with N,N-dimethylformamide (700ml ⁇ 5), no droplets dripped out, the filter cake was collected, and the resin obtained was directly used in the next step.
  • This comparative example provides trilysine acetate, the preparation method of which is different from Example 1 only in step 8, which is as follows: Step 8: salt transfer
  • the trilysine acetate obtained in this comparative example is a mixture of trilysine acetate and trilysine trifluoroacetate.
  • the invention provides a polylysine salt and a purification method thereof.
  • the method for purifying polylysine salts includes: providing a crude product to be purified; the crude product to be purified includes crude polylysine salts, polylysine trifluoroacetate, and polylysine salts combined with three or two acetic acids.
  • Polylysine salt at least one of polylysine salts combined with three or two hydrochloric acids; mix the mixed solution of the crude product to be purified and the solvent with a strong basic anion exchange resin, and separate the filtrate;
  • the polylysine salt is obtained by adding an acidic solvent to the filtrate to convert the salt into salt and then removing the solvent. , has good economic value and application prospects.

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Abstract

提供了一种多赖氨酸盐及其纯化方法。多赖氨酸盐的纯化方法包括：提供待纯化的粗品；所述待纯化的粗品包括多赖氨酸盐的粗品、多赖氨酸三氟乙酸盐、结合了三个或两个醋酸的多赖氨酸盐、结合了三个或两个盐酸的多赖氨酸盐中的至少一种；将待纯化的粗品和溶剂的混合溶液与强碱性阴离子交换树脂混合，分离滤液；向所述滤液中加入酸性溶剂转盐后除去溶剂即制得所述多赖氨酸盐。

Description

一种多赖氨酸盐及其制备方法和纯化方法
交叉引用
本申请要求2022年5月17日提交的专利名称为“三赖氨酸醋酸盐及其制备方法与应用”的第202210542271.4号中国专利申请和2022年5月17日提交的专利名称为“一种多赖氨酸盐及其纯化方法”的第202210542240.9号中国专利申请的优先权，其全部公开内容通过引用整体并入本文。
技术领域
本发明涉及化学合成，具体涉及多赖氨酸盐及其制备方法和纯化方法。
背景技术
三赖氨酸醋酸盐是赖氨酸的三肽，作为水凝胶的交联剂。三赖氨酸醋酸盐属于三肽，肽类的合成主要采用固相合成法。目前国内外，Fmoc固相合成法已被广泛用于各种性能多肽的合成，具有方便、快捷、操作简单、产率高的优势。但是目前固相合成法合成多肽，纯化时多采用色谱纯化，产品干燥时使用冻干的方法，这样就增加了生产成本。
另一方面，现有的制备方法通过柱色谱纯化后，通过HPLC转盐法制备三赖氨酸醋酸盐，制备的三赖氨酸醋酸盐为三赖氨酸三醋酸盐，而三赖氨酸三醋酸盐的吸湿性强，物理性质不稳定，不方便后期使用，不方便保存。
基于上述现状，急需提出一种新的三赖氨酸醋酸盐的制备方法，以克服现有工艺的缺点，并制备出高品质的三赖氨酸醋酸盐。
发明内容
本发明旨在至少解决以上技术问题之一。
本发明首先提供一种如下式(I)所示的三赖氨酸醋酸盐，
还提供如下式TLYS所示的三赖氨酸醋酸盐，
根据本发明实施例，所述三赖氨酸醋酸盐的分子式为C18H38N6O4·CH3CO2H。
现有三赖氨酸醋酸盐为结合了三个醋酸的三赖氨酸三醋酸盐，吸湿性较强，不易通过浓缩和干燥等方式得到较好的固体，应用受到限制。本发明制备得到的三赖氨酸醋酸盐仅结合了一个醋酸，可通过浓缩和干燥等方式得到较好的固体，利于称量和使用，且三赖氨酸的含量更高。
本发明还提供上述式TLYS所示三赖氨酸醋酸盐的晶型A。
根据本发明实施例，所述三赖氨酸醋酸盐的晶型A使用Cu-Kα辐射，以衍射角2θ角度表示的X-射线粉末衍射在20.227、23.194、23.686处有特征峰，其中，每个特征峰2θ角的误差范围为±0.2。
根据本发明实施例，所述三赖氨酸醋酸盐的晶型A使用Cu-Kα辐射，以衍射角2θ角度表示的X-射线粉末衍射在4.632、19.695、20.227、22.172、23.194、23.686、28.079处有特征峰，其中，每个特征峰2θ角的误差范围为±0.2。
根据本发明实施例，所述三赖氨酸醋酸盐的晶型A使用Cu-Kα辐射，以衍射角2θ角度表示的X-射线粉末衍射在4.632、7.951、11.825、13.980、17.667、18.307、19.695、20.227、21.686、22.172、23.194、23.686、28.079处有特征峰，其中，每个特征峰2θ角的误差范围为±0.2。
根据本发明实施例，所述三赖氨酸醋酸盐的晶型A使用Cu-Kα辐射，以衍射角2θ角度表示的X-射线粉末衍射在4.632、7.951、9.272、11.825、13.980、16.544、17.343、17.667、18.307、19.695、20.227、21.686、22.172、23.194、23.686、27.327、28.079、29.270、32.239处有特征峰，其中，每个特征峰2θ角的误差范围为±0.2。
根据本发明实施例，所述三赖氨酸醋酸盐的晶型A使用Cu-Kα辐射，以衍射角2θ角度表示的X-射线粉末衍射在4.632、5.478、7.951、9.272、9.742、11.825、13.980、16.192、16.544、17.343、17.667、18.307、18.716、19.695、20.227、21.686、22.172、23.194、23.686、24.672、27.327、28.079、29.270、32.239处有特征峰，其中，每个特征峰2θ角的误差范围为±0.2。
根据本发明实施例，所述三赖氨酸醋酸盐的晶型A使用Cu-Kα辐射，以衍射角2θ角度表示的X-射线粉末衍射峰如表1所示，误差范围±0.2：
表1

根据本发明实施例，所述三赖氨酸醋酸盐的晶型A具有基本如图1所示的粉末X射线衍射图。
根据本发明实施例，所述三赖氨酸醋酸盐的晶型A通过热重分析测量，176.10℃热量损失为8.1wt％±2wt％，可选8.1wt％±1wt％。
根据本发明实施例，所述三赖氨酸醋酸盐的晶型A的差示扫描量热分析图谱的DSC曲线在147.81℃处有吸热峰。
根据本发明实施例，所述三赖氨酸醋酸盐的晶型A具有基本如图7所示的DSC图。
本发明提供的三赖氨酸醋酸盐(为三赖氨酸一醋酸盐)及上述晶型A具有良好的物理稳定性、吸湿性低，方便保存，能避免药物开发和生成过程中转晶的风险。
本发明还提供上式(I)或式TLYS所示三赖氨酸醋酸盐的制备方法，包括：将式TLYS-4所示化合物(三赖氨酸三氟乙酸盐)采用强碱性阴离子交换树脂脱去三氟乙酸，然后与醋酸反应，生成TLYS所示化合物；
反应如下：
优选地，所述强碱性阴离子交换树脂选自201×4型或201×7型。研究发现，若使用其他树脂，得到三赖氨酸醋酸盐为三赖氨酸醋酸盐与三赖氨酸三氟乙酸盐的混合物，产品吸湿性较强。
发明人在实践中发现，使用弱阴离子交换树脂制备，需要将树脂装成树脂柱进行洗脱，将三赖氨酸吸附在树脂上，去除三氟乙酸，然后使用醋酸水溶液将三赖氨酸洗脱下来并形成三赖氨酸 三醋酸盐。这种转盐方式操作繁琐，需要进行装柱分离，且只能制备三赖氨酸三醋酸盐，无法制备出三赖氨酸一醋酸盐。
发明人意外地发现，将强碱性阴离子交换树脂加入TLYS-4所示化合物(三赖氨酸三氟乙酸盐)的水溶液中搅拌一定时间，即可脱去三氟乙酸，然后通过过滤去除树脂，滤液加入冰醋酸成盐，形成三赖氨酸醋酸盐(上式I或TLYS所示)。这种方法操作简单，容易控制，无需使用装柱分离。
在一些实施例，上述制备方法在脱去三氟乙酸后还包括将溶液使用活性炭脱色、过滤的步骤，然后再在滤液加入冰醋酸成盐，形成三赖氨酸一醋酸盐(上式I或TLYS所示)。
在一些实例中，还包括制备式TLYS-4所示化合物的步骤，反应路线如下：
在一些实例中，上式(I)或式TLYS所示三赖氨酸醋酸盐的制备方法包括：首位氨基酸固定化、脱-Fmoc基团、第二个氨基酸偶联、脱-Fmoc基团、第三个氨基酸偶联、脱-Fmoc基团、切割、转盐。该方法不需使用色谱纯化和冻干干燥。该方法制备的式(I)、式TLYS所示三赖氨酸醋酸盐及晶型，具有良好的物理稳定性、吸湿性低，方便保存，能避免药物开发和生成过程中转晶的风险。
本文中，Fmoc为9-芴甲氧羰基。Boc为叔丁基氧羰基。DIPEA为二异丙基乙胺。TFA为三氟乙酸。DMF为N,N-二甲基甲酰胺。HBTU为O-苯并三唑-N,N,N,N-四甲基脲六氟磷酸盐。HOBt为1-羟基苯并三唑。2-CTC为2-氯三苯甲基氯。TLYS-A为N-芴甲氧羰基-N'-叔丁基氧羰基-L-赖氨酸。
在一些实施例，上述反应路线，首位氨基酸固定化的步骤中，所使用的2-CTC树脂的取代度为1.0-1.2mmol/g，例如1.10mmol/g。
在一些实施例，所述首位氨基酸固定化的步骤中，所使用的2-CTC树脂需要在N,N-二甲基甲酰胺中于20～30℃浸泡溶胀12h以上，这样可以使树脂充分溶胀利于首位氨基酸固定化。
在一些实施例，所述首位氨基酸固定化的步骤中，首位氨基酸固定化时的反应温度为25～35℃，优选为28～30℃；优选反应时间15～18h。如果反应温度低于25℃，反应进行不彻底，收 率低；如果温度高于35℃，会造成产品纯度降低。另外，如果反应温度低于25℃，得到的三赖氨酸醋酸盐为三赖氨酸二醋酸盐和三赖氨酸三醋酸盐的混合物，产品吸湿性较强。反应时间低于15h，反应进行不彻底，收率低，反应时间高于18h，对产品质量和收率无影响，但是增加生产成本。
在一些实施例，所述首位氨基酸固定化的步骤中，还包括使用无水甲醇于对2-CTC树脂进行封端，例如使用无水甲醇于20～30℃搅拌1h对树脂进行封端。如果不进行封端，会降低产品的纯度。
在一些实施例，上述制备方法中首位氨基酸固定化的方法如下：
树脂预处理：将取代度为1.10mmol/g的2-CTC树脂加入到不锈钢桶中，继续加入N,N-二甲基甲酰胺，于20～30℃下浸泡，使树脂充分溶胀，溶胀时间≥12h，过滤，滤饼用N,N-二甲基甲酰胺洗涤。
反应釜中加入N,N-二甲基甲酰胺，搅拌加入N-芴甲氧羰基-N'-叔丁基氧羰基-L-赖氨酸，DIPEA，搅拌溶解，加入上述溶胀后的2-CTC树脂，于25～35℃，搅拌反应15～18h。过滤，滤饼用N,N-二甲基甲酰胺洗涤，收集滤饼，转入反应釜中加入无水甲醇，于20～30℃搅拌1h树脂封端。抽滤，滤饼用N,N-二甲基甲酰胺洗涤，无液滴滴出，收集滤饼，所得树脂直接用于下一步。
在一些实施例，上述三赖氨酸醋酸盐的晶型A的制备方法包括：将上述三赖氨酸醋酸盐(上式I所示或TLYS)加入乙醇，分离得到三赖氨酸醋酸盐的晶型A。其中，优选在加入乙醇后保持温度20～30℃搅拌打浆，然后分离得到三赖氨酸醋酸盐的晶型A。在该温度条件下有利于更好地去除杂质。过滤后，即可将滤饼干燥例如真空干燥得到三赖氨酸醋酸盐的晶型A。
在一些实施例，转盐和精制同时完成，即将式TLYS-4所示化合物(三赖氨酸三氟乙酸盐)采用强碱性阴离子交换树脂脱去三氟乙酸，使用活性炭脱色、过滤，然后加入醋酸(冰醋酸)成盐，真空浓缩(35～40℃)，浓缩物中加入乙醇继续真空浓缩至无液滴滴出，浓缩剩余物中再加入无水乙醇，20～30℃搅拌打浆(例如2h)，过滤，滤饼采用真空干燥(干燥温度为30～35℃，真空度≤-0.095Mpa)，样品过筛(45目筛，0.4mm)后，继续真空干燥(30～35℃，3～5h，真空度≤-0.095MPa)，可稳定的制备出高纯度的三赖氨酸醋酸盐的晶型A。
在一些实施例，上述制备方法转盐和精制同时完成，并可得到物理稳定性好、吸湿性低的三赖氨酸醋酸盐的晶型A，这种晶型A方便保存，能避免药物开发和生成过程中转晶的风险。
本发明还包括上述方法制备的三赖氨酸醋酸盐及其晶型A。
本发明还包括上述三赖氨酸醋酸盐或其晶型A在制备药物中的应用，例如作为水凝胶的交联剂。
本发明还提供多赖氨酸盐及其纯化方法。
多赖氨酸盐如下式(II)所示，

其中，Lys代表赖氨酸；A代表酸，包括醋酸、盐酸等；n为≥3的自然数，例如n为3-20，或3-11，或3-5。
在一些实施例，多赖氨酸盐包括多赖氨酸醋酸盐、多赖氨酸盐酸盐。具体地，所述多赖氨酸醋酸盐可选为三赖氨酸醋酸盐、四赖氨酸醋酸盐、五赖氨酸醋酸盐、六赖氨酸醋酸盐、七赖氨酸醋酸盐、八赖氨酸醋酸盐、九赖氨酸醋酸盐、十赖氨酸醋酸盐、十一赖氨酸醋酸盐、三赖氨酸盐酸盐、四赖氨酸盐酸盐、五赖氨酸盐酸盐、六赖氨酸盐酸盐、七赖氨酸盐酸盐、八赖氨酸盐酸盐、九赖氨酸盐酸盐、十赖氨酸盐酸盐、十一赖氨酸盐酸盐等。
在一些实施例，所述多赖氨酸盐仅结合了一个醋酸或一个盐酸，与结合了三个(或两个)醋酸或三个(或两个)盐酸的多赖氨酸盐相比，更容易通过浓缩和干燥等方式得到较好的固体，利于称量和使用，且三赖氨酸的含量更高。实验证明，上述多赖氨酸盐在采用内包装药用低密度聚乙烯袋，外套聚酯/铝/聚乙烯药品包装用复合膜袋包装条件下，无明显吸湿现象。
上述多赖氨酸盐的纯化方法包括：提供待纯化的粗品；将待纯化的粗品和溶剂的混合溶液与强碱性阴离子交换树脂混合，分离滤液；向所述滤液中加入酸性溶剂转盐后除去溶剂，即可制得所述多赖氨酸盐。
在一些实施例，所述溶剂为水。
在一些实施例，所述待纯化的粗品包括所述多赖氨酸盐的粗品、多赖氨酸三氟乙酸盐、结合了三个或两个醋酸的多赖氨酸盐、结合了三个或两个盐酸的多赖氨酸盐中的至少一种。
在一些实施例，多赖氨酸三氟乙酸盐结构如式PLYS-N所示：
其中，Lys代表赖氨酸；n为≥3的自然数，例如n为3-20，或3-11，或3-5。
在一些实施例，所述待纯化的粗品包括三赖氨酸三醋酸盐、三赖氨酸二醋酸盐、四赖氨酸三醋酸盐、四赖氨酸二醋酸盐、三赖氨酸三盐酸盐、三赖氨酸二盐酸盐、四赖氨酸三盐酸盐、四赖氨酸二盐酸盐中的至少一种。
在一些实施例，所述多赖氨酸盐粗品可市售购得，或按现有方法或本发明方法制备。
在一些实施例，所述待纯化的粗品中还含有强酸性杂质。
在一些实施例，所述强酸性杂质为三氟乙酸。
在一些实施例，除去溶剂的方法包括冻干、浓缩。
在一些实施例，所述酸性溶剂选自醋酸、盐酸。
上述多赖氨酸盐的纯化方法避免了使用色谱柱，方法简便，成本低，利于工业化生产。
以待纯化的粗品为多赖氨酸三氟乙酸盐(式PLYS-N所示化合物)为例，其纯化的反应路线如下：
其中，LYS、n、A的含义与上文相同。
与上文制备上式(I)或式TLYS所示三赖氨酸醋酸盐的方法类似，所述强碱性阴离子交换树脂选自201×4型或201×7型。若使用其他树脂，得到三赖氨酸醋酸盐为三赖氨酸醋酸盐与三赖氨酸三氟乙酸盐的混合物，产品吸湿性较强。
式PLYS-N所示化合物可参照上文PLYS-4所示化合物的方法制备，即在主链上继续偶联赖氨酸。
在制得上述式(II)所示多赖氨酸盐后还包括加入乙醇中，分离得到其晶型的步骤。其中，优选在加入乙醇后保持温度20～30℃搅拌打浆，然后分离得到该晶型。具体操作可参见上文。
本发明还提供的多赖氨酸盐制备方法另一优势是方法简便，成本低，利于工业化生产。
本发明还提供式(II)所示多赖氨酸盐或其晶型在制备药物中的应用；例如作为水凝胶的交联剂。
附图说明
图1和图2：本发明实施例1制备的三赖氨酸醋酸盐的晶型A粉末X射线衍射图谱及峰数据。
图3和图4：本发明实施例1制备的三赖氨酸醋酸盐的晶型A高分辨质谱图谱。
图5：本发明实施例1制备的三赖氨酸醋酸盐的晶型A红外图谱。
图6：本发明实施例1制备的三赖氨酸醋酸盐的晶型A的TGA热分析图谱。
图7：本发明实施例1制备的三赖氨酸醋酸盐的晶型A的DSC热分析图谱。
具体实施方式
以下实施例用于说明本发明，但不用来限制本发明的范围。实施例中未注明具体技术或条件者，按照本领域内的文献所描述的技术或条件，或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可通过正规渠道商购买得到的常规产品。
在一些实施例，三赖氨酸醋酸盐及其晶型A的制备方法路线如下：
在一些实施例，三赖氨酸醋酸盐及其晶型A的制备方法：
工序1：首位氨基酸固定化
树脂预处理：将取代度为1.10mmol/g的2-CTC树脂加入到不锈钢桶中，继续加入N,N-二甲基甲酰胺，于20～30℃下浸泡，使树脂充分溶胀，溶胀时间≥12h，过滤，滤饼用N,N-二甲基甲酰胺洗涤。
反应釜中加入N,N-二甲基甲酰胺，搅拌加入N-芴甲氧羰基-N'-叔丁基氧羰基-L-赖氨酸，DIPEA，搅拌溶解，加入上述溶胀后的2-CTC树脂，于25～35℃，搅拌反应15～18h。过滤，滤饼用N,N-二甲基甲酰胺洗涤，收集滤饼，转入反应釜中加入无水甲醇，于20～30℃搅拌1h树脂封端。抽滤，滤饼用N,N-二甲基甲酰胺洗涤，无液滴滴出，收集滤饼，所得树脂直接用于下一步。
上述反应的优选投料摩尔比为2-CTC树脂：N-芴甲氧羰基-N'-叔丁基氧羰基-L-赖氨酸：DIPEA＝1.0:(1.1～1.8):(3.0～5.0)。
上述反应的优选温度为25～35℃。
工序2：脱-Fmoc基团
反应釜中加入N,N-二甲基甲酰胺，哌啶，搅拌均匀，加入上述滤饼树脂，于20～30℃搅拌0.5～1h，过滤，滤饼用N,N-二甲基甲酰胺洗涤，无液滴滴出，收集滤饼，所得树脂直接用于下一步。
上述反应的优选投料质量比为2-CTC树脂:哌啶＝1.0:(1.5～3.0)。
上述反应的优选温度为20～30℃。
工序3：第二个氨基酸偶联
反应釜中加入N,N-二甲基甲酰胺，搅拌加入N-芴甲氧羰基-N'-叔丁基氧羰基-L-赖氨酸，HBTU，DIPEA，于20～30℃，搅拌0.5h，加入工序2所得树脂，于25～35℃，搅拌反应12～15h，过滤，滤饼用N,N-二甲基甲酰胺洗涤，无液滴滴出，收集滤饼，所得树脂直接用于下一步。
上述反应的优选投料摩尔比为2-CTC树脂：N-芴甲氧羰基-N'-叔丁基氧羰基-L-赖氨酸： HBTU：DIPEA＝1.0:(1.1～1.8):(1.5～2.0):(1.5～2.0)。
上述反应的优选温度为25～35℃。
工序4：脱-Fmoc基团
反应釜中加入N,N-二甲基甲酰胺，哌啶，搅拌均匀，加入上述滤饼树脂，于20～30℃搅拌0.5h，过滤，滤饼用N,N-二甲基甲酰胺洗涤，无液滴滴出，收集滤饼，所得树脂直接用于下一步。
上述反应的优选投料质量比为2-CTC树脂:哌啶＝1.0:(1.5～3.0)。
上述反应的优选温度为20～30℃。
工序5：第三个氨基酸偶联
反应釜中加入N,N-二甲基甲酰胺，搅拌加入N-芴甲氧羰基-N'-叔丁基氧羰基-L-赖氨酸，HBTU，DIPEA，于20～30℃，搅拌0.5h，加入工序4所得树脂，于25～35℃，搅拌反应12～15h，过滤，滤饼用N,N-二甲基甲酰胺洗涤，无液滴滴出，收集滤饼，所得树脂直接用于下一步。
上述反应的优选投料摩尔比为2-CTC树脂：N-芴甲氧羰基-N'-叔丁基氧羰基-L-赖氨酸：HBTU：DIPEA＝1.0:(1.1～1.8):(1.5～2.0):(1.5～2.0)。
上述反应的优选温度为25～35℃。
工序6：脱-Fmoc基团
反应釜中加入N,N-二甲基甲酰胺，哌啶，搅拌均匀，加入上述滤饼树脂，于20～30℃搅拌0.5h，过滤，滤饼用N,N-二甲基甲酰胺洗涤，无液滴滴出，收集滤饼。反应釜中加入异丙醇，搅拌加入上述滤饼，于20～30℃，搅拌浸泡0.5h，过滤；反应釜中加入二氯甲烷，搅拌加入上述滤饼，于20～30℃，搅拌浸泡0.5h，过滤至无液滴滴出；反应釜中加入异丙醇，搅拌加入上述滤饼，于20～30℃，搅拌浸泡0.5h，过滤至无液滴滴出，滤饼于20～30℃鼓风干燥6～8h。
上述反应的优选投料质量比为2-CTC树脂:哌啶＝1.0:(1.5～3.0)。
上述反应的优选温度为20～30℃。
工序7：切割
反应釜中加入甲基叔丁基醚，搅拌降温至0℃(备用)。
反应釜中加入三氟乙酸，三异丙基硅烷，纯化水，控温＜30℃，搅拌下分批加入上述树脂(加入过程放热)，保持温度20～30℃，搅拌4h，过滤，滤饼用三氟乙酸洗涤，无液滴滴出，收集滤液。将滤液滴加至甲基叔丁基醚中，保温0～10℃搅拌2h，过滤，滤饼用甲基叔丁基醚浆洗，无液滴滴出，收集滤饼。(产品易吸潮，不宜长时间暴露在空气中)
上述反应的优选投料质量比为2-CTC树脂：三氟乙酸：三异丙基硅烷：纯化水＝1.0:(8.0～15.0):(0.05～0.20):(0.05～0.20)。
上述反应的优选温度为20～30℃。
工序8：转盐
反应釜中加入纯化水，加入工序7所得滤饼，搅拌溶清，静置、分液(去除上层甲基叔丁基醚相)，保留下层水相。水相搅拌下加入强碱性阴离子交换树脂(201×4型或201×7型)调节pH＝9-10，保温20～30℃，搅拌1h。过滤，滤饼用纯化水洗涤，收集滤液，滤液中加入活性炭，保持温度20～30℃，搅拌10min，用0.45μm滤膜过滤，收集滤液。滤液中加入冰醋酸调节pH＝2-4，于35～40℃真空浓缩至无液滴滴出，浓缩物(即为上式I或式TLYS所示三赖氨酸醋酸盐)中加入乙醇，继续减压浓缩至无液滴滴出。浓缩剩余物中加入无水乙醇，保持20～30℃搅拌打浆2h，过滤，滤饼用无水乙醇洗涤，无液滴滴出，滤饼于30～35℃真空干燥3h，真空度≤-0.095Mpa，样品过45目筛(0.4mm)后，于30～35℃继续真空干燥3～5h，真空度≤-0.095MPa，得到三赖氨酸醋酸盐的晶型A。
上述转盐工序中使用强碱性阴离子交换树脂(201×4型或201×7型)调节pH＝9-10，优选9.5～9.8，冰醋酸调节pH＝2-4，优选2.5～3.5。
上述转盐工序中使用真空干燥，温度30～35℃，真空度≤-0.095Mpa。
上述转盐工序干燥过程中样品过45目筛(0.4mm)，主要目的是将颗粒粉碎，便于干燥彻底。
实施例1
本实施例提供三赖氨酸醋酸盐及其晶型A，其制备方法如下：
工序1：首位氨基酸固定化
树脂预处理：将取代度为1.10mmol/g的2-CTC树脂(350.00g)加入到10L不锈钢桶中，继续加入N,N-二甲基甲酰胺(3.5L)，于25℃下浸泡，使树脂充分溶胀，溶胀时间15h，过滤，滤饼用N,N-二甲基甲酰胺(3.5L)洗涤。
10L玻璃反应釜中加入N,N-二甲基甲酰胺(3.5L)，搅拌加入TLYS-A(270.59g)，DIPEA(199.05g)，搅拌溶解，加入上述溶胀后的2-CTC树脂，于35℃，搅拌反应16h。过滤，滤饼用N,N-二甲基甲酰胺(700ml×3)洗涤，收集滤饼，转入10L玻璃反应釜中加入3.5L无水甲醇，于25℃搅拌1h树脂封端。抽滤，滤饼用N,N-二甲基甲酰胺(700ml×5)洗涤，无液滴滴出，收集滤饼，所得树脂直接用于下一步。
工序2：脱-Fmoc基团
10L玻璃反应釜中加入N,N-二甲基甲酰胺(2.8L)，哌啶(700g)，搅拌均匀，加入上述滤饼树脂，于20℃搅拌0.5h，过滤，滤饼用N,N-二甲基甲酰胺(700ml×5)洗涤，无液滴滴出，收集滤饼，所得树脂直接用于下一步。
工序3：第二个氨基酸偶联
10L玻璃反应釜中加入N,N-二甲基甲酰胺(3.5L)，搅拌加入TLYS-A(270.59g)，HBTU(240.91g)，DIPEA(82.11g)，于25℃，搅拌0.5h，加入工序2所得树脂，于25℃，搅拌反应12h，过滤，滤饼用N,N-二甲基甲酰胺(700ml×5)洗涤，无液滴滴出，收集滤饼，所得树脂直接用于下一步。
工序4：脱-Fmoc基团
10L玻璃反应釜中加入N,N-二甲基甲酰胺(2.8L)，哌啶(700g)，搅拌均匀，加入上述滤饼树脂，于20℃搅拌0.5h，过滤，滤饼用N,N-二甲基甲酰胺(700ml×5)洗涤，无液滴滴出，收集滤饼，所得树脂直接用于下一步。
工序5：第三个氨基酸偶联
10L玻璃反应釜中加入N,N-二甲基甲酰胺(3.5L)，搅拌加入TLYS-A(270.59g)，HBTU(240.91g)，DIPEA(82.11g)，于25℃，搅拌0.5h，加入工序2所得树脂，于25℃，搅拌反应12h，过滤，滤饼用N,N-二甲基甲酰胺(700ml×5)洗涤，无液滴滴出，收集滤饼，所得树脂直接用于下一步。
工序6：脱-Fmoc基团
10L玻璃反应釜中加入N,N-二甲基甲酰胺(2.8L)，哌啶(700g)，搅拌均匀，加入上述滤饼树脂，于20℃搅拌0.5h，过滤，滤饼用N,N-二甲基甲酰胺(700ml×5)洗涤，无液滴滴出，收集滤饼。10L不锈钢桶中加入异丙醇(3.5L)，搅拌加入上述滤饼，于25℃，搅拌浸泡0.5h，过滤；10L不锈钢桶中加入二氯甲烷(3.5L)，搅拌加入上述滤饼，于25℃，搅拌浸泡0.5h，过滤至无液滴滴出；10L不锈钢桶中加入异丙醇(3.5L)，搅拌加入上述滤饼，于25℃，搅拌浸泡0.5h，过滤至无液滴滴出，滤饼于20℃鼓风干燥7h。
工序7：切割
50L玻璃反应釜中加入甲基叔丁基醚(21.1L)，搅拌降温至0℃(备用)
10L玻璃反应釜中加入三氟乙酸(3.5kg)，三异丙基硅烷(35g)，纯化水(35g)，控温＜30℃，搅拌下分批加入上述树脂(加入过程放热)，保持温度25℃，搅拌4h，过滤，滤饼用350ml三氟乙酸洗涤，无液滴滴出，收集滤液。将滤液滴加至21.1L甲基叔丁基醚中(50L反应釜)，保温5℃搅拌2h，过滤，滤饼用1000ml×2甲基叔丁基醚浆洗，无液滴滴出，收集滤饼。(产品易吸潮，不宜长时间暴露在空气中)
工序8：转盐
5L玻璃反应器中加入1050ml纯化水，加入工序7所得滤饼，搅拌溶清，静置、分液(去除上层甲基叔丁基醚相)，保留下层水相。水相搅拌下加入630.00g强碱性阴离子交换树脂(201×4型)调节pH＝9.5，保温25℃，搅拌1h。过滤，滤饼用408ml纯化水洗涤，收集滤液，滤液中加入10.5g活性炭，保持温度25℃，搅拌10min，用0.45μm滤膜过滤，收集滤液。滤液中加入冰醋酸调节pH＝2.5，于36℃真空浓缩至无液滴滴出，浓缩物(即为上式I或式TLYS所示三赖氨酸醋酸盐)中加入410ml乙醇，继续减压浓缩至无液滴滴出。浓缩剩余物中加入700ml无水乙醇，保持25℃搅拌打浆2h，过滤，滤饼用105ml×2无水乙醇洗涤，无液滴滴出，滤饼于32℃真空干燥3h，真空度≤-0.095Mpa，样品过45目筛(0.4mm)后，于32℃继续真空干燥3～5h，真空度≤-0.095MPa，得到100g三赖氨酸醋酸盐的晶型A，产品纯度99.52％，醋酸含量12.9％，收率为56.1％。
实施例1制备的三赖氨酸醋酸盐的晶型A如下式TLYS所示，分子式为C18H38N6O4·CH3CO2H。
本实施例制备的三赖氨酸醋酸盐的晶型A粉末X射线衍射图谱如图1所示，峰数据如图2所示。
实施例2
本实施例提供三赖氨酸醋酸盐及其晶型A，其制备方法与实施例1的区别仅在于工序1不同，具体如下：
工序1：首位氨基酸固定化
树脂预处理：将取代度为1.10mmol/g的2-CTC树脂(350.00g)加入到10L不锈钢桶中，继续加入N,N-二甲基甲酰胺(3.5L)，于20℃下浸泡，使树脂充分溶胀，溶胀时间20h，过滤，滤饼用N,N-二甲基甲酰胺(3.5L)洗涤。
10L玻璃反应釜中加入N,N-二甲基甲酰胺(3.5L)，搅拌加入TLYS-A(265.0g)，DIPEA(195.0g)，搅拌溶解，加入上述溶胀后的2-CTC树脂，于25℃，搅拌反应15h。过滤，滤饼用N,N-二甲基甲酰胺(700ml×3)洗涤，收集滤饼，转入10L玻璃反应釜中加入3.5L无水甲醇，于20℃搅拌1h树脂封端。抽滤，滤饼用N,N-二甲基甲酰胺(700ml×5)洗涤，无液滴滴出，收集滤饼，所得树脂直接用于下一步。
本实施例得到99.5g三赖氨酸醋酸盐的晶型A，产品纯度99.42％，醋酸含量12.8％，收率为55.9％。
本实施例制备的三赖氨酸醋酸盐的晶型A粉末X射线衍射图谱与图1近似，其结构如上式 TLYS所示。
实施例3
本实施例提供三赖氨酸醋酸盐及其晶型A，其制备方法与实施例1的区别仅在于工序1不同，具体如下：
工序1：首位氨基酸固定化
树脂预处理：将取代度为1.10mmol/g的2-CTC树脂(350.00g)加入到10L不锈钢桶中，继续加入N,N-二甲基甲酰胺(3.5L)，于30℃下浸泡，使树脂充分溶胀，溶胀时间20h，过滤，滤饼用N,N-二甲基甲酰胺(3.5L)洗涤。
10L玻璃反应釜中加入N,N-二甲基甲酰胺(3.5L)，搅拌加入TLYS-A(280.0g)，DIPEA(205.0g)，搅拌溶解，加入上述溶胀后的2-CTC树脂，于35℃，搅拌反应18h。过滤，滤饼用N,N-二甲基甲酰胺(700ml×3)洗涤，收集滤饼，转入10L玻璃反应釜中加入3.5L无水甲醇，于30℃搅拌1h树脂封端。抽滤，滤饼用N,N-二甲基甲酰胺(700ml×5)洗涤，无液滴滴出，收集滤饼，所得树脂直接用于下一步。
本实施例99.2g三赖氨酸醋酸盐的晶型A，产品纯度99.45％，醋酸含量12.9％，收率为55.7％。
本实施例制备的三赖氨酸醋酸盐的晶型A粉末X射线衍射图谱与图1近似，其结构如上式TLYS所示。
实施例4
本实施例提供四赖氨酸醋酸盐，其制备方法如下：
工序1：首位氨基酸固定化
树脂预处理：将取代度为1.10mmol/g的2-CTC树脂(350.00g)加入到10L不锈钢桶中，继续加入N,N-二甲基甲酰胺(3.5L)，于20℃下浸泡，使树脂充分溶胀，溶胀时间20h，过滤，滤饼用N,N-二甲基甲酰胺(3.5L)洗涤。
10L玻璃反应釜中加入N,N-二甲基甲酰胺(3.5L)，搅拌加入TLYS-A(265.0g)，DIPEA(195.0g)，搅拌溶解，加入上述溶胀后的2-CTC树脂，于25℃，搅拌反应15h。过滤，滤饼用N,N-二甲基甲酰胺(700ml×3)洗涤，收集滤饼，转入10L玻璃反应釜中加入3.5L无水甲醇，于20℃搅拌1h树脂封端。抽滤，滤饼用N,N-二甲基甲酰胺(700ml×5)洗涤，无液滴滴出，收集滤饼，所得树脂直接用于下一步。
工序2：脱-Fmoc基团
10L玻璃反应釜中加入N,N-二甲基甲酰胺(2.8L)，哌啶(600g)，搅拌均匀，加入上述滤饼树脂，于20℃搅拌0.5h，过滤，滤饼用N,N-二甲基甲酰胺(700ml×5)洗涤，无液滴滴出，收集滤饼，所得树脂直接用于下一步。
工序3：第二个氨基酸偶联
10L玻璃反应釜中加入N,N-二甲基甲酰胺(3.5L)，搅拌加入TLYS-A(265.0g)，HBTU(231.0g)，DIPEA(79.0g)，于20℃，搅拌0.5h，加入工序2所得树脂，于20℃，搅拌反应12h，过滤，滤饼用N,N-二甲基甲酰胺(700ml×5)洗涤，无液滴滴出，收集滤饼，所得树脂直接用于下一步。
工序4：脱-Fmoc基团
10L玻璃反应釜中加入N,N-二甲基甲酰胺(2.8L)，哌啶(700g)，搅拌均匀，加入上述滤饼树脂，于20～30℃搅拌0.5h，过滤，滤饼用N,N-二甲基甲酰胺(700ml×5)洗涤，无液滴滴出，收集滤饼，所得树脂直接用于下一步。
工序5：第三个氨基酸偶联
10L玻璃反应釜中加入N,N-二甲基甲酰胺(3.5L)，搅拌加入TLYS-A(265.0g)，HBTU(231.0g)，DIPEA(79.0g)，于20℃，搅拌0.5h，加入工序2所得树脂，于20℃，搅拌反应15h，过滤，滤饼用N,N-二甲基甲酰胺(700ml×5)洗涤，无液滴滴出，收集滤饼，所得树脂直接用于下一步。
工序6：脱-Fmoc基团
10L玻璃反应釜中加入N,N-二甲基甲酰胺(2.8L)，哌啶(700g)，搅拌均匀，加入上述滤饼树脂，于20～30℃搅拌0.5h，过滤，滤饼用N,N-二甲基甲酰胺(700ml×5)洗涤，无液滴滴出，收集滤饼，所得树脂直接用于下一步。
工序7：第四个氨基酸偶联
10L玻璃反应釜中加入N,N-二甲基甲酰胺(3.5L)，搅拌加入TLYS-A(265.0g)，HBTU(231.0g)，DIPEA(79.0g)，于20℃，搅拌0.5h，加入工序2所得树脂，于20℃，搅拌反应15h，过滤，滤饼用N,N-二甲基甲酰胺(700ml×5)洗涤，无液滴滴出，收集滤饼，所得树脂直接用于下一步。
工序8：脱-Fmoc基团
10L玻璃反应釜中加入N,N-二甲基甲酰胺(2.8L)，哌啶(600g)，搅拌均匀，加入上述滤饼树脂，于20℃搅拌0.5h，过滤，滤饼用N,N-二甲基甲酰胺(700ml×5)洗涤，无液滴滴出，收集滤饼。10L不锈钢桶中加入异丙醇(3.5L)，搅拌加入上述滤饼，于20℃，搅拌浸泡0.5h，过滤；10L不锈钢桶中加入二氯甲烷(3.5L)，搅拌加入上述滤饼，于20℃，搅拌浸泡0.5h，过滤至无液滴滴出；10L不锈钢桶中加入异丙醇(3.5L)，搅拌加入上述滤饼，于20℃，搅拌浸泡0.5h，过滤至无液滴滴出，滤饼于20℃鼓风干燥6h。
工序9：切割
50L玻璃反应釜中加入甲基叔丁基醚(21.1L)，搅拌降温至0℃(备用)
10L玻璃反应釜中加入三氟乙酸(2.8kg)，三异丙基硅烷(18g)，纯化水(18g)，控温＜30℃，搅拌下分批加入上述树脂(加入过程放热)，保持温度20℃，搅拌4h，过滤，滤饼用350ml三氟乙酸洗涤，无液滴滴出，收集滤液。将滤液滴加至21.1L甲基叔丁基醚中(50L反应釜)，保温0℃搅拌2h，过滤，滤饼用1000ml×2甲基叔丁基醚浆洗，无液滴滴出，收集滤饼。(产品易吸潮，不宜长时间暴露在空气中，过滤结束后，滤饼应立即放入烧杯中，2～8℃储存)
工序10：转盐
5L玻璃反应器中加入1050ml纯化水，加入工序7所得滤饼，搅拌溶清，静置、分液(去除上层甲基叔丁基醚相)，保留下层水相。水相搅拌下加入630.00g强碱性阴离子交换树脂(201×4型)调节pH＝9.5，保温20℃，搅拌1h。过滤，滤饼用408ml纯化水洗涤，收集滤液，滤液中加入10.5g活性炭，保持温度20℃，搅拌10min，用0.45μm滤膜过滤，收集滤液。滤液中加入冰醋酸调节pH＝3.0，于35℃真空浓缩至无液滴滴出，浓缩物中加入410ml乙醇，继续减压浓缩至无液滴滴出。浓缩剩余物中加入700ml无水乙醇，保持20℃搅拌打浆2h，过滤，滤饼用105ml×2无水乙醇洗涤，无液滴滴出，滤饼于30℃真空干燥3h，真空度≤-0.095Mpa，样品过45目筛(0.4mm)后，于30℃继续真空干燥3h，真空度≤-0.095MPa，得到125g四赖氨酸醋酸盐，产品纯度99.18％。
所得四赖氨酸醋酸盐为白色粉末，在采用内包装药用低密度聚乙烯袋，外套聚酯/铝/聚乙烯药品包装用复合膜袋包装条件下，无明显吸湿现象。
实施例5
本实施例提供五赖氨酸醋酸盐，其制备方法如下：
工序1：首位氨基酸固定化
树脂预处理：将取代度为1.10mmol/g的2-CTC树脂(350.00g)加入到10L不锈钢桶中，继续 加入N,N-二甲基甲酰胺(3.5L)，于20℃下浸泡，使树脂充分溶胀，溶胀时间20h，过滤，滤饼用N,N-二甲基甲酰胺(3.5L)洗涤。
10L玻璃反应釜中加入N,N-二甲基甲酰胺(3.5L)，搅拌加入TLYS-A(265.0g)，DIPEA(195.0g)，搅拌溶解，加入上述溶胀后的2-CTC树脂，于25℃，搅拌反应15h。过滤，滤饼用N,N-二甲基甲酰胺(700ml×3)洗涤，收集滤饼，转入10L玻璃反应釜中加入3.5L无水甲醇，于20℃搅拌1h树脂封端。抽滤，滤饼用N,N-二甲基甲酰胺(700ml×5)洗涤，无液滴滴出，收集滤饼，所得树脂直接用于下一步。
工序2：脱-Fmoc基团
10L玻璃反应釜中加入N,N-二甲基甲酰胺(2.8L)，哌啶(600g)，搅拌均匀，加入上述滤饼树脂，于20℃搅拌0.5h，过滤，滤饼用N,N-二甲基甲酰胺(700ml×5)洗涤，无液滴滴出，收集滤饼，所得树脂直接用于下一步。
工序3：第二个氨基酸偶联
10L玻璃反应釜中加入N,N-二甲基甲酰胺(3.5L)，搅拌加入TLYS-A(265.0g)，HBTU(231.0g)，DIPEA(79.0g)，于20℃，搅拌0.5h，加入工序2所得树脂，于20℃，搅拌反应12h，过滤，滤饼用N,N-二甲基甲酰胺(700ml×5)洗涤，无液滴滴出，收集滤饼，所得树脂直接用于下一步。
工序4：脱-Fmoc基团
10L玻璃反应釜中加入N,N-二甲基甲酰胺(2.8L)，哌啶(700g)，搅拌均匀，加入上述滤饼树脂，于20～30℃搅拌0.5h，过滤，滤饼用N,N-二甲基甲酰胺(700ml×5)洗涤，无液滴滴出，收集滤饼，所得树脂直接用于下一步。
工序5：第三个氨基酸偶联
10L玻璃反应釜中加入N,N-二甲基甲酰胺(3.5L)，搅拌加入TLYS-A(265.0g)，HBTU(231.0g)，DIPEA(79.0g)，于20℃，搅拌0.5h，加入工序2所得树脂，于20℃，搅拌反应15h，过滤，滤饼用N,N-二甲基甲酰胺(700ml×5)洗涤，无液滴滴出，收集滤饼，所得树脂直接用于下一步。
工序6：脱-Fmoc基团
10L玻璃反应釜中加入N,N-二甲基甲酰胺(2.8L)，哌啶(700g)，搅拌均匀，加入上述滤饼树脂，于20～30℃搅拌0.5h，过滤，滤饼用N,N-二甲基甲酰胺(700ml×5)洗涤，无液滴滴出，收集滤饼，所得树脂直接用于下一步。
工序7：第四个氨基酸偶联
10L玻璃反应釜中加入N,N-二甲基甲酰胺(3.5L)，搅拌加入TLYS-A(265.0g)，HBTU(231.0g)，DIPEA(79.0g)，于20℃，搅拌0.5h，加入工序2所得树脂，于20℃，搅拌反应15h，过滤，滤饼用N,N-二甲基甲酰胺(700ml×5)洗涤，无液滴滴出，收集滤饼，所得树脂直接用于下一步。
工序8：脱-Fmoc基团
10L玻璃反应釜中加入N,N-二甲基甲酰胺(2.8L)，哌啶(700g)，搅拌均匀，加入上述滤饼树脂，于20～30℃搅拌0.5h，过滤，滤饼用N,N-二甲基甲酰胺(700ml×5)洗涤，无液滴滴出，收集滤饼，所得树脂直接用于下一步。
工序9：第五个氨基酸偶联
10L玻璃反应釜中加入N,N-二甲基甲酰胺(3.5L)，搅拌加入TLYS-A(265.0g)，HBTU(231.0g)，DIPEA(79.0g)，于20℃，搅拌0.5h，加入工序2所得树脂，于20℃，搅拌反应15h，过滤，滤饼用N,N-二甲基甲酰胺(700ml×5)洗涤，无液滴滴出，收集滤饼，所得树脂直接用于下一步。
工序10：脱-Fmoc基团
10L玻璃反应釜中加入N,N-二甲基甲酰胺(2.8L)，哌啶(600g)，搅拌均匀，加入上述滤饼树 脂，于20℃搅拌0.5h，过滤，滤饼用N,N-二甲基甲酰胺(700ml×5)洗涤，无液滴滴出，收集滤饼。10L不锈钢桶中加入异丙醇(3.5L)，搅拌加入上述滤饼，于20℃，搅拌浸泡0.5h，过滤；10L不锈钢桶中加入二氯甲烷(3.5L)，搅拌加入上述滤饼，于20℃，搅拌浸泡0.5h，过滤至无液滴滴出；10L不锈钢桶中加入异丙醇(3.5L)，搅拌加入上述滤饼，于20℃，搅拌浸泡0.5h，过滤至无液滴滴出，滤饼于20℃鼓风干燥6h。
工序11：切割
50L玻璃反应釜中加入甲基叔丁基醚(21.1L)，搅拌降温至0℃(备用)
10L玻璃反应釜中加入三氟乙酸(2.8kg)，三异丙基硅烷(18g)，纯化水(18g)，控温＜30℃，搅拌下分批加入上述树脂(加入过程放热)，保持温度20℃，搅拌4h，过滤，滤饼用350ml三氟乙酸洗涤，无液滴滴出，收集滤液。将滤液滴加至21.1L甲基叔丁基醚中(50L反应釜)，保温0℃搅拌2h，过滤，滤饼用1000ml×2甲基叔丁基醚浆洗，无液滴滴出，收集滤饼。(产品易吸潮，不宜长时间暴露在空气中，过滤结束后，滤饼应立即放入烧杯中，2～8℃储存)
工序12：转盐
5L玻璃反应器中加入1050ml纯化水，加入工序7所得滤饼，搅拌溶清，静置、分液(去除上层甲基叔丁基醚相)，保留下层水相。水相搅拌下加入630.00g强碱性阴离子交换树脂(201×4型)调节pH＝9.5，保温20℃，搅拌1h。过滤，滤饼用408ml纯化水洗涤，收集滤液，滤液中加入10.5g活性炭，保持温度20℃，搅拌10min，用0.45μm滤膜过滤，收集滤液。滤液中加入冰醋酸调节pH＝3.0，于35℃真空浓缩至无液滴滴出，浓缩物中加入410ml乙醇，继续减压浓缩至无液滴滴出。浓缩剩余物中加入700ml无水乙醇，保持20℃搅拌打浆2h，过滤，滤饼用105ml×2无水乙醇洗涤，无液滴滴出，滤饼于30℃真空干燥3h，真空度≤-0.095Mpa，样品过45目筛(0.4mm)后，于30℃继续真空干燥3h，真空度≤-0.095MPa，得到153g五赖氨酸醋酸盐，产品纯度99.58％。
所得五赖氨酸醋酸盐为白色粉末，在采用内包装药用低密度聚乙烯袋，外套聚酯/铝/聚乙烯药品包装用复合膜袋包装条件下，无明显吸湿现象。
实施例6
本实施例提供四赖氨酸盐酸盐，其制备方法与实施例4的区别仅在于工序10不同，具体如下：工序10：转盐
5L玻璃反应器中加入1050ml纯化水，加入工序7所得滤饼，搅拌溶清，静置、分液(去除上层甲基叔丁基醚相)，保留下层水相。水相搅拌下加入630.00g强碱性阴离子交换树脂(201×4型)调节pH＝9.5，保温20℃，搅拌1h。过滤，滤饼用408ml纯化水洗涤，收集滤液，滤液中加入10.5g活性炭，保持温度20℃，搅拌10min，用0.45μm滤膜过滤，收集滤液。滤液中加入盐酸调节pH＝3.0，于35℃真空浓缩至无液滴滴出，浓缩物中加入410ml乙醇，继续减压浓缩至无液滴滴出。浓缩剩余物中加入700ml无水乙醇，保持20℃搅拌打浆2h，过滤，滤饼用105ml×2无水乙醇洗涤，无液滴滴出，滤饼于30℃真空干燥3h，真空度≤-0.095Mpa，样品过45目筛(0.4mm)后，于30℃继续真空干燥3h，真空度≤-0.095MPa，得到115g四赖氨酸盐酸盐，产品纯度99.25％。
所得四赖氨酸盐酸盐为白色粉末，在采用内包装药用低密度聚乙烯袋，外套聚酯/铝/聚乙烯药品包装用复合膜袋包装条件下，无明显吸湿现象。
实施例7
本实施例提供五赖氨酸盐酸盐，其制备方法与实施例5的区别仅在于工序12不同，具体如下：工序12：转盐
5L玻璃反应器中加入1050ml纯化水，加入工序7所得滤饼，搅拌溶清，静置、分液(去除上 层甲基叔丁基醚相)，保留下层水相。水相搅拌下加入630.00g强碱性阴离子交换树脂(201×4型)调节pH＝9.5，保温20℃，搅拌1h。过滤，滤饼用408ml纯化水洗涤，收集滤液，滤液中加入10.5g活性炭，保持温度20℃，搅拌10min，用0.45μm滤膜过滤，收集滤液。滤液中加入盐酸调节pH＝3.0，于35℃真空浓缩至无液滴滴出，浓缩物中加入410ml乙醇，继续减压浓缩至无液滴滴出。浓缩剩余物中加入700ml无水乙醇，保持20℃搅拌打浆2h，过滤，滤饼用105ml×2无水乙醇洗涤，无液滴滴出，滤饼于30℃真空干燥3h，真空度≤-0.095Mpa，样品过45目筛(0.4mm)后，于30℃继续真空干燥3h，真空度≤-0.095MPa，得到150g五赖氨酸盐酸盐，产品纯度99.49％。
所得五赖氨酸盐酸盐为白色粉末，在采用内包装药用低密度聚乙烯袋，外套聚酯/铝/聚乙烯药品包装用复合膜袋包装条件下，无明显吸湿现象。
实验1
测试条件
粉末X射线衍射测试条件：Bruker D8 Advance X-射线衍射分析仪、扫描步长0.02°，光源：CuKa，管压＝40kV，管流＝40mA。
高分辨测试条件：LCMS-IT/TOF，离子源ESI，扫描范围100～1000m/z。
红外测试条件：傅立叶变换红外光谱仪，采用KBr压片法录制光谱图。
TGA热分析测试条件：TG/DTA6200型热重分析仪，升温速率10℃/min。
DSC热分析测试条件：DSC6220型差示扫描量热仪，升温速率:10℃/min。
实施例1制备的三赖氨酸醋酸盐的晶型A粉末X射线衍射图谱如图1所示，峰数据如图2所示。
实施例1制备的三赖氨酸醋酸盐的晶型A高分辨质谱图谱如图3和图4所示。
实施例1制备的三赖氨酸醋酸盐的晶型A红外图谱如图5所示。
实施例1制备的三赖氨酸醋酸盐的晶型A的TGA热分析图谱如图6所示。
实施例1制备的三赖氨酸醋酸盐的晶型A的DSC热分析图谱如图7所示。
实验2
实施例1-3制备的三赖氨酸醋酸盐样品采用内包装药用低密度聚乙烯袋，外套聚酯/铝/聚乙烯药品包装用复合膜袋，在温度6±2℃条件下放样，放置6个月，分别于0、1、2、3、6、12、18月取样测定。结果如下：

本品在温度6±2℃条件下放置18个月，各项检测指标与0天比较无明显变化，说明本品在6±2℃条件下稳定性良好。
对比例1
三赖氨酸三醋酸盐的制备方法：重复实施例1工序1至工序7，将所得滤饼使用HPLC进行转盐(醋酸)，将得到的溶液浓缩，冻干，得到三赖氨酸三醋酸盐。经检测，其三肽含量69.79％，说明其醋酸含量约30％，为三醋酸盐。
本对比例1通过色谱纯化和转盐制得的三赖氨酸三醋酸盐的性能测试结果如下，其吸湿性强，难以保存。
对比例2
本对比例提供三赖氨酸醋酸盐，其制备方法与实施例1的区别仅在于工序1不同，具体如下：工序1：首位氨基酸固定化
树脂预处理：将取代度为1.10mmol/g的2-CTC树脂(350.00g)加入到10L不锈钢桶中，继续加入N,N-二甲基甲酰胺(3.5L)，于20℃下浸泡，使树脂充分溶胀，溶胀时间20h，过滤，滤饼用N,N-二甲基甲酰胺(3.5L)洗涤。
10L玻璃反应釜中加入N,N-二甲基甲酰胺(3.5L)，搅拌加入TLYS-A(265.0g)，DIPEA(195.0g)，搅拌溶解，加入上述溶胀后的2-CTC树脂，于18℃，搅拌反应15h。过滤，滤饼用N,N-二甲基甲酰胺(700ml×3)洗涤，收集滤饼，转入10L玻璃反应釜中加入3.5L无水甲醇，于18℃搅拌1h树脂封端。抽滤，滤饼用N,N-二甲基甲酰胺(700ml×5)洗涤，无液滴滴出，收集滤饼，所得树脂直接用于下一步。
本对比例得到40.5g三赖氨酸醋酸盐，产品吸湿性较强，纯度97.5％，收率为％，醋酸含量25.8％， 醋酸含量介于二醋酸盐和三醋酸盐之间。
实验证明，本对比例得到三赖氨酸醋酸盐为三赖氨酸二醋酸盐和三醋酸盐的混合物。
对比例3
本对比例提供三赖氨酸醋酸盐，其制备方法与实施例1的区别仅在于工序8不同，具体如下：工序8：转盐
5L玻璃反应器中加入1050ml纯化水，加入工序7所得滤饼，搅拌溶清，静置、分液(去除上层甲基叔丁基醚相)，保留下层水相。水相搅拌下加入630.00g D201大孔强碱性苯乙烯系阴离子交换树脂调节pH＝9.5，保温20℃，搅拌1h。过滤，滤饼用408ml纯化水洗涤，收集滤液，滤液中加入10.5g活性炭，保持温度20℃，搅拌10min，用0.45μm滤膜过滤，收集滤液。滤液中加入冰醋酸调节pH＝3.0，于35℃真空浓缩至无液滴滴出，浓缩物中加入410ml乙醇，继续减压浓缩至无液滴滴出。浓缩剩余物中加入700ml无水乙醇，保持20℃搅拌打浆2h，过滤，滤饼用105ml×2无水乙醇洗涤，无液滴滴出，滤饼于30℃真空干燥3h，真空度≤-0.095Mpa，样品过45目筛(0.4mm)后，于30℃继续真空干燥3h，真空度≤-0.095MPa，得到75g三赖氨酸醋酸盐，产品吸湿性较强，产品纯度98.5％，醋酸含量7.8％，不符合理论量，三氟乙酸含量22.7％。
实验证明，本对比例得到三赖氨酸醋酸盐为三赖氨酸醋酸盐与三赖氨酸三氟乙酸盐的混合物。
虽然，上文中已经用一般性说明及具体实施方案对本发明作了详尽的描述，但在本发明基础上，可以对之作一些修改或改进，这对本领域技术人员而言是显而易见的。因此，在不偏离本发明精神的基础上所做的这些修改或改进，均属于本发明要求保护的范围。
工业实用性
本发明提供一种多赖氨酸盐及其纯化方法。多赖氨酸盐的纯化方法包括：提供待纯化的粗品；所述待纯化的粗品包括多赖氨酸盐的粗品、多赖氨酸三氟乙酸盐、结合了三个或两个醋酸的多赖氨酸盐、结合了三个或两个盐酸的多赖氨酸盐中的至少一种；将待纯化的粗品和溶剂的混合溶液与强碱性阴离子交换树脂混合，分离滤液；向所述滤液中加入酸性溶剂转盐后除去溶剂即制得所述多赖氨酸盐。，具有较好的经济价值和应用前景。

Claims (19)

  1. 下式(I)或式TLYS所示的三赖氨酸醋酸盐，
  2. 下式TLYS所示的三赖氨酸醋酸盐的晶型A，其特征在于，所述晶型A使用Cu-Kα辐射，以衍射角2θ角度表示的X-射线粉末衍射在20.227、23.194、23.686处有特征峰，其中，每个特征峰2θ角的误差范围为±0.2；
  3. 根据权利要求2所述三赖氨酸醋酸盐的晶型A，其特征在于，以衍射角2θ角度表示的X-射线粉末衍射在4.632、19.695、20.227、22.172、23.194、23.686、28.079处有特征峰，其中，每个特征峰2θ角的误差范围为±0.2；
    可选地，以衍射角2θ角度表示的X-射线粉末衍射在4.632、7.951、11.825、13.980、17.667、18.307、19.695、20.227、21.686、22.172、23.194、23.686、28.079处有特征峰，其中，每个特征峰2θ角的误差范围为±0.2；
    可选地，所述三赖氨酸醋酸盐的晶型A使用Cu-Kα辐射，以衍射角2θ角度表示的X-射线粉末衍射在4.632、7.951、9.272、11.825、13.980、16.544、17.343、17.667、18.307、19.695、20.227、21.686、22.172、23.194、23.686、27.327、28.079、29.270、32.239处有特征峰，其中，每个特征峰2θ角的误差范围为±0.2；
    可选地，所述三赖氨酸醋酸盐的晶型A使用Cu-Kα辐射，以衍射角2θ角度表示的X-射线粉 末衍射在4.632、5.478、7.951、9.272、9.742、11.825、13.980、16.192、16.544、17.343、17.667、18.307、18.716、19.695、20.227、21.686、22.172、23.194、23.686、24.672、27.327、28.079、29.270、32.239处有特征峰，其中，每个特征峰2θ角的误差范围为±0.2。
  4. 根据权利要求2或3所述三赖氨酸醋酸盐的晶型A，其特征在于，所述晶型A具有基本如图1所示的粉末X射线衍射图。
  5. 根据权利要求2或3所述三赖氨酸醋酸盐的晶型A，其特征在于，所述晶型A通过热重分析测量，176.10℃热量损失为8.1wt％±2wt％，可选8.1wt％±1wt％。
  6. 根据权利要求2或3所述三赖氨酸醋酸盐的晶型A，其特征在于，所述晶型A差示扫描量热分析图谱的DSC曲线在147.81℃处有吸热峰。
  7. 根据权利要求2或3所述三赖氨酸醋酸盐的晶型A，其特征在于，所述晶型A具有基本如图7所示的DSC图。
  8. 权利要求1所述三赖氨酸醋酸盐的制备方法，其特征在于，包括：将式TLYS-4所示化合物采用强碱性阴离子交换树脂脱去三氟乙酸，然后与醋酸反应，生成TLYS所示化合物；
    反应如下：
    优选地，所述强碱性阴离子交换树脂选自201×4型或201×7型；
    优选地，还包括制备式TLYS-4所示化合物的步骤，反应路线如下：
    优选在首位氨基酸固定化的步骤中，首位氨基酸固定化时的反应温度为25～35℃，更优选为28～30℃；更优选反应时间15～18h；
    优选在首位氨基酸固定化的步骤中，还包括使用无水甲醇于对2-CTC树脂进行封端。
  9. 权利要求2-7任一项所述三赖氨酸醋酸盐的晶型A的制备方法，其特征在于，包括：将权利要求1所述三赖氨酸醋酸盐加入乙醇，分离得到三赖氨酸醋酸盐的晶型A；优选在加入乙醇后保持温度20～30℃搅拌打浆；
    优选所述制备方法包括：将式TLYS-4所示化合物采用强碱性阴离子交换树脂脱去三氟乙酸，使用活性炭脱色、过滤，然后加入醋酸成盐，真空浓缩，浓缩物中加入乙醇继续真空浓缩至无液滴滴出，浓缩剩余物中再加入无水乙醇，20～30℃搅拌打浆，过滤，滤饼采用真空干燥，样品过筛后，继续真空干燥，制备得到三赖氨酸醋酸盐的晶型A；更优选所述强碱性阴离子交换树脂选自201×4型或201×7型；
  10. 一种多赖氨酸盐的纯化方法，其特征在于，其包括：
    提供待纯化的粗品；所述待纯化的粗品包括多赖氨酸盐的粗品、多赖氨酸三氟乙酸盐、结合了三个或两个醋酸的多赖氨酸盐、结合了三个或两个盐酸的多赖氨酸盐中的至少一种；
    将待纯化的粗品和溶剂的混合溶液与强碱性阴离子交换树脂混合，分离滤液；
    向所述滤液中加入酸性溶剂转盐后除去溶剂。
  11. 根据权利要求10所述多赖氨酸盐的纯化方法，其特征在于，多赖氨酸三氟乙酸盐结构如式PLYS-N所示：
    其中，Lys代表赖氨酸；n为≥3的自然数，例如n为3-20，或3-11，或3-5。
  12. 根据权利要求10或11所述多赖氨酸盐的纯化方法，其特征在于，多赖氨酸盐如下式(II)所示，
    其中，Lys代表赖氨酸；A代表酸，包括醋酸、盐酸；n为≥3的自然数，例如n为3-20，或3-11，或3-5。
  13. 根据权利要求10所述多赖氨酸盐的纯化方法，其特征在于，所述待纯化的粗品包括三赖氨酸三醋酸盐、三赖氨酸二醋酸盐、四赖氨酸三醋酸盐、四赖氨酸二醋酸盐、三赖氨酸三盐酸盐、三赖氨酸二盐酸盐、四赖氨酸三盐酸盐、四赖氨酸二盐酸盐。
  14. 根据权利要求10-13任一项所述多赖氨酸盐的纯化方法，其特征在于，所述待纯化的粗品中还含有强酸性杂质；可选地，所述强酸性为三氟乙酸。
  15. 根据权利要求10-14任一项所述多赖氨酸盐的纯化方法，其特征在于，所述强碱性阴离子交换树脂选自201×4型或201×7型。
  16. 一种多赖氨酸盐，其特征于，由权利要求10-15任一项所述方法制得。
  17. 下式(II)所示的多赖氨酸盐，
    Lys代表赖氨酸；A代表酸，包括醋酸、盐酸；n为≥3的自然数，例如n为3-20，或3-11，或3-5。
  18. 根据权利要求17所述的多赖氨酸盐，其特征于，它选自三赖氨酸醋酸盐、四赖氨酸醋酸盐、五赖氨酸醋酸盐、六赖氨酸醋酸盐、七赖氨酸醋酸盐、八赖氨酸醋酸盐、九赖氨酸醋酸盐、十赖氨酸醋酸盐、十一赖氨酸醋酸盐、三赖氨酸盐酸盐、四赖氨酸盐酸盐、五赖氨酸盐酸盐、六赖氨酸盐酸盐、七赖氨酸盐酸盐、八赖氨酸盐酸盐、九赖氨酸盐酸盐、十赖氨酸盐酸盐、十一赖氨酸盐酸盐。
  19. 权利要求1所述三赖氨酸醋酸盐、权利要求2-7任一项所述三赖氨酸醋酸盐晶型A或权利要求16-18所述多赖氨酸盐或其晶型在制备药物中的应用；可选作为水凝胶的交联剂。
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Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102911493A (zh) * 2012-09-28 2013-02-06 山东赛克赛斯药业科技有限公司 可生物降解的医用水凝胶及其制备方法与应用
CN105778124A (zh) * 2012-09-28 2016-07-20 山东赛克赛斯药业科技有限公司 可生物降解的医用水凝胶及其制备方法与应用
CN109077993A (zh) * 2018-08-31 2018-12-25 北京诺康达医药科技股份有限公司 一种泪小管缓释水凝胶植入剂及其制备方法
WO2018233591A1 (zh) * 2017-06-20 2018-12-27 江苏恒瑞医药股份有限公司 一种苯并哌啶类衍生物的盐、其晶型及盐、其晶型的制备方法
CN112028784A (zh) * 2020-09-03 2020-12-04 北京哈三联科技有限责任公司 一种游离l-赖氨酸固体的制备方法
CN113171463A (zh) * 2021-03-31 2021-07-27 北京诺康达医药科技股份有限公司 原位载药水凝胶及其制备方法与应用
WO2021253305A1 (zh) * 2020-06-18 2021-12-23 苏州园方生物科技有限公司 一种dl-赖氨酸的制备方法

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102911493A (zh) * 2012-09-28 2013-02-06 山东赛克赛斯药业科技有限公司 可生物降解的医用水凝胶及其制备方法与应用
CN105778124A (zh) * 2012-09-28 2016-07-20 山东赛克赛斯药业科技有限公司 可生物降解的医用水凝胶及其制备方法与应用
WO2018233591A1 (zh) * 2017-06-20 2018-12-27 江苏恒瑞医药股份有限公司 一种苯并哌啶类衍生物的盐、其晶型及盐、其晶型的制备方法
CN109077993A (zh) * 2018-08-31 2018-12-25 北京诺康达医药科技股份有限公司 一种泪小管缓释水凝胶植入剂及其制备方法
WO2021253305A1 (zh) * 2020-06-18 2021-12-23 苏州园方生物科技有限公司 一种dl-赖氨酸的制备方法
CN112028784A (zh) * 2020-09-03 2020-12-04 北京哈三联科技有限责任公司 一种游离l-赖氨酸固体的制备方法
CN113171463A (zh) * 2021-03-31 2021-07-27 北京诺康达医药科技股份有限公司 原位载药水凝胶及其制备方法与应用

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
LIU SA, HONGXIA DONG, JIANWEI GUO: "Synthesis and Characterization of Poly-L-Lysine Dendrimers", POLYMER MATERIALS SCIENCE & ENGINEERING, vol. 27, no. 5, 15 August 2011 (2011-08-15), pages 20 - 23, XP093108185 *
VERBARO DANIEL J., MATHIEU DANIEL, TOAL SIOBHAN E., SCHWALBE HARALD, SCHWEITZER-STENNER REINHARD: "Ionized trilysine: a model system for understanding the nonrandom structure of poly-L-lysine and lysine-containing motifs in proteins", JOURNAL OF PHYSICAL CHEMISTRY PART B, AMERICAN CHEMICAL SOCIETY, US, vol. 116, no. 28, 19 July 2012 (2012-07-19), US , pages 8084 - 8094, XP093108186, ISSN: 1520-6106, DOI: 10.1021/jp303794s *

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