Ultrahigh molecular weight polyglutamic acid and preparation method thereof

Abstract

The invention provides ultra-high molecular weight polyglutamic acid and a preparation method thereof, gamma-protecting group-L-glutamic acid-N-carboxyl anhydride (glutamate-N-carboxyl anhydride) is used as a raw material, and polyglutamic acid with molecular weight of 1500-4000 kDa is prepared by using a metal hydride to promote an initiator to initiate polymerization. The anionic active ring-opening polymerization process is adopted, the production cost is low, the reaction condition is mild, and the obtained polyglutamic acid is large in molecular weight and can be applied to the fields of drug delivery, tissue engineering materials, soft tissue repair and the like.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedical material preparation, and in particular relates to ultra-high molecular weight polyglutamic acid and a preparation method thereof. Background Art

[0002] Polyglutamic acid (PGA) is a polymer with glutamic acid as a structural unit. Its structure is similar to that of polypeptides in organisms and has good biocompatibility, biodegradability and biosafety. In addition, due to the presence of a large number of free carboxyl groups on the polyglutamic acid molecular chain, on the one hand, active molecules can be modified for drug delivery, and cross-linking can also be performed through polyhydroxy or polyamine cross-linking agents to prepare gels for use in biological tissue engineering. On the other hand, carboxyl groups have the ability to bind water, which makes polyglutamic acid have high water absorption and moisturizing properties, and can be used in cosmetics as a moisturizing ingredient. Due to the many advantages of polyglutamic acid, it has become one of the most actively studied biomedical polymers.

[0003] At present, there are three main methods for synthesizing polyglutamic acid, namely extraction, microbial fermentation and chemical synthesis. The extraction method is to extract polyglutamic acid from natto. Due to the low content of polyglutamic acid in natto, the extraction process is complicated, the production cost is high and it is difficult to produce on a large scale. The microbial fermentation method is currently an industrialized method for preparing polyglutamic acid, which can produce 10-2000kDa polyglutamic acid, but its production process is complicated, the molecular weight is not easy to control and the production capacity is low. The chemical synthesis method using γ-protecting group-L-glutamic acid-N-carboxyl cyclic anhydride (i.e. glutamate NCA, the same below) through ring-opening polymerization is an important synthesis method of kilogram-level polyglutamic acid. The process is simple, the product is easy to purify, and the molecular weight of the obtained polyglutamic acid is controllable and the distribution coefficient is small.

[0004] In the prior art, in chemical synthesis, polyglutamic acid is prepared by using alkaline reagents (such as triethylamine, sodium borohydride, sodium hydride, etc.) as initiators under the protection of inert gas, and the preparation process usually needs to be carried out in a closed glove box. For example, patent document CN102666566A discloses a method in which glutamate monomer is used as raw material, triethylamine, sodium borohydride or a combination thereof is used as initiator to prepare starting polyglutamic acid benzyl ester under the protection of argon, and then hydrolyzed to obtain a product polyglutamic acid having a molecular weight of 12-72kDa, but its product molecular weight is relatively low, and there is still a large gap with the molecular weight of the product obtained by microbial fermentation, and an anhydrous and oxygen-free environment needs to be provided, the process is complex, the reaction conditions are harsh, and the preparation cost is relatively high.

[0005] In the method of preparing polyglutamic acid using ring-opening polymerization, previous work has mainly focused on developing new initiators to prepare high molecular weight polyglutamic acid esters (PBG), and then removing the protective ester group to obtain polyglutamic acid of the corresponding molecular weight. Amine compounds such as sodium hydride, potassium hydride, primary amines, secondary amines, transition metal complexes, hexamethyldisilazane, trimethylsilyl derivatives, Al-Schiff base complexes, rare earth metal complexes, ammonium salts and amine-borane Lewis acid-base pairs have been developed successively. The above initiators are used alone or in combination to greatly improve the molecular weight of polyglutamic acid. Patent US4161948A reported that triethylamine and sodium hydride were used to initiate the polymerization of polyglutamic acid and leucine NCA monomers, and a 25kDa polymer could be prepared. Patent CN101511862A also reported the use of potassium hydride, sodium hydride and cyclohexylamine mixed initiator to prepare amino acid NCA copolymers, and a copolymer with a molecular weight of up to 25kDa could be obtained. Recently, Lv Hua et al. from Peking University published a paper titled "J Yuan, Z Yi, Z Li, Y Wang, & L Hua. (2018). A s-sn Lewis pair-mediated ring-opening polymerization of α-amino acid n-carboxyanhydrides: fast kinetics, high molecular weight, and facile bioconjugation. ACS Macro Letters, 7, 892-897." which reported the use of trimethyltin-thiophenol as an initiator to prepare poly(benzyl glutamate) with a molecular weight of up to 800 kDa. However, the initiator thiophenol is a highly toxic compound, and the poly(glutamic acid) obtained by initiation is not suitable for biomedical applications. In addition, the molecular weight of the product obtained is still somewhat different from that of the product obtained by microbial fermentation.

[0006] In addition, the monomer of γ-protected-L-glutamic acid-N-carboxyl ring anhydride (glutamate NCA) is sensitive to water vapor, which leads to its synthesis being mostly carried out in a glove box or using a vacuum line with experimental equipment. In order to solve this problem, Liu Runhui et al. from East China University of Science and Technology published "Wu Y, Zhang D, Ma P, et al. Lithium hexamethyldisilazide initiated superfast ring opening polymerization of alpha-amino acid N-carboxyanhydrides [J]. Nature Communications, 2018, 9 (1)". They used lithium bis(trimethylsilyl)amide as an initiator to react in an open system to obtain polyglutamic acid with a molecular weight of 280 kDa, which solved the problem of synthesizing polyglutamic acid in an air atmosphere. However, the molecular weight of the product obtained was relatively low, which was still far behind the molecular weight of the product obtained by microbial fermentation. Summary of the invention

[0007] In order to solve the above problems, the present invention provides a method for preparing ultrahigh molecular weight polyglutamic acid by forming a composite initiation system in an air atmosphere by using hydrides corresponding to highly electronegative metals (such as magnesium hydride, calcium hydride, cuprous hydride) and initiators (such as triethylamine, pyridine) to initiate a ring-opening polymerization reaction.

[0008] The first aspect of the present invention provides an ultra-high molecular weight polyglutamic acid, which is obtained by a ring-opening polymerization reaction initiated by a composite initiation system formed by a metal hydride and an initiator and a monomer to obtain the ultra-high molecular weight polyglutamic acid.

[0009] Furthermore, the metal hydride is selected from one or more of magnesium hydride, calcium hydride, and cuprous hydride.

[0010] Furthermore, the initiator is an organic amine compound and / or a heterocyclic compound.

[0011] Preferably, the organic amine compound is selected from one or more of triethylamine, dimethylamine, diethylamine, di-n-propylamine, diisopropylamine, N-ethylmethylamine, di-n-butylamine, diisobutylamine, di-sec-butylamine, di-tert-butylamine, dipentylamine, di-n-octylamine, diallylamine, dicyclohexylamine and diphenylamine.

[0012] Preferably, the heterocyclic compound is selected from one or more of pyridine, aziridine, pyrrole, pyrrolidine, imidazole, indole, piperidine and purine.

[0013] In some embodiments of the present invention, the initiator is triethylamine.

[0014] In some embodiments of the present invention, the initiator is pyridine.

[0015] Furthermore, in the polymerization reaction, the molar ratio of monomer to initiator is (1-50):1; in the composite initiation system, the content of metal hydride is 1-30wt% of the initiator, specifically 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, preferably 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%.

[0016] Furthermore, the monomer is glutamate-N-carboxyl cyclic anhydride.

[0017] Furthermore, the glutamate-N-carboxyl ring anhydride is a γ-alkyl-L-glutamate-N-carboxyl ring anhydride or a γ-aryl-L-glutamate-N-carboxyl ring anhydride, wherein the alkyl group is selected from methyl, ethyl, tert-butyl, allyl or tert-butyl dimethyl, etc.; the aryl group is selected from benzyl, or benzyl containing halogen atoms, phenolic hydroxyl groups, nitro groups, sulfonic acid groups and other substituents, or diphenylmethyl, triphenylmethyl, ethylphenylbenzene, naphthylmethyl, anthracenemethyl, etc., and the above groups can all be used as the carboxyl protecting group at the γ position of glutamate.

[0018] In some embodiments of the present invention, the glutamate-N-carboxyl ring anhydride is γ-benzyl-L-glutamate-N-carboxyl ring anhydride, γ-tert-butyl-L-glutamate-N-carboxyl ring anhydride or γ-ethyl-L-glutamate-N-carboxyl ring anhydride.

[0019] Furthermore, the molecular weight of the ultra-high molecular weight polyglutamic acid is 1500-4000 kDa, and its molecular weight distribution coefficient is 1 <PD<2。

[0020] The second aspect of the present invention provides a method for preparing ultra-high molecular weight polyglutamic acid, which comprises the following steps:

[0021] (1) Synthesis of polyglutamate: glutamate-N-carboxyl cyclic anhydride monomer (glutamate NCA), organic solvent A, metal hydride, and initiator are mixed and reacted to obtain polyglutamate;

[0022] (2) Synthesis of polyglutamic acid: the polyglutamate obtained in step (1), an organic solvent B, and a deprotecting agent are mixed and reacted to obtain ultrahigh molecular weight polyglutamic acid;

[0023] Wherein, the molecular weight of the ultra-high molecular weight polyglutamic acid is 1500-4000 kDa, such as 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000 kDa.

[0024] Further, the glutamate-N-carboxyl ring anhydride monomer is γ-alkyl-L-glutamate-N-carboxyl ring anhydride or γ-aryl-L-glutamate-N-carboxyl ring anhydride;

[0025] Preferably, the alkyl group is selected from methyl, ethyl, tert-butyl, allyl or tert-butyldimethyl; the aryl group is selected from benzyl, benzyl containing halogen atoms, phenolic hydroxyl, nitro, sulfonic acid substituents, diphenylmethyl, triphenylmethyl, ethylphenylbenzene, naphthylmethyl or anthracenemethyl;

[0026] More preferably, the glutamate-N-carboxyl ring anhydride monomer is γ-benzyl-L-glutamate-N-carboxyl ring anhydride, γ-tert-butyl-L-glutamate-N-carboxyl ring anhydride or γ-ethyl-L-glutamate-N-carboxyl ring anhydride.

[0027] The present invention finds that metal hydride is selective. In the process of ring-opening polymerization, metal and monomer anion (i.e., glutamate NCA anion) form MN and MO bond forms of ion pairs. The stronger the electronegativity of the metal in the metal hydride, such as calcium (1.00), magnesium (1.31), and copper (1.90) in the present invention, the weaker the bond energy of the formed ion bond and the more inclined to covalent bond. At this time, the ion pairs formed are loose ion pairs, and the ion pairs have higher stability and good solubility in the system, which can better promote the initiator to carry out the chain transfer process, so that ultra-high molecular weight polyglutamic acid can be prepared. Loose ion pair chain initiation and transfer process are shown in formula (I).

[0028] The weaker the electronegativity of the metal, the easier it is to form an ionic bond. The ion pair formed is a tight ion pair, which has poor solubility in the system. The metal ion and the monomer anion have strong interaction forces and are tightly bound, which is not conducive to the chain transfer process. For example, the use of sodium hydride or potassium hydride and initiator triethylamine alone or in combination reported in the patent US4161948A and CN101511862A mentioned in the background technology has a small molecular weight of the polymer obtained, which is only 25kDa at most. This may be because it uses metal sodium (0.93) and potassium (0.82) with weak electronegativity, forming a tight ion pair, which is not conducive to chain transfer, and thus the prepared polyglutamic acid has a small degree of polymerization and a low molecular weight. Tight ion pair chain initiation and transfer process are shown in formula (II).

[0029] Formula (I):

[0030]

[0031] Formula (II):

[0032]

[0033] Furthermore, in step (1), glutamate-N-carboxyl intracyclic anhydride monomer is first dissolved in an organic solvent A, and then a premix of a metal hydride and an initiator is added, mixed and reacted to obtain polyglutamate;

[0034] In step (2), the polyglutamate obtained in step (1) is first dissolved in an organic solvent B, and then a deprotecting agent is added and mixed to react to obtain ultrahigh molecular weight polyglutamic acid.

[0035] Furthermore, in step (1), the organic solvent A is selected from one or more of dioxane, n-hexane, cyclohexane, dichloromethane, chloroform, tetrahydrofuran, N,N-dimethylformamide, dimethyl sulfoxide, ether, diisopropyl ether, acetonitrile, methanol, ethanol, ethyl acetate, benzene and toluene, preferably dioxane.

[0036] Furthermore, in step (1), the metal hydride is selected from one or more of magnesium hydride, calcium hydride, and cuprous hydride.

[0037] Furthermore, in step (1), the initiator is an organic amine compound and / or a heterocyclic compound.

[0038] Preferably, the organic amine compound is selected from one or more of triethylamine, dimethylamine, diethylamine, di-n-propylamine, diisopropylamine, N-ethylmethylamine, di-n-butylamine, diisobutylamine, di-sec-butylamine, di-tert-butylamine, dipentylamine, di-n-octylamine, diallylamine, dicyclohexylamine and diphenylamine.

[0039] Preferably, the heterocyclic compound is selected from one or more of pyridine, aziridine, pyrrole, pyrrolidine, imidazole, indole, piperidine and purine.

[0040] In some embodiments of the present invention, the initiator is triethylamine.

[0041] In some embodiments of the present invention, the initiator is pyridine.

[0042] Furthermore, in step (1), the molar ratio of the glutamate-N-carboxyl cyclic anhydride monomer to the initiator is (1-50):1, specifically 1:1, 5:1, 10:1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1, preferably (5-20):1.

[0043] Further, in step (1), the content of the metal hydride is 1-30wt% of the initiator, specifically 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, preferably 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%.

[0044] Furthermore, in step (1), the reaction is carried out at room temperature, such as 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30° C.; and the reaction time is 1 to 5 days, such as 1, 2, 3, 4, or 5 days.

[0045] Furthermore, the step (1) further comprises the steps of alcohol precipitation and drying of the polyglutamate.

[0046] Furthermore, the solvent used in the alcohol precipitation is ethanol.

[0047] Furthermore, in step (2), the organic solvent B is selected from one or more of dichloromethane, chloroform, tetrahydrofuran, N,N-dimethylformamide, dimethyl sulfoxide, dioxane, n-hexane, cyclohexane, ether, diisopropyl ether, acetonitrile, methanol, ethanol, ethyl acetate, benzene and toluene, preferably dichloromethane.

[0048] Furthermore, in step (2), the deprotecting agent is a silane protecting agent selected from one or more of trimethylsilyl iodide, trimethylsilane, tert-butyldimethylsilyl chloride, triisopropylsilyl chloride, tert-butyldimethylsilyl trifluorosulfonate, and triisopropylsilyl trifluorosulfonate, preferably trimethylsilyl iodide.

[0049] Furthermore, in step (2), the molar ratio of the polyglutamate to the deprotecting agent is 1:(1-3), specifically 1:1, 1:1.2, 1:1.4, 1:1.6, 1:1.8, 1:2, 1:2.5, 1:3, preferably 1:(1-2.5), more preferably 1:1.6.

[0050] Furthermore, in step (2), the reaction is carried out at room temperature, such as 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30°C; and the reaction time is 12-24h, such as 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24h.

[0051] Furthermore, the step (2) further comprises the steps of hydrolyzing the polyglutamate, stratifying, washing, adjusting the pH of the system, alcohol precipitation, redissolving, dialysis and freeze-drying.

[0052] Furthermore, the solvent used in the hydrolysis is water.

[0053] Furthermore, the detergent used in the washing is dichloromethane.

[0054] Furthermore, the pH value of the adjustment system is 7-9, specifically 7, 7.2, 7.4, 7.6, 7.8, 8, 8.2, 8.4, 8.6, 8.8, and 9.

[0055] Furthermore, the solvent used in the alcohol precipitation is ethanol.

[0056] Furthermore, the solvent used for the re-dissolution is water.

[0057] The technical principle of the present invention is as follows (taking the case where the initiator is triethylamine and the protective agent is trimethyl iodosilane as an example):

[0058] Polyglutamate solution polymerization mechanism:

[0059] Chain Initiation:

[0060]

[0061] Chain transfer and termination:

[0062]

[0063] As an alkaline initiator, triethylamine first captures the proton of the glutamate-N-carboxyl ring anhydride monomer amide to form an active initiating ion pair, and then the ion pair reacts with the solid metal hydride of the system at the interface to form a metal ion-monomer active intermediate ion pair, which continues to attack the NCA monomer to form a dimer. The glutamate NCA anion in the dimer forms a loose ion pair with the metal ion. The presence of the loose ion pair is beneficial to stabilize the glutamate NCA active intermediate and avoid the occurrence of side reactions. On the other hand, the loose ion pair has good solubility in organic solvents, and the metal ion and glutamate NCA anion have weak interaction, which is beneficial for the glutamate NCA anion end to attack new monomers to form monomer active intermediates and promote chain transfer. Therefore, ultra-high molecular weight polyglutamate can be prepared.

[0064] In one embodiment of the present invention, the steps of synthesizing polybenzyl glutamate include: cleaning the Schneck tube with chromic acid and capping it with trimethylsilyl chloride, first adding γ-benzyl-L-glutamic acid-N-carboxyl ring anhydride, then adding dioxane, and then adding triethylamine containing metal hydride, after fully shaking, reacting at room temperature for 3 days, then diluting the obtained viscous solution with dioxane, dripping ethanol precipitation, and drying the precipitate to obtain polybenzyl glutamate.

[0065] In one embodiment of the present invention, the steps of synthesizing polyglutamic acid include: adding the obtained polyglutamic acid benzyl ester to dichloromethane, then adding trimethylsilyl iodide and stirring at room temperature overnight, wherein the molar ratio of trimethylsilyl iodide: polyglutamic acid benzyl ester is 1.6:1, adding water and stirring the next day to hydrolyze the trimethylsilyl polyglutamate, separating the lower layer to remove impurities, washing with dichloromethane for multiple times to remove impurities in the system, using starch potassium iodide solution to test whether the iodine in the system is completely removed, adjusting the pH of the system to 7-9, and then using 5 times the volume of ethanol for precipitation, re-dissolving the obtained precipitate with purified water, removing the salt after dialysis for three days, and then freeze-drying to obtain polyglutamic acid.

[0066] In another embodiment of the present invention, the steps of synthesizing poly(tert-butyl glutamate) include: cleaning the Schneck tube with chromic acid and capping it with trimethylsilyl chloride, first adding γ-tert-butyl-L-glutamic acid-N-carboxyl ring anhydride, then adding dioxane, and then adding pyridine containing a metal hydride, shaking well, reacting at room temperature for 3 days, then diluting the obtained viscous solution with dioxane, dropping ethanol into it for precipitation, and drying the precipitate to obtain poly(tert-butyl glutamate).

[0067] In another embodiment of the present invention, the steps of synthesizing polyglutamic acid include: adding the obtained polyglutamic acid tert-butyl ester to dichloromethane, then adding trimethylsilyl iodide and stirring at room temperature overnight, wherein the molar ratio of trimethylsilyl iodide to polyglutamic acid tert-butyl ester is 2.8:1, adding water and stirring the next day to hydrolyze the trimethylsilyl polyglutamic acid tert-butyl ester, separating the lower layer to remove impurities, washing with dichloromethane for multiple times to remove impurities in the system, using starch potassium iodide solution to test whether the iodine in the system is completely removed, adjusting the pH of the system to 7-9, and then using 5 times the volume of ethanol for precipitation, re-dissolving the obtained precipitate with purified water, removing the salt after dialysis for three days, and then freeze-drying to obtain polyglutamic acid.

[0068] The third aspect of the present invention provides an application of the ultra-high molecular weight polyglutamic acid described in the first aspect or the ultra-high molecular weight polyglutamic acid prepared by the preparation method described in the second aspect in drug delivery, tissue engineering material preparation, and soft tissue repair.

[0069] Compared with the prior art, the present invention has the following outstanding features:

[0070] (1) The present invention selects specific magnesium hydride, calcium hydride, cuprous hydride metal hydride and initiator (such as organic amine or heterocyclic compound) to form a composite initiation system, and uses anionic active ring-opening polymerization technology (Anionic Active Ring-opening Polymerization, AARP) to prepare polyglutamic acid. In the process of ring-opening polymerization, metal and monomer anions form MN and MO bond forms of ion pairs, and the metal ion-monomer active intermediate can be formed with the monomer active intermediate by utilizing the strong electronegativity of the metal in the metal hydride. On the one hand, it is beneficial to stabilize the glutamate NCA active intermediate and avoid the occurrence of side reactions; on the other hand, the loose ion pair has good solubility in organic solvents, and the metal ion and polyglutamate NCA anion force are weak, which is beneficial for the glutamate NCA anion end to attack new monomers to form monomer active intermediates, promote chain transfer, and thus be able to prepare ultra-high molecular weight polyglutamic acid.

[0071] (2) The loose ion pair intermediates formed by the metal hydride-promoting initiation system used in the present invention can exist stably in non-polar solvents. When the monomers in the system are completely reacted, the monomers are continuously added without terminating the chain growth, and the chain growth reaction can be further carried out to expand the molecular weight. The anionic living ring-opening polymerization technology of the present invention can promote chain growth and chain transfer continuously and with high stability, and the molecular weight of the prepared polyglutamic acid can reach 1500-4000 kDa, providing more possibilities for the application scenarios of polyglutamic acid.

[0072] (3) The composite initiation system using metal hydride to promote initiation can be carried out in an open system at room temperature and pressure. Under the synthesis conditions without a glove box, ultra-high molecular weight polyglutamic acid (1500-4000 kDa) polyglutamic acid can be obtained, which is much higher than the molecular weight of polyglutamic acid synthesized by the existing glove box ring-opening polymerization technology (30-280 kDa). The synthesis process of polyglutamic acid of the present invention can be carried out without a glove box, the reaction conditions are mild, and the production cost is greatly reduced.

[0073] (4) The molecular weight of the polyglutamic acid prepared by the present invention is superior to that of the polyglutamic acid produced by the traditional microbial fermentation method, the molecular weight is controllable, and the molecular distribution coefficient is small; and compared with the microbial fermentation method, the process method of the present invention is simple to operate and the product is easy to purify.

[0074] (5) The ultra-high molecular weight polyglutamic acid synthesized by the present invention has a wide range of uses and can be used in the fields of drug delivery, tissue engineering materials, and soft tissue repair. BRIEF DESCRIPTION OF THE DRAWINGS

[0075] Figure 1 Shown is the GPC test molecular weight diagram of poly-L-glutamic acid-γ-benzyl ester prepared in Example 4 of the present invention.

[0076] Figure 2 Shown is the molecular weight graph of the GPC test of poly-L-glutamic acid prepared in Example 4 of the present invention. DETAILED DESCRIPTION

[0077] Unless otherwise defined, all scientific and technical terms used in the present invention have the same meanings as commonly understood by one of ordinary skill in the art to which the present invention relates.

[0078] In the present invention, the term "room temperature" refers to that the temperature of an article is close to or the same as the temperature of a space (e.g., a place in a fume hood in which the article is located). Typically, the room temperature is about 20°C to about 30°C, or about 22°C to 27°C, or about 25°C.

[0079] In the present invention, the term "air atmosphere" refers to the mixed gas that permeates around the earth, mainly including oxygen, carbon dioxide, nitrogen, rare gases and other substances (such as water vapor, etc.).

[0080] In the present invention, the term "PD" refers to the polydispersity index, which is used to describe the molecular weight distribution of a polymer. PD is a quantitative indicator of the molecular weight distribution in a polymer, and is specifically calculated by dividing the weight average molecular weight (Mw) of the polymer by the number average molecular weight (Mn). The smaller the value of PD, the narrower the molecular weight distribution in the polymer, that is, the smaller the difference between the molecular weights in the polymer; conversely, the larger the value of PD, the wider the molecular weight distribution in the polymer, that is, the greater the difference between the molecular weights in the polymer. Generally, the PD value range is 1-10, and the PD of a better polymer material is generally between 1-2.

[0081] The main raw materials used in this article, such as metal hydride, triethylamine, γ-benzyl-L-glutamic acid-N-carboxyl ring anhydride, γ-tert-butyl-L-glutamic acid-N-carboxyl ring anhydride, γ-ethyl-L-glutamic acid-N-carboxyl ring anhydride, and dioxane, are all commercially available products.

[0082]

[00136] Various publications, patents, and published patent specifications are cited herein, the disclosures of which are incorporated by reference in their entireties.

[0083] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0084] Example 1: Preparation of poly-L-glutamic acid 1 by triethylamine promoted by 1% magnesium hydride

[0085] 1. Preparation of Poly-γ-benzyl L-glutamate 1

[0086] 2.0 g of γ-benzyl-L-glutamic acid-N-carboxyl ring anhydride was added to a Schneck tube cleaned with chromic acid and capped with trimethylchlorosilane under air atmosphere, followed by 50 ml of ultra-dry dioxane and a composite initiator system of ultra-dry triethylamine and magnesium hydride (magnesium hydride was added to triethylamine to form a premix), wherein the amount of triethylamine added was 0.1537 g, and the amount of magnesium hydride added was 1% of the mass of triethylamine; the molar ratio of monomer to initiator triethylamine was 5:1, and then the mixture was shaken and reacted at room temperature for three days. After three days, the solution was viscous, diluted with dioxane, and then dropped into anhydrous ethanol for precipitation. After the precipitate was filtered and vacuum dried for 24 hours, poly-L-glutamic acid-γ-benzyl ester 1 was obtained.

[0087] 2. Preparation of Poly-L-Glutamate 1

[0088] The obtained benzyl polyglutamate is added to dichloromethane, and then trimethylsilyl iodide is stirred at room temperature overnight, wherein the molar ratio of benzyl polyglutamate to trimethylsilyl iodide is 1:1.6, and then water is added and stirred the next day to hydrolyze the trimethylsilyl polyglutamate, and then the lower layer is separated to remove impurities, and dichloromethane is used for multiple washing to remove impurities in the system, and then starch potassium iodide solution is used to test whether the iodine in the system is completely removed, the pH of the system is adjusted to 7-9, and then 5 times the volume of ethanol is used for precipitation, the precipitate is re-dissolved with purified water, and the salt is removed after dialysis for three days, and then freeze-dried to obtain poly-L-glutamic acid 1.

[0089] Example 2: Preparation of poly-L-glutamic acid 2 by triethylamine promoted by 5% magnesium hydride

[0090] 1. Preparation of poly-L-glutamic acid-γ-benzyl ester 2

[0091] Add 2.0g of γ-benzyl-L-glutamic acid-N-carboxyl ring anhydride to the Schneck tube cleaned with chromic acid and capped with trimethylchlorosilane under air atmosphere, then add 50ml of ultra-dry dioxane and a composite initiator system of ultra-dry triethylamine and magnesium hydride (magnesium hydride is added to triethylamine to form a premix), wherein the amount of triethylamine added is 0.1537g, the amount of magnesium hydride added is 5% of the mass of triethylamine, and the molar ratio of monomer to initiator triethylamine is 5:1, then shake well and react at room temperature for three days. After three days, the solution becomes viscous, and is diluted with dioxane and then dripped into anhydrous ethanol for precipitation. After the precipitate is filtered and vacuum dried for 24 hours, poly-L-glutamic acid-γ-benzyl ester 2 can be obtained.

[0092] 2. Preparation of Poly-L-Glutamate 2

[0093] The same method as step 2 in Example 1 was used to finally obtain poly-L-glutamic acid 2.

[0094] Example 3: Preparation of poly-L-glutamic acid 3 by triethylamine promoted by 10% magnesium hydride

[0095] 1. Preparation of poly-L-glutamic acid-γ-benzyl ester 3

[0096] 2.0 g of γ-benzyl-L-glutamic acid-N-carboxyl ring anhydride was added to the Schneck tube cleaned with chromic acid and capped with trimethylchlorosilane under air atmosphere, followed by 50 ml of ultra-dry dioxane and a composite initiator system of ultra-dry triethylamine and magnesium hydride (magnesium hydride was added to triethylamine to form a premix), wherein the amount of triethylamine added was 0.1537 g, the amount of magnesium hydride added was 10% of the mass of triethylamine, and the molar ratio of the monomer to the initiator triethylamine was 5:1, and then the mixture was shaken and reacted at room temperature for three days. After three days, the solution was viscous, diluted with dioxane, and then dropped into anhydrous ethanol for precipitation. After the precipitate was filtered and vacuum dried for 24 hours, poly-L-glutamic acid-γ-benzyl ester 3 was obtained.

[0097] 2. Preparation of Poly-L-Glutamate 3

[0098] The same method as step 2 in Example 1 was followed to finally obtain poly-L-glutamic acid 3.

[0099] Example 4: Preparation of Poly-L-Glutamic Acid 4 by Triethylamine Promoted by 20% Magnesium Hydride

[0100] 1. Preparation of poly-γ-benzyl L-glutamate 4

[0101] 2.0 g of γ-benzyl-L-glutamic acid-N-carboxyl ring anhydride was added to the Schneck tube cleaned with chromic acid and capped with trimethylchlorosilane under air atmosphere, followed by 50 ml of ultra-dry dioxane and a composite initiator system of ultra-dry triethylamine and magnesium hydride (magnesium hydride was added to triethylamine to form a premix), wherein the amount of triethylamine added was 0.1537 g, the amount of magnesium hydride added was 20% of the mass of triethylamine, and the molar ratio of the monomer to the initiator triethylamine was 5:1, and then the mixture was shaken and reacted at room temperature for three days. After three days, the solution was viscous, diluted with dioxane, and then dropped into anhydrous ethanol for precipitation. After the precipitate was filtered and vacuum dried for 24 hours, poly-L-glutamic acid-γ-benzyl ester 4 was obtained.

[0102] 2. Preparation of Poly-L-Glutamate 4

[0103] The same method as step 2 in Example 1 was followed to finally obtain poly-L-glutamic acid 4.

[0104] Example 5: Preparation of Poly-L-Glutamic Acid 5 by Triethylamine Promoted by 30% Magnesium Hydride

[0105] 1. Preparation of Poly-γ-benzyl L-glutamate 5

[0106] 2.0 g of γ-benzyl-L-glutamic acid-N-carboxyl ring anhydride was added to the Schneck tube cleaned with chromic acid and capped with trimethylchlorosilane under air atmosphere, followed by 50 ml of ultra-dry dioxane and a composite initiator system of ultra-dry triethylamine and magnesium hydride (magnesium hydride was added to triethylamine to form a premix), wherein the amount of triethylamine added was 0.1537 g, the amount of magnesium hydride added was 30% of the mass of triethylamine, and the molar ratio of the monomer to the initiator triethylamine was 5:1, and then the mixture was shaken and reacted at room temperature for three days. After three days, the solution was viscous, diluted with dioxane, and then dropped into anhydrous ethanol for precipitation. After the precipitate was filtered and vacuum dried for 24 hours, poly-L-glutamic acid-γ-benzyl ester 5 was obtained.

[0107] 2. Preparation of Poly-L-Glutamate 5

[0108] The same method as step 2 in Example 1 was followed to finally obtain poly-L-glutamic acid 5.

[0109] Example 6: Preparation of poly-L-glutamic acid 6 by triethylamine promoted by 2% calcium hydride

[0110] 1. Preparation of poly-γ-benzyl L-glutamate 6

[0111] Add 2.0g of γ-benzyl-L-glutamic acid-N-carboxyl ring anhydride to the Schneck tube cleaned with chromic acid and capped with trimethylchlorosilane under air atmosphere, then add 50ml of ultra-dry dioxane and a composite initiator system of ultra-dry triethylamine and calcium hydride (calcium hydride is added to triethylamine to form a premix), wherein the amount of triethylamine added is 0.1537g, the amount of calcium hydride added is 2% of the mass of triethylamine, and the molar ratio of monomer to initiator triethylamine is 5:1, then shake well and react at room temperature for three days. After three days, the solution becomes viscous, and is diluted with dioxane and then dripped into anhydrous ethanol for precipitation. After the precipitate is filtered and vacuum dried for 24 hours, poly-L-glutamic acid-γ-benzyl ester 6 can be obtained.

[0112] 2. Preparation of Poly-L-Glutamate 6

[0113] The same method as step 2 in Example 1 was followed to finally obtain poly-L-glutamic acid 6.

[0114] Example 7: Preparation of poly-L-glutamic acid 7 by triethylamine promoted by 2% cuprous hydride

[0115] 1. Preparation of poly-γ-benzyl L-glutamate 7

[0116] 2.0 g of γ-benzyl-L-glutamic acid-N-carboxyl ring anhydride was added to the Schneck tube cleaned with chromic acid and capped with trimethylchlorosilane under air atmosphere, followed by 50 ml of ultra-dry dioxane and a composite initiator system of ultra-dry triethylamine and cuprous hydride (copper hydride was added to triethylamine to form a premix), wherein the amount of triethylamine added was 0.1537 g, the amount of cuprous hydride added was 2% of the mass of triethylamine, and the molar ratio of the monomer to the initiator triethylamine was 5:1, and then the mixture was shaken and reacted at room temperature for one day. After one day, the solution was viscous, diluted with dioxane, and then dropped into anhydrous ethanol for precipitation. After the precipitate was filtered and vacuum dried for 24 hours, poly-L-glutamic acid-γ-benzyl ester 7 was obtained.

[0117] 2. Preparation of Poly-L-Glutamate 7

[0118] The same method as step 2 in Example 1 was followed to finally obtain poly-L-glutamic acid 7.

[0119] Example 8: Preparation of Poly-L-Glutamic Acid 8 by Pyridine Promoted by 25% Calcium Hydride

[0120] 1. Preparation of Poly-γ-tert-butyl L-glutamate

[0121] Add 2.0g of γ-tert-butyl-L-glutamic acid-N-carboxyl ring anhydride to the Schneck tube cleaned with chromic acid and capped with trimethylchlorosilane under air atmosphere, then add 50ml of ultra-dry dioxane and a composite initiator system of ultra-dry pyridine and calcium hydride (calcium hydride is added to pyridine to form a premix), wherein the amount of pyridine added is 0.1381g, the amount of calcium hydride added is 25% of the mass of pyridine, and the molar ratio of monomer to initiator pyridine is 5:1, then shake well and react at room temperature for three days. After three days, the solution becomes viscous, and is diluted with dioxane and then dripped into anhydrous ethanol for precipitation. After the precipitate is filtered and vacuum dried for 24 hours, poly-L-glutamic acid-γ-tert-butyl ester can be obtained.

[0122] 2. Preparation of Poly-L-Glutamate 8

[0123] The obtained polyglutamate tert-butyl ester was added to dichloromethane, and then trimethylsilyl iodide was stirred at room temperature overnight, wherein the molar ratio of polyglutamate tert-butyl ester to trimethylsilyl iodide was 1:2.8. Then water was added and stirred the next day to hydrolyze the trimethylsilyl polyglutamate. Then the lower layer was separated to remove impurities. Dichloromethane was used for washing to remove impurities in the system several times. Then starch potassium iodide solution was used to test whether the iodine in the system was completely removed. The pH of the system was adjusted to 7-9. Then 5 volumes of ethanol were used for precipitation. The precipitate was re-dissolved with purified water, dialyzed for three days to remove salt, and then freeze-dried to obtain poly-L-glutamic acid 8.

[0124] Example 9: Preparation of poly-L-glutamic acid 9 by triethylamine promoted by 2% calcium hydride, secondary addition of monomer

[0125] 1. Preparation of poly-γ-benzyl L-glutamate 8

[0126] 2.0 g of γ-benzyl-L-glutamic acid-N-carboxyl ring anhydride was added to the Schneck tube cleaned with chromic acid and capped with trimethylchlorosilane under air atmosphere, followed by 50 ml of ultra-dry dioxane and a composite initiator system of ultra-dry triethylamine and calcium hydride (calcium hydride was added to triethylamine to form a premix), wherein the amount of triethylamine added was 0.1537 g, the amount of calcium hydride added was 2% of the mass of triethylamine, and the molar ratio of the monomer to the initiator triethylamine was 5:1, and then the mixture was shaken and reacted at room temperature for three days. After three days, the solution became viscous, and the reaction was not terminated at this time. 2.0 g of γ-benzyl-L-glutamic acid-N-carboxyl ring anhydride monomer was dissolved in ultra-dry dioxane and then added to the reaction system, and then reacted for another two days. At this time, the obtained product was diluted with dioxane and then dropped into anhydrous ethanol for precipitation. The precipitate was filtered and vacuum dried for 24 hours to obtain poly-L-glutamic acid-γ-benzyl ester 8.

[0127] 2. Preparation of Poly-L-Glutamate 9

[0128] The same method as step 2 in Example 1 was followed to finally obtain poly-L-glutamic acid 9.

[0129] Example 10: Preparation of Poly-L-Glutamic Acid 10 by Triethylamine Promoted by 10% Magnesium Hydride

[0130] 1. Preparation of Poly-γ-ethyl L-glutamate

[0131] Add 2.0g of γ-ethyl-L-glutamic acid-N-carboxyl ring anhydride to the Schneck tube cleaned with chromic acid and capped with trimethylchlorosilane under air atmosphere, then add 50ml of ultra-dry dioxane and a composite initiator system of ultra-dry triethylamine and magnesium hydride (magnesium hydride is added to triethylamine to form a premix), wherein the amount of triethylamine added is 0.2008g, and the amount of magnesium hydride added is 10% of the mass of triethylamine; the molar ratio of monomer to initiator triethylamine is 20:1, then shake well and react at room temperature for three days. After three days, the solution becomes viscous, and is diluted with dioxane and then dripped into anhydrous ethanol for precipitation. After the precipitate is filtered and vacuum dried for 24 hours, poly-L-glutamic acid-γ-ethyl ester can be obtained.

[0132] 2. Preparation of Poly-L-Glutamate 10

[0133] The obtained polyethyl glutamate is added to dichloromethane, and then trimethylsilyl iodide is stirred at room temperature overnight, wherein the molar ratio of polyethyl glutamate to trimethylsilyl iodide is 1:1.6, and then water is added and stirred the next day to hydrolyze the trimethylsilyl polyglutamate, and then the lower layer is separated to remove impurities, and dichloromethane is used for multiple washings to remove impurities in the system, and then starch potassium iodide solution is used to test whether the iodine in the system is completely removed, the pH of the system is adjusted to 7-9, and then 5 times the volume of ethanol is used for precipitation, the precipitate is re-dissolved with purified water, and the salt is removed after dialysis for three days, and then freeze-dried to obtain poly-L-glutamic acid 10.

[0134] Comparative Example 1: Preparation of Poly-L-Glutamic Acid 11 by Triethylamine Promoted by 35% Magnesium Hydride

[0135] 1. Preparation of poly-γ-benzyl-L-glutamate 9

[0136] 2.0 g of γ-benzyl-L-glutamic acid-N-carboxyl ring anhydride was added to the Schneck tube cleaned with chromic acid and capped with trimethylchlorosilane under air atmosphere, followed by 50 ml of ultra-dry dioxane and a composite initiator system of ultra-dry triethylamine and magnesium hydride (magnesium hydride was added to triethylamine to form a premix), wherein the amount of triethylamine added was 0.1537 g, the amount of magnesium hydride added was 35% of the mass of triethylamine, and the molar ratio of the monomer to the initiator triethylamine was 5:1, and then the mixture was shaken and reacted at room temperature for three days. After three days, the solution was viscous, diluted with dioxane, and then dropped into anhydrous ethanol for precipitation. After the precipitate was filtered and vacuum dried for 24 hours, poly-L-glutamic acid-γ-benzyl ester 9 was obtained.

[0137] 2. Preparation of Poly-L-Glutamic Acid 11

[0138] The same method as step 2 in Example 1 was followed to finally obtain poly-L-glutamic acid 11.

[0139] Comparative Example 2: Preparation of Poly-L-Glutamic Acid 12 Catalyzed by Triethylamine

[0140] 1. Preparation of Poly-γ-benzyl L-glutamate 10

[0141] In the Schneck tube cleaned with chromic acid and capped with trimethylchlorosilane, 2.0 g of γ-benzyl-L-glutamic acid-N-carboxyl cyclic anhydride was added under air atmosphere, followed by 50 ml of ultra-dry dioxane and 0.1537 g of ultra-dry triethylamine, with a molar ratio of monomer to initiator triethylamine of 5:1, and then shaken and reacted at room temperature for three days. After three days, the solution became viscous, diluted with dioxane, and then dropped into anhydrous ethanol for precipitation. After the precipitate was filtered and vacuum dried for 24 hours, poly-L-glutamic acid-γ-benzyl ester 10 was obtained.

[0142] 2. Preparation of Poly-L-Glutamate 12

[0143] The same method as step 2 in Example 1 was followed to finally obtain poly-L-glutamic acid 12.

[0144] Comparative Example 3: Preparation of Poly-L-Glutamic Acid 13 Catalyzed by Magnesium Hydride

[0145] 1. Preparation of poly-L-glutamic acid-γ-benzyl ester 11

[0146] In an air atmosphere, add 2.0 g of γ-benzyl-L-glutamic acid-N-carboxyl cyclic anhydride to a Schneck tube cleaned with chromic acid and capped with trimethylsilyl chloride, followed by 50 ml of ultra-dry dioxane and 0.0399 g of magnesium hydride, with a molar ratio of monomer to magnesium hydride of 5:1, and then shake well to react at room temperature for three days. After three days, the solution becomes viscous, and is diluted with dioxane and then dripped into anhydrous ethanol for precipitation. After the precipitate is filtered and vacuum dried for 24 hours, poly-L-glutamic acid-γ-benzyl ester 11 can be obtained.

[0147] 2. Preparation of Poly-L-Glutamate 13

[0148] The same method as step 2 in Example 1 was followed to finally obtain poly-L-glutamic acid 13.

[0149] Comparative Example 4: Preparation of poly-L-glutamic acid 14 catalyzed by triethylamine containing (2%) potassium hydride

[0150] 1. Preparation of Poly-γ-benzyl L-glutamate 12

[0151] 2.0 g of γ-benzyl-L-glutamic acid-N-carboxyl ring anhydride was added to the Schneck tube cleaned with chromic acid and capped with trimethylchlorosilane under air atmosphere, followed by 50 ml of ultra-dry dioxane and a composite initiator system of ultra-dry triethylamine and potassium hydride (potassium hydride was added to triethylamine to form a premix), wherein the amount of triethylamine added was 0.1537 g, the amount of potassium hydride added was 2% of the mass of triethylamine, and the molar ratio of the monomer to the initiator triethylamine was 5:1, and then the mixture was shaken and reacted at room temperature for three days. After three days, the solution was viscous, diluted with dioxane, and then dropped into anhydrous ethanol for precipitation. After the precipitate was filtered and vacuum dried for 24 hours, poly-L-glutamic acid-γ-benzyl ester 12 was obtained.

[0152] 2. Preparation of Poly-L-Glutamate 14

[0153] The same method as step 2 in Example 1 was followed to finally obtain poly-L-glutamic acid 14.

[0154] Comparative Example 5: Preparation of Poly-L-Glutamic Acid 15 Catalyzed by Triethylamine Containing (2%) Sodium Hydride

[0155] 1. Preparation of poly-L-glutamic acid-γ-benzyl ester 13

[0156] 2.0 g of γ-benzyl-L-glutamic acid-N-carboxyl ring anhydride was added to the Schneck tube cleaned with chromic acid and capped with trimethylchlorosilane under air atmosphere, followed by 50 ml of ultra-dry dioxane and a composite initiator system of ultra-dry triethylamine and sodium hydride (sodium hydride was added to triethylamine to form a premix), wherein the amount of triethylamine added was 0.1537 g, the amount of sodium hydride added was 2% of the mass of triethylamine, and the molar ratio of monomer to initiator was 5:1, and then the mixture was shaken and reacted at room temperature for three days. After three days, the solution was viscous, diluted with dioxane, and then dropped into anhydrous ethanol for precipitation. After the precipitate was filtered and vacuum dried for 24 hours, poly-L-glutamic acid-γ-benzyl ester 13 was obtained.

[0157] 2. Preparation of Poly-L-Glutamate 15

[0158] The same method as step 2 in Example 1 was followed to finally obtain poly-L-glutamic acid 15.

[0159] Molecular weight performance test

[0160] The polyglutamate obtained in the above examples and comparative examples used 0.1 M LiBr in DMF solution as the mobile phase, the test temperature was 45° C., and polystyrene was used as the internal standard. The molecular weight of the polyglutamate was tested by GPC / SEC.

[0161] The obtained poly-L-glutamic acid 1-15 was continuously tested for molecular weight using 0.1 M sodium hydrogen phosphate / sodium dihydrogen phosphate buffer as mobile phase at a test temperature of 45° C. and a GPC / SEC triple detector.

[0162] The test results are shown in Table 1.

[0163] Table 1: Molecular weight test of polyglutamate and poly-L-glutamic acid

[0164]

[0165]

[0166] Based on the above experimental data, it can be seen from the comparison of Examples 1-5 and Comparative Example 1 that the molecular weight of the polymer increases with the increase in the mass fraction of metal magnesium hydride, and the increase is obvious before 20%, and it is basically unchanged between 20-30%. This is because when the mass fraction is below 30%, magnesium hydride can react with the activity of triethylamine to form magnesium ions and NCA active intermediate ion pairs, and a good ion balance is achieved. With the increase in the number of ion pairs, it is beneficial to accelerate the chain transfer process and reduce side reactions. Therefore, the molecular weight of the prepared polyglutamic acid is relatively large, and it has reached the plateau period of equilibrium between 20%-30%, and the increase in the number of ion pairs can no longer dominate the reaction. When the ratio exceeds 30%, the introduction of excessive magnesium ions into the system will destroy the original ion balance. The destruction of the ion balance will cause free metal ions to attack the NCA monomer, forming too many active ends, and there are more side reactions, resulting in a decrease in the molecular weight of the obtained product.

[0167] It can be seen from Examples 6-7 and Comparative Examples 4-5 that under the same metal hydride content (2%), the molecular weight of the polyglutamic acid product prepared in the Examples is much higher than the molecular weight of the polyglutamic acid obtained in the Comparative Examples. This indicates that the reaction system is selective for metal hydrides, and by selecting hydrides of metal ions with strong electronegativity, such as calcium and copper, these metal ions can form loose ion pairs with the NCA active intermediates to promote chain transfer, thereby obtaining high molecular weight polyglutamic acid; while hydrides of metal ions with weak electronegativity, such as sodium and potassium, will form tight ion pairs with the NCA active intermediates, which is not conducive to the chain growth process, and the obtained polyglutamic acid has a low molecular weight.

[0168] It can be seen from Example 8 that by screening specific metal hydrides, a composite initiation system can be formed with different organic base initiators such as a heterocyclic compound pyridine, and glutamate NCA with different carboxyl protecting agents, such as γ-tert-butyl-L-glutamic acid-N-carboxyl ring anhydride, can be used as a monomer to obtain polyglutamic acid with a higher molecular weight (greater than 2000 KDa).

[0169] It can be seen from Example 9 that the use of metal hydride to promote triethylamine-initiated polymerization forms a stable loose ion pair in a non-polar organic solvent. Without terminating the system, continuing to add monomers can further extend the chain and expand the molecular weight.

[0170] It can be seen from Example 2 and Comparative Examples 2 and 3 that the comparative examples with only metal hydride and only triethylamine cannot produce high molecular weight polyglutamic acid. This is because magnesium hydride is in a solid state in the reaction system, and without the effect of triethylamine, it is difficult to form an active initiating ion pair, and the extent of chain transfer and chain growth is limited, so the molecular weight of the prepared polyglutamic acid is low. In Comparative Example 3, only triethylamine is present without calcium hydride, so loose active intermediate ion pairs cannot be formed, and higher polyglutamic acid cannot be produced.

[0171] Therefore, the preparation of ultra-high polyglutamic acid is based on the use of organic amines as initiators and the use of hydrides corresponding to highly electronegative metal ions as catalysts to form a composite initiation system, which can prepare ultra-high molecular weight polyglutamic acid while ensuring ion balance.

[0172] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein by equivalents. However, these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. An ultra-high molecular weight polyglutamic acid, characterized in that: The composite initiator system formed by metal hydride and initiator initiates a ring-opening polymerization reaction with monomers to obtain ultra-high molecular weight polyglutamic acid; The metal hydride is selected from one or more of magnesium hydride, calcium hydride and cuprous hydride; The initiator is an organic amine compound and / or a heterocyclic compound.

2. The ultra-high molecular weight polyglutamic acid according to claim 1, characterized in that The organic amine compound is selected from one or more of triethylamine, dimethylamine, diethylamine, di-n-propylamine, diisopropylamine, N-ethylmethylamine, di-n-butylamine, diisobutylamine, di-sec-butylamine, di-tert-butylamine, dipentylamine, di-n-octylamine, diallylamine, dicyclohexylamine and diphenylamine; Preferably, the heterocyclic compound is selected from one or more of pyridine, aziridine, pyrrole, pyrrolidine, imidazole, indole, piperidine and purine; More preferably, the initiator is triethylamine or pyridine; Preferably, the monomer is glutamate-N-carboxyl cyclic anhydride; Preferably, in the polymerization reaction, the molar ratio of the monomer to the initiator is (1-50):1; And / or, in the composite initiation system, the content of the metal hydride is 1-30wt% of the initiator.

3. A method for preparing ultra-high molecular weight polyglutamic acid, characterized in that: The steps include: (1) Synthesis of polyglutamate: glutamate-N-carboxyl ring anhydride monomer, organic solvent A, metal hydride, and initiator are mixed and reacted to obtain polyglutamate; (2) Synthesis of polyglutamic acid: the polyglutamate obtained in step (1), an organic solvent B, and a deprotecting agent are mixed and reacted to obtain ultrahigh molecular weight polyglutamic acid; The metal hydride is selected from one or more of magnesium hydride, calcium hydride and cuprous hydride.

4. The preparation method according to claim 3, characterized in that: In step (1), the content of the metal hydride is 1-30wt% of the initiator.

5. The preparation method according to claim 3, characterized in that: In step (1), the glutamate-N-carboxyl ring anhydride monomer is γ-alkyl-L-glutamate-N-carboxyl ring anhydride or γ-aryl-L-glutamate-N-carboxyl ring anhydride; Preferably, the alkyl group is selected from methyl, ethyl, tert-butyl, allyl or tert-butyldimethyl; the aryl group is selected from benzyl, benzyl containing halogen atoms, phenolic hydroxyl, nitro, sulfonic acid substituents, diphenylmethyl, triphenylmethyl, ethylphenylbenzene, naphthylmethyl or anthracenemethyl; More preferably, the glutamate-N-carboxyl ring anhydride monomer is γ-benzyl-L-glutamate-N-carboxyl ring anhydride, γ-tert-butyl-L-glutamate-N-carboxyl ring anhydride or γ-ethyl-L-glutamate-N-carboxyl ring anhydride.

6. The preparation method according to claim 3, characterized in that: In step (1), the initiator is an organic amine compound and / or a heterocyclic compound; Preferably, the organic amine compound is selected from one or more of triethylamine, dimethylamine, diethylamine, di-n-propylamine, diisopropylamine, N-ethylmethylamine, di-n-butylamine, diisobutylamine, di-sec-butylamine, di-tert-butylamine, dipentylamine, di-n-octylamine, diallylamine, dicyclohexylamine, and diphenylamine; Preferably, the heterocyclic compound is selected from one or more of pyridine, aziridine, pyrrole, pyrrolidine, imidazole, indole, piperidine and purine; More preferably, the initiator is triethylamine or pyridine.

7. The preparation method according to claim 3, characterized in that: In step (1), the molar ratio of the glutamate-N-carboxyl intracyclic anhydride monomer to the initiator is (1-50):1, preferably (5-20):1; And / or, in step (1), the reaction is carried out at room temperature for 1-5 days.

8. The preparation method according to claim 3, characterized in that: In step (2), the molar ratio of the polyglutamate to the deprotecting agent is 1:(1-3).

9. The preparation method according to claim 3, characterized in that: The organic solvent A is selected from one or more of dioxane, n-hexane, cyclohexane, dichloromethane, chloroform, tetrahydrofuran, N,N-dimethylformamide, dimethyl sulfoxide, ether, diisopropyl ether, acetonitrile, methanol, ethanol, ethyl acetate, benzene, and toluene, preferably dioxane; And / or, the deprotecting agent is a silane protecting agent selected from one or more of trimethylsilyl iodide, trimethylsilane, tert-butyldimethylsilyl chloride, triisopropylsilyl chloride, tert-butyldimethylsilyl trifluorosulfonate, triisopropylsilyl trifluorosulfonate, preferably trimethylsilyl iodide; And / or, the organic solvent B is selected from one or more of dichloromethane, chloroform, tetrahydrofuran, N,N-dimethylformamide, dimethyl sulfoxide, dioxane, n-hexane, cyclohexane, ether, diisopropyl ether, acetonitrile, methanol, ethanol, ethyl acetate, benzene and toluene, preferably dichloromethane.

10. Use of the ultra-high molecular weight polyglutamic acid according to any one of claims 1 to 2 or the ultra-high molecular weight polyglutamic acid prepared by the preparation method according to any one of claims 3 to 9 in drug delivery, preparation of tissue engineering materials, and soft tissue repair.

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