WO2021089056A1

WO2021089056A1 - SYNTHESIS OF β,γ-AMINO ACID N-CARBOXYL THIOCARBONYL ANHYDRIDE MONOMER, POLYMERIZATION REACTION, PREPARATION OF POLYMER, AND APPLICATION THEREOF

Info

Publication number: WO2021089056A1
Application number: PCT/CN2020/127637
Authority: WO
Inventors: 刘润辉; 周敏
Original assignee: 华东理工大学
Priority date: 2019-11-08
Filing date: 2020-11-09
Publication date: 2021-05-14
Also published as: CN112778236A

Abstract

Synthesis of a β,γ-amino acid N-carboxyl thiocarbonyl anhydride monomer, a polymerization reaction, a polymer, a polypeptide, preparation of a polymer and a polypeptide derivative, and an application thereof. Preparation conditions for conventional β- and γ-amino acid polymers are improved, the polymers are not sensitive to moisture, exposure aggregation can be performed, and synthesis of an α-amino acid, β-amino acid, and γ-amino acid blended or block polymer is implemented; the prepared amino acid polymer can be used in fields of antibacterial, antifungal, antiviral, anti-mite, anti-tumor, cell adhesion, tissue engineering, drug modification, protein modification, protein protectant, drug synergistic, drug delivery, gene delivery, self-assembly materials, and the like.

Description

Synthesis, polymerization, polymer preparation and application of β, γ-amino acid N-carboxythiocarbonyl ring anhydride monomer

Technical field

The invention belongs to the technical field of amino acid polymers, and particularly relates to β-, γ-amino acid N-carboxythiocarbonyl ring intracyclic anhydride (β-NTA, γ-NTA) monomers, derivative polymers thereof, and preparation methods and applications thereof. More specifically, it relates to β-NTA monomers, γ-NTA monomers, amino acid polymers prepared by polymerization initiated by amine or organic base initiators, and various biological functional applications.

Background technique

β- and γ-amino acid polymers have a secondary structure and biocompatibility similar to those of natural proteins, peptides and α-amino acid polymers, as well as excellent resistance to protease degradation, so they are widely used in the field of biomedical materials. The application prospects of protein simulation, antibacterial materials, drugs and gene delivery, stimulus response peptides, tissue engineering and other fields. At present, the main method for synthesizing β-amino acid polymer is β-lactam strong base ring-opening method, mainly using acid chloride (t-BuBzCl) as initiator, LiHMDS as co-initiator, ring-opening polymerization under the protection of nitrogen to prepare β- Amino acid polymer (also called nylon-3 polymer). The conditions for the synthesis of γ-amino acid polymers are more stringent. The main method is to polymerize γ-lactam in a strictly dry solvent under strong alkali, high temperature and under the protection of nitrogen. However, this method still has the following shortcomings that need to be improved: 1. For some monomers containing ester groups and other functional groups, they cannot be polymerized normally under the t-BuBzCl/LiHMDS system; 2. The conditions are harsh and require extremely strict anhydrous Under oxygen conditions, the reaction cannot be carried out under open (containing air and water) conditions, and it is difficult to realize the industrialized mass production of amino acid polymers.

At the same time, amino acid sequence peptides and copolymers such as α/β, α/γ, β/γ, α/β/γ play an extremely important role in the field of mimic proteins, peptides, and protein-protein interactions (PPIs). Role, but because of its different skeleton structure makes the preparation more difficult, only α-amino acid and β- or γ-amino acid can be prepared by solid-phase method through condensation reaction. The cycle is long and the cost is high, so α-, β -, The development of methods for preparing copolymers of γ-amino acids is extremely important. At present, the common method for preparing α-amino acid polymers is to initiate the polymerization of α-NCA monomers by primary amines, but primary amines cannot undergo β-lactam ring-opening polymerization; as mentioned above, t-BuBzCl is required to prepare β-amino acid polymers. /LiHMDS initiates β-lactam polymerization, and the preparation of γ-amino acid polymer requires strong alkali and high temperature conditions to initiate γ-lactam polymerization. This polymerization condition does not match the ring-opening copolymerization of α-NCA, and α-, β cannot be realized. -, Preparation of binary or multi-component copolymers of γ-amino acids. Therefore, the current polymerization method cannot be used to prepare α-, β-, γ-amino acid copolymers.

Therefore, there is a need to develop more gentle methods for preparing β-, γ-amino acid polymers, and methods capable of preparing α-, β-, γ-amino acid sequence peptides and binary and multi-component copolymers.

Summary of the invention

In order to overcome the shortcomings of the prior art, the inventors designed a class of β-NTA monomers with novel structure through long-term, extensive and in-depth research, and at the same time developed a preparation method for β-NTA monomers, and used initiators to open the ring. Polymerizing β-NTA monomers to obtain a series of β-amino acid polymers with novel structures; the present invention also prepares α/β-amino acid copolymers by introducing α-NCA monomers and α-NTA; Β-amino acid polymers and α/β-amino acid copolymers for various applications (such as antibacterial, anti-tumor, tissue engineering scaffolds and self-assembly); the method of the present invention avoids the harsh conditions of β-lactam ring-opening polymerization, It is not sensitive to moisture and can be operated without the glove box, and can be successfully operated in an open container without any protection. On this basis, the inventor completed the present invention.

The first aspect of the present invention provides a β- or γ-amino acid N-carboxythiocarbonyl intracyclic anhydride (β-NTA, γ-NTA) monomer, which has a structure as shown in formula (I):

among them,

s is 0 or 1;

R ₁ , R ₂ , R ₃ , R _4, R ₁₁ and R ₂₁ are each independently selected from the following group of substituted or unsubstituted groups: hydrogen, C ₁ -C ₆ alkyl, C ₁ -C ₆ haloalkyl, C ₁ -C ₆ alkylhydroxyl group, C ₁ -C ₆ alkoxy group, C ₁ -C ₆ alkylsulfonyl group, C ₂ -C ₆ alkenyl group, C ₂ -C ₆ alkynyl group, C ₃ -C ₁₂ ring Alkyl, C ₆ -C ₁₂ aryl, 5-12 membered heteroaryl, 5-12 membered heterocyclic group, C ₁ -C ₆ alkyl-C ₆ -C ₁₂ aryl, amino,

C ₁ -C ₆ alkylguanidino group, C ₁ -C ₆ alkyl ester group, thio C ₁ -C ₆ alkyl ester group; and when s is 0, R ₁ , R ₂ , R ₃ and R ₄ Can not be hydrogen at the same time;

Or R ₁ and R ₂ and the carbon atoms to which they are connected together form a substituted or unsubstituted C ₃ -C ₁₂ cycloalkyl group, a substituted or unsubstituted C ₄ -C ₁₂ cycloalkenyl group or a 5-12 membered heterocyclic group ；

Or R ₃ and R ₄ and the carbon atoms to which they are connected together form a substituted or unsubstituted C ₃ -C ₁₂ cycloalkyl group, a substituted or unsubstituted C ₄ -C ₁₂ cycloalkenyl group or a substituted or unsubstituted 5- 12-membered heterocyclic group;

Or R ₁ and R ₃ and the carbon atom to which they are connected together form a substituted or unsubstituted C ₆ -C ₁₂ aryl group, a substituted or unsubstituted C ₃ -C ₁₂ cycloalkyl group, a substituted or unsubstituted C _4- C ₁₂ cycloalkenyl or 5-12 membered heterocyclic group;

Or when s is 1, R ₁₁ and R ₂₁ and the carbon atom to which they are connected together form a substituted or unsubstituted C ₃ -C ₁₂ cycloalkyl group, a substituted or unsubstituted C ₄ -C ₁₂ cycloalkenyl group or 5 -12 membered heterocyclic group;

Or when s is 1, R ₃ and R ₁₁ and the carbon atom to which they are connected together form a substituted or unsubstituted C ₆ -C ₁₂ aryl group, a substituted or unsubstituted C ₃ -C ₁₂ cycloalkyl group, a substituted or Unsubstituted C ₄ -C ₁₂ cycloalkenyl or substituted or unsubstituted 5-12 membered heterocyclic group;

P ₁ is a protecting group, selected from the following group: tert-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz), fluorenylmethyloxycarbonyl (Fmoc), phthaloyl (Pht), acetyl (Ac), three Fluoroacetyl (Tfa), benzyl (Bn), triphenylmethyl (Tr), P _{2 is} selected from the following group: hydrogen, substituted or unsubstituted C ₁ -C ₆ alkyl, substituted or unsubstituted C ₆ -C ₁₂ aryl, substituted or unsubstituted 5-12 membered heteroaryl, substituted or unsubstituted 5-12 membered heterocyclic group;

The substitution refers to substitution by one or more substituents selected from the group consisting of halogen, hydroxy, amino, phenyl, C ₁ -C ₆ alkyl, C ₁ -C ₆ haloalkyl, C ₁ -C ₆ alkoxy Group, C ₁ -C ₆ haloalkoxy, C ₃ -C ₈ cycloalkyl.

In another preferred example, the β- or γ-amino acid N-carboxythiocarbonyl intracyclic anhydride (β-NTA, γ-NTA) monomer has a monomer as shown in formula II or III structure:

In the formula, ring A is independently selected from the following group: substituted or unsubstituted C ₆ -C ₁₂ aryl, substituted or unsubstituted C ₃ -C ₁₂ cycloalkyl, substituted or unsubstituted C ₄ -C ₁₂ ring Alkenyl or substituted or unsubstituted 5-12 membered heterocyclic group, said substitution refers to substitution by one or more substituents selected from the group consisting of halogen, hydroxy, amino, C ₁ -C ₆ alkyl, C ₁ -C ₆ haloalkyl, C ₁ -C ₆ alkoxy, C ₁ -C ₆ haloalkoxy;

In the formula, n is an integer of 0-8;

The definitions of R ₃ , R ₄ , R ₁₁ , R ₂₁ and s are as described above.

In another preferred embodiment, the monomer of formula I is selected from: L-aspartic acid 1-benzyl ester N-carboxythiocarbonyl ring anhydride, DL-β-phenylpropylamino N-carboxythiocarbonyl ring Internal acid anhydride, DL-β-norleucine N-carboxythiocarbonyl ring internal acid anhydride, N(α)-ZL-2,3-diaminopropionic acid N-carboxythiocarbonyl ring internal acid anhydride, β ^2,3 -Norbornene N-carboxythiocarbonyl intracyclic anhydride, β ^2,3 -cyclohexyl N-carboxythiocarbonyl intracyclic anhydride, γ ^2,3 -phenyl N-carboxythiocarbonyl intracyclic anhydride.

The second aspect of the present invention provides a method for synthesizing the β- or γ-amino acid N-carboxythiocarbonyl ring intracyclic anhydride (β-NTA, γ-NTA) monomer of the first aspect, including the steps:

(2) Reacting compound 3 with phosphorus halide in a second inert solvent to obtain a monomer of formula I;

Where

R _{5 is} selected from: substituted or unsubstituted C ₁ -C ₆ alkyl or substituted or unsubstituted benzyl;

The substitution refers to substitution by a substituent selected from the following group: halogen, hydroxyl, amino, C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy;

The definitions of R ₁ , R ₂ , R ₃ , R ₄ , R ₁₁ , R ₂₁ and s are as described above.

In another preferred embodiment, the phosphorus halide is selected from the following group: phosphorus tribromide, phosphorus trichloride, phosphorus pentabromide or phosphorus pentachloride; preferably phosphorus tribromide or phosphorus trichloride.

In another preferred embodiment, the method for preparing the monomer of formula I further comprises the steps:

(1) In the first inert solvent, reacting compound 2 with compound 1 in the presence of a base to obtain compound 3;

Where

R _{6 is} selected from: substituted or unsubstituted C ₁ -C ₆ alkyl or substituted or unsubstituted C ₁ -C ₆ alkylcarboxy;

The substitution refers to substitution by one or more substituents selected from the group consisting of halogen, hydroxyl, amino, -C ₁ -C ₆ alkyl, -C ₁ -C ₆ haloalkyl, -C ₁ -C ₆ alkoxy Group, -C ₁ -C ₆ haloalkoxy;

The definitions of R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , R ₁₁ , R ₂₁ , and s are as described above.

In another preferred example, in the step (1), the reaction temperature is 10-70°C; preferably, it is 20°C-60°C.

In another preferred example, in the step (1), the reaction time is 10 hours to 4 days.

In another preferred example, in the step (1), the reaction time is generally 2-3 days at room temperature.

In another preferred example, in the step (1), the reaction time is about 18 hours at 60°C.

In another preferred example, the step (1) is to dissolve compound 2 and the base in the first inert solvent, add compound 1 and stir the reaction; after the reaction, the reaction solution is adjusted to pH 3 with hydrochloric acid, and ethyl acetate is used. The ester was extracted three times, and the organic phase was dried and concentrated, and then separated and purified by column to obtain compound 3.

In another preferred example, in the step (1), the base is an inorganic base.

In another preferred embodiment, in the step (1), the base is independently selected from the following group: sodium hydroxide, potassium hydroxide, lithium hydroxide, potassium carbonate, sodium carbonate, cesium carbonate, sodium bicarbonate, Or a combination thereof; preferably sodium hydroxide or sodium bicarbonate.

In another preferred embodiment, in the step (1), the first inert solvent is independently selected from the following group: water (such as deionized water), methanol, ethanol, isopropanol, n-butanol, or Combination; preferably water (such as deionized water), methanol, or a combination thereof.

In another preferred embodiment, in the step (2), the reaction temperature is 0°C±25°C.

In another preferred example, in the step (2), the reaction time is 1-12 hours.

In another preferred example, the step (2) is to dissolve the compound 3 obtained in (1) in a second inert solvent, and add phosphorus halide to react at 0°C under the protection of an inert gas; after the reaction, The solvent was removed, ethyl acetate was added to dissolve, and quickly washed with ice water 3 times, the organic phase was dried and concentrated, and then purified to obtain the monomer of formula I.

In another preferred embodiment, in the step (2), the second inert solvent is dry.

In another preferred embodiment, in the step (2), the second inert solvent is independently selected from the following group: dichloromethane, tetrahydrofuran, ethyl acetate, dioxane, acetonitrile, or a combination thereof; preferably The ground is selected from dichloromethane, ethyl acetate, or a combination thereof.

In another preferred embodiment, in the step (2), the reaction is performed under the protection of an inert gas; the inert gas is preferably nitrogen, argon, or a combination thereof.

In another preferred example, in the step (2), the purification system is selected from the following group: ethyl acetate/n-hexane, ethyl acetate/petroleum ether, methylene chloride/n-hexane, methylene chloride/petroleum Ether, tetrahydrofuran/n-hexane, tetrahydrofuran/petroleum ether, or a combination thereof are preferably selected from the ethyl acetate/n-hexane system.

The third aspect of the present invention provides a β- or γ-amino acid homopolymer, the monomer of which is selected from the β- or γ-NTA monomer having the structure shown in formula (I) described in the first aspect.

In another preferred example, the homopolymer has a structure shown in formula IV, V, VI:

among them,

m is 5～2000;

R and R'are each independently selected from the following group: H, substituted or unsubstituted C ₁ -C ₆ alkyl, substituted or unsubstituted C ₆ -C ₁₂ aryl, substituted or unsubstituted C ₂ -C ₆ Alkenyl, substituted or unsubstituted C ₂ -C ₆ alkynyl;

Wherein, the substitution refers to substitution by one or more substituents selected from the following group: C ₁ -C ₆ alkyl, C ₆ -C ₁₂ aryl, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl; -N ₃ , C ₁ -C ₆ alkyl-(C=O)-O-, C ₁ -C ₆ alkyl-(C=O)-N-, amino, hydroxy, mercapto,

Q ₃ -O-, Q ₄ -S-, 5-6 membered heterocyclic group, adamantane, calixpyrrole, cyclodextrin, polyethylene glycol; wherein, Q ₁ and Q ₂ are each independently selected from the following group: H, C ₁ -C ₆ alkyl, C ₁ -C ₆ alkyl-O-(C=O)-, C ₆ -C ₁₄ aryl-C ₁ -C ₆ alkyl-O-(C=O) -, and Q ₁ and Q _{2 are} not H at the same time; Q _{3 is} independently selected from the following group: C ₁ -C ₆ alkyl, C ₆ -C ₁₂ aryl-C ₁ -C ₆ alkyl; Q _{4 is} independently Selected from the following group: C ₁ -C ₆ alkyl, C ₆ -C ₁₂ aryl-C ₁ -C ₆ alkyl, C ₆ -C ₁₂ aryl-C ₁ -C ₆ alkyl-(C=O) -O-; and R and R'are not H at the same time;

Or R, R'and the adjacent N atoms form a substituted or unsubstituted 5-12 membered heterocyclic group; the substitution refers to substitution by one or more substituents selected from the group consisting of halogen, hydroxyl, amino, C ₁ -C ₆ alkyl, C ₁ -C ₆ haloalkyl, C ₁ -C ₆ alkoxy, C ₁ -C ₆ haloalkoxy;

In another preferred embodiment, the homopolymer has the structure shown in formula VII, VIII, IX:

Among them, m, n, R, R', R ₃ , R ₄ , R ₁₁ , R ₂₁ , and s are as defined above.

In another preferred example, the homopolymer has a structure represented by formula X, XI, XII:

The definitions of m, R, R', A, R ₁₁ , R ₂₁ and s are as described above.

In another preferred embodiment, the homopolymer monomer is selected from the following group: L-aspartic acid 1-benzyl ester N-carboxythiocarbonyl ring anhydride, DL-β ³ -phenylpropylamino N-carboxyl Thiocarbonyl intracyclic anhydride, DL-β ³ -norleucine N-carboxythiocarbonyl intracyclic anhydride, N(α)-ZL-2,3-diaminopropionic acid N-carboxythiocarbonyl intracyclic anhydride , Β ^2,3 -Norbornene N-carboxythiocarbonyl intracyclic anhydride, β ^2,3 -cyclohexyl N-carboxythiocarbonyl intracyclic anhydride, γ ^2,3 -phenyl N-carboxythiocarbonyl ring Internal acid anhydride, or a combination thereof.

In another preferred example, the GPC spectrum of the homopolymer is a single peak, and the molecular weight distribution PDI is preferably 1.01-1.5.

In another preferred example, in the Maldi-Tof-Ms spectrum of the homopolymer, the molecular weight distribution PDI is preferably 1.01-1.5.

In another preferred embodiment, the homopolymer is prepared by a method including the following steps:

In the third inert solvent, in the presence of an organic base initiator, any one of the β- or γ-NTA monomers having the structure represented by formula (I) described in the first aspect are polymerized to form the β- or γ-amino acid homopolymer;

Wherein, the organic base is independently selected from: amines, amine salts, or other organic bases, or combinations thereof;

The amine is selected from the group consisting of R-NH ₂ , R-NH-R',

Or a combination;

The salt of the amine is independently selected from the following group: hydrochloride, hydrobromide, formate, acetate, or trifluoroacetate;

R, R', and R" are each independently selected from the following group: H, substituted or unsubstituted C ₁ -C ₆ alkyl, substituted or unsubstituted C ₆ -C ₁₂ aryl, substituted or unsubstituted C ₂ -C ₆ alkenyl, substituted or unsubstituted C ₂ -C ₆ alkynyl;

Wherein, the substitution refers to substitution by one or more substituents selected from the following group: C ₁ -C ₆ alkyl, C ₆ -C ₁₂ aryl, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl; -N ₃ , C ₁ -C ₆ alkyl-(C=O)-O-, C ₁ -C ₆ alkyl-(C=O)-N-, amino, hydroxyl, mercapto,

Or R, R'and the adjacent N atoms form a substituted or unsubstituted 5-12 membered heterocyclic group; the substitution refers to substitution by one or more substituents selected from the group consisting of halogen, hydroxyl, amino, C ₁ -C ₆ alkyl, C ₁ -C ₆ haloalkyl, C ₁ -C ₆ alkoxy, C ₁ -C ₆ haloalkoxy.

In another preferred example, the molar ratio of monomer to amine initiator is 1-2000:1.

In another preferred embodiment, the polymerization reaction is carried out in an environment with or without inert gas protection.

In another preferred embodiment, the polymerization reaction is carried out in any open or non-open reactor.

In another preferred example, the other organic base is selected from: DBU, HMDS, LiHMDS, NaHMDS, KHMDS, EtONa.

In another preferred embodiment, the third inert solvent is independently selected from: THF, DMF, DMAc, MeCN, Dioxane, and DMSO.

In another preferred embodiment, the third inert solvent is DMF.

The fourth aspect of the present invention provides a copolymer or sequence peptide, the monomers of which are selected from: a) the β- or γ-amino acid N-carboxythiocarbonyl intracyclic anhydride (β-NTA, γ) described in the first aspect -NTA) two or more of monomers; and optionally b) one or more of α-NCA and α-NTA monomers.

In another preferred example, the mass ratio of α-NCA or α-NTA in the copolymer is: 100%>α-NCA≥0% or 100%>α-NTA≥0%.

In another preferred example, the copolymer or sequence peptide has a structural unit of _{C n1} D _n2 _C'n3 or D _n1 C _n2 D' _n3:

Wherein, n _{3 is} independently an integer of 0-2000; n ₁ and n ₂ are each independently an integer of 1-2000;

C and C'are different, and each independently is

D and D'are each independently

R ₇ and R ₈ are each independently H, -C ₁ -C ₆ alkyl-R ₉ , -N ₃ , -(C=O)-OR ₉ , -(C=O)-NH-R ₉ ,- NH-R ₉ , -OR ₉ , -SR ₉ , -Ph-R ₁₀ or 5-6 membered heterocyclic group;

Or R ₈ is connected to the C atom and the N atom adjacent to the C atom to form a 5-6 membered heterocyclic group;

R _{9 is} selected from the group consisting of one or more substituted or unsubstituted substituents: hydrogen, C ₁ -C ₆ alkyl, phenyl, indolyl, 5-6 membered heteroaryl, C ₂ -C ₆ alkene Group, C ₂ -C ₆ alkynyl group, guanidyl group, benzyl group, tert-butoxycarbonyl group, benzyloxycarbonyl group, tert-butyl group, trityl group or fluorenyl methoxycarbonyl group; the substituted substituent is selected from the following group One or more groups of: halogen, nitro, C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkenyloxy or CH ₃ ( O-CH ₂ -CH ₂ )y, and y is an integer of 1-6;

R _{10 is} selected from: H, -OH;

In another preferred example, the copolymer or sequence peptide has a C _n1 D _n2 _C'n3 structural unit, preferably n ₁ and n ₂ are each independently an integer from 1 to 1000, and n _{3 is} independently 0 -1000 is an integer.

In another preferred example, the copolymer is a block polymer with C _n1 D _n2 _C'n3 structural units, preferably n ₁ and n ₂ are each independently an integer of 1-1000, and n _{3 is} independently It is an integer of 0-1000.

In another preferred example, the copolymer or sequence peptide has D _n1 C _n2 D' _n3 structural unit, preferably n ₁ and n ₂ are each independently an integer of 1-1000, and n _{3 is} independently 0 -1000 is an integer.

In another preferred embodiment, the polymer blend or sequence peptide has [C _n'1 D _n'2 _C'n'3 ] _Y or [D _n'1 C _n'2 D'_n'3 ] structure _Y, Y is an integer of 10-2000; defining C, C ', D, D ' as described above; wherein, n _'3 independently 1>n' ₃ ≥0 decimal; n _'1, n' ₂ are each independently a decimal> 0 and less than 1.

In another preferred embodiment, the copolymer has the following structural units:

The definitions of R ₁ , R ₂ , R ₃ , R ₄ , R ₁₁ , R ₂₁ and s are as described above; the definition of R ₁ 'is the same as that of R ₁ , and R ₁ ' is _{different from R 1} , R ₂ ', R ₃ ' The definitions of, R ₄ ', R ₁₁ ', and R ₂₁ 'can be deduced by analogy.

In another preferred example, the copolymer or sequence peptide has the following structural units:

R ₁ , R ₂ , R ₃ , R ₄ , R ₇ , R ₈ , R ₁₁ , R ₂₁ , R ₁ ', R ₂ ', R ₃ ', R ₄ ', R ₁₁ ', R ₂₁ 'and s The definition is as described above.

R ₁ , R ₂ , R ₃ , R ₄ , R ₇ , R ₈ , R ₁₁ , R ₂₁ and s are as defined above, R ₇ ′ is the same as R ₇ , and R ₈ ′ is the same as R ₈ .

In another preferred embodiment, the homopolymer or copolymer is selected from the homopolymer or copolymer described in the embodiment.

In another preferred embodiment, the selected homopolymer or copolymer or sequence peptide is selected from:

In another preferred embodiment, the copolymer is prepared by a method including the following steps:

(i) In a third inert solvent, mixing two or more of the monomers described in the first aspect; and optionally one or more of α-NCA and α-NTA monomers,

(ii) Perform a polymerization reaction in the presence of an organic base initiator to form a statistical coplymer amino acid copolymer;

Or (i') in the third inert solvent, in the presence of an organic base initiator, first polymerize the first monomer,

(ii') After the polymerization reaction in (i') is over, the second monomer is added to carry out the polymerization reaction,

And optionally (iii') repeat step (ii') q times,

So as to form a block copolymer amino acid copolymer;

Wherein, q is an integer ≥ 1;

The first monomer and the second monomer are different and independently selected from: any of the β-NTA or γ-NTA monomers, α-NCA monomers or α-NTA monomers described in the first aspect;

The amine is selected from the group consisting of R-NH ₂ , R-NH-R',

Or a combination;

The salt of the amine is independently selected from: hydrochloride, hydrobromide, formate, acetate, or trifluoroacetate;

The definitions of R, R', and R" are as described above.

The fifth aspect of the present invention provides a method for preparing the copolymer of the fourth aspect, the method comprising the steps of: (i) combining two or more of the monomers described in the first aspect in an inert solvent ; And optionally b) a mixture of one or more of α-NCA and α-NTA monomers,

(ii) Perform a polymerization reaction in the presence of an organic base initiator to form a statistical coplymer β-amino acid copolymer;

Or (i') in an inert solvent, in the presence of an organic base initiator, first polymerize the first monomer,

(ii') After the polymerization reaction in (i') is completed, the second monomer is added to carry out the polymerization reaction, thereby forming a block copolymer β-amino acid copolymer;

And optionally (iii') repeat step (ii') q times,

So as to form a block copolymer β-amino acid copolymer;

Wherein, q is an integer ≥ 1;

The amine is selected from the group consisting of R-NH ₂ , R-NH-R',

Or a combination;

The definitions of R, R', and R" are as described above.

In another preferred example, in the step (iii'), 50≥q≥1, preferably 20≥q≥2, preferably q is 1, 2, 3, 4, 5, 6.

In another preferred embodiment, the first monomer is any of the aforementioned β-NTA or γ-NTA monomers.

In another preferred example, the second monomer is α-NCA monomer or α-NTA monomer.

In another preferred example, the first monomer is any of the aforementioned β-NTA or γ-NTA monomers, and the second monomer is an α-NCA monomer or α-NTA monomer.

In another preferred embodiment, the β-NTA or γ-NTA monomer is selected from: L-aspartic acid 1-benzyl ester N-carboxythiocarbonyl ring anhydride, DL-β ³ -phenylpropylamino N -Carboxythiocarbonyl ring anhydride, DL-β ³ -Norleucine N-carboxythiocarbonyl ring anhydride, N(α)-ZL-2,3-diaminopropionic acid N-carboxythiocarbonyl ring Internal acid anhydride, β ^2,3 -norbornene N-carboxythiocarbonyl intracyclic anhydride, β ^2,3 -cyclohexyl N-carboxythiocarbonyl intracyclic anhydride, γ ^2,3 -phenyl N-carboxythio Carbonyl ring anhydride or a combination thereof.

In another preferred embodiment, the α-NCA monomer or α-NTA monomer is selected from: L-glutamic acid-5-benzyl ester-N-carboxyl ring anhydride, N-ε-tert-butoxycarbonyl- DL-Lysine-N-carboxyl anhydride, N-ε-tert-butoxycarbonyl-L-lysine-N-carboxyl anhydride, N-ε-tert-butoxycarbonyl-DL-ornithine- N-carboxyl ring anhydride, O-tert-butyl-L-serine-N-carboxyl ring anhydride, DL-norleucine-N-carboxyl ring anhydride, L-glutamic acid-5-benzyl ester-N -Carboxythiocarbonyl inner anhydride, N-ε-tert-butoxycarbonyl-DL-lysine-N-carboxythiocarbonyl inner anhydride, N-ε-tert-butoxycarbonyl-L-lysine-N -Carboxythiocarbonyl intracyclic anhydride, N-ε-tert-butoxycarbonyl-DL-ornithine-N-carboxythiocarbonyl intracyclic anhydride, O-tert-butyl-L-serine-N-carboxythiocarbonyl Intracyclic anhydride, DL-norleucine-N-carboxythiocarbonyl intracyclic anhydride, or a combination thereof.

In another preferred embodiment, in the polymerization reaction, the second monomer is added first, and the first monomer is added after the reaction is completed.

In another preferred example, in the polymerization reaction, after the first monomer is added, after the reaction is completed, another first monomer is added.

In another preferred embodiment, in the polymerization reaction, after the first monomer is added, after the reaction is completed, continue to add the second monomer.

In another preferred embodiment, in the polymerization reaction, after at least one first monomer participates in the reaction, after the reaction is completed, the subsequent feeding includes any of the foregoing feeding sequences.

In another preferred embodiment, in the polymerization reaction, the inert solvent is selected from the following group: tetrahydrofuran, DMF, DMAc, acetonitrile, dioxane, dimethyl sulfoxide, or a combination thereof. Preferably it is tetrahydrofuran.

The sixth aspect of the present invention provides a use of the polymer of the third or fourth aspect for anti-bacterial, anti-fungal, anti-viral, anti-mite, anti-tumor, cell adhesion, tissue engineering, and drug modification , Protein modification, protein protection, drug synergy, drug delivery, gene delivery and self-assembly materials.

In another preferred embodiment, the antibacterial material is in the form of a solution or a surface coating.

In another preferred embodiment, the antibacterial objects are microorganisms such as bacteria and fungi, and may include Escherichia coli (E.coli), Pseudomonas aeruginosa (P.aeruginosa), Acinetobacter baumannii (A.baumannii), Enterobacter aerogenes (E. aerogenes) ), Klebsiella pneumoniae(K.pneumoniae), Serratia marcescens(S.marcescens), Entebacter Cloacae(E.cloacae), Bacillus subtilis(B.subtilis), Staphylococcus aureus(S.aureus), Stamidphylocisoccus. Candida albicans (C.albicans), Cryptococcus neoformans (C.neoformans).

In another preferred embodiment, the antibacterial use includes free cells of microorganisms, biofilms, and spores.

In another preferred embodiment, the polymer is used to treat tumors.

In another preferred embodiment, the tumor is selected from the group consisting of melanoma, skin cancer, glioma, mesothelioma, lymphoma, leukemia, breast cancer, ovarian cancer, cervical cancer, glioblastoma, Multiple myeloma, prostate cancer, Burkitt lymphoma, head and neck cancer, colon cancer, colorectal cancer, non-small cell lung cancer, small cell lung cancer, esophageal cancer, gastric cancer, pancreatic cancer, hepatobiliary cancer, gallbladder cancer, small intestine cancer, Rectal cancer, kidney cancer, bladder cancer, prostate cancer, penile cancer, urethral cancer, testicular cancer, vaginal cancer, uterine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, pancreatic endocrine cancer, carcinoid cancer, bone cancer, retinoblastoma Cell tumor, Hodgkin’s lymphoma, non-Hodgkin’s lymphoma, Kaposi’s sarcoma, multicentric Casterman’s disease, AIDS-related primary exudative lymphoma, neuroectodermal tumor, or rhabdomyosarcoma.

It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described in the following (such as the embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, I will not repeat them here.

Detailed ways

the term

As used in the text, the term "sequence peptide (ie polypeptide)" refers to polypeptides and polypeptide derivatives with a fixed sequence prepared by a stepwise reaction of β- or γ-NTA monomers.

Throughout the specification, the terms "optionally substituted" or "may be substituted" are used to indicate that the group may or may not be further substituted or fused with one or more non-hydrogen substituents (to form a polycyclic ring). system). The substituents for suitable chemically suitable specific functional groups will be obvious to those skilled in the art.

As used in the text, the term "C ₁ -C ₆ alkyl" refers to a straight or branched chain alkyl group having 1 to 6 carbon atoms, such as methyl, ethyl, propyl, isopropyl, n-butyl, Isobutyl, tert-butyl, pentyl, hexyl, etc., preferably ethyl.

As used in the text, the term "C ₁ -C ₆ alkoxy" refers to a straight or branched chain alkoxy group having 1 to 6 carbon atoms, such as methoxy, ethoxy, propoxy, isopropoxy Group, n-butoxy, isobutoxy, tert-butoxy, pentoxy, hexoxy, etc., preferably ethoxy.

As used in the text, the term "C ₆ -C ₁₂ aryl" refers to aromatic cyclic hydrocarbon groups with 6-12 carbon atoms, especially monocyclic and bicyclic groups such as phenyl, biphenyl or Naphthyl. Where there are two or more aromatic rings (bicyclic, etc.), the aromatic ring of the aryl group can be connected by a single bond (such as biphenyl) or condensed (such as naphthalene, anthracene, etc.).

As used in the text, the term "5-12 membered heteroaryl" refers to a 5-12 membered heteroaromatic system containing 1-3 atoms selected from N, O, and S. The heteroaryl group is preferably a 5- to 10-membered ring, more preferably 5-membered or 6-membered, such as pyrrolyl, pyrazolyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, thiadiazolyl, isothiazolyl , Furyl, pyridinyl, pyrazinyl, pyrimidinyl, pyridazinyl, triazinyl, triazole and tetrazolyl, etc. "Heteroaryl" may be substituted or unsubstituted. When substituted, the substituent is preferably one or more of the following groups, which are independently selected from alkyl, deuterated alkyl, haloalkyl, alkoxy , Haloalkoxy, alkenyl, alkynyl, alkylthio, alkylamino, halogen, amino, nitro, hydroxyl, mercapto, cyano, cycloalkyl, heterocyclic, aryl, heteroaryl, cycloalkane Sulfur group, oxo group, carboxyl group and carboxylate group. .

As used in the text, the term "C ₃ -C ₁₂ cycloalkyl" refers to a fully saturated cyclic hydrocarbon group with 3-12 carbon atoms, preferably a cycloalkyl group with 3-8 carbon atoms, each ring It contains 3-8 carbon atoms. "Substituted C ₃ -C ₁₂ cycloalkyl" means that one or more positions in the cycloalkyl group are substituted, especially 1-4 substituents, which can be substituted at any position. Including but not limited to cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl and the like.

As used in the text, the term "C ₄ -C ₁₂ cycloalkenyl" refers to an unsaturated cyclic hydrocarbon group having 4-12 carbon atoms with 1-3 double bonds, such as cyclobutenyl, cyclic Pentenyl, cyclohexenyl, etc.

As used in the text, the term "5-12 membered heterocyclyl" refers to a fully saturated or partially unsaturated cyclic group having 5-12 ring atoms and 1-3 heteroatoms (including but not limited to Such as 3-7 membered monocyclic ring, 6-11 membered bicyclic ring, or 8-12 membered tricyclic ring system). Among them, nitrogen atoms or sulfur atoms can be oxidized, and nitrogen atoms can also be quaternized. The heterocyclic group can be attached to any heteroatom or carbon atom residue of the ring or ring system molecule. Typical monocyclic heterocycles include, but are not limited to, azetidinyl, pyrrolidinyl, oxetanyl, pyrazolinyl, imidazolinyl, imidazolidinyl, oxazolidinyl, isoxazolidine Group, thiazolidinyl, isothiazolidinyl, tetrahydrofuranyl, piperidinyl, piperazinyl, 2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolidinyl, hexahydroazepine Inyl group, 4-piperidinone group, tetrahydropyranyl group, morpholino group, thiomorpholino group, thiomorpholine sulfoxide group, thiomorpholine sulfone group, 1,3-dioxanyl group and Tetrahydro-1,1-dioxythiophene, etc. Polycyclic heterocyclic groups include spiro, condensed and bridged heterocyclic groups; the spiro, condensed and bridged heterocyclic groups involved are optionally connected to other groups through a single bond, or through a ring Any two or more atoms above are further connected to other cycloalkyl groups, heterocyclic groups, aryl groups and heteroaryl groups; the heterocyclic group may be substituted or unsubstituted. When substituted, The substituent is preferably one or more of the following groups, which are independently selected from alkyl, deuterated alkyl, haloalkyl, alkoxy, haloalkoxy, alkenyl, alkynyl, alkylthio, alkylamino, Halogen, amino, nitro, hydroxyl, mercapto, cyano, cycloalkyl, heterocyclic, aryl, heteroaryl, cycloalkylthio, oxo, carboxy, and carboxylate. , Such as tetrahydropyrrolyl, tetrahydrofuranyl, piperidinyl, piperazinyl and the like.

As used in the text, the term "C ₁ -C ₆ alkylhydroxy" refers to -C ₁ -C ₆ alkyl-OH, for example, -CH ₂ OH, -CH ₂ CH ₂ OH.

As used in the text, the term "C1-C6 alkoxy" refers to -C ₁ -C ₆ alkyl-O-, for example, methoxy, ethoxy, propoxy and the like.

As used in the text, the term "C ₁ -C ₆ alkylsulfonyl" refers to C ₁ -C ₆ alkyl S(=O) ₂ -.

As used in the text, the term "C ₁ -C ₆ alkyl-C ₆ -C ₁₂ aryl" refers to -C ₁ -C ₆ alkyl-C ₆ -C ₁₂ aryl, such as -CH ₂ CH ₂ CH ₂ Ph , -Bn.

As used in the text, the term "C ₁ -C ₆ alkyl ester group" refers to C ₁ -C ₆ alkyl C(=O)-O- or -(=O)-OC ₁ -C ₆ alkyl.

As used in the text, the term "thio C1-C6 alkyl ester group" refers to C ₁ -C ₆ alkyl C(=S)-O- or -(=S)-OC ₁ -C ₆ alkyl.

As used in the text, the term "C ₁ -C ₆ alkylguanidino" C ₁ -C ₆ alkyl NHC(=NH)NH-.

When the substituent is non-terminal, the substituent is a subunit of the corresponding substituent, for example, an alkyl group, and the non-terminal substituent is an alkylene group. In this case, the alkyl group and the alkylene group can be used interchangeably.

As used in the text, "structural unit" refers to a structural fragment, and the polymer or sequence peptide may include one or more structural units.

As used in the text, the term "statistic coplymer" refers to the irregularly connected polymer formed by the simultaneous polymerization of two or more monomers, including: β/β-, γ/γ-, β /γ-, α/γ-, α/β/γ amino acid copolymer.

As used in the text, the term "block type" refers to a polymer formed by linking different chain segments obtained by successively polymerizing two or more monomers, including β-β-, γ-γ-, γ-β- , Α-β-, α-γ-, α-β-γ-amino acid copolymer.

As used in the text, the term "substituted benzyl" refers to a benzyl group substituted with a substituent selected from C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, phenyl, such as 4-methylbenzyl Group, 3,5-dimethylbenzyl, 2,4,6-trimethylbenzyl, 4-methoxybenzyl, 3,5-dimethoxybenzyl, 2,4,6-trimethyl Oxybenzyl, 1-(4-methylphenyl)ethyl, 1-(4-methoxyphenyl)ethyl and the like.

As used in the text, the term "C ₁ -C ₆ alkylcarboxy" refers to -C ₁ -C ₆ alkyl COOH, such as -CH ₂ COOH, -CH ₂ CH ₂ COOH, -CH ₂ CH ₂ CH ₂ COOH,- CH ₂ CH ₂ CH ₂ CH ₂ COOH, preferably -CH ₂ COOH, -CH ₂ CH ₂ COOH. Among them, the alkyl group may be substituted.

The "substituent" as used herein refers to a part of a molecule that is covalently bonded to an atom in the target molecule. For example, a "ring substituent" may be a moiety such as a halogen, an alkyl group, or other substituents described herein, which is covalently bonded to an atom that is a ring member, preferably a carbon or nitrogen atom. As used herein, the term "substituted" means that any one or more hydrogens on the designated atom are replaced by a designated substituent, provided that the normal valence of the designated atom is not exceeded, and the compound resulting from the substitution is stable, That is, compounds that can be separated, characterized and tested for biological activity.

The structural formula or name of the monomer given in the present invention may only exemplify a specific configuration or no specific configuration. The monomer may also include all other configurations corresponding to the given configuration. .

Β-NTA and γ-NTA monomers of the present invention

The β-NTA and γ-NTA have the structure shown in formula I:

among them,

s is 0 or 1;

Preferably, the monomer structure is as shown in formula II:

In the formula, ring A is substituted or unsubstituted aryl, substituted or unsubstituted C ₃ -C ₁₂ cycloalkyl, substituted or unsubstituted C ₄ -C ₁₂ cycloalkenyl, or substituted or unsubstituted 5-12 A membered heterocyclic group, said substitution means being substituted by one or more substituents selected from the group consisting of halogen, hydroxy, amino, -C ₁ -C ₆ alkyl, -C ₁ -C ₆ haloalkyl, -C ₁ -C ₆ alkoxy, -C ₁ -C ₆ haloalkoxy;

Preferably, the monomer structure is as shown in formula III:

Wherein, n is an integer of 0-8.

The configuration of the β-NTA monomer may be one of L type, D type, and DL mixed type. Among them, DL mixed type can be L type and D type mixed in any ratio. For example, it can be mixed in a ratio of 1:1, but it is not limited to this ratio.

The side chain of the β-NTA monomer also contains one or more of amino, carboxyl, hydroxyl, mercapto, aliphatic, aromatic and the like.

The β-NTA monomer can be selected from the following group: L-aspartic acid 1-benzyl ester N-carboxythiocarbonyl intracyclic anhydride, DL-β-phenylpropylamino N-carboxythiocarbonyl intracyclic anhydride, DL-β-norleucine N-carboxythiocarbonyl ring anhydride, or a combination thereof.

Any suitable α-NCA monomer can be used in the present invention. The exemplary α-NCA monomer of the present invention is one or more of the compounds represented by formula A:

Where

n ₁ "is an integer from 0 to 4;

X is none, azide group (N ₃ ), ester group (-(C=O)-O-), amide group (-(C=O)-N-), -NH-, -O-,- S-, phenyl or 5-6 membered heterocyclic group;

R _A is hydrogen or C ₁ -C ₆ alkyl;

R _B and R _C are each independently none, hydrogen, C ₁ -C ₆ alkyl, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, benzyl, tert-butoxycarbonyl, benzyloxycarbonyl, tertiary Butyl, trityl or fluorenylmethyloxycarbonyl;

One or more hydrogen atoms in R _B and R _C may be substituted by a group selected from the group consisting of halogen, nitro, C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkenyloxy or CH ₃ (O-CH ₂ -CH ₂ )y, and y is an integer of 1-6.

Preferably, the α-NCA monomer is selected from the following group: L-glutamic acid-5-benzyl ester-N-carboxyl ring anhydride, N-ε-tert-butoxycarbonyl-DL-lysine-N-carboxyl ring Internal acid anhydride, N-ε-tert-butoxycarbonyl-L-lysine-N-carboxyl anhydride, N-ε-tert-butoxycarbonyl-DL-ornithine-N-carboxyl anhydride, O-tert Butyl-L-serine-N-carboxyl anhydride, DL-norleucine-N-carboxyl anhydride, or a combination thereof.

Preferably, the α-NCA monomer can also be in other configurations (L-type, D-type, or DL mixed-type) corresponding to various monomers. For example, corresponding to L-glutamate-5-benzyl ester-N-carboxyl ring anhydride, the amino acid N-carboxyl ring inner anhydride monomer can also be D-glutamate-5-benzyl ester-N-carboxyl ring Internal acid anhydride or DL-glutamic acid-5-benzyl ester-N-carboxyl ring internal acid anhydride.

Any suitable α-NTA monomer can be used in the present invention. The exemplary α-NTA monomer of the present invention is one or more of the compounds represented by formula B:

Where

n ₁ "is an integer from 0 to 4;

X is none, azide group (N ₃ ), ester group (-(C=O)-O-), amide group (-(C=O)-N-), -NH-, -O-,- S-, phenyl or 5-6 membered heterocyclic ring;

R _A 'is hydrogen or C1-C6 alkyl;

R _B 'and R _C ' are each independently none, hydrogen, C ₁ -C ₆ alkyl, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, benzyl, tert-butoxycarbonyl, benzyloxycarbonyl , Tert-butyl, trityl or fluorenylmethyloxycarbonyl;

One or more hydrogen atoms in R _B 'and R _C ' may be substituted by a group selected from the group consisting of halogen, nitro, C1-C6 alkyl, C ₁ -C ₆ alkoxy, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkenyloxy or CH ₃ (O-CH ₂ -CH ₂ )y, and y is an integer of 1-6.

Preferably, the α-NTA monomer is selected from the following group: L-glutamic acid-5-benzyl ester-N-carboxythiocarbonyl ring anhydride, N-ε-tert-butoxycarbonyl-DL-lysine-N -Carboxythiocarbonyl intracyclic anhydride, N-ε-tert-butoxycarbonyl-L-lysine-N-carboxythiocarbonyl intracyclic anhydride, N-ε-tert-butoxycarbonyl-DL-ornithine-N -Carboxythiocarbonyl intracyclic anhydride, O-tert-butyl-L-serine-N-carboxythiocarbonyl intracyclic anhydride, DL-norleucine-N-carboxythiocarbonyl intracyclic anhydride, or a combination thereof.

Preferably, the α-NTA monomer can also be in other configurations (L-type, D-type, or DL mixed-type) corresponding to various monomers. For example, corresponding to L-glutamic acid-5-benzyl ester-N-carboxythiocarbonyl ring anhydride, the amino acid N-carboxythiocarbonyl ring anhydride monomer can also be D-glutamate-5-benzyl Ester-N-carboxythiocarbonyl intracyclic anhydride or DL-glutamic acid-5-benzyl ester-N-carboxythiocarbonyl intracyclic anhydride.

The structure and synthesis of the α-NTA and α-NCA monomers in the present invention have been reported in the literature and are available for purchase, which is not within the protection scope of this patent.

Preparation method of amino acid polymer of the present invention

1. A preparation method of β- or γ-amino acid polymer

The method includes the steps: in an organic solvent, in the presence of primary amine initiator RNH ₂ , secondary amine initiator RR'NH, tertiary amine initiator R ₃ N and their salts, the β-, γ-NTA One or more of the monomers (such as two, three, or four or more) are polymerized to form the β-, γ-amino acid polymer;

Wherein, R is selected from the following group: substituted or unsubstituted C ₁ -C ₆ alkyl, substituted or unsubstituted C ₆ -C ₁₂ aryl, substituted or unsubstituted C ₂ -C ₆ alkenyl, substituted or unsubstituted C 2 -C 6 alkenyl A substituted C ₂ -C ₆ alkynyl group; wherein, the substitution refers to substitution by a substituent selected from the following group: C ₁ -C ₆ alkyl group, C ₆ -C ₁₂ aryl group, C ₂ -C ₆ alkene , C ₂ -C ₆ alkynes; azide group (N ₃ ), C ₁ -C ₆ alkyl ester group (C ₁ -C ₆ alkyl-(C=O)-O-), C ₁ -C ₆ alkyl amide group (C ₁ -C ₆ alkyl-(C=O)-N-), amino (NH ₂ -), hydroxyl (HO-), mercapto (HS-), 5-6 membered heterocycle, Adamantane, calixpyrrole, cyclodextrin, polyethylene glycol; wherein _{the hydrogen atom on the amino group (NH 2} -) can be C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxycarbonyl (C _1- C ₆ alkyl-O-(C=O)-), C ₆ -C ₁₄ aryl C ₁ -C ₆ alkoxycarbonyl (C ₆ -C ₁₄ aryl-C ₁ -C ₆ alkyl- O-(C=O)-) substitution; the hydrogen atom on the hydroxyl group (HO-) can be substituted by C ₁ -C ₆ alkyl, C ₆ -C ₁₂ aryl-C ₁ -C ₆ alkyl; The hydrogen atom on the mercapto group (HS-) can be substituted by C ₁ -C ₆ alkyl, C ₆ -C ₁₂ aryl-C ₁ -C ₆ alkyl, C ₆ -C ₁₂ aryl-C ₁ -C ₆ alkane Alkyl ester group (C ₆ -C ₁₂ aryl-C ₁ -C ₆ alkyl-(C=O)-O-) substitution;

The salt-forming form is selected from the group consisting of hydrochloride, hydrobromide, formate, acetate, and trifluoroacetate.

The method can replace the primary amine initiator RNH ₂ with any one of the organic bases described below or a combination form thereof. The organic base is preferably selected from DBU, HMDS, LiHMDS, NaHMDS, KHMDS, NaEtO, and the like.

The reaction can be carried out in an instrument or device without any protection (for example, an open beaker, a flask, or various open reactors commonly used in the industry).

The reaction can also be carried out in a nitrogen-protected instrument or device (such as a glove box).

In the reaction, one type of β-NTA can be used for polymerization, or two, three, or four different types of β-NTA can be used for polymerization.

2. Preparation method of α-, β-, γ-amino acid blend polymer

X = S or O; s = 0 or 1; n ₁ ≥ 0, n ₂ ≥ 0, n ₃ ≥ 0; n ₁ , n _{3 are} not 0 at the same time

R ₁ , R ₂ , R ₃ , R ₄ , R ₁₁ , R ₂₁ , R ₁ ', R ₂ ', R ₃ ', R ₄ ', R ₁₁ ', R ₂₁ 'and s are as defined above;

R ₇ or R ₈ is the side chain structure of α-amino acid and its derivatives, wherein the α-amino acid is selected from the group consisting of glycine, alanine, valine, leucine, isoleucine, and phenylalanine , Tryptophan, tyrosine, aspartic acid, asparagine, glutamic acid, lysine, glutamine, methionine, serine, threonine, cysteine, proline, Histidine, arginine, and derivatives derived from the above-mentioned amino acids; the derivatives derived from the above-mentioned amino acids are those whose carboxylic acid groups on the amino acids are esterified (for example, benzyl esterification, tert-butyl esterification, methyl esterification) The hydrogen atom on the amino group of the amino acid is substituted (for example, the derivative substituted by tert-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz), fluorenylmethyloxycarbonyl (Fmoc), etc.) , The hydrogen atom on the hydroxyl group on the amino acid is substituted (for example, a derivative of tert-butyl (tBu) substitution), and the hydrogen atom on the free sulfhydryl group on the amino acid is substituted (for example, by the trityl group ( Trt), benzyl, benzyl ester and other substituted derivatives). These derivatives are stable under the polymerization reaction system.

The method includes the steps: in an organic solvent, in the presence of primary amine initiator RNH ₂ , secondary amine initiator RR'NH, tertiary amine initiator R ₃ N and their salts, the β-, γ-NTA One or more of the monomers (such as two, three, or four or more) and one or more of α-NTA or α-NCA are polymerized together to form the blend Type α/β-, α/γ-, β/γ-, α/β/γ- and other amino acid polymers;

The method includes the steps of: first polymerizing a β- or γ-NTA monomer in an organic solvent in the presence of an initiator;

After the above polymerization reaction is over, another α-NCA or α-NTA monomer is added to carry out the polymerization reaction, thereby forming block type α-β-, α-γ-, β-γ-, α-β- γ-amino acid copolymer.

The obtained α/β-, α/γ-, β/γ-, α/β/γ-, α-β-, α-γ-, β-γ-, α-β-γ-amino acid copolymer The type depends on the type of α-NCA or α-NTA, β-NTA, γ-NTA monomer and the order of addition.

The copolymer is a polymer or a block copolymer obtained by copolymerizing two or more monomers in a set ratio.

The organic solvent may be selected from the following group: tetrahydrofuran, DMF, DMAc, acetonitrile, dioxane, dimethyl sulfoxide, preferably tetrahydrofuran.

The reaction is carried out at room temperature or under heating conditions.

The amount of the initiator is determined according to the chain length of the polymer to be prepared.

The polymerization reaction time varies according to the needs of different monomers, and also varies according to the length of the polymer to be prepared.

The preparation of the amino acid polymer in the present invention can remove the COS _(g) cleanly after the post-treatment purification step, and the content is less than 5‰ by GC detection.

The main advantages of the present invention are:

1. The use of the new β-NTA or γ-NTA monomer of the present invention fundamentally solves the shortcomings of traditional β-lactam strong base ring-opening polymerization conditions, which are harsh, sensitive to moisture, and difficult to open reaction; avoid strict in the glove box Operate the polymerization reaction in an anhydrous environment.

2. Using the new β-NTA or γ-NTA monomer and polymer preparation method of the present invention can quickly and easily synthesize a large number of polypeptide libraries, which can be used for biological activity such as antibacterial activity, cell activity screening, and other polypeptide polymer functions.

The present invention will be further explained below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods that do not indicate specific conditions in the following examples usually follow the conventional conditions or the conditions recommended by the manufacturer. Unless otherwise specified, percentages and parts are weight percentages and parts by weight. The experimental materials and reagents used in the following examples can be obtained from commercial channels unless otherwise specified.

Method for detecting polymer molecular weight and molecular weight distribution

Gel Permeation Chromatography (GPC) method

¹ H NMR

Maldi-Tof-Ms

Example 1 Synthesis of L-aspartic acid 1-benzyl N-carboxythiocarbonyl ring anhydride monomer (Bn-β ³ -LCHG NTA)

Dissolve S-ethoxythiocarbonylthioglycolic acid (9.0g, 50.0mmol) in a 200mL flask of deionized water, add solid sodium bicarbonate (16.8g, 200.0mmol) to the flask, mix and stir, then add after dissolution L-aspartic acid 1-benzyl ester (11.2g, 50.0mmol) was mixed and stirred for reaction, and stirred at room temperature for about 48 hours; then 1M hydrochloric acid solution was slowly added dropwise, the pH was adjusted to about 3, and ethyl acetate was used The organic matter in the aqueous phase was extracted three times, and the organic phases were combined. The organic phase was then quenched with saturated brine and dried over anhydrous magnesium sulfate; the crude product was recrystallized and purified with ethyl acetate and n-hexane to obtain a white solid intermediate (12.0g) , The yield is 77%).

Under the protection of nitrogen, the dry white intermediate (1.6g, 5.0mmol) was dissolved in a dry dichloromethane solution (50mL), mixed and stirred at 0°C in an ice bath, and then phosphorus tribromide (1.4 g, 5.0mmol) solution. After the dripping is completed, the temperature is raised to 25°C, and the reaction is continued for 6 hours. After the reaction is over, the organic phase is washed three times with deionized water at 0°C, the organic phase is dried with anhydrous magnesium sulfate, filtered, and concentrated The solvent was removed to obtain the crude product, which was purified by recrystallization with ethyl acetate and n-hexane under the protection of nitrogen to obtain a white solid product (1.0 g, yield 75%). ¹ H NMR (400MHz, CDCl ₃ ): δ7.33-7.41 (m, 5H, Ph), 6.95 (s, 1H, NH), 5.27 (s, 2H, PhCH ₂ ), 4.44 (dt, J = 3.2, 10.0Hz, 1H, NHC H ), 3.22 (dd, J = 3.2, 16.4 Hz, 1H, COC H H), 2.97 (dd, J = 10.0, 16.4 Hz, 1H, COCH H ). ¹³ C NMR (400MHz, CDCl ₃ ):δ193.70,168.02,163.62,134.15,129.30,129.02,128.80,69.02,51.23,42.83.HRMS(ESI-TOF)m/z:[M+Na] ⁺ calculated for C ₁₂ H ₁₁ NO ₄ S 288.03065 ;Found 288.0305.

Example 2 Synthesis of DL-β-phenylpropylamino N-carboxythiocarbonyl ring anhydride monomer (β ³ -HPhg NTA)

Dissolve S-ethoxythiocarbonylthioglycolic acid (9.0g, 50.0mmol) in a flask of 200mL deionized water, add solid sodium hydroxide (8.0g, 200.0mmol) to the flask, mix and stir, and then add it after dissolving DL-β-Phenylpropylamino (8.3g, 50.0mmol) was mixed and stirred for reaction, and stirred at room temperature for about 48 hours; then 1M hydrochloric acid solution was slowly added dropwise, the pH was adjusted to about 3, and the aqueous phase was extracted with ethyl acetate After three organics, the organic phases were combined, the organic phases were then quenched with saturated brine, and dried over anhydrous magnesium sulfate; the crude product was purified by recrystallization with ethyl acetate and n-hexane to obtain a white solid intermediate (11.0g, product The rate is 87%).

Under the protection of nitrogen, the dry white intermediate (2.5g, 10.0mmol) was dissolved in a dry dichloromethane solution (100mL), mixed and stirred at 0°C in an ice bath, and then phosphorus tribromide ( 2.7g, 10.0mmol) solution. After the dripping is completed, the temperature is raised to 25°C and the reaction is continued for 6 hours. After the reaction is over, the organic phase is washed three times with 0°C deionized water, the organic phase is dried with anhydrous magnesium sulfate, filtered, and concentrated The solvent was removed to obtain the crude product, which was recrystallized and purified with ethyl acetate and n-hexane under the protection of nitrogen to obtain a white solid product (1.8 g, yield 85%). ¹ H NMR (400MHz, CDCl ₃ ): δ7.20-7.38 (m, 5H, Ph), 6.00 (s, 1H, NH), 4.50 (dd, J = 3.6, 9.2 Hz, 1H, NHC H ), 3.32 (dd, J = 3.6, 14.0 Hz, 1H, COC H H), 2.91 (dd, J = 9.6, 13.6 Hz, 1H, COCH H ). ¹³ C NMR (400MHz, CDCl ₃ ): δ197.90,166.89,134.52, 129.48,129.16,127.84,68.03,39.00.HRMS(EI-TOF)m/z:[M] ⁺ calculated for C ₁₀ H ₉ NO ₂ S 207.03540; Found 207.0353.

Example 3 Synthesis of N(α)-ZL-2,3-diaminopropionic acid N-carboxythiocarbonyl ring anhydride monomer (Cbz-β ² -LDAP NTA)

Dissolve S-ethoxythiocarbonylthioglycolic acid (9.0g, 50.0mmol) in a flask of 200mL deionized water, add solid sodium hydroxide (8.0g, 200.0mmol) to the flask, mix and stir, and then add it after dissolving N(α)-ZL-2,3-Diaminopropionic acid (11.9g, 50.0mmol) was mixed and stirred for reaction, and stirred at room temperature for about 48 hours; then 1M hydrochloric acid solution was slowly added dropwise to adjust the pH to about 3. , The organics in the aqueous phase were extracted three times with ethyl acetate, and then the organic phases were combined. The organic phase was then quenched with saturated brine and dried with anhydrous magnesium sulfate; the crude product was separated and purified with ethyl acetate and petroleum ether system silica gel column to obtain Colorless oily intermediate (13.9 g, 85% yield).

Under the protection of nitrogen, the dried colorless oil (3.3g, 10.0mmol) was dissolved in a dry dichloromethane solution (100mL), mixed and stirred in an ice bath at 0℃, and then phosphorus tribromide was added dropwise (2.7g, 10.0mmol) solution. After the dripping is completed, the temperature is raised to 25°C and the reaction is continued for 6 hours. After the reaction is over, the organic phase is washed three times with 0°C deionized water, and the organic phase is dried with anhydrous magnesium sulfate and filtered. The crude product was obtained by concentrating and removing the solvent. Under the protection of nitrogen, it was purified by recrystallization with ethyl acetate and n-hexane to obtain a white solid product (1.7 g, yield 60%). ¹ H NMR (400MHz, CDCl ₃ ): δ 7.33-7.40 (m, 5H, Ph), 6.60 (s, 1H, NH), 5.59 (s, 1H, CbzN H ), 5.13 (s, 2H, PhCH ₂ ), 4.56-4.61 (m, 1H, NHC H H), 3.83-3.88 (m, 1H, CH ₂ , NHCH H ), 3.42 (t, J = 12.4 Hz, 1H, COCH). ¹³ C NMR (400MHz, CDCl ₃ ):δ194.63,163.72,155.82,135.64,128.81,128.68,128.38,67.87,58.02,42.02.HRMS(EI-TOF)m/z:(M) ⁺ calculated for C ₁₂ H ₁₂ N ₂ O ₄ S 280.05178 ,found 280.0520.

Example 4 Synthesis of DL-β-norleucine N-carboxythiocarbonyl ring anhydride monomer (β ³ -HNle NTA)

Dissolve S-ethoxythiocarbonylthioglycolic acid (9.0g, 50.0mmol) in a flask of 200mL deionized water, add solid sodium hydroxide (8.0g, 200.0mmol) to the flask, mix and stir, and then add it after dissolving DL-β-norleucine (7.3g, 50.0mmol) was mixed and stirred for reaction, and the reaction was stirred at room temperature for about 48 hours; then 1M hydrochloric acid solution was slowly added dropwise, the pH was adjusted to about 3, and the water was extracted with ethyl acetate. The organics of the two phases were combined three times. The organic phases were then quenched with saturated brine and dried over anhydrous magnesium sulfate; the crude product was recrystallized and purified with ethyl acetate and n-hexane to obtain a white solid intermediate (10.5g, product). The rate is 90%).

Under the protection of nitrogen, the dry white intermediate (2.3g, 10.0mmol) was dissolved in a dry dichloromethane solution (100mL), mixed and stirred under an ice bath at 0℃, and then phosphorus tribromide (2.7 g, 10.0mmol) solution. After the dropwise addition is completed, the temperature is raised to 25°C and the reaction is continued for 6 hours. After the reaction is over, wash the organic phase with 0°C deionized water three times, dry the organic phase with anhydrous magnesium sulfate, filter, and concentrate to remove The crude product obtained from the solvent was purified by recrystallization with ethyl acetate and n-hexane under the protection of nitrogen to obtain a white solid product (1.2 g, yield 65%). ¹ H NMR (400MHz, CDCl ₃ ): δ7.11 (s, 1H, NH), 3.66-3.73 (m, 1H, CH, NHC H ), 2.87 (dd, J = 2.8, 16Hz, 1H, COC H H ), 2.65 (dd, J = 10.0, 16.4 Hz, 1H, COCH H ), 1.58-1.75 (m, 2H, CH ₃ CH ₂ CH ₂ C H ₂ CH), 1.34-1.41 (m, 4H, CH ₃ C H ₂ C H ₂ ), 0.92 (t, J=6.8 Hz, 3H, CH ₃ ). ¹³ C NMR (400MHz, CDCl ₃ ): δ196.01,165.75,49.42,45.78,33.63,27.45,22.38,13.94.HRMS( EI-TOF)m/z:[M] ⁺ calculated for C ₈ H ₁₃ NO ₂ S 187.06670; Found 187.0665.

Example 5 Synthesis of β ^2,3 -Norbornene N-carboxythiocarbonyl ^{Intracyclic Anhydride Monomer (β 2,3-} NB NTA)

Dissolve S-ethoxythiocarbonylthioglycolic acid (6.6g, 36.5mmol) in a 200mL flask of anhydrous DMF, add triethylamine (25.5mL, 182.6mmol) and DMAP (0.9g, 7.3mmol) into the flask After mixing, add β ^2,3 -norbornene amino acid (7.0g, 36.5mmol), mix and stir to react, and stir at room temperature for about 30 hours; then slowly add 1M hydrochloric acid solution dropwise to adjust the pH to After spinning off the DMF solvent, add 100 mL of deionized water, extract the organics in the aqueous phase three times with ethyl acetate, combine the organic phases, and then quench the organic phase with saturated brine, and dry with anhydrous magnesium sulfate; crude product It was separated and purified with ethyl acetate and n-hexane to obtain a white solid intermediate (3.5 g, yield 39.5%).

The dry white intermediate (1g, 4.1mmol) was dissolved in a dry dichloromethane solution (50mL) under the protection of nitrogen, mixed and stirred at 0°C in an ice bath, and then phosphorus tribromide (0.43mL, 4.5mmol) solution. After the dripping is completed, the temperature is raised to 25°C and the reaction is continued for 6 hours. After the reaction is over, the organic phase is washed three times with 0°C deionized water, the organic phase is dried with anhydrous magnesium sulfate, filtered, and concentrated to remove the solvent. The crude product was purified by recrystallization with ethyl acetate and n-hexane under the protection of nitrogen to obtain a white solid product (0.5 g, yield 61.1%). ¹ H NMR (400MHz, CDCl ₃ ): δ7.61 (s, 1H), 3.77 (d, J = 8.3 Hz, 1H), 2.79 (s, 1H), 2.73 (d, J = 8.3 Hz, 1H), 2.42 (s, 1H), 1.68 (s, 3H), 1.43 (s, 1H), 1.31 (d, J = 10.6 Hz, 2H). ¹³ C NMR (400MHz, CDCl ₃ ): δ197.41,164.10,77.27,58.44 ,54.59,45.82,43.69,34.24,29.70,25.24.HRMS(ESI-TOF)m/z:[M] ⁺ calculated for C ₉ H ₁₁ NO ₂ S 198.05887; Found 198.0597.

Example 6 Synthesis of β ^2,3 -Cyclohexyl N-carboxythiocarbonyl Intracyclic Anhydride Monomer (β ^2,3- CH NTA)

Dissolve S-ethoxythiocarbonylthioglycolic acid (4.8g, 26.7mmol) in a 150mL flask of anhydrous DMF, add triethylamine (18.5mL, 133.5mmol) and DMAP (0.7g, 5.4mmol) into the flask After mixing, add β ^2,3 -cyclohexyl amino acid (4.8g, 26.7mmol) and stir to react, and stir at room temperature for about 30 hours; then slowly add 1M hydrochloric acid solution dropwise to adjust the pH to 3. After spinning off the DMF solvent, add 100 mL of deionized water, extract the organics in the aqueous phase three times with ethyl acetate, then combine the organic phases, and then quench the organic phase with saturated brine and dry with anhydrous magnesium sulfate; Column separation and purification of ethyl acetate and n-hexane gave a colorless oily intermediate (2.8 g, yield 39.7%).

The dry colorless oily intermediate (1g, 4.33mmol) was dissolved in a dry dichloromethane solution (50mL) under the protection of nitrogen, mixed and stirred under an ice bath at 0℃, and then phosphorus tribromide (0.45 mL, 4.76mmol) solution. After the dripping is completed, the temperature is raised to 25°C and the reaction is continued for 6 hours. After the reaction is over, the organic phase is washed three times with 0°C deionized water, the organic phase is dried with anhydrous magnesium sulfate, filtered, and concentrated to remove The crude product obtained from the solvent was purified by recrystallization with ethyl acetate and n-hexane under nitrogen protection to obtain a white solid product (0.42 g, yield 45.3%). ¹ H NMR (400MHz, CDCl ₃ ): δ7.56 (s, 1H), 3.90–3.78 (m, 1H), 2.81 (d, J = 9.1Hz, 1H), 2.06 (d, J = 8.9Hz, 1H ), 1.88 (d, J = 3.4 Hz, 1H), 1.69 (dd, J = 32.5, 11.3 Hz, 4H), 1.49 (d, J = 45.6 Hz, 2H). ¹³ C NMR (400MHz, CDCl ₃ ): δ198.94,166.14,77.27,50.32,49.48,29.36,24.23,23.19.δ197.41,164.10,77.27,58.44,54.59,45.82,43.69,34.24,29.70,25.24.HRMS(ESI-TOF)m/z: [M] ⁺ calculated for C ₈ H ₁₁ NO ₂ S 186.05887; Found 186.0603.

Example 7 Synthesis of γ ^2,3 -Phenyl N-carboxythiocarbonyl Intracyclic Anhydride (γ ^2,3- Phe-NTA)

Dissolve S-benzyloxythiocarbonylthioglycolic acid (6.5g, 26.7mmol) in a 150mL flask of anhydrous DMF, add triethylamine (18.5mL, 133.5mmol) and DMAP (0.7g, 5.4mmol) into the flask Mix and stir in the mixture, after dissolving, add 2-(aminomethyl)benzoic acid (4.0g, 26.7mmol), mix and stir the reaction, and stir the reaction at room temperature under nitrogen protection for about 72 hours; then slowly add 1M hydrochloric acid solution dropwise, Adjust the pH to about 3, spin off the DMF solvent, add 100 mL of deionized water, extract the organics in the aqueous phase three times with ethyl acetate, then combine the organic phases, then the organic phases are quenched with saturated brine and dried with anhydrous magnesium sulfate The crude product was separated and purified with ethyl acetate and n-hexane to obtain a colorless oily intermediate (2.5g, yield 30.8%).

The dried colorless oily intermediate (1g, 3.32mmol) was dissolved in a dry dichloromethane solution (50mL) under the protection of nitrogen, mixed and stirred at 0℃ in an ice bath, and then phosphorus tribromide (0.45 mL, 4.76mmol) solution. After the dripping is completed, the temperature is raised to 25°C and the reaction is continued for 6 hours. After the reaction is over, the organic phase is washed three times with 0°C deionized water, the organic phase is dried with anhydrous magnesium sulfate, filtered, and concentrated to remove The crude product obtained from the solvent was purified by recrystallization with ethyl acetate and n-hexane under the protection of nitrogen to obtain 0.1 g of a white solid product.

Example 8 N-hexylamine initiates the polymerization of ^{Bn-β 3 -LCHG NTA}

In a glove box protected by nitrogen, accurately weigh n-hexylamine (101.2 mg, 1.0 mmol) and prepare a 0.2M solution with dry N,N-dimethylformamide for use.

The Bn-β ³ -LCHG NTA monomer (53.0 mg, 0.2 mmol) was accurately weighed, and dried N,N-dimethylformamide (1 mL) was dissolved in a reaction flask equipped with a stir bar.

In the stirred reaction flask, add 0.2M n-hexylamine (50 μL) solution. The mixture was stirred at room temperature for 3 days in a glove box, and the resulting solution was transferred out of the glove box.

In the above reaction mixture, pour cold n-hexane (45 mL), the precipitated white flocs are collected by centrifugation, dried in a gas stream, and re-dissolved in tetrahydrofuran (2.0 mL), and then a large amount of cold n-hexane (45 mL) is added ,precipitation. This dissolution-precipitation process was repeated three times in total to obtain poly-L-aspartic acid 1-benzyl ester homopolymer (41.3 mg, yield 96.0%).

The polymer identified by gel permeation chromatography (GPC) had a molecular weight Mn=4270 and a molecular weight distribution PDI=1.18.

Example 9 3-azidopropylamine initiates the polymerization of ^{Bn-β 3 -LCHG NTA}

In a glove box protected by nitrogen, accurately weigh 3-azidopropylamine (100.1 mg, 1.0 mmol), and prepare a 0.2M solution with dry N,N-dimethylformamide for use.

In a stirred reaction flask, add a 0.2M solution of 3-azidopropylamine (100 μL). The mixture was stirred and reacted in a glove box at room temperature for 3 days, and the resulting solution was transferred out of the glove box.

Pour cold n-hexane (45 mL) into the above reaction mixture. The precipitated white flocs are collected by centrifugation, dried in air flow, and re-dissolved in tetrahydrofuran (2.0 mL), and then a large amount of cold n-hexane (45 mL) is added for precipitation . This dissolution-precipitation process was repeated three times in total to obtain poly-L-aspartic acid 1-benzyl ester homopolymer (40.3 mg, yield 93.5%).

The molecular weight Mn of the polymer identified by GPC was 2230 and the molecular weight distribution PDI was 1.12.

Example 10 Alkynylpropylamine initiates the polymerization of Bn-β ^{3 -LCHG NTA}

In a glove box protected by nitrogen, accurately weigh alkynyl propylamine (55.1 mg, 1.0 mmol) and prepare a 0.2M solution with dry N,N-dimethylformamide for use.

The Bn-β ³ -LCHG NTA (53.0 mg, 0.2 mmol) was accurately weighed, and the dried N,N-dimethylformamide (1 mL) was dissolved in a reaction flask equipped with a stir bar.

In a stirred reaction flask, add a 0.2M alkynyl propylamine (100 μL) solution. The mixture was stirred and reacted in a glove box at room temperature for 3 days, and the resulting solution was transferred out of the glove box.

Pour cold n-hexane (45 mL) into the above reaction mixture. The precipitated white flocs are collected by centrifugation, dried in air flow, and re-dissolved in tetrahydrofuran (2.0 mL), and then a large amount of cold n-hexane (45 mL) is added for precipitation . This dissolution-precipitation process was repeated three times in total to obtain poly-L-aspartic acid 1-benzyl ester homopolymer (40.2 mg, yield 95.3%).

The polymer identified by GPC had a molecular weight Mn=2010 and a molecular weight distribution PDI=1.12.

Example 11 2-Triphenylmethylmercaptoethylamine initiates the polymerization of ^{Bn-β 3 -LCHG NTA}

In a glove box protected by nitrogen, accurately weigh 2-triphenylmethyl mercaptoethylamine (319.5 mg, 1.0 mmol) and prepare a 0.2M solution with dry N,N-dimethylformamide for use.

In a stirred reaction flask, add a 0.2M solution of 2-triphenylmethylmercaptoethylamine (50μL). The mixture was stirred and reacted in a glove box at room temperature for 3 days, and the resulting solution was transferred out of the glove box.

Pour cold n-hexane (45 mL) into the above reaction mixture. The precipitated white flocs are collected by centrifugation, dried in air flow, and re-dissolved in tetrahydrofuran (2.0 mL), and then a large amount of cold n-hexane (45 mL) is added for precipitation . This dissolution-precipitation process was repeated three times in total to obtain poly-L-aspartate 1-benzyl ester homopolymer (40.1 mg, yield 90.7%).

The polymer identified by GPC had a molecular weight Mn=3950 and a molecular weight distribution PDI=1.13.

Example 12 p-tert-butylbenzylamine initiates β ³ -HPhg NTA polymerization under open conditions

Under outdoor open conditions, accurately weigh p-tert-butylbenzylamine (16.3g, 100mmol), and prepare a solution with a concentration of 0.5M with dry N,N-dimethylformamide for use.

The β ³ -HPhg NTA monomer (10 g, 48.3 mmol) was accurately weighed, and the undried N,N-dimethylformamide was dissolved in a reaction flask equipped with a stir bar to prepare a 0.5 M solution.

In a stirred reaction flask, a solution of p-tert-butylbenzylamine (4.8 mL) with a concentration of 0.5 M was added. The mixture was stirred and reacted in a glove box at room temperature for 3 days, and the resulting solution was transferred out of the glove box.

Pour cold n-hexane (200 mL) into the above reaction mixture. The precipitated white flocs are collected by centrifugation, dried in an air stream, and re-dissolved in tetrahydrofuran (20 mL), then a large amount of cold n-hexane is added for precipitation, and the mixture is stirred overnight. Then, it was filtered to obtain 5.73 g (yield 76.5%) of a polyhomopolymer.

The molecular weight of the polymer identified by ¹ H NMR was Mn=3106 and DP=20.

Example 13 N-hexylamine initiates random copolymerization of ^{β 3} -HNle NTA and β ^{3 -HPhg NTA monomers}

Accurately weigh β ³ -HNle NTA monomer (37.5 mg, 0.2 mmol) and β ³ -HPhg NTA monomer (41.4 mg, 0.2 mmol), and dissolve in dry N,N-dimethylformamide (2 mL) In a reaction flask equipped with a stir bar.

In the stirred reaction flask, add 100 μL of 0.2M n-hexylamine solution. The mixture was stirred and reacted in a glove box at room temperature for 3 days, and the resulting solution was transferred out of the glove box.

Pour cold n-hexane (45 mL) into the above reaction mixture. The precipitated white flocs were collected by centrifugation, dried in air flow, and re-dissolved in tetrahydrofuran (2.0 mL), and then a large amount of cold n-hexane was added for precipitation. This dissolution-precipitation process was repeated three times in total to obtain 52.4 mg (yield 92.0%) of random copolymer.

The polymer identified by Maldi-Tof-Ms had a molecular weight Mn=2750 and a molecular weight distribution PDI=1.15.

Example 14 2-Triphenylmethylmercaptoethylamine initiates β ³ -HNle NTA and β ² -LDAP NTA monomers to prepare deprotected blend type β-amino acid copolymer

Accurately weigh 2-triphenylmethyl mercaptoethylamine (319.5 mg, 1.0 mmol) and prepare a 0.2M solution with dry N,N-dimethylformamide for use.

Accurately weigh β ³ -HNle NTA monomer ( ^{37.5 mg, 0.2 mmol) and β 2} -LDAP NTA monomer (56.0 mg, 0.2 mmol), and dissolve in dry N,N-dimethylformamide (2 mL) In a reaction flask equipped with a stir bar.

In a stirred reaction flask, add a 0.2M solution of 2-triphenylmethylmercaptoethylamine (100 μL). The mixture was stirred and reacted in a glove box at room temperature for 3 days, and the resulting solution was transferred out of the glove box.

Pour cold n-hexane (45 mL) into the above reaction mixture. The precipitated white flocs were collected by centrifugation, dried in air flow, and re-dissolved in tetrahydrofuran (2.0 mL), and then a large amount of cold n-hexane was added for precipitation. This dissolution-precipitation process was repeated three times in total to obtain a random copolymer (68.3 mg, yield 90%).

The polymer identified by ¹ H NMR and Maldi-Tof-Ms had a molecular weight Mn=3800 and a molecular weight distribution PDI=1.15.

Furthermore, the obtained block-type β-amino acid copolymer was dissolved in 1.0 mL of trifluoroacetic acid and 1.0 mL of acetic acid solution containing 33% hydrogen bromide. After reacting at room temperature for 3 hours, the solvent was blown dry and re-dissolved in methanol (1.0 mL), then add a large amount of cold ether to precipitate. This dissolution-precipitation process was repeated three times in total to obtain a deprotected blended β-amino acid copolymer.

Example 15 Preparation of deprotected block type β-amino acid copolymer with 2-triphenylmethylmercaptoethylamine β ³ -HPhG NTA monomer and Cbz-β ^{2 -LDAP NTA monomer}

Accurately weigh β ³ -HPhG NTA monomer (41.4 mg, 0.2 mmol) and Cbz-β ² -LDAP NTA monomer (56.0 mg, 0.2 mmol), and use dry N,N-dimethylformamide (1 mL) Dissolve in two reaction flasks equipped with a stir bar.

Add a 0.2M solution of 2-triphenylmethylmercaptoethylamine (100μL) to a stirred reaction flask containing Cbz-β ^{2 -LDAP NTA monomer.} Stir the mixture in a glove box at room temperature. After the reaction is over, the prepared β ³ -HPhG NTA monomer is added to the reaction flask and the reaction is continued for 3 days. After the reaction is over, the resulting solution is transferred out of the glove box.

Pour cold n-hexane (45 mL) into the above reaction mixture. The precipitated white flocs were collected by centrifugation, dried in air flow, and re-dissolved in tetrahydrofuran (2.0 mL), and then a large amount of cold n-hexane was added for precipitation. This dissolution-precipitation process was repeated three times in total to obtain β-amino acid block polymer (70.3 mg, yield 88.0%).

The polymer identified by GPC had a molecular weight Mn=3380 and a molecular weight distribution PDI=1.25.

Furthermore, the obtained block-type β-amino acid copolymer was dissolved in 1.0 mL of trifluoroacetic acid and 1.0 mL of acetic acid solution containing 33% hydrogen bromide. After reacting at room temperature for 3 hours, the solvent was blown dry and re-dissolved in methanol (1.0 mL), then add a large amount of cold ether to precipitate. This dissolution-precipitation process was repeated three times in total to obtain the deprotected block-type β-amino acid copolymer.

Example 16 A blended α/β amino acid copolymer prepared by polymerization of 2-triphenylmethyl mercaptoethylamine initiated L-Lys(Boc)α-NCA monomer and β ^{3 -HPhG NTA monomer}

In a glove box protected by nitrogen, the L-Lys(Boc)α-NCA monomer (54.4mg, 0.2mmol) and β ³ -HPhG NTA (41.4mg, 0.2mmol) were accurately weighed, and dried with dry N, N- Dimethylformamide (2 mL) was dissolved in a reaction flask equipped with a stir bar. Then a solution of 0.2M 2-triphenylmethylmercaptoethylamine (100 μL) was added. The mixture was stirred and reacted in a glove box at room temperature for 3 days, and the resulting solution was transferred out of the glove box.

Pour cold n-hexane (45 mL) into the above reaction mixture. The precipitated white flocs were collected by centrifugation, dried in air flow, and re-dissolved in tetrahydrofuran (2.0 mL), and then a large amount of cold n-hexane was added for precipitation. This dissolution-precipitation process was repeated three times in total to obtain a blended α/β-amino acid polymer (75.0 mg, yield 92.2%). The polymer identified by GPC had a molecular weight Mn=4250 and a molecular weight distribution PDI=1.17.

Furthermore, the obtained blended α/β-amino acid polymer was dissolved in 2.0 mL of trifluoroacetic acid, and after reacting at room temperature for 3 hours, the solvent was blown dry and re-dissolved in methanol (1.0 mL), and then a large amount of cold ether was added. precipitation. This dissolution-precipitation process was repeated three times in total to obtain a deprotected blended α/β-amino acid polymer.

Example 17 Block type α/β amino acid copolymer prepared by polymerization of L-Lys(Boc)α-NCA monomer and β ^{3 -HPhG NTA monomer by 2-triphenylmethyl mercaptoethylamine}

Refer to Example 16, after 2-triphenylmethylmercaptoethylamine has initiated the polymerization of L-Lys(Boc)α-NCA, then add β ³ -HPhG NTA monomer, and wait for the second monomer to react. , Post-processing the polymer and deprotecting the polymer to prepare a block-type α/β amino acid copolymer. The first block polymer α amino acid polymer molecular weight Mn = 2500 and molecular weight distribution PDI = 1.18 was identified by GPC. The block type α/β amino acid copolymer obtained after connecting β amino acid polymer has molecular weight Mn = 3960 and molecular weight Distribution PDI=1.26.

Example 18 The application of 2-triphenylmethyl mercaptoethylamine initiated binary copolymerization of a mixture of L-Lys(Boc)α-NCA monomer and β ³ -HPhG NTA monomer as a solution antibacterial material

The polymer synthesis method is the same as in Example 16, wherein ^{the ratio between the L-Lys(Boc) α-NCA monomer and β 3} -HPhG NTA monomer is 5:5. After the reaction, the deprotected polymer purified by post-treatment was dissolved again in 5 mL of ultrapure water, filtered, and lyophilized for the next biological activity test.

The minimum inhibitory concentration (MIC) test uses the following method. Bacteria are cultured overnight with LB liquid medium (Luria-Bertani Broth) in a 37°C shaker at 150 rpm. The cultured bacterial cells are collected by centrifugation and redispersed to In MH (Mueller-Hinton Broth) medium, read the absorbance (OD ₆₀₀ ) at 600 nm with a microplate reader (when OD ₆₀₀ =1, the concentration of Staphylococcus aureus is approximately 1.5×10 ⁹ cfu/mL). Dilute the bacterial solution with MH medium to 2×10 ⁵ cfu/mL for later use. The polymer was diluted with MH medium in a 96-well plate at a concentration ranging from 400 to 3.13 μg/mL. Then add 50 μL of the diluted bacterial solution to each well to make the total volume of the bacterial solution and polymer 100 μL, shake it slightly for 10 seconds, and incubate it in a mold incubator at 37°C for 9 hours. Then read the OD ₆₀₀ with a microplate reader. In the same 96-well plate, there are 4 wells with only MH medium added as a negative control, and 4 wells with MH medium and bacterial liquid (without polymer) as a positive control . Two parallel samples are tested each time and repeated twice at different times. Using the formula for the percentage of bacterial growth in each hole

Calculation. Then draw a line graph of the calculated data, and the MIC value is the lowest concentration of the polymer that inhibits the growth of bacteria.

The minimum inhibitory concentration of the tested polymer against a variety of bacteria, including methicillin-resistant Staphylococcus aureus USA300 (Staphylococcus aureus USA300), methicillin-resistant Staphylococcus aureus Mu50 (Staphylococcus aureus Mu50), Bacillus subtilis ( Bacillus subtilis BR-151), Escherichia coli JM109, Pseudomonas aeruginosa ATCC ₉₀ 27, Multidrug-resistant Pseudomonas aeruginosa ATCC ₁₅ 442, Sulfamethoxazole and Tetracycline is naturally resistant to Pseudomonas aeruginosa O1 and Acinetobacter baumannii ATCC BAA-747. The minimum inhibitory concentrations of the tested amino acid polymers against the positive bacteria Staphylococcus aureus USA300, Staphylococcus aureus Mu50 and Bacillus subtilis BR-151 were 12.5μg/mL, 12.5μg/mL and 3.13μg/mL, respectively, against the negative bacteria Pseudomonas aeruginosa The minimum inhibitory concentrations of Pseudomonas aeruginosa ATCC ₁₅ 442, Pseudomonas aeruginosa ATCC ₉₀ 27 and Pseudomonas aeruginosa O1 are all 50.0μg/mL; the lowest against the negative bacteria Acinetobacter baumannii ATCC BAA-747 and Escherichia coli JM109 The inhibitory concentrations were 25.0μg/mL and 100.0μg/mL, respectively. The obtained MIC results prove that such amino acid polymers can have strong and broad-spectrum antibacterial activity.

Example 19 The application of 2-triphenylmethyl mercaptoethylamine initiated ^{binary copolymerization of a mixture of β 3} -HNle NTA and β ² -LDAP NTA as a solution antifungal material

The polymer synthesis method was the same as in Example 14. The deprotected polymer was dissolved in ultrapure water again, filtered and lyophilized for the next biological activity test. The minimum inhibitory concentration (MFC) of the tested amino acid polymer against Candida albicans K1 and Cryptococcus neoformans was 3.13μg/mL (C.albicans K1) and 1.56μg/mL (C.neoformans).

Example 20 A blended α/β amino acid copolymer prepared by polymerization of L-Lys(Boc) α-NCA monomer and β ^{3 -HPhG NTA monomer as a surface coating antibacterial} Material application

The polymer synthesis method is the same as that in Example 16. The difference is that the deprotected C-terminal amino acid polymer with a sulfhydryl group is grafted on the surface of the gold sheet. The surface sterilization test adopts the following method. The bacteria use LB liquid medium (Luria- Bertani Broth) was cultured overnight in a shaker at 37°C at 150 rpm. After the culture is completed, take 7.5 mL of the bacterial solution from the Erlenmeyer flask and centrifuge at 4000 rpm for 5 minutes to collect the bacteria, and re-disperse it in PBS and re-centrifuge. Repeat the PBS dispersion bacterial solution centrifugation three times to collect the bacterial solution, and read at 600nm with a microplate reader The absorbance (OD ₆₀₀ ) was quantified to the number of colonies. The bacterial solution was diluted with PBS to 1×10 ⁵ cfu/mL for later use. Put the prepared polymer antibacterial surface into a 24-well plate with PBS as a control. Add 80 μL of the above-mentioned concentration of bacterial solution to the surface of the polymer gold piece, of which 80 μL of bacterial solution was directly added to the well plate as a blank control, and PBS was added to the blank well plate to control humidity, and the mold was placed in a 37 ℃ mold incubator for static cultivation for 2.5 After hours, take out the orifice plate, add 1920μL PBS to the orifice to dilute, sonicate for 3min, mix for 2min under a homogenizer, take out 30μL with a pipette and add to the LB agar medium to spread, and place it in a 37℃ mold incubator Inside the culture. After the colony is counted, the surface antibacterial activity is analyzed. The experimental group is recorded as C _sample and the blank control is recorded as C _control . The antibacterial activity (bacterial killing rate) of the substrate surface is calculated by the following formula:

Test the surface sterilization of the blended α/β amino acid copolymer on Methicillin-resistant Staphylococcus aureus (MRSA, methicillin-resistant Staphylococcus aureus). The experimental results prove that the surface of the blended α/β amino acid copolymer has a bactericidal rate of 99.9% against MRSA and has excellent surface bactericidal efficacy.

Example 21 2-Triphenylmethylmercaptoethylamine initiates the ^{binary copolymerization of a mixture of β 3} -HNle NTA and β ² -LDAP NTA monomers as a cell adhesion material

The polymer synthesis method is the same as that in Example 14, except that the ^{ratio x:y of β 3} -HNle NTA and β ² -LDAP NTA monomers is 0:10 to 7:3. After the polymerization reaction, the reaction solution was transferred to a 50 mL centrifuge tube, and 45 mL of petroleum ether was added to make a white precipitate. The resulting precipitate was separated by centrifugation and re-dissolved in 1.5 mL of tetrahydrofuran, and another 45 mL of n-hexane was added to make it precipitate. Precipitation: Two or more monomers synthesized are purified through the three-time dissolution-precipitation process and mixed in a set ratio to copolymerize the resulting polymer. Add 2 mL of trifluoroacetic acid and 5% (v/v) triethylsilane to the drained polymer. After shaking gently overnight at room temperature, blow off the excess trifluoroacetic acid to obtain a viscous liquid that dissolves in After 0.5 mL of methanol, 45 mL of frozen ether was added to make a white precipitate precipitate, and the dissolution-precipitation process was repeated three times to obtain a random polymer with side chain amino groups and terminal sulfhydryl groups deprotected. The deprotected polymer was again dissolved in 5 mL of ultrapure water, filtered and lyophilized, and used for the next biological activity test.

Graft the amino acid polymer onto the surface of the glass substrate. The specific method is as follows. Use 3-aminopropyltriethoxysilane as the amino modifier on the glass surface to modify and clean the surface-activated glass slide, and then modify the aminated glass slide with PEG , And finally connect the amino acid polymer and the positive control polypeptide (RGD). Trypsin digestion to collect the cells in a centrifuge tube, adjust the cell density to 8×10 ⁴ cells/mL; inoculate the cells into the holes on the surface of the amino acid polymer; put the polymer surface in a petri dish and place it in a 37°C incubator In the cultivation. After the cells are incubated for 2 hours, observe the attachment, spreading and agglomeration of the cells on the polymer surface under an inverted microscope; then immerse the polymer surface with the cells in the culture medium and continue culturing for 24 to 48 hours, and treat multiple areas An inverted fluorescence microscope was used to observe the morphology of cells adhering to the surface of the amino acid polymer, and to calculate the coverage area (%) of the cell surface. The experimental results show that on the glass surface grafted with amino acid polymer, mouse embryonic fibroblasts (NIH 3T3) exhibited different adhesion effects at 48 hours ^{. The ratio of β 3} -HNle and β ² -LDAP is 6:4. The cell adhesion effect is similar to that of the positive control RGD. At the same time, the number of cells grown on the surface ^{of Poly[(β 3} -HNle) _0.6 -(β ² -LDAP) _{0.4] polymer grafted with the positive control RGD is obtained by counting.} 80% of the number of cells on the surface. Cell adhesion is a key step in the role of cells and materials in tissue engineering. Only through adhesion can cells undergo a series of behaviors such as proliferation, migration, and differentiation. Therefore, supporting cell adhesion is a must in the application of biomaterials in tissue engineering. Indispensable nature.

Example 22 The application of 2-triphenylmethyl mercaptoethylamine initiating the ^{binary copolymerization of a mixture of β 3} -HNle NTA and β ² -LDAP NTA monomers as an anti-tumor material

The polymer synthesis method is the same as that in Example 14, except that the ^{ratio x:y of β 3} -HNle NTA and β ² -LDAP NTA monomers is 0:10 to 7:3. Cytotoxicity test (MTT cell proliferation detection) adopts the following method to inoculate NCI-H460 cells, U87 cells, and B16 cells with a ^{density of 3×10 4 into 96-well plates, each with a volume of 100 μL.} The cells were cultured at 37°C for 24 hours. After removing the old medium, add medium containing amino acid polymers of different concentrations, and set three multiple wells for each concentration. After culturing the cells at 37°C for 24 hours, 10 μL of MTT solution (5 mg/mL, prepared in PBS) was added to each well, and the incubation was continued for 4 hours to terminate the culture. Carefully aspirate the culture supernatant in the wells, add DMSO (150 μL) to each well, and shake for 10 minutes on a shaker to fully dissolve the crystals. On the same 96-well plate, cells treated with no amino acid polymer were included as a control group, and a blank group without cells inoculated with only DMSO added. Select the wavelength of 570nm, measure the light absorption value (OD value) of each well on the microplate reader, and calculate the cell survival rate:% cell survival=(OD ^polymer- OD ^blank )/(OD ^control- OD ^blank )×100. On this basis, a curve of cell survival rate with the concentration of amino acid polymer is drawn, and the lowest amino acid concentration (IC ₅₀ ) that causes 50% of mammalian cell death is obtained from the curve.

Test a series of polymers with different amino acid ratios (ratio from 0% β ³ -HNle+100% β ² -LDAP to 30% β ³ -HNle+70% β ² -LDAP) for a variety of tumor cells (NCI-H460 lung cancer) Cells, U87 glioma cells, B16 melanoma cells), the experimental results show that when β ³ -HNle: β ² _{-LDAP = 6:4, the IC 50} of the amino acid polymer to NCI-H460 lung cancer cells is 100 μg/ mL, showing anti-tumor effect.

Example 23 Preparation of 2-triphenylmethylmercaptoethylamine β ³ -HPhG NTA monomer and Cbz-β ² -LDAP NTA monomer. Application of deprotected block type β-amino acid copolymer as self-assembly material

The polymer synthesis method was the same as in Example 15. The deprotected polymer was dissolved again in 5 mL of ultrapure water, filtered and lyophilized, and used for the next self-assembly test.

The preparation of the polymer self-assembled structure adopts the following method. Dissolve 1 mg of the deprotected amphiphilic polymer in a corresponding volume of ultrapure water, configure it as a 0.2 mg/mL or 0.5 mg/mL solution, and keep the solution at 390 rpm After stirring at medium speed for 2h at the rotating speed, it was left still for 12h. The self-assembly solution was filtered with a 0.8μm filter and DLS test was performed.

The self-assembled samples were tested for particle size and dispersion by dynamic light scattering (DLS). The sample is placed in a PS cuvette, the volume of each test sample is about 1.5 mL, and the test is repeated three times for each sample, the test temperature is 25°C, and the test angle is set to 90 degrees. Data processing uses the cumulative analysis of the experimental correlation function, and uses the Stokes-Einstein equation to calculate the diffusion coefficient.

The results of the DLS experiment show that the amino acid polymer forms a relatively stable self-assembled structure in water, with a particle size of 85 nm and a dispersive PD of 0.375.

Comparative Example 1 Polymerization of (S)-4-oxo-2-azetidinecarboxylic acid benzyl ester initiated by p-tert-butylbenzoyl chloride

For the polymerization method of β-lactam, refer to the reported literature (J.Am.Chem.Soc.2009,131,1589-1597), and accurately weigh (S)-4-oxo- in a glove box protected by nitrogen. Benzyl 2-azetidine carboxylate (41.0 mg, 0.2 mmol) was used to prepare a 0.2M solution with dry tetrahydrofuran (1 mL) for later use.

Accurately weigh the p-tert-butyl benzoyl chloride (3.9mg, 0.02mmol), dissolve it in tetrahydrofuran to form a 0.2M solution, and prepare the spare (S)-4-oxo-2-azetidine carboxylate. Add 100 μL of p-tert-butyl benzoyl chloride solution to the benzyl acid solution, add magnets and stir.

In a stirred reaction flask, a 0.2M lithium hexamethyldisilazide (Li(NSiMe ₃ ) ₂ , 250 μL) solution in tetrahydrofuran was added. The mixture was stirred and reacted in a glove box at room temperature for 6 hours, and the resulting solution was transferred out of the glove box.

Pour cold n-hexane (45 mL) into the above reaction mixture. The precipitated white flocs are collected by centrifugation, dried in air flow, and re-dissolved in tetrahydrofuran (2.0 mL), and then a large amount of cold n-hexane (45 mL) is added for precipitation . This dissolution-precipitation process was repeated three times in total to obtain 13.7 mg (yield 31.5%) of product.

The loss of the benzene ring of the polymer detected by proton nuclear magnetic resonance ( ¹ H NMR) indicates that the ester group cannot exist stably under strong base ring-opening conditions.

Comparative Example 2 p-tert-butyl benzoyl chloride initiated polymerization of 4-phenyl-2-azetidinone under open conditions

Under outdoor open conditions, accurately weigh 4-phenyl-2-azetidinone (29.5 mg, 0.2 mmol) and prepare a 0.2M concentration solution with dry tetrahydrofuran (1 mL) for use.

Accurately weigh the p-tert-butyl benzoyl chloride (3.9mg, 0.02mmol), dissolve it in tetrahydrofuran to form a 0.2M solution, and add the p-tert-butyl benzoyl chloride to the prepared 4-phenyl-2-azetidinone solution. A solution of tert-butyl benzoyl chloride (50 μL) was added to the magnet and stirred.

In a stirred reaction flask, a 0.2M lithium hexamethyldisilazide (Li(NSiMe ₃ ) ₂ , 125 μL) solution in tetrahydrofuran was added. The mixture was stirred and reacted in a glove box at room temperature for 6 hours, and the resulting solution was transferred out of the glove box.

Cold n-hexane (45 mL) was poured into the above reaction mixture, and no precipitated polymer product was obtained.

Therefore, compared with the traditional β-lactam ring-opening polymerization system, the advantages of the present invention are:

1. The traditional β-lactam ring-opening polymerization system has harsh polymerization conditions, such as the commonly used t-BuBzCl/LiHMDS initiating system to polymerize some monomers containing ester groups and other functional groups, which are usually not normally controllable. This makes it very difficult for the traditional β-lactam ring-opening polymerization method to prepare β-amino acid polymers with side chains containing functional groups unstable to alkalis, which limits the use of this type of side chain β-amino acid polymers in biology. Applications in the field of medicine and biomaterials.

The amine initiates the ring-opening polymerization of β-NTA or γ-NTA of the present invention, and the polymerization conditions are mild, especially for the polymerization of alkali-labile β-NTA or γ-NTA monomers. A side reaction.

2. The traditional β-lactam ring-opening polymerization system is very sensitive to moisture. Therefore, it needs ultra-dry solvent and ultra-dry environment to react. This extremely stringent requirement on reaction conditions and reaction environment not only places high requirements on the synthesis technology of researchers, but also greatly hinders the synthetic screening and large-scale synthesis of peptide libraries.

The amine initiates the ring-opening polymerization of β-NTA or γ-NTA of the present invention, which can be operated in a glove box without any protection, in a conventional solvent that does not remove water, and succeeds in an open container. operating. This reduces the technical and experience requirements of reaction operators a lot, which is conducive to the wide application of more researchers.

3. During the amine-initiated β-NTA or γ-NTA ring-opening polymerization process of the present invention, other types of α-NCA or α-NTA can be added at the same time to prepare a blended or block type α/β/γ Amino acid copolymer. This greatly increases the cycle and cost of the traditional preparation method (a-amino acid and β-amino acid are prepared by a solid-phase method through a condensation reaction).

It should be understood that the β-amino acid polymer prepared by the β-NTA ring-opening polymerization of the present invention is completely different from the β-amino acid polymer prepared by the traditional β-lactam ring-opening polymerization. This is mainly because the polymerization kinetics of these two ring-opening polymerization methods are completely different; at the same time, the new β-amino acid polymer prepared by β-NTA ring-opening polymerization is polymerization from the C-terminal to the N-terminal of the polymerized monomer, and β- The β-amino acid polymer prepared by the ring-opening polymerization of lactam is the polymerization from the N-terminus to the C-terminus of the polymerized monomer. These two points lead to differences in the β-amino acid polymer itself, molecular weight distribution, and physical and chemical properties prepared by the two methods. In addition, the two β-amino acid polymers prepared by β-NTA ring-opening polymerization and β-lactam ring-opening polymerization have different structures at both ends. The front and rear groups of the polymer of β-NTA ring-opening polymerization are primary amine functional groups and The front and back end groups of the polymer of β-lactam ring-opening polymerization are acid chloride functional groups and lactam.

All documents mentioned in the present invention are cited as references in this application, as if each document was individually cited as a reference. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

Claims

A β- or γ-amino acid N-carboxythiocarbonyl intracyclic anhydride (β-NTA, γ-NTA) monomer, which has the structure shown in formula (I):

among them,

s is 0 or 1;

R 1 , R 2 , R 3 , R 4 , R 11 and R 21 are each independently selected from the following group of substituted or unsubstituted groups: hydrogen, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, C 1 -C 6 alkylhydroxyl group, C 1 -C 6 alkoxy group, C 1 -C 6 alkylsulfonyl group, C 2 -C 6 alkenyl group, C 2 -C 6 alkynyl group, C 3 -C 12 ring Alkyl, C 6 -C 12 aryl, 5-12 membered heteroaryl, 5-12 membered heterocyclic group, C 1 -C 6 alkyl-C 6 -C 12 aryl, amino,
C 1 -C 6 alkylguanidino group, C 1 -C 6 alkyl ester group, thio C 1 -C 6 alkyl ester group; and when s is 0, R 1 , R 2 , R 3 and R 4 Can not be hydrogen at the same time;

Or R 1 and R 2 and the carbon atoms to which they are connected together form a substituted or unsubstituted C 3 -C 12 cycloalkyl group, a substituted or unsubstituted C 4 -C 12 cycloalkenyl group or a 5-12 membered heterocyclic group ；

Or R 3 and R 4 and the carbon atoms to which they are connected together form a substituted or unsubstituted C 3 -C 12 cycloalkyl group, a substituted or unsubstituted C 4 -C 12 cycloalkenyl group or a substituted or unsubstituted 5- 12-membered heterocyclic group;

Or R 1 and R 3 and the carbon atom to which they are connected together form a substituted or unsubstituted C 6 -C 12 aryl group, a substituted or unsubstituted C 3 -C 12 cycloalkyl group, a substituted or unsubstituted C 4- C 12 cycloalkenyl or 5-12 membered heterocyclic group;

Or when s is 1, R 11 and R 21 and the carbon atom to which they are connected together form a substituted or unsubstituted C 3 -C 12 cycloalkyl group, a substituted or unsubstituted C 4 -C 12 cycloalkenyl group or 5 -12 membered heterocyclic group;

Or when s is 1, R 3 and R 11 and the carbon atom to which they are connected together form a substituted or unsubstituted C 6 -C 12 aryl group, a substituted or unsubstituted C 3 -C 12 cycloalkyl group, a substituted or Unsubstituted C 4 -C 12 cycloalkenyl or substituted or unsubstituted 5-12 membered heterocyclic group;

P 1 is a protecting group, selected from the following group: tert-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz), fluorenylmethyloxycarbonyl (Fmoc), phthaloyl (Pht), acetyl (Ac), three Fluoroacetyl (Tfa), benzyl (Bn), triphenylmethyl (Tr), P 2 is selected from the following group: hydrogen, substituted or unsubstituted C 1 -C 6 alkyl, substituted or unsubstituted C 6 -C 12 aryl, substituted or unsubstituted 5-12 membered heteroaryl, substituted or unsubstituted 5-12 membered heterocyclic group;

The substitution refers to substitution by one or more substituents selected from the group consisting of halogen, hydroxy, amino, phenyl, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, C 1 -C 6 alkoxy Group, C 1 -C 6 haloalkoxy, C 3 -C 8 cycloalkyl.
The β- or γ-amino acid N-carboxythiocarbonyl intracyclic anhydride (β-NTA, γ-NTA) monomer according to claim 1, wherein the monomer has the formula II or III Structure:

In the formula, ring A is independently selected from the following group: substituted or unsubstituted C 6 -C 12 aryl, substituted or unsubstituted C 3 -C 12 cycloalkyl, substituted or unsubstituted C 4 -C 12 ring Alkenyl or substituted or unsubstituted 5-12 membered heterocyclic group, said substitution refers to substitution by one or more substituents selected from the group consisting of halogen, hydroxy, amino, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, C 1 -C 6 alkoxy, C 1 -C 6 haloalkoxy;

In the formula, n is an integer of 0-8;

The definitions of R 3 , R 4 , R 11 , R 21 , and s are as described in claim 1.
A method for synthesizing β- or γ-amino acid N-carboxythiocarbonyl ring anhydride (β-NTA, γ-NTA) monomer according to claim 1, characterized in that it comprises the steps:

(2) Reacting compound 3 with phosphorus halide in a second inert solvent to obtain a monomer of formula I;

Where

R 5 is selected from: substituted or unsubstituted C 1 -C 6 alkyl or substituted or unsubstituted benzyl;

The substitution refers to substitution by a substituent selected from the following group: halogen, hydroxyl, amino, C 1 -C 6 alkyl, C 1 -C 6 alkoxy;

The definitions of R 1 , R 2 , R 3 , R 4 , R 11 , R 21 and s are as described in claim 1.
A β- or γ-amino acid homopolymer, characterized in that its monomer is selected from the β- or γ-NTA monomer having the structure shown in formula (I) according to claim 1.
The homopolymer according to claim 4, wherein the homopolymer has the structure shown in formula IV, V, VI:

among them,

m is 5～2000;

R and R'are each independently selected from the following group: H, substituted or unsubstituted C 1 -C 6 alkyl, substituted or unsubstituted C 6 -C 12 aryl, substituted or unsubstituted C 2 -C 6 Alkenyl, substituted or unsubstituted C 2 -C 6 alkynyl;

Wherein, the substitution refers to substitution by one or more substituents selected from the following group: C 1 -C 6 alkyl, C 6 -C 12 aryl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl; -N 3 , C 1 -C 6 alkyl-(C=O)-O-, C 1 -C 6 alkyl-(C=O)-N-, amino, hydroxy, mercapto,
Q 3 -O-, Q 4 -S-, 5-6 membered heterocyclic group, adamantane, calixpyrrole, cyclodextrin, polyethylene glycol; wherein, Q 1 and Q 2 are each independently selected from the following group: H, C 1 -C 6 alkyl, C 1 -C 6 alkyl-O-(C=O)-, C 6 -C 14 aryl-C 1 -C 6 alkyl-O-(C=O) -, and Q 1 and Q 2 are not H at the same time; Q 3 is independently selected from the following group: C 1 -C 6 alkyl, C 6 -C 12 aryl-C 1 -C 6 alkyl; Q 4 is independently Selected from the following group: C 1 -C 6 alkyl, C 6 -C 12 aryl-C 1 -C 6 alkyl, C 6 -C 12 aryl-C 1 -C 6 alkyl-(C=O) -O-; and R and R'are not H at the same time;

Or R, R'and the adjacent N atoms form a substituted or unsubstituted 5-12 membered heterocyclic group; the substitution refers to substitution by one or more substituents selected from the group consisting of halogen, hydroxyl, amino, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, C 1 -C 6 alkoxy, C 1 -C 6 haloalkoxy;

The definitions of R 1 , R 2 , R 3 , R 4 , R 11 , R 21 and s are as described in claim 1.
The homopolymer of claim 4, wherein the homopolymer has a structure represented by formula VII, VIII, IX, X, XI, XII:

among them,

Ring A is independently selected from the following group: substituted or unsubstituted C 6 -C 12 aryl, substituted or unsubstituted C 3 -C 12 cycloalkyl, substituted or unsubstituted C 4 -C 12 cycloalkenyl or A substituted or unsubstituted 5-12 membered heterocyclic group, said substitution means being substituted by one or more substituents selected from the group consisting of halogen, hydroxy, amino, C 1 -C 6 alkyl, C 1 -C 6 Haloalkyl, C 1 -C 6 alkoxy, C 1 -C 6 haloalkoxy;

The definitions of m, n, R, and R'are as described in claim 5;

The definitions of R 3 , R 4 , R 11 , R 21 , and s are as described in claim 1.
The homopolymer of claim 4, wherein the homopolymer monomer is selected from the group consisting of L-aspartic acid 1-benzyl ester N-carboxythiocarbonyl intracyclic anhydride, DL-β 3 -Phenylpropylamino N-carboxythiocarbonyl ring anhydride, DL-β 3 -norleucine N-carboxythiocarbonyl ring anhydride, N(α)-ZL-2,3-diaminopropionic acid N -Carboxythiocarbonyl intracyclic anhydride, β 2,3 -norbornene N-carboxythiocarbonyl intracyclic anhydride, β 2,3 -cyclohexyl N-carboxythiocarbonyl intracyclic anhydride, γ 2,3 -benzene N-carboxyl thiocarbonyl ring anhydride, or a combination thereof.
The homopolymer of claim 4, wherein the homopolymer is prepared by a method including the following steps:

In the third inert solvent, in the presence of an organic base initiator, any one of the β- or γ-NTA monomers having the structure shown in formula (I) according to claim 1 is polymerized to form the β- or γ-amino acid homopolymer;

Wherein, the organic base is independently selected from: amines, amine salts, or other organic bases, or combinations thereof;

The amine is selected from the group consisting of R-NH 2 , R-NH-R',
Or a combination;

The salt of the amine is independently selected from the following group: hydrochloride, hydrobromide, formate, acetate, or trifluoroacetate;

R, R', and R" are each independently selected from the following group: H, substituted or unsubstituted C 1 -C 6 alkyl, substituted or unsubstituted C 6 -C 12 aryl, substituted or unsubstituted C 2 -C 6 alkenyl, substituted or unsubstituted C 2 -C 6 alkynyl;

Wherein, the substitution refers to substitution by one or more substituents selected from the following group: C 1 -C 6 alkyl, C 6 -C 12 aryl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl; -N 3 , C 1 -C 6 alkyl-(C=O)-O-, C 1 -C 6 alkyl-(C=O)-N-, amino, hydroxyl, mercapto,
Q 3 -O-, Q 4 -S-, 5-6 membered heterocyclic group, adamantane, calixpyrrole, cyclodextrin, polyethylene glycol; wherein, Q 1 and Q 2 are each independently selected from the following group: H, C 1 -C 6 alkyl, C 1 -C 6 alkyl-O-(C=O)-, C 6 -C 14 aryl-C 1 -C 6 alkyl-O-(C=O) -, and Q 1 and Q 2 are not H at the same time; Q 3 is independently selected from the following group: C 1 -C 6 alkyl, C 6 -C 12 aryl-C 1 -C 6 alkyl; Q 4 is independently Selected from the following group: C 1 -C 6 alkyl, C 6 -C 12 aryl-C 1 -C 6 alkyl, C 6 -C 12 aryl-C 1 -C 6 alkyl-(C=O) -O-; and R and R'are not H at the same time;

Or R, R'and the adjacent N atoms form a substituted or unsubstituted 5-12 membered heterocyclic group; the substitution refers to substitution by one or more substituents selected from the group consisting of halogen, hydroxyl, amino, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, C 1 -C 6 alkoxy, C 1 -C 6 haloalkoxy.
A copolymer or sequence peptide, characterized in that its monomer is selected from: a) the β- or γ-amino acid N-carboxythiocarbonyl ring intracyclic anhydride (β-NTA, γ-NTA) of claim 1 Two or more of monomers; and optionally b) one or more of α-NCA and α-NTA monomers.
The copolymer or sequence peptide according to claim 9, characterized in that it has a structural unit of C n1 D n2 C'n3 or D n1 C n2 D' n3:

Wherein, n 3 is independently an integer of 0-2000; n 1 and n 2 are each independently an integer of 1-2000;

C and C'are different, and each independently is
D and D'are each independently

R 7 and R 8 are each independently H, -C 1 -C 6 alkyl-R 9 , -N 3 , -(C=O)-OR 9 , -(C=O)-NH-R 9 ,- NH-R 9 , -OR 9 , -SR 9 , -Ph-R 10 or 5-6 membered heterocyclic group;

Or R 8 is connected to the C atom and the N atom adjacent to the C atom to form a 5-6 membered heterocyclic group;

R 9 is selected from the group consisting of one or more substituted or unsubstituted substituents: hydrogen, C 1 -C 6 alkyl, phenyl, indolyl, 5-6 membered heteroaryl, C 2 -C 6 alkene Group, C 2 -C 6 alkynyl group, guanidyl group, benzyl group, tert-butoxycarbonyl group, benzyloxycarbonyl group, tert-butyl group, trityl group or fluorenyl methoxycarbonyl group; the substituted substituent is selected from the following group One or more groups of: halogen, nitro, C 1 -C 6 alkyl, C 1 -C 6 alkoxy, C 2 -C 6 alkenyl, C 2 -C 6 alkenyloxy or CH 3 ( O-CH 2 -CH 2 )y, and y is an integer of 1-6;

R 10 is selected from: H, -OH;

The definitions of R 1 , R 2 , R 3 , R 4 , R 11 , R 21 and s are as described in claim 1.
The copolymer or sequence peptide of claim 9, wherein the polymer blend or sequence peptide has [C n'1 D n'2 C'n'3 ] Y or [D n'1 C n'2 D'n'3 ] Y structure,

Wherein, Y is an integer from 10 to 2000; C, C', D, and D'are as defined in claim 10;

Wherein, n '3 independently 1>n' 3 ≥0 decimal; n '1, n' 2 are each independently a> 0 is a decimal, and less than 1.12 of the copolymer as claimed in claim 9 or. The sequence peptide is characterized in that the copolymer is prepared by a method including the following steps:

(i) In a third inert solvent, mixing two or more of the monomers described in claim 1; and optionally one or more of α-NCA and α-NTA monomers,

(ii) Perform a polymerization reaction in the presence of an organic base initiator to form a statistical coplymer amino acid copolymer;

Or (i') in the third inert solvent, in the presence of an organic base initiator, first polymerize the first monomer,

(ii') After the polymerization reaction in (i') is over, the second monomer is added to carry out the polymerization reaction,

And optionally (iii') repeat step (ii') q times,

So as to form a block copolymer amino acid copolymer;

Wherein, q is an integer ≥ 1;

The first monomer and the second monomer are different and independently selected from: any β-NTA or γ-NTA monomer, α-NCA monomer or α-NTA monomer according to claim 1;

Wherein, the organic base is independently selected from: amines, amine salts, or other organic bases, or combinations thereof;

The amine is selected from the group consisting of R-NH 2 , R-NH-R',
Or a combination;

The salt of the amine is independently selected from: hydrochloride, hydrobromide, formate, acetate, or trifluoroacetate;

The definitions of R, R', and R" are as set forth in claim 8.
The homopolymer according to claim 4 or the copolymer or sequence peptide according to claim 9, wherein the homopolymer or copolymer or sequence peptide is selected from:
The method for preparing the copolymer according to claim 9, wherein the method comprises the steps: (i) combining two or more of the monomers according to claim 1 in an inert solvent; and optionally Ground b) a mixture of one or more of α-NCA and α-NTA monomers,

(ii) Perform a polymerization reaction in the presence of an organic base initiator to form a statistical coplymer β-amino acid copolymer;

Or (i') in an inert solvent, in the presence of an organic base initiator, first polymerize the first monomer,

(ii') After the polymerization reaction in (i') is completed, the second monomer is added to carry out the polymerization reaction, thereby forming a block copolymer β-amino acid copolymer;

And optionally (iii') repeat step (ii') q times,

So as to form a block copolymer β-amino acid copolymer;

Wherein, q is an integer ≥ 1;

The first monomer and the second monomer are different and independently selected from: any β-NTA or γ-NTA monomer, α-NCA monomer or α-NTA monomer according to claim 1;

Wherein, the organic base is independently selected from: amines, amine salts, or other organic bases, or combinations thereof;

The amine is selected from the group consisting of R-NH 2 , R-NH-R',
Or a combination;

The salt of the amine is independently selected from: hydrochloride, hydrobromide, formate, acetate, or trifluoroacetate;

The definitions of R, R', and R" are as set forth in claim 8.
The use of the polymer or sequence peptide according to any one of claims 4-13, characterized in that it is used for anti-bacterial, anti-fungal, anti-viral, anti-mite, anti-tumor, cell adhesion, tissue engineering, medicine Modification, protein modification, protein protection, drug synergy, drug delivery, gene delivery, and self-assembled materials.