Polyamino acid, preparation method thereof and application of polyamino acid as biocompatible filling material

Abstract

The invention discloses a glycine-proline-hydroxyproline terpolymer, the terpolymer has a structure as shown in the following formula (I), and R1, L1, L2, L3, R2, x, y and z are as defined in the specification. The invention also discloses a method for preparing the glycine-proline-hydroxyproline terpolymer and an application of the glycine-proline-hydroxyproline terpolymer as a biocompatible filling material. The invention further discloses the glycine-proline-hydroxyproline terpolymer and the application of the glycine-proline-hydroxyproline terpolymer as the biocompatible filling material. R1-(L1) x-(L2) y-(L3) z-R2 Formula (I)

Description

[0001] This application is a divisional application of Chinese Patent Application No. 202410263708.X, filed on March 7, 2024, entitled "Polyamino acids, preparation methods thereof and use as biocompatible fillers". Technical Field

[0002] This disclosure relates to the field of polymers, and more particularly to a polyamino acid, a method for preparing the polyamino acid, and the use of the polyamino acid as a biocompatible filler. Background Technology

[0003] In modern life, with people's pursuit of beauty and the development of technology, more and more people are seeking to improve their appearance and retain their youth through medical aesthetic procedures. Using fillers to improve facial depressions or reduce or even remove wrinkles is one of the most popular medical aesthetic methods. Common medical aesthetic filler methods include hyaluronic acid fillers, autologous fat fillers, and collagen fillers. Collagen fillers are favored by many medical aesthetic enthusiasts due to their excellent biocompatibility, moisturizing properties, anti-wrinkle properties, repairing properties, and shaping performance.

[0004] Collagen is the most abundant protein type in animals, playing multiple roles including structural support, tissue repair, and physical protection. The most distinctive feature of collagen's unique structural component—the helical segment—is its amino acid composition: -[glycine (Gly)-XY]. n The periodic arrangement of X and Y represents any amino acid, but typically X is proline (Pro) and Y is hydroxyproline (Hyp). Hyp is crucial for proper collagen formation because its trans-hydroxyl groups can stabilize the secondary helical structure of the collagen molecule by forming intramolecular hydrogen bonds.

[0005] Currently, the collagen used in medicine mainly includes natural collagen extracted from animal tissues (animal-derived collagen) and recombinant humanized collagen obtained through biosynthetic pathways. Animal-derived collagen has limited production capacity and its immunogenicity cannot be eliminated. The production of recombinant humanized collagen requires extensive screening of suitable bacterial strains, which is complex and costly; moreover, the synthesized recombinant humanized collagen does not contain Hyp, which is crucial for the proper formation of collagen, resulting in a significant difference in composition from natural collagen.

[0006] Therefore, there is a need to develop a polyamino acid product that can be chemically synthesized, has a similar composition and / or properties to natural collagen, and avoids problems such as immunogenicity and endotoxins. Summary of the Invention

[0007] To address the above and other issues, the inventors designed and synthesized a polyamino acid, which is a terpolymer of glycine (Gly)-proline (Pro)-hydroxyproline (Hyp).

[0008] The polyamino acids provided in this application are prepared by ring-opening copolymerization (chemical synthesis) of N-carboxylic acid anhydride (NCA), and have similar composition, sequence, and physicochemical properties to collagen (especially its helical segments). Compared to animal-derived collagen, these polyamino acids avoid immunogenicity; compared to recombinant humanized collagen, they avoid the problem of endotoxin removal during bacterial expression; and their sequence structure contains an important Hyp, where the trans-hydroxyl group on the side chain provides a stable PPII helical structure. The polyamino acids provided in this application exhibit strong cell adhesion, low cytotoxicity and immunogenicity, and the ability to provide nutrients to promote collagen regeneration, making them effective substitutes (collagen-like substances) for animal-derived collagen and recombinant humanized collagen. Furthermore, these polyamino acids also have many advantages, including simple synthesis steps, low cost, high yield, and the ability to be modified after polymerization to upgrade the product.

[0009] At least one embodiment of this disclosure provides a glycine-proline-hydroxyproline terpolymer having the structure shown in formula (I):

[0010] R1-(L1) x -(L2) y -(L3) z -R2

[0011] Formula (I)

[0012] in,

[0013] L1 is ;

[0014] L2 is ;

[0015] L3 is ;

[0016] R A Each occurrence is independently selected from H, acetyl, and -(L1). x -(L2) y -(L3) z -R2, provided that at least some R... A For -(L1) x -(L2) y -(L3) z -R2;

[0017] R1 and R2 are independently H or terminal groups, wherein the terminal group is a hydroxyl, carboxyl, amino, amide, or ester group;

[0018] x, y, and z represent the percentages of L1, L2, and L3 units relative to the total number of monomer units in the ternary copolymer, respectively. Each occurrence of x, y, and z is an independent fraction greater than 0 and less than 1, provided that the sum of all x, y, and z is 1.

[0019] The terpolymer has a main chain formed by connecting three different monomers in a random order and proportion, and the monomer units in the main chain further have one or more -(L1) groups as substituting side groups. x -(L2) y -(L3) z -R2.

[0020] In some examples, L1 is L2 is L3 is .

[0021] In some examples, R A Each occurrence is independently selected from H, acetyl, and -(L1). x -(L2) y -(L3) z -R2, provided that at least some R... A For -(L1) x -(L2) y -(L3) z -R2, and at least some R A It is an acetyl group.

[0022] In some examples, the weight-average molecular weight of the terpolymer is 5 to 100 kg / mol.

[0023] Another embodiment of this disclosure also provides a method for preparing the terpolymer described herein, the method comprising: polymerizing the compounds represented by formulas (1) to (3) in an organic solvent in the presence of a base to obtain the terpolymer:

[0024] (1)

[0025] (2)

[0026] (3),

[0027] Where R A1 Selected from H and acetyl groups.

[0028] In some examples, the base is selected from potassium hydride, sodium hydride, potassium methoxide, sodium methoxide, potassium tert-butoxide, sodium tert-butoxide, triazabicyclo[4.4.0]dec-5-ene, 1,8-diazabicyclo[5.4.0]undec-7-ene, N,N-diisopropylethylamine, 4-dimethylaminopyridine, aniline, and any mixture thereof; and / or the organic solvent is selected from dimethyl sulfoxide, dimethylformamide, acetonitrile, dichloromethane, ethyl acetate, tetrahydrofuran, acetone, and mixtures thereof.

[0029] Another embodiment of this disclosure also provides the use of the terpolymer described herein as a biocompatible filler material.

[0030] In some examples, the biocompatible filler material is a medical aesthetic filler material.

[0031] In addition to the above, this disclosure also provides at least the following aspects:

[0032] At least one embodiment of this disclosure provides a polyamino acid having the structure shown in formula (I):

[0033] R1-(L1) x -(L2) y -(L3) z -R2

[0034] Formula (I)

[0035] in,

[0036] L1 is ;

[0037] L2 is ;

[0038] L3 is ;

[0039] R a R b and R c Independently selected from H, halogens, C 1-6 Alkyl, C 1-6 Alkoxy and C 1-6 Alkoxyalkyl;

[0040] R A Each time it appears, it is independently selected from H and C. 1-6 Alkyl group, C 6-10 Aromatic acyl group and -(L1) x -(L2) y -(L3) z -R2;

[0041] R1 and R2 are either H groups or terminal groups;

[0042] Each occurrence of x, y, and z is an independent fraction greater than 0 and less than 1, provided that the sum of all x, y, and z is 1.

[0043] In some examples, L1 in the polyamino acid is L2 is L3 is .

[0044] In some examples, L1 in the polyamino acid is L2 is L3 is .

[0045] In some examples, in the polyamino acid, R A Each occurrence is independently selected from H and -(L1). x -(L2) y -(L3) z -R2, provided that at least some R... A For -(L1) x -(L2) y -(L3) z -R2.

[0046] In some examples, in the polyamino acid, R A Each time it appears, it is independently selected from H and C. 1-6 Alkyl group, C 6-10 Aromatic acyl group and -(L1) x -(L2) y -(L3) z -R2, provided that at least some R... A For -(L1) x -(L2) y -(L3) z -R2, and at least some R A Selected from C 1-6 Alkyl group, C 6-10 Aroyl.

[0047] In some examples, at least some R in the polyamino acids A For -(L1) x -(L2) y -(L3) z -R2, and at least some R A It is an acetyl group.

[0048] In some examples, in the polyamino acid, R A Each time it appears, it is selected independently from C. 1-6 Alkyl and C 6-10 Aroyl.

[0049] In some examples, in the polyamino acid, R A It is an acetyl group.

[0050] In some examples, the polyamino acid has a weight-average molecular weight of 5 to 100 kg / mol.

[0051] This disclosure also provides a method for preparing polyamino acids, comprising: preparing compounds of formulas (II) to (IV) as follows:

[0052]

[0053] in,

[0054] R a R b and R c Independently selected from H, halogens, C 1-6 Alkyl, C 1-6 Alkoxy and C 1-6 Alkoxyalkyl

[0055] R A1 Selected from H, C 1-6 Alkyl and C 6-10 Aroyl;

[0056] Polymerization in an organic solvent in the presence of a base yields the polyamino acid shown in formula (I):

[0057] R1-(L1) x -(L2) y -(L3) z -R2

[0058] Formula (I)

[0059] in,

[0060] L1 is ;

[0061] L2 is ;

[0062] L3 is ;

[0063] R a R b and R c Independently selected from H, halogens, C 1-6 Alkyl, C 1-6 Alkoxy and C 1-6 Alkoxyalkyl;

[0064] R A Each time it appears, it is independently selected from H and C. 1-6 Alkyl group, C 6-10Aromatic acyl group and -(L1) x -(L2) y -(L3) z -R2;

[0065] R1 and R2 are either H groups or terminal groups;

[0066] Each occurrence of x, y, and z is an independent fraction greater than 0 and less than 1, provided that the sum of all x, y, and z is 1.

[0067] In some examples, R a R b and R c All are H.

[0068] In some examples, R A1 For H, and R A Each occurrence is independently selected from H and -(L1). x -(L2) y -(L3) z -R2.

[0069] In some examples, the method further includes acylation of the polymerized polyamino acid of formula (I) to obtain at least partially acylated polyamino acid.

[0070] In some examples, at least some R A1 For H, and at least some R A1 Selected from C 1-6 Alkyl and C 6-10 Aromatic acyl group, and at least some R A Selected from H, C 1-6 Alkyl group, C 6-10 Aromatic acyl group and -(L1) x -(L2) y -(L3) z -R2.

[0071] In some examples, R A1 Selected from C 1-6 Alkyl and C 6-10 Aromatic acyl group, and R A Selected from C 1-6 Alkyl and C 6-10 Aroyl.

[0072] In some examples, R A1 and R A All are acetyl groups.

[0073] In some examples, the base is selected from potassium hydride, sodium hydride, potassium methoxide, sodium methoxide, potassium tert-butoxide, sodium tert-butoxide, triazabicyclo[4.4.0]dec-5-ene, 1,8-diazabicyclo[5.4.0]undec-7-ene, N,N-diisopropylethylamine, 4-dimethylaminopyridine, aniline, and any mixture thereof.

[0074] In some examples, the organic solvent is selected from dimethyl sulfoxide, dimethylformamide, acetonitrile, dichloromethane, ethyl acetate, tetrahydrofuran, acetone, and mixtures thereof.

[0075] This disclosure also provides the use of any of the above-mentioned polyamino acids as biocompatible filler materials.

[0076] In some examples, the biocompatible filler material is a medical aesthetic filler material. Attached Figure Description

[0077] To more clearly illustrate the technical solutions of the embodiments of this disclosure, the accompanying drawings of the embodiments will be briefly described below. Obviously, the drawings described below only relate to some embodiments of this disclosure and are not intended to limit this disclosure.

[0078] Figure 1 Size exclusion chromatography (SEC) curves of products prepared according to an embodiment of the present disclosure using N,N-diisopropylethylamine (DIPEA) as a base and adding different equivalent amounts of raw materials are shown.

[0079] Figure 2 The SEC curves of products prepared according to another embodiment of this disclosure using 4-dimethylaminopyridine (DMAP) as a base and adding different equivalent amounts of raw materials are shown.

[0080] Figure 3 The SEC curves of products prepared using different bases according to yet another embodiment of this disclosure are shown, wherein blue represents DIPEA, red represents 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), and orange represents DMAP.

[0081] Figure 4 The SEC curves of copolymers prepared according to another embodiment of this disclosure using acetylated hydroxyproline N-carboxylactam (AcHypNCA) to replace all or 50% of hydroxyproline N-carboxylactam (HypNCA) are shown.

[0082] Figure 5This illustrates a glycine-proline-hydroxyproline terpolymer (GPO terpolymer, where G represents glycine units, P represents proline units, and O represents hydroxyproline units) prepared using DIPEA as a base and a Gly:Pro:Hyp:base feed ratio of 25:25:25:1 (DIPEA-25-25-25). 1 H-NMR (D2O);

[0083] Figure 6 The GPO terpolymer prepared using DIPEA-40-30-30 is shown. 1 H-NMR (D2O);

[0084] Figure 7 This shows a GPO terpolymer made using DIPEA-100-100-100. 1 Circular dichroism spectrum of H-NMR (D2O);

[0085] Figure 8 The infrared absorption spectra of the GPO terpolymer (blue) prepared using DIPEA-25-25-25 and its hydrolysis (red) are shown.

[0086] Figure 9 The changes in the aqueous phase SEC curves of the GPO terpolymer (red) prepared using DBU as alkali and a Gly:Pro:Hyp:alkali feed ratio of 100:100:100:1 (DBU-100-100-100) are shown.

[0087] Figure 10 The diagram shows a GPO terpolymer prepared using DIPEA-25-25-25 and the effect of adding NaOH to it. 1 Changes in H-NMR (D2O);

[0088] Figure 11 This is a bar chart showing the comparison of cell adhesion ability of GPO terpolymers and binary copolymers (GO-DIPEA: glycine-hydroxyproline binary copolymer; PO: proline-hydroxyproline binary copolymer) made using DIPEA-25-25-25, as well as hyaluronic acid and bovine Achilles tendon collagen, with phosphate-buffered saline (PBS) used as a control;

[0089] Figure 12This is a bar chart comparing the cell adhesion abilities of GPO terpolymers with different amino acid ratios and bovine Achilles tendon collagen and PBS as controls, where 11 - DIPEA-40-30-30; 12 - DIPEA-30-35-35; 13 - DIPEA-20-40-40; 14 - DIPEA-40-40-20; 15 - DIPEA-40-20-40; 16 - DIPEA-40-10-50;

[0090] Figure 13 This is a bar chart comparing the cell adhesion abilities of GPO terpolymers with different degrees of acetylation and bovine Achilles tendon collagen and PBS as controls, where 1 # - GPO-DIPEA-45:20:35;2 # - GPO-DIPEA-45:25:30; 3 # - GPO-DIPEA-45:27.5:27.5;4 # - GP(AcO)-BnNH2-40:30:30;5 # - GP(AcO)-DIPEA-40:30:30; 6 # - GPO-DIPEA-50:15:35; 7 # - GPO(AcO)-DIPEA-40:30:15:15, where AcO represents acetylated hydroxyproline units. Detailed Implementation

[0091] For the purposes of the detailed description below, it should be understood that this disclosure may take various alternative variations and sequences of steps unless expressly stated otherwise. Furthermore, except in any operational example, or otherwise indicated, all figures representing the amounts of ingredients used, for example, in the specification and claims, should be understood to be modified in all cases by the term “about.” Therefore, unless otherwise stated, the numerical parameters set forth in the following specification and appended claims are approximations varying according to the desired performance to be obtained from this disclosure. At least not in an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should be interpreted at least according to the number of significant figures reported and by applying ordinary rounding techniques.

[0092] Although the numerical ranges and parameters described in this disclosure are approximate, the values ​​listed in the specific examples are reported as precisely as possible. However, any numerical value inherently contains some error that is necessarily caused by the standard deviation found in their respective test measurements.

[0093] Furthermore, it should be understood that any numerical range described herein is intended to include all subranges falling within it. For example, the range “1 to 10” is intended to include all subranges between the minimum value 1 and the maximum value 10, i.e., a minimum value equal to or greater than 1 and a maximum value equal to or less than 10.

[0094] In this disclosure, unless otherwise expressly stated, the use of the singular includes the plural and the plural includes the singular. Furthermore, in this disclosure, unless otherwise expressly stated, “or” is used to mean “and / or,” even if “and / or” may be explicitly used in certain circumstances. Additionally, in this disclosure, unless otherwise expressly stated, “a” or “an” is used to mean “at least one.” For example, “a” polymer, “a” composition, etc., refer to any one or more of these articles.

[0095] In recent years, cosmetic medical procedures, especially filler treatments, have increasingly become a popular way for beauty enthusiasts to improve their appearance. Collagen is an essential nutrient naturally present in the skin. Due to its excellent biocompatibility, moisturizing properties, anti-wrinkle effects, repairing properties, and shaping capabilities, collagen is considered a suitable filler material for cosmetic medical procedures. Currently, collagen commonly used for medical / cosmetic purposes mainly includes natural collagen extracted from animal tissues (animal-derived collagen) and recombinant humanized collagen obtained through biosynthetic pathways. The drawbacks of animal-derived collagen include limited production capacity and the inability to eliminate immunogenicity; while the problem with recombinant humanized collagen is that its production requires extensive screening of suitable bacterial strains, resulting in complex processes and high costs; moreover, synthetic recombinant humanized collagen does not contain Hyp, which is crucial for proper collagen formation, and its composition differs significantly from natural collagen.

[0096] The inventors of this application, through extensive efforts, designed and synthesized a polyamino acid, which is a glycine-proline-hydroxyproline terpolymer.

[0097] As described in this article, the term "polyamino acid" refers to a compound composed of multiple amino acid molecules linked by peptide bonds. It can be classified into homopolymers (polymerized from a single amino acid monomer) and copolymers (polymerized from two or more amino acid monomers). The basic building blocks (monomer units) of polyamino acids are amino acid molecules, each consisting of an amino group, a carboxyl group, and one or more side chains, linked by peptide bonds into a linear chain. Polyamino acids exhibit low immunogenicity, good degradability and mechanical properties, and their performance is controllable, thus they are widely used in fields such as controlled drug release, tissue engineering, and regenerative medicine.

[0098] In some examples, the polyamino acids provided in this application can be ternary copolymers. As used herein, the term "ternary copolymer" refers to a copolymer formed by copolymerizing three different amino acid monomers. For example, the ternary copolymer provided in this application can be an unbranched linear copolymer, that is, the copolymer can have a main chain formed by connecting three different monomers in any order and proportion, and the main chain does not have branched polyamino acid side chains (but may have other functional groups, such as hydroxyl or amino protecting groups, such as acyl groups, etc.). Alternatively, the ternary copolymer provided in this application can also be a branched copolymer, that is, the polymer can have a main chain formed by connecting one or more or all of three different monomers in any order and proportion, and the monomer units in the main chain further have one or more polyamino acid side chains as substituted side groups. The one or more polyamino acid side chains may further have one or more polyamino acid side chains as substituted side groups, thereby forming a dendritic polymer structure.

[0099] In some examples, the polyamino acids described herein may be glycine-proline-hydroxyproline terpolymers, also known as Gly-Pro-Hyp terpolymers or GPO terpolymers, and these terms are used interchangeably herein. For example, the polyamino acids described herein may have the structure shown in formula (I):

[0100] R1-(L1) x -(L2) y -(L3) z -R2

[0101] Formula (I)

[0102] in,

[0103] L1 can be ;

[0104] L2 can be ;

[0105] L3 can be ;

[0106] R a R b and R c It can be independently selected from H, halogen, C 1-6 Alkyl, C 1-6 Alkoxy and C 1-6 Alkoxyalkyl;

[0107] R A Each occurrence can be independently selected from H and C. 1-6 Alkyl group, C 6-10 Aromatic acyl group and -(L1) x -(L2)y -(L3) z -R2;

[0108] R1 and R2 can be H or end groups independently;

[0109] Each occurrence of x, y, and z can be an independent fraction greater than 0 and less than 1, provided that the sum of all x, y, and z is 1.

[0110] * indicates the connection point between the related group and other groups.

[0111] As used in this article, the term "halogen" can also be referred to as a halogen atom or halogen group, which refers to a monovalent group formed by a halogen atom fluorine (F), chlorine (Cl), bromine (Br) or iodine (I), namely -F, -Cl, -Br or -I.

[0112] As used herein, the term "alkyl" refers to a straight-chain or branched monovalent hydrocarbon group formed by removing a hydrogen atom from a saturated aliphatic hydrocarbon, with the chemical formula -C n H 2n+1 In some examples, suitable alkyl groups for use in this disclosure may include, but are not limited to, straight-chain or branched alkyl groups having 1 to 20 carbon atoms, i.e., straight-chain or branched C164-20 ... 1-20 Alkyl, for example, C 1-18 Alkyl, C 1-16 Alkyl, C 1-12 Alkyl, C 1-10 Alkyl, C 1-6 Alkyl, C 1-4 Alkyl groups, etc. Alternatively, alkyl groups suitable for use in this disclosure may include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, 2-ethylbutyl, etc.

[0113] As used herein, the term "alkoxy" refers to -O-alkyl, with the chemical formula -OC. n H 2n+1 In some examples, suitable alkoxy groups for use in this disclosure may include, but are not limited to, alkoxy groups having 1 to 6 carbon atoms, i.e., C646. 1-6 Alkyl groups, such as, but not limited to, methoxy, ethoxy, 1-propoxy, 2-propoxy, tert-butoxy, etc.

[0114] As used herein, the term "alkoxyalkyl" refers to an alkoxy-substituted alkyl group with the chemical formula -C n H 2n -OC n H 2n+1 In some examples, suitable alkoxyalkyl groups for use in this disclosure may include, but are not limited to, alkoxyalkyl groups having 1 to 6 carbon atoms, i.e., C64 ... 1-6Alkoxyalkyl, for example, but not limited to, methoxymethyl, ethoxymethyl, 1-propoxymethyl, 2-propoxymethyl, tert-butoxymethyl, ethoxymethyl, etc.

[0115] As used herein, the term "acyl" refers to a group of atoms remaining after removing one or more hydroxyl groups from an organic or inorganic oxyacid, having the general formula RC(=O)-, where R can be H, alkyl, or aryl. In some aspects, the acyl group described in this application can be an alkylacyl group (i.e., R is H or alkyl), for example, C 1-6 Alkyl group; in other aspects, the acyl group described in this application may be an aromatic acyl group (i.e., R is an aryl group), for example, C 6-10 Aromatic acyl group. In some examples, the acyl group described in this disclosure may include, but is not limited to, formyl, acetyl, propionyl, butyryl, valeryl, hexanoyl, benzoyl, naphthoyl, etc.

[0116] As used herein, the term "terminal group" refers to a non-hydrogen group at the end of a polyamino acid molecular chain, i.e., the terminal group / capping group of the polyamino acid chain. The terminal group described in the embodiments of this disclosure can be any terminal group commonly found in the art, including but not limited to hydroxyl, carboxyl, amino, amide, ester, etc., and this disclosure does not impose any specific limitations on it.

[0117] In equation (I) above, the symbols x, y, and z represent the percentages of glycine (Gly or G) units, proline (Pro or P) units, and hydroxyproline (Hyp or O) units relative to the total number of monomer units in the polyamino acid molecular chain, respectively. Each occurrence of x, y, and z is an independent fraction greater than 0 and less than 1, provided that the sum of all x, y, and z is 1.

[0118] In some embodiments, L1 in the polyamino acid may be... L2 can be L3 can be .

[0119] In other embodiments, L1 in the polyamino acid may be... L2 can be L3 can be .

[0120] In other embodiments, the polyamino acid may have an unacylated branched structure, i.e., in the structure of formula (I) above, R A Each occurrence is independently selected from H and -(L1). x -(L2) y -(L3) z -R2, provided that at least some R... A For -(L1) x -(L2) y -(L3)z -R2.

[0121] In other embodiments, the polyamino acid may have at least a partially acylated branched structure, i.e., in the structure of formula (I) above, R A Each time it appears, it is independently selected from H and C. 1-6 Alkyl group, C 6-10 Aromatic acyl group and -(L1) x -(L2) y -(L3) z -R2, provided that at least some R... A For -(L1) x -(L2) y -(L3) z -R2, and at least some R A Selected from C 1-6 Alkyl group, C 6-10 Aromatic acyl group. For example, the polyamino acid can be at least partially acetylated branched structure, i.e., in the structure of formula (I) above, at least some R... A For -(L1) x -(L2) y -(L3) z -R2, and at least some R A It is an acetyl group.

[0122] In other embodiments, the polyamino acid may have a fully acylated, unbranched structure, i.e., in the structure of formula (I) above, R A Each time it appears, it is selected independently from C. 1-6 Alkyl and C 6-10 Aromatic acyl group. For example, the polyamino acid can be a fully acetylated, unbranched structure. That is, in the structure of formula (I) above, R A It is an acetyl group.

[0123] In some embodiments, any of the polyamino acids described above may have a suitable weight-average molecular weight, for example, 5 to 100 kg / mol.

[0124] At least another embodiment of this disclosure also provides a method for preparing polyamino acids, comprising: taking compounds of formulas (II) to (IV):

[0125] in,

[0126] R a R b and R c Independently selected from H, halogens, C 1-6 Alkyl, C 1-6 Alkoxy and C 1-6 Alkoxyalkyl

[0127] R A1 Selected from H, C 1-6 Alkyl and C 6-10 Aroyl;

[0128] Polymerization in an organic solvent in the presence of a base yields the polyamino acid shown in formula (I):

[0129] R1-(L1) x -(L2) y -(L3) z -R2

[0130] Formula (I)

[0131] in,

[0132] L1 is ;

[0133] L2 is ;

[0134] L3 is ;

[0135] R a R b and R c Independently selected from H, halogens, C 1-6 Alkyl, C 1-6 Alkoxy and C 1-6 Alkoxyalkyl;

[0136] R A Each time it appears, it is independently selected from H and C. 1-6 Alkyl group, C 6-10 Aromatic acyl group and -(L1) x -(L2) y -(L3) z -R2;

[0137] R1 and R2 are either H groups or terminal groups;

[0138] Each occurrence of x, y, and z is an independent fraction greater than 0 and less than 1, provided that the sum of all x, y, and z is 1.

[0139] In some implementations, R in equations (I) to (IV) above a R b and R c Both can be H.

[0140] In other embodiments, the method may include using a non-acylated form of monomer (IV) as a starting material to synthesize polyamino acids. That is, R in formulas (II) to (IV) above... A1 It can be H, and R in equation (I) above AEach occurrence is independently selected from H and -(L1). x -(L2) y -(L3) z -R2. Further, the method may also include further acylation of the polyamino acid of formula (I) obtained by polymerization to obtain at least partially acylated polyamino acid, for example, further acetylation of the polyamino acid of formula (I) to obtain at least partially acetylated polyamino acid.

[0141] In other embodiments, the method may include using a mixture of acylated and non-acylated monomers (IV) as raw materials to synthesize polyamino acids. That is, at least some of the Rs in formulas (II) to (IV) above. A1 For H, and at least some R A1 Selected from C 1-6 Alkyl and C 6-10 The aromatic acyl group causes the polymerized polyamino acid of formula (I) to be at least partially acylated. That is, at least some of the R groups in formula (I) above... A Selected from H, C 1-6 Alkyl group, C 6-10 Aromatic acyl group and -(L1) x -(L2) y -(L3) z -R2. For example, at least some R A1 (and at least some R) A () can be an acetyl group.

[0142] In other embodiments, the method may include using an acylated monomer (IV) as a starting material to synthesize polyamino acids. That is, R in formulas (II) to (IV) above... A1 Selected from C 1-6 Alkyl and C 6-10 Aromatic acyl group, and R in formula (I) above A Selected from C 1-6 Alkyl and C 6-10 Aromatic acyl group. For example, R A1 and R A Both can be acetyl groups.

[0143] In some embodiments of the method, the base used may be selected from potassium hydride, sodium hydride, potassium methoxide, sodium methoxide, potassium tert-butoxide, sodium tert-butoxide, triazabicyclo[4.4.0]dec-5-ene, 1,8-diazabicyclo[5.4.0]undec-7-ene, N,N-diisopropylethylamine, 4-dimethylaminopyridine, aniline, and any mixture thereof. Furthermore, the organic solvent used may be selected from dimethyl sulfoxide, dimethylformamide, acetonitrile, dichloromethane, ethyl acetate, tetrahydrofuran, acetone, and mixtures thereof.

[0144] This disclosure also provides, in at least another embodiment, the use of any of the polyamino acids described herein as biocompatible fillers. In some examples, the biocompatible filler may be a medical aesthetic filler.

[0145] Example

[0146] The embodiments of this disclosure will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of this disclosure. Unless otherwise specified, specific conditions in the examples are performed under conventional conditions or conditions recommended by the manufacturer. Reagents or instruments used, unless otherwise specified, are all commercially available conventional products. Unless otherwise indicated, all quantities listed are described in parts by weight based on total weight. This disclosure should not be construed as being limited to the specific embodiments described.

[0147] Example 1. Preparation of Gly-Pro-Hyp terpolymer (GPO terpolymer)

[0148] In a glove box, glycine-N-carboxylic anhydride (GlyNCA) (101 mg, 1.00 mmol, 100 equivalents), L-proline-N-carboxylic anhydride (ProNCA) (141 mg, 1.00 mmol, 100 equivalents), and L-hydroxyproline-N-carboxylic anhydride (HypNCA) (157 mg, 1.00 mmol, 100 equivalents) were added to a 5 mL glass vial and dissolved in 1500 μL of anhydrous DMSO. Then, N,N-diethylisopropylamine (DIPEA) (0.5 M × 20 μL, 0.01 mmol, 1 equivalent) was added, and the reaction was allowed to proceed for 30 min. After diluting to 10 mL with water, the solution was passed through a PD10 desalting column and lyophilized to obtain the final product.

[0149] GPO terpolymers were synthesized using the same methods described above, employing different base types and charges, as well as varying monomer ratios, as listed in Table 1. Representative molecular weights and dispersities were then determined. Molecular weight and dispersity were measured in 1 × PBS (pH = 7.4) using an aqueous SEC and a multi-angle light scattering / differential detector. The refractive index (dn / dc) (658 nm) was measured to be 0.0762 ml / g using the DIPEA-10-10-10 copolymer.

[0150] Table 1. Synthesis of GPO copolymers with different types and equivalents of alkali and different monomer ratios, along with some representative molecular weights and dispersities.

[0151]

[0152] like Figure 1-4 As shown, SEC curves of the products obtained using DMAP (4-dimethylaminopyridine), DBU (1,8-diazabicyclo[5.4.0]undec-7-ene), and DIPEA (N,N-diisopropylethylamine) as bases and with different types and ratios of raw materials are presented. Figure 1 and Figure 4 The Cytiva Superdex 200 column was used. Figure 2 and Figure 3 A Cytiva Superdex 75 column was used. The above reaction conditions were used to screen different types of organic bases and their equivalents. The results of characterizing the copolymer elution volume using aqueous SEC showed that the higher the base equivalent, the larger the molecular weight of the resulting polymer, with DIPEA exhibiting the largest molecular weight when used as the base.

[0153] The polyamino acid products prepared above were tested using methods such as proton nuclear magnetic resonance spectroscopy, circular dichroism spectroscopy, infrared spectroscopy, and hydrolysis.

[0154] Specifically, such as Figure 6-10 As shown, Figure 6 The GPO terpolymer prepared using DIPEA-40-30-30 is shown. 1 H-NMR (D2O); Figure 7 This shows a GPO terpolymer made using DIPEA-100-100-100. 1 Circular dichroism spectrum of H-NMR (D2O); Figure 8 The infrared absorption spectra of the GPO terpolymer (blue) prepared using DIPEA-25-25-25 and its hydrolysis (red) are shown. Figure 9 The changes in the aqueous phase SEC curves of the GPO terpolymer (red) prepared using DBU as alkali and a Gly:Pro:Hyp:alkali feed ratio of 100:100:100:1 (DBU-100-100-100) are shown. Figure 10 The diagram shows a GPO terpolymer prepared using DIPEA-25-25-25 and the effect of adding NaOH to it. 1 H-NMR (D2O) changes.

[0155] As can be seen from the figures above, the synthesized copolymers with HypNCA components exhibit a branched structure and a PPII helical conformation. These copolymers are stable in water for up to 4 days, and the ester bonds can be hydrolyzed by the addition of alkali.

[0156] Example 2. Cell adhesion performance test of the synthesized GPO terpolymer

[0157] The cell adhesion properties of the GPO terpolymer synthesized in Example 1 were tested, using PBS, hyaluronic acid, and bovine Achilles tendon collagen as controls. The specific testing methods are as follows:

[0158] Cell adhesion performance was tested using the BALB / 3T3 cell line. The test sample was dissolved in dH2O to a concentration of 200 μg / mL. Bovine Achilles tendon collagen was dissolved in 0.5 M acetic acid to a concentration of 200 μg / mL; the mixture was then stirred for 48 hours, and the supernatant was collected by centrifugation at 12000 rpm.

[0159] Take a 96-well cell culture plate, add 100 μL of the prepared test sample solution to each well, and seal at 4°C in the dark for at least 11 hours. Remove the coated cell culture plate and incubate at room temperature for 1 hour. Discard the coating solution, wash the cell culture plate with PBS, and then block it with 0.1% BSA solution at room temperature for 1 hour. Wash again with PBS. Adjust the BALB / 3T3 cell concentration to 2-5 μL using complete culture medium. 5 After seeding the cells onto a plate at a density of 100 μL / mL, incubate the plate in a 5% CO2 incubator for 1 hour, then remove it. Wash the plate four times with PBS, discard the PBS, and incubate it again with 10% CCK8 solution for 1 hour. Then, read the values ​​at 450 nm using a microplate reader.

[0160] Figure 11 The cell adhesion properties of GPO terpolymer and GH and PH binary copolymers are shown in comparison. Figure 12 The cell adhesion properties of GPO terpolymers with different amino acid contents are shown, with PBS, hyaluronic acid, and / or bovine Achilles tendon collagen used as controls. Figure 11 and 12 As shown, the GPO terpolymer (DIPEA-25-25-25) is significantly higher than the binary copolymer and comparable to hyaluronic acid, exhibiting strong cell adhesion ability. Moreover, the cell adhesion ability of the GPO terpolymer decreases with decreasing glycine content, and the optimal ratio of proline to hydroxyproline is 1:1.

[0161] Example 3. Synthesis of acylated polyamino acids and testing of their cell adhesion properties.

[0162] Using a method similar to that described in Example 1, copolymer 1 was prepared according to the composition and initiator (base) listed in Table 2 below. # ~7 # .

[0163] Table 2. Composition of GPO copolymers and the alkali used

[0164]

[0165] The cell adhesion properties of the prepared GPO copolymer were tested using the same method as that used in Example 2. Figure 13 This study compares the cell adhesion abilities of GPO terpolymers with different degrees of acetylation, bovine Achilles tendon collagen, and PBS controls. Figure 13 It is evident that copolymers with varying degrees of acylation also exhibit good cell adhesion properties, approximately 70% of those found in bovine Achilles tendon and 90% of those found in hyaluronic acid.

[0166] Therefore, the polyamino acids provided in this disclosure have similar composition and sequence to collagen, and have strong cell adhesion similar to collagen, making them suitable as biocompatible fillers, especially for medical aesthetic applications.

Claims

1. A glycine-proline-hydroxyproline terpolymer, said terpolymer having the structure shown in formula (I): R1-(L1) x -(L2) y -(L3) z -R2 Formula (I) in, L1 is ; L2 is ; L3 is ; R A Each occurrence is independently selected from H, acetyl, and -(L1). x -(L2) y -(L3) z -R2, provided that at least some R... A For -(L1) x -(L2) y -(L3) z -R2; R1 and R2 are independently H or terminal groups, wherein the terminal group is a hydroxyl, carboxyl, amino, amide, or ester group; x, y, and z represent the percentages of L1, L2, and L3 units relative to the total number of monomer units in the ternary copolymer, respectively. Each occurrence of x, y, and z is an independent fraction greater than 0 and less than 1, provided that the sum of all x, y, and z is 1. The terpolymer has a main chain formed by connecting three different monomers in a random order and proportion, and the monomer units in the main chain further have one or more -(L1) groups as substituting side groups. x -(L2) y -(L3) z -R2.

2. The terpolymer as described in claim 1, wherein... L1 is ; L2 is ; L3 is .

3. The terpolymer as described in claim 2, wherein, R A Each occurrence is independently selected from H, acetyl, and -(L1). x -(L2) y -(L3) z -R2, provided that at least some R... A For -(L1) x -(L2) y -(L3) z -R2, and at least some R A It is an acetyl group.

4. The terpolymer according to any one of claims 1 to 3, wherein the weight-average molecular weight of the terpolymer is 5 to 100 kg / mol.

5. The method for preparing the terpolymer according to any one of claims 1 to 4, wherein the method comprises: The compounds shown in formulas (1) to (3) were polymerized in an organic solvent in the presence of a base to obtain the terpolymer: (1) (2) (3) Where R A1 Selected from H and acetyl groups.

6. The method of claim 5, wherein, The base is selected from potassium hydride, sodium hydride, potassium methoxide, sodium methoxide, potassium tert-butoxide, sodium tert-butoxide, triazabicyclo[4.4.0]dec-5-ene, 1,8-diazabicyclo[5.4.0]undec-7-ene, N,N-diisopropylethylamine, 4-dimethylaminopyridine, aniline, and any mixture thereof; and / or The organic solvent is selected from dimethyl sulfoxide, dimethylformamide, acetonitrile, dichloromethane, ethyl acetate, tetrahydrofuran, acetone, and mixtures thereof.

7. Use of the terpolymer according to any one of claims 1 to 4 as a biocompatible filler material.

8. The use as described in claim 7, wherein, The biocompatible filler material is a medical aesthetic filler material.

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