Polymers having reactive functional groups

Abstract

To provide a polymer having the enlarged introduction amount of a reactive functional group.SOLUTION: Disclosed is a polymer having a molecular weight of 5,000-30,000 g / mol and having 2-15 repeating units represented by the below-mentioned formula (1) (in the formula, each of plurally existing R1 is independently a 2-12C alkylene group or a group represented by the below-mentioned formula (2); R4 is a polyoxyethylene group having the molecular weight of 200-5,000 g / mol; each of plurally existing R2 is independently a group represented by the above formula (2) or the below-mentioned formula (3); and R5 is a 2-12C alkylene group).SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to polymers having reactive functional groups. More specifically, the present invention relates to polyether ester amides and polyether ester urethanes having reactive functional groups. [Background technology]

[0002] Conventionally, polymers such as polyether ester amides and polyether ester urethanes are known to be applicable to fields such as nanobiotechnology and biomaterials when molded into gels, micelles, fibers, films, etc. For example, Patent Document 1 provides an example of a polyether ester amide type medical adhesive. Patent Document 2 shows an example relating to the activation of PEG (polyethylene glycol), which is sometimes used in drug delivery. Patent Document 3 describes a polymer having a polyester ether amide skeleton that can be used for transporting drugs, etc. Patent Document 4 describes a compound having a polyester amide skeleton that can transport drugs, etc.

[0003] Furthermore, examples of polymers containing amino acid residues have also been reported. For example, Non-Patent Document 1 exemplifies a polyesteramide synthesized using a monomer containing phenylalanine, a type of amino acid. Non-Patent Document 2 reports a polyesteramide synthesized using phenylalanine. Non-Patent Document 3 exemplifies a polyesteramide obtained by polycondensation of a monomer containing phenylalanine and a monomer containing allylglycine, etc. Non-Patent Document 4 reports a polyesteramide synthesized using arginine.

[0004] Furthermore, examples of polyesteramide compounds being conjugated with reactive functional groups have been reported in Non-Patent Documents 5 and 6. [Prior art documents] [Patent Documents]

[0005] [License 1] Patent No. 3642912 [License 2] U.S. Patent No. 7125558 [License 3] U.S. Patent and Trademark Publication No. 2011 / 0027379 [License 4] International Patent Application No. PCT / US2011 / 027721 [Non-licensed literature]

[0006] [Non-licensed Document 1] Guo, K., Chu, CC, et al. (2005), Synthesis and characterization of novel biodegradable unsaturated poly(ester amide)s. J. Polym. Sci. A Polym. Chem., 43:1463. [Non-licensed Document 2] Chen X., Zhao L., et al. (2018)Significant Suppression of Non-small-cell Lung Cancer by Hydrophobic Poly(ester amide) Nanoparticles with High Docetaxel Loading. Front.,Pharmacol.,9:118. [Non-licensed Document 3] Pang,X., Chu,CC,(2010), Synthesis, characterization and biodegradation of poly(ester amide)s based hydrogels. Polymer,51:4200. [Non-licensed Document 4] Gu Z., Wu J. et al. (2018) Poly(ester amide)-based hybrid hydrogels for efficient transdermal insulin delivery. J. Mater. Chem. B, 6:6723. [Non-Patent Document 5] Wit, M.A., Wang, Z. et al. (2008). Syntheses, characterization, and functionalization of poly(ester amide)s with pendant amine functional groups. J. of Polym. Sci. Part A, 46:6376. [Non-Patent Document 6] Ji, Y., Shan, S. et al. (2017) A Novel Pseudo-Protein-Based Biodegradable Nanomicellar Platform for the Delivery of Anticancer Drugs. Small 13:1601491. [Summary of the Invention] [Problems to be Solved by the Invention]

[0007] However, when applying polymers as described in Patent Documents 1 to 4 and Non-Patent Documents 1 to 4 to the fields of nanobiology, biomaterials, etc., the scope of application was limited.

[0008] In addition, the polymers as described in Non-Patent Documents 5 and Non-Patent Documents 6 have reactive functional groups on the polymer side chains, but there is room for increasing the introduction amount of the reactive functional groups.

[0009] An object of the present invention is to provide a polymer with an increased introduction amount of reactive functional groups. [Means for Solving the Problems]

[0010] The present invention relates to the following [1] to [5]. [1] A polymer having a molecular weight of 5,000 to 30,000 g / mol and having 2 to 15 repeating units represented by the following formula (1). [Chemical formula] [In the formula, A plurality of R 1 are each independently an alkylene group having 2 to 12 carbon atoms or a group represented by the following formula (2), [Chemical formula] R 4 is a polyoxyethylene group having a molecular weight of 200 to 5,000 g / mol, A plurality of R 2 are each independently a group represented by the above formula (2) or a group represented by the following formula (3), [Chemical formula] R 5 is an alkylene group having 2 to 12 carbon atoms, R 1 and R 2 at least one of which is a group represented by the above formula (2), A plurality of R 3 are each independently a reactive side chain of an amino acid, The content of R 3 in the above repeating unit is 1.5 to 2 equivalents, x + y is 1, x and y are both greater than 0] [2] The polymer according to [1], wherein R 3 has at least one selected from the group consisting of a hydroxy group, a carboxy group, a thiol group, and an amino group. [3] The polymer according to [1] or [2], wherein R 3 is modified with an activating functional group. [4] The polymer according to [3], wherein the activating functional group is a maleimide group. A bioconjugation product that is a complex of a polymer described in any of [5][1] to [4] with at least one selected from the group consisting of peptides, antibodies, antigens, RNA, and DNA. [Effects of the Invention]

[0011] According to the present invention, it is possible to provide a polymer in which the amount of reactive functional groups introduced is increased.

[0012] By utilizing reactive functional groups in amino acid side chains, a biodegradable polymer matrix material with abundant reaction sites and capable of various reactions can be provided. The polymer of the present invention facilitates the introduction of various biomolecules, linkers, polymers, and second reactive functional groups, enabling the provision of polymer materials with novel properties. The polymer of the present invention is expected to open up new applications in a wide range of fields, including nanobiotechnology and biomaterials.

[0013] The polymer of the present invention allows for the simple introduction of new functional groups and the conferring of properties to the side chains of the polymer through organic chemical reactions of the reactive functional groups present in the polymer. In particular, it is considered applicable to bioconjugation, which involves forming complexes with biomolecules. Examples of biomolecules include antibodies, antigens, peptides, proteins, DNA, and RNA. Possible applications of the polymer of the present invention include cell culture scaffolds, drug delivery systems (DDS), ELISA (enzyme-linked immunosorbent assay), immunoassays, antibody-drug conugates (ADCs), drug discovery reagents, biosensor media, and pathogen diagnostic reagents. [Brief explanation of the drawing]

[0014] [Figure 1] Figure 1 shows the 1H-NMR spectrum of hydrophobic monomer I. [Figure 2]Figure 2 shows the 1H-NMR spectrum of hydrophilic monomer I. [Figure 3] Figure 3 shows the 1H-NMR spectrum of hydrophobic monomer II. [Figure 4] Figure 4 shows the 1H-NMR spectrum of hydrophilic monomer II. [Figure 5] Figure 5 shows the 1H-NMR spectrum of a polyether ester amide (maleimide group modified). [Figure 6] Figure 6 shows the GPC measurement results for polyether ester amide. [Figure 7] Figure 7 shows the 1H-NMR spectrum of polyether ester urethane (I) (maleimide group modified). [Figure 8] Figure 8 shows the GPC measurement results for polyether ester urethane (I). [Figure 9] Figure 9 is a conceptual diagram of gelation by polymers (maleimide group modification) and peptide linkers. [Figure 10] Figure 10(A) shows 15 w / v% polyether ester urethane (I) gels using peptide linkers as crosslinking agents (polymer:peptide linker ratios from left to right: 1:1, 2:1, 3:1, 4:1). Figure 10(B) shows 20 w / v% polyether ester urethane (I) gels using peptide linkers as crosslinking agents (polymer:peptide linker ratios from left to right: 1:1, 2:1, 3:1, 4:1). Figure 10(C) shows 25 w / v% polyether ester urethane (I) gels using peptide linkers as crosslinking agents (polymer:peptide linker ratios from left to right: 1:1, 2:1, 3:1, 4:1). [Figure 11] Figure 11(A) is an SEM image (500x magnification) of a 20 w / v% polyether ester urethane (I) gel (polymer:peptide linker ratio = 3:1). Figure 11(B) is an SEM image (500x magnification) of a 25 w / v% polyether ester urethane (I) gel (polymer:peptide linker ratio = 3:1). [Figure 12]Figure 12(A) shows a fluorescence image of cells cultured on a cell culture substrate using polymer-containing cell culture medium (cell nucleus). Figure 12(B) shows a fluorescence image of cells cultured on a cell culture substrate using polymer-containing cell culture medium (actin fiber). Figure 12(C) shows the results of measuring the viability of cells cultured on a cell culture substrate using polymer-containing cell culture medium. [Figure 13] Figure 13(A) shows fluorescence images obtained by visualizing cells cultured in a gel formed with polymer and peptide linker using the LIVE / DEAD method (left: LIVE, right: DEAD). Figure 13(B) shows fluorescence images of cells cultured on a gel formed with polymer and peptide linker (left: cell nucleus, right: actin fiber). [Modes for carrying out the invention]

[0015] The following describes in detail embodiments for carrying out the present invention. The following embodiments are illustrative for explaining the present invention and are not intended to limit the present invention to the following. The present invention can be appropriately modified and implemented within the scope of its spirit.

[0016] One embodiment of the present invention is a polymer having a molecular weight of 5,000 to 30,000 g / mol and having 2 to 15 repeating units represented by the following formula (1). [ka] [In the formula, Multiple Rs exist 1 Each of these is independently an alkylene group having 2 to 12 carbon atoms or a group represented by the following formula (2): [ka] R 4 This is a polyoxyethylene group with a molecular weight of 200 to 5000 g / mol. Multiple Rs exist 2 Each of these is independently a group represented by formula (2) above or a group represented by formula (3) below, [ka] R 5 This is an alkylene group having 2 to 12 carbon atoms. R 1 and R 2 At least one of them is a group represented by the above formula (2), Multiple Rs exist 3 These are, independently, reactive side chains of amino acids, R in the repeating unit above 3 The content is 1.5 to 2 equivalents. x+y is 1, Both x and y are greater than 0.

[0017] The molecular weight of the polymer according to this embodiment is 5,000 to 30,000 g / mol, may be 7,000 to 20,000 g / mol, or 10,000 to 15,000 g / mol. The polymer according to this embodiment has 2 to 15 repeating units represented by the above formula (1), may have 5 to 12 units, or may have 7 to 10 units.

[0018] R 1 The group may be an alkylene group having 2 to 12 carbon atoms, an alkylene group having 4 to 10 carbon atoms, or an alkylene group having 6 to 9 carbon atoms. Examples of alkylene groups having 2 to 12 carbon atoms include ethylene, propylene, butylene, pentylene, hexylene, heptylene, octylene, nonylene, decylene, undecylene, and dodecylene groups, and these may be linear or branched.

[0019] R 1 and / or R 2 R is the group represented by the above formula (2). In the above formula (2), 4The molecular weight is 200 to 5000 g / mol, may be 500 to 4500 g / mol, or 1000 to 4000 g / mol. The degree of polymerization of the oxyethylene group in the polyoxyethylene group may be 4 to 115, may be 10 to 100, or may be 20 to 80.

[0020] R 2 R may be a group represented by the above formula (3). In the above formula (3), 5 This group may be an alkylene group having 2 to 12 carbon atoms, an alkylene group having 4 to 10 carbon atoms, or an alkylene group having 6 to 9 carbon atoms. Examples of alkylene groups having 2 to 12 carbon atoms include ethylene, propylene, butylene, pentylene, hexylene, heptylene, octylene, nonylene, decylene, undecylene, and dodecylene groups, and these may be linear or branched.

[0021] R 3 R is a reactive side chain of an amino acid. In this specification, "amino acid" or "amino acid residue" refers to both naturally occurring amino acids and amino acids that do not exist naturally. Amino acids are broadly classified into D-forms and L-forms according to their stereochemistry, but the amino acid residues constituting the polymer in this specification are preferably L-forms. A "reactive side chain" is a side chain having a reactive functional group. Examples of reactive functional groups include hydroxyl groups, thiol groups, carboxyl groups, and amino groups. 3 The group may have at least one selected from the group consisting of a hydroxyl group, a carboxyl group, a thiol group, and an amino group. Examples of amino acids having a reactive side chain include serine (Ser, S), threonine (Thr, T), cysteine ​​(Cys, C), methionine (Met, M), aspartic acid (Asp, D), glutamic acid (Glu, E), lysine (Lys, K), arginine (Arg, R), asparagine (Asn, N), glutamine (Gln, Q), tyrosine (Tyr, Y), histidine (His, H), and tryptophan (Trp, W).

[0022] R 3The compound may be modified with an activated functional group. An activated functional group is a functional group that can react with amino groups, thiol groups, hydroxyl groups, carboxyl groups, etc., and form crosslinks or linkers via the resulting covalent bonds. Examples of activated functional groups include amine-reactive groups, hydroxyl-reactive groups, thiol-reactive groups, aldehyde or ketone-reactive groups, and carboxyl-reactive groups. More specifically, examples of activated functional groups include functional groups that can react with amine groups such as amines, epoxy, isothiocyanates, isocyanates, succinimidyl esters, sulfonyl halides, aryl halides, aldehydes, glutaraldehyde, anhydrides, and imidazole carbamates; functional groups that can react with thiol groups such as alkylene halides, aryl halides, maleimides, peroxides, vinyl sulfones, and disulfides; functional groups that can react with carboxylic acid groups such as amines and hydroxyls; and functional groups that can react with hydroxyl groups such as epoxy and azides. The activated functional group is preferably a maleimide group. When the activating functional group is a maleimide group, no extra catalyst is required during reactions with thiol groups, etc., and no by-products that have adverse effects on the human body and the environment are produced, thus improving biocompatibility. In addition, even if the bond between the maleimide group and thiol groups, etc. is broken down in the body, there is the advantage that it has little effect on the body. When the activating functional group is a maleimide group, the polymer according to this embodiment has superior biodegradability and biocompatibility, making it more suitable for use in the fields of nanobiotechnology and biomaterials.

[0023] R in the repeating unit represented by the above formula (1) 3 The content is 1.5 to 2 equivalents, may be 1.6 to 1.9 equivalents, or may be 1.7 to 1.8 equivalents. In this specification, "equivalent" means the amount of R contained in 1 mole of the repeating unit represented by the above formula (1). 3 This refers to the number of moles.

[0024] x and y may be 0.1 and 0.9, 0.2 and 0.8, 0.3 and 0.7, 0.4 and 0.6, 0.45 and 0.55, 0.5 and 0.5, 0.55 and 0.45, 0.6 and 0.4, 0.7 and 0.3, 0.8 and 0.2, or 0.9 and 0.1, respectively. Preferably, x and y are 0.45 and 0.55, or 0.65 and 0.45, and more preferably 0.5 and 0.5, respectively.

[0025] The polyether ester amide and polyether ester urethane according to this embodiment are polymers obtained by polycondensation of at least two monomers. The polyether ester amide and polyether ester urethane according to this embodiment can be produced by polycondensation using raw materials containing a monomer represented by the following formula (4) (monomer (A)) and a monomer represented by the following formula (5) (monomer (B)). The polycondensation method will be described later. [ka] [ka]

[0026] In equation (4) above, R 1 As for the R that the above equation (1) has 1 The same group as the one exemplified is shown as R. 1 If is an alkylene group having 2 to 12 carbon atoms, monomer (A) is a hydrophobic monomer in the activated form of a dicarboxylic acid. 1 When is the group represented by formula (2) above, monomer (A) is a hydrophilic monomer in the activated form of a dicarboxylic acid.

[0027] In the above formula (4), there are multiple LG 1This is an independently leaving electron-withdrawing substituent. Examples of leaving electron-withdrawing substituents include groups derived from p-fluorophenol, p-chlorophenol, p-bromophenol, p-nitrophenol, p-cyanophenol, p-fluorothiophenol, p-chlorothiophenol, p-bromothiophenol, p-nitrothiophenol, p-cyanothiophenol, N-hydroxysuccinimide, N-trimethylsilylimidasol, and the like.

[0028] Monomer (A) can be produced by known esterification reactions in general organic synthesis methods. The method for producing monomer (A) is described by LG. 1 Let us explain using the example where both groups are derived from p-nitrophenol. In a solvent, the esterification reaction between the dichloride of a saturated dicarboxylic acid and p-nitrophenol is carried out in the presence of a coupling agent and a catalyst. The solvent in the esterification reaction may contain an organic base reagent (e.g., pyridine). By recrystallizing and purifying the resulting ester compound, monomer (A) can be obtained.

[0029] Examples of saturated dicarboxylic acids include butanediic acid, glutaric acid, adipic acid, pimelic acid, suberic acid, azelaic acid, sebacic acid, undecanediic acid, dodecanediic acid, tridecanediic acid, and tetradecanediic acid. Suitable saturated dicarboxylic acid dichlorides include butanediic acid dichloride, glutaric acid dichloride, adipic acid dichloride, pimelic acid dichloride, suberic acid dichloride, azelaic acid dichloride, sebacic acid dichloride, undecanediic acid dichloride, dodecanediic acid dichloride, tridecanediic acid dichloride, and tetradecanediic acid dichloride.

[0030] In equation (5) above, R 2 As for the R that the above equation (1) has 2 The same group as the one exemplified is shown as R. 2 If the group is represented by formula (2) above, monomer (B) is a hydrophilic monomer. 2If the group is represented by formula (3) above, monomer (B) is a hydrophobic monomer.

[0031] In equation (5) above, R 6 The R that is present in the above equation (1) 3 It may also be the case that the reactive functional group is protected by a protecting group. 3 That's fine.

[0032] In the above formula (5), there are multiple LG 2 These are acids that can independently form salt compounds with the N-terminus of amino acid residues. Examples of acids that can form salt compounds with the N-terminus of amino acid residues include sulfonic acids, trifluoroacetic acid, and hydrochloric acid.

[0033] Monomer (B) can be synthesized, for example, by the first and second steps described later. The first and second steps are performed by LG 2 Let us explain using the case where p-toluenesulfonic acid is used as an example. In the first step, an amino acid and a saturated hydrocarbon diol or polyoxyethylene compound are dissolved in a solvent, and the esterification reaction is carried out in the presence of a condensing agent and a catalyst. This causes the esterification reaction between the carboxyl group at the C-terminus of the amino acid and the hydroxyl group of the saturated hydrocarbon diol or polyoxyethylene compound to proceed, and an intermediate monomer (B) is produced. Either or both of the N-terminus of the amino acid used in the esterification reaction and the reactive functional group present in the side chain may be protected by a protecting group.

[0034] In the second step, the N-terminus of the intermediate amino acid residue is subjected to a chloride reaction with p-toluenesulfonic acid, resulting in the deprotection of the Boc group and yielding monomer (B). If a protecting group is attached to the reactive side chain, the protecting group may be removed according to conventional methods.

[0035] Examples of saturated hydrocarbon diols include 1,2-ethanediol, propanediol, 1,3-propanediol, 1,4-butanediol, 1,5-pentanediol, 1,6-hexanediol, 1,7-heptanediol, 1,8-octanediol, 1,9-nonanediol, 1,10-decanediol, 1,11-undecanediol, and 1,12-dodecanediol.

[0036] Examples of amino acid protecting groups include the tert-butoxycarbonyl group (Boc group), benzyl group (Bzl group), methyl group (Me group), trityl group (Trt group), acetamidomethyl group (Acm group), sulfoxide group (SO group), methyl ester group (OMe group), benzyl ester group (OBzl group), 9-fluorenylmethyloxycarbonyl group (Fmoc group), benzyloxycarbonyl group (Cbz group), 4-methyltrityl group (Mtt group), 4-methoxy-2,3,6-trimethylbenzenesulfonyl group (Mtr group), methanethiosulfonate group (Mts group), 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl group (Pbf group), and xantyl group (Xan group).

[0037] For example, an amino acid in which the N-terminus is protected by a Boc group and the reactive functional group in the side chain is protected by a Bzl group is denoted as Boc-Ser(Bzl)-OH. Examples of amino acids in which the N-terminus and the reactive functional group in the side chain are protected by protecting groups include Boc-Ser(Bzl)-OH, Boc-Ser(Me)-OH, Boc-Ser(Trt)-OH, Boc-Thr(Bzl)-OH, Boc-Thr(Me)-OH, Boc-Cys(Acm)-OH, Boc-Cys(Trt)-OH, Boc-Cys(Bzl)-OH, Boc-Cys(Me)-OH, Boc-Met(SO)-OH, Boc-Asp(OMe)-OH, Boc-Asp(OBzl)-OH, Boc-Glu(OBzl)-OH, Boc-Glu(OMe)-OH, Boc-Lys(Fmoc)-OH, Boc-Lys(Cbz)-OH, Boc-Lys(Mtt)-OH, Boc-Arg(Mtr)-OH, Boc-Arg(Mts)-OH, Boc-Arg(Cbz)2-OH, Boc-Arg(Pb f) -OH, Boc-Asn(Xan)-OH, Boc-Asn(Trt)-OH, Boc-Gln(Trt)-OH, Boc-Tyr(Bzl)-OH, Boc-Tyr(Me)-OH, Boc-His(Cbz)-OH, Boc-Trp(Mts)-OH, and the like. In this embodiment, the amino acid used as a raw material is preferably lysine (Boc-Lys(Cbz)-OH), in which the N-terminus is protected by a Boc group and the reactive functional group of the side chain is protected by a Cbz group.

[0038] The polyether ester amide and polyether ester urethane according to this embodiment may contain only the monomer represented by formula (4) and the monomer represented by formula (5) as constituent units, or may contain a third monomer other than the monomers represented by formulas (4) and (5) as constituent units. Both ends of the third monomer may be the same as either end of the monomer represented by formulas (4) and (5). Based on the mass of the polyether ester amide and polyether ester urethane according to this embodiment, the content of monomers having amino acid residues with reactive functional groups (monomer (B)) is preferably 50% by mass or more, more preferably 60% by mass or more, and even more preferably 70% by mass or more.

[0039] The polyether ester amide and polyether ester urethane according to this embodiment may be polymers having a repeating structure represented by the following formula (6). n is 2 to 15, may be 5 to 12, or may be 7 to 10. [ka]

[0040] The polycondensation for producing the polyether ester amide and polyether ester urethane according to this embodiment can be carried out, for example, as follows. First, monomers (A) and (B), and optionally a third monomer, are dispersed in a solvent and completely dissolved at 60°C. Examples of solvents include dimethyl sulfoxide (DMSO), dimethylformamide (DMF), and dimethylacetamide (DMAc). Next, an organic base reagent is added to the obtained monomer solution and the polycondensation reaction is carried out at 70°C for 48 hours. Examples of organic base reagents include diazabicyclononene, diazabicycloundecene, N-ethyldiisopropylamine, triethylamine, 2,6-lutidine, and pyridine. From the obtained reaction solution, a polymer having repeating units represented by the following formula (7) can be obtained by dialysis and / or freeze-drying. [ka]

[0041] In the repeating unit represented by the above formula (7), R 6 R 3 If so, the polymer obtained by the polycondensation reaction of the above monomers is the polyether ester amide and polyether ester urethane according to this embodiment. In the repeating unit represented by formula (7) above, R 6 However, R where the reactive functional group is protected by a protecting group 3 If this is the case, the polyether ester amide and polyether ester urethane according to this embodiment can be obtained by removing the protecting group and deprotecting the product. The deprotection reaction can be carried out by a known deprotection method appropriate to the type of protecting group. For example, side chains protected with a Cbz group, a Bzl group, etc. can be deprotected using a trifluoroacetic acid-based cocktail reagent, a methanesulfonic acid-based cocktail reagent, a hydrobromic acid-based cocktail reagent, a palladium / carbon-hydrogen reducing agent, etc. Side chains protected with a methyl ester group can be deprotected by treatment with a basic reagent cocktail. Side chains protected with a Trt group, a Pbf group, etc. can be deprotected by treatment with a trifluoroacetic acid-based cocktail reagent.

[0042] If at least one of monomer (A) and monomer (B) is a hydrophilic monomer, the resulting polymer can be made hydrophilic. When monomer (A) is a hydrophobic monomer and monomer (B) is a hydrophilic monomer, the resulting polymer is called a polyether ester amide. When monomer (A) is a hydrophilic monomer and monomer (B) is a hydrophobic monomer, the resulting polymer is called a polyether ester urethane (I). When both monomer (A) and monomer (B) are hydrophilic monomers, the resulting polymer is called a polyether ester urethane (II).

[0043] The polyether ester amides and polyether ester urethanes (polyether ester urethanes (I) and (II)) according to this embodiment can easily provide polymers having reactive functional groups and / or activating reactive groups. The polyether ester amides and polyether ester urethanes (I) and (II) according to this embodiment can be suitably used for conjugation of biomolecules (bioponjugation). Bioconjugation is a chemical means of linking multiple biomolecules by stable covalent bonds. The polyether ester amides and polyether ester urethanes (I) and (II) according to this embodiment can be bonded to various biomolecules directly or indirectly via low molecular weight linkers.

[0044] The polyether ester amides and polyether ester urethanes (I) and (II) according to this embodiment have high biocompatibility and biodegradability because they use amino acids as raw materials. Furthermore, they have excellent hydrophilicity because they have polyoxyethylene groups in their main chain. For these reasons, they can be suitably used in the field of biomaterials as described above. In addition, by introducing amino acid residues having one equivalent or more of reactive side chains per mole of monomer raw materials, the amount and / or type of reactive functional groups introduced increases. As a result, the polymers according to this embodiment are expected to react more readily with various compounds, and thus have a wider range of applications.

[0045] Examples of biomolecules include antibodies, antigens, peptides, proteins, DNA, RNA, enzymes, and lipids. The complexes of these biomolecules with the polyether ester amide and polyether ester urethanes (I) and (II) according to this embodiment are referred to as "bioponjugation products." The bioconjugation products may also be complexes of biomolecules, the polymers according to this embodiment, and other molecules. Examples of other molecules include low-molecular-weight linkers, and more specifically, peptide linkers. Bioconjugation products can be applied, for example, to cell culture scaffolds, drug delivery systems, ELISA (enzyme-linked immunosorbent assay), immunoassays, antibody-drug conjugates, drug discovery reagents, biosensor media, and pathogen diagnostic reagents.

[0046] As a peptide linker, for example, a peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 (Ac-GCRGGPAGMRGKGRCG-NH2: SEQ ID NO: 1) can be used. In SEQ ID NO: 1, the N-terminal Ac- means that the N-terminus is acetylated, and the C-terminal -NH2 means that the C-terminus is amidated. The amino acid sequence represented by SEQ ID NO: 1 is an amino acid sequence that can be cleaved by various proteases (e.g., MMP-2, MMP-3, and MMP-9) and can be produced as follows.

[0047] <Composition of reaction solution, buffer solution, etc.> • Deprotection solution: Piperidine (Fujifilm Wako Pure Chemical Industries, Ltd., 25 vol%), Dimethylformamide; DMF (Fujifilm Wako Pure Chemical Industries, Ltd., 75 vol%) · Amino acid cocktail: N-terminal Fmoc-protected amino acids (Fmoc-Ala-OH, Fmoc-Gly-OH, Fmoc-Ile-OH, Fmoc-Leu-OH, Fmoc-Pro-OH, Fmoc-Gln(OtBu)-OH, Fmoc-Val-OH and Fmoc-Trp(Boc)-OH are manufactured by Merck KGaA, Fmoc-Met-OH is manufactured by Sigma-Aldrich, Fmoc-Lys(Boc)-OH, Fmoc-Arg(pbf)-OH and Fmoc-Cys(Trt)-OH are manufactured by Chemscene, 6 equivalents relative to the reaction point), ethyl cyano(hydroxyimino)acetate; Oxyma pure (trademark, manufactured by Merck KGaA, 6 equivalents relative to the reaction point), N,N'-diisopropylcarbodiimide (manufactured by FUJIFILM Wako Pure Chemical Corporation, 6 equivalents relative to the reaction point), 2,4,6-trimethylpyridine (manufactured by Tokyo Chemical Industry Co., Ltd., 12 equivalents relative to the reaction point), DMF (amount of solvent) · Inactivation solution: acetic anhydride (manufactured by FUJIFILM Wako Pure Chemical Corporation, 25 vol%), DMF (75 vol%) · Deprotection solution: trifluoroacetic acid; TFA (manufactured by Tokyo Chemical Industry Co., Ltd., 95 mass%), triisopropylsilane (manufactured by FUJIFILM Wako Pure Chemical Corporation, 2.5 mass%), distilled water (2.5 mass%) · Eluent A: 0.1% TFA / distilled water · Eluent B: 0.1% TFA / acetonitrile (for high performance liquid chromatography, manufactured by FUJIFILM Wako Pure Chemical Corporation)

[0048] <Fmoc Peptide Solid Phase Synthesis> A peptide consisting of the amino acid sequence represented by Sequence ID No. 1 is synthesized by the Fmoc solid-phase synthesis method. An Econopack column (20 mL, BIO RAD) is set in a multi-solid-phase synthesizer (KMS-3, Kokusan Chemical Co., Ltd.), and Rink amide AM resin (100-200 mesh, 0.7 mmol / g, Merck KGaA) is weighed to a 0.05 mmol scale. Then, 2 mL of dichloromethane (DCM, peptide synthesis grade, Fujifilm Wako Pure Chemical Industries, Ltd.) is added and the mixture is swollen under supercurrent stirring for 12-24 hours. After removing the swollen solution, 2 mL of deprotection solution is added to deprotect the Fmoc groups on the resin (2 mL x 2 times, 10 minutes each). After removing the deprotection solution, the mixture is washed with DMF and DCM, and then an amino acid cocktail is added and reacted under supercurrent stirring for 2-3 hours to couple the amino acids onto the resin support. After removing the reaction solution, 2 mL of inactivating solution is added and treated under supercurrent stirring for 30 minutes to cap any unreacted sites. After removing the inactivating solution, the mixture is washed with DMF and DCM to obtain an Fmoc amino acid-introduced resin support. Subsequently, the above deprotection, coupling reaction, and inactivation of unreacted sites are repeated to synthesize a peptide consisting of the amino acid sequence represented by SEQ ID NO: 1.

[0049] <Rough purification, LC separation and purification> After synthesizing a peptide consisting of the amino acid sequence represented by Sequence ID No. 1, the Fmoc group at the N-terminus is deprotected, and the peptide is washed with DMF, DCM, and methanol. A dry resin support is obtained by standing the mixture in a desiccator for 24 hours. Two mL of chilled deresination solution is added to the dry resin support, and the mixture is treated under supercurrent stirring for 3-4 hours to achieve deresination and deprotection of the side chain protecting group. After collecting the supernatant containing the peptide, another two mL of the deresination solution is added to wash the resin support. Tenfold of cold diethyl ether is added to the supernatant and washing solution (total 4 mL), and the precipitate is collected by centrifugation (2,000 × g, 10 min). The precipitate is washed again with cold diethyl ether and then dried under reduced pressure to obtain a crude polypeptide. The obtained crude polypeptide is dissolved in eluent A, and then the target component is fractionated using eluent A and eluent B with a medium-pressure liquid chromatograph (model: EPCLC-AI-580S, Yamazen Corporation). After removing acetonitrile under reduced pressure, the peptide is freeze-dried to obtain the purified peptide. [Examples]

[0050] The present invention will be described in detail below with reference to embodiments for carrying out the present invention, but these are merely examples for illustrating the present invention and are not intended to limit the present invention to the following. Furthermore, the following examples can be modified as appropriate within the scope of the present invention. Unless otherwise stated, commercially available reagents were used. "NMR measurement results" were obtained by proton nuclear magnetic resonance spectroscopy using a nuclear magnetic resonance spectrometer (JEOL Ltd., product name JNM-ECZ400S / LI, 400MHz). 1 This is the result of the 1H-NMR spectral analysis. The "Yield" (%) is calculated as 100 × yield (mol) of the target substance (hydrophobic monomer I or II, or hydrophilic monomer I and II) / amount (mol) of raw materials used (azelaic acid chloride, polyethylene glycol (PEG), or 1,9-nonanediol).

[0051] <Synthesis of hydrophobic monomer I> In a flask purged with nitrogen gas, 3 equivalents of p-nitrophenol (starting material) and 4 equivalents of pyridine were dissolved in acetone (100 mL). 1 equivalent of azelaic acid chloride was added dropwise to the same flask, and the mixture was stirred at room temperature for 5 hours to react p-nitrophenol with azelaic acid. The resulting crude product was washed with aqueous hydrochloric acid and brine, and purified by recrystallization. These operations yielded a white, powdery hydrophobic monomer I in 95% yield. Hydrophobic monomer I is a compound represented by the following formula (8). Figure 1 shows the NMR measurement results. In Figure 1, the vertical axis represents the relative intensity of the proton signal, and the horizontal axis represents the chemical shift (ppm). From Figure 1, it was confirmed that the target monomer had been synthesized. [ka]

[0052] <Synthesis of hydrophilic monomer I> In a flask purged with nitrogen gas, 1 equivalent of PEG (molecular weight: 2,000 g / mol, degree of polymerization a (constant)) and 4 equivalents of pyridine were dissolved in 100 mL of dichloromethane. 4 equivalents of 4-nitrophenyl chloroformate were added dropwise to the same flask, and the mixture was stirred at room temperature for 18 hours to react the PEG with 4-nitrophenyl chloroformate. The resulting crude product was washed with aqueous hydrochloric acid and purified by precipitation with diethyl ether. These operations yielded a white, waxy hydrophilic monomer I in 90% yield. Hydrophilic monomer I is a compound represented by the following formula (9). Figure 2 shows the NMR measurement results. In Figure 2, the vertical axis represents the relative intensity of the proton signal, and the horizontal axis represents the chemical shift (ppm). Figure 2 shows that the target monomer was synthesized. [ka]

[0053] <Synthesis of hydrophobic monomer II> In a flask purged with nitrogen gas, 1 equivalent of 1,9-nonanediol and 2.2 equivalents of Boc-Lys(Cbz)-OH were dissolved in 100 mL of dichloromethane. The coupling agent and catalyst were added to the same flask, and the reaction was allowed to proceed at room temperature for 18 hours. The resulting crude intermediate was washed with acidic aqueous solution, basic aqueous solution, and brine to obtain an intermediate. The Boc groups, which are the protecting groups at both ends of the obtained intermediate, were removed by substitution under toluene with 2.1 equivalents of p-toluenesulfonic acid to synthesize the p-toluenesulfonate compound. The resulting crude product was obtained by recrystallization. These operations yielded a white powdery hydrophobic monomer II in 85% yield. Hydrophobic monomer II is a compound represented by the following formula (10). Figure 3 shows the NMR measurement results. In Figure 3, the vertical axis is the relative intensity of the proton signal, and the horizontal axis is the chemical shift (ppm). From Figure 3, it can be seen that the target monomer has been synthesized. [ka]

[0054] <Synthesis of hydrophilic monomer II> In a flask purged with nitrogen gas, 1 equivalent of PEG (molecular weight: 2,000 g / mol, degree of polymerization a (constant)) and 2.2 equivalents of Boc-Lys(Cbz)-OH were dissolved in 100 mL of dichloromethane. The condensing agent and catalyst were added to the same flask and reacted at room temperature for 18 hours. The resulting crude intermediate was washed with acidic aqueous solution, basic aqueous solution, and brine to obtain an intermediate. The Boc groups, which are the protecting groups at both ends of the intermediate, were removed by substitution under toluene with 2.1 equivalents of p-toluenesulfonic acid to synthesize a p-toluenesulfonate compound. The resulting crude product was purified by precipitation with diethyl ether. These operations yielded a white to yellow waxy hydrophilic monomer II in 85% yield. Hydrophilic monomer II is a compound represented by the following formula (11). Figure 4 shows the NMR measurement results. In Figure 4, the vertical axis is the relative intensity of the proton signal, and the horizontal axis is the chemical shift (ppm). Figure 4 shows that the target monomer has been synthesized.

[0055] [ka]

[0056] <Polymer polycondensation> The polyether ester urethane (I) (Cbz-polyether ester urethane (I)), polyether ester amide (Cbz-polyether ester amide), and polyether ester urethane (II) (Cbz-polyether ester urethane (II)), which are included in the compound represented by the following general formula (12), were obtained by polycondensation of the above-mentioned hydrophobic monomer I or hydrophilic monomer I with hydrophobic monomer II or hydrophilic monomer II. Cbz-polyether ester amide was obtained by polycondensation of hydrophobic monomer I and hydrophilic monomer II, where x was b (constant), y was c (constant), and n was d (constant) in the following general formula (12). Cbz-polyether ester urethane (I) was obtained by polycondensation of hydrophilic monomer I and hydrophobic monomer II, where x was e (constant), y was f (constant), and n was g (constant) in the following general formula (12). Cbz-polyether ester urethane (II) was obtained by polycondensation of hydrophilic monomer I and hydrophilic monomer II. The compounds represented by the following general formula (12) do not include polyesteramide (Cbz-polyesteramide), which is a compound obtained by polycondensation of hydrophobic monomer I and hydrophobic monomer II. The polycondensation was carried out by dissolving 1 equivalent of hydrophobic monomer I or hydrophilic monomer I and 1 equivalent of hydrophobic monomer II or hydrophilic monomer II in DMSO and DMAc at 60°C, and then adding TEA. The polycondensation reaction was carried out at 70°C for 48 hours, and the mixture was purified by dialysis to obtain a yellow waxy polymer. [ka]

[0057] <Polymer side chain delinking protection> The Cbz groups of Cbz-polyether ester urethane (I), Cbz-polyether ester amide, Cbz-polyether ester urethane (II), and Cbz-polyester amide were removed by acid treatment, and each polymer was deprotected. The resulting crude products were purified by dialysis to obtain a yellow, waxy polymer. The polyether ester amide and polyether ester urethane (I) obtained by these operations are examples and have repeating units represented by the following general formula (13). The polyester amide obtained by these operations is a comparative example and does not have repeating units represented by the following general formula (13). [ka]

[0058] <Conjugation: Maleimide group> Maleimide groups are widely used in fields such as nanobiotechnology and biomaterials because they can undergo Michael reactions with molecules containing thiol groups, produce no byproducts, and are non-cytotoxic. In this example, polymers containing maleimide groups were produced. Maleimide groups were introduced into lysine-free amines of polyether ester urethane (I), polyether ester amide, polyether ester urethane (II), and polyester amide obtained by "<polymer side chain deprotection>".

[0059] To introduce maleimide groups into hydrophilic polyether ester urethane (I), polyether ester amide, and polyether ester urethane (II), these polymers were first dissolved in MES (2-morpholinoethanesulfonic acid) buffer at pH 4.5-5. 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl), N-hydroxysuccinimide (NHS), and 6-maleimidohexanoic acid were added, and the mixture was stirred for 24 hours to carry out the maleimide group addition reaction. After the reaction, the mixture was dialyzed with an acidic aqueous solution (pH 4.5-5), and then freeze-dried to obtain a white to yellow sponge-like polymer.

[0060] In the case of hydrophobic polyesteramides, polyesteramide and N-(6-maleimidocaproyloxy)succinimide (EMCS) were first dissolved in DMF and DMSO. These compounds were reacted for 24 hours and purified by dialysis to obtain a yellow, waxy polymer.

[0061] Figure 5 shows the NMR measurement results of a polyether ester amide (Mal-polyether ester amide, x=b (constant), y=c (constant), n=d (constant), hydrophobic) to which a maleimide group was introduced. In Figure 5, the vertical axis represents the relative intensity of the proton signal, and the horizontal axis represents the chemical shift (ppm). From the peaks shown in Figure 5, it was found that the target polymer was obtained.

[0062] Figure 6 shows the molecular weight measurement results by GPC (gel filtration chromatography). In Figure 6, the vertical axis represents the detection signal intensity, and the horizontal axis represents the elution time (minutes). From the results in Figure 6 and the results calculated using the calibration curve of the polystyrene standard sample, it was found that the molecular weight (Mw) of the Cbz-polyether ester amide obtained by "<polymer polycondensation>" is approximately 33,000 g / mol (PDI: 1.75), the molecular weight (Mw) of the polyether ester amide obtained by "<polymer side chain deprotection>" is approximately 21,000 g / mol (PDI: 1.39), and the molecular weight (Mw) of the Mal-polyether ester amide is approximately 22,000 g / mol (PDI: 1.37).

[0063] Figure 7 shows the NMR measurement results of a polyether ester urethane (I) (Mal-polyether ester urethane (I), x=e (constant), y=f (constant), n=g (constant), hydrophilic) with introduced maleimide groups. In Figure 7, the vertical axis represents the relative intensity of the proton signal, and the horizontal axis represents the chemical shift (ppm). From the peaks shown in Figure 7, it was found that the target polymer was obtained.

[0064] Figure 8 shows the molecular weight measurement results by GPC (gel filtration chromatography). In Figure 8, the vertical axis represents the detection signal intensity, and the horizontal axis represents the elution time (minutes). From the results in Figure 8 and the results calculated using the calibration curve of the polystyrene standard sample, it was found that the molecular weight (Mw) of Cbz-polyether ester urethane (I) obtained by "<polymer polycondensation>" is approximately 45,000 g / mol (PDI: 1.90), the molecular weight (Mw) of polyether ester urethane (I) obtained by "<polymer side chain deprotection>" is approximately 30,000 g / mol (PDI: 1.49), and the molecular weight (Mw) of Mal-polyether ester urethane (I) is approximately 40,000 g / mol (PDI: 1.42).

[0065] <Confirmation of the reactivity of the maleimide group: Gellation reaction using thiols> Maleimide groups are widely used in fields such as nanobiotechnology and biomaterials because they can undergo Michael reactions with molecules containing thiol groups, produce no byproducts, and are non-cytotoxic. As an example of the application of the present invention, reactivity was verified by gelation using a polymer containing maleimide groups and a peptide having dithiol groups at both ends. A conceptual diagram of gelation is shown in Figure 9.

[0066] A maleimide-modified polyether ester urethane (I) was dissolved in phosphate-buffered saline (PBS) to a concentration of 15, 20, or 25% by mass / volume (w / v%), and the pH was adjusted to 7-8. The resulting solution was called the polymer solution. The dithiol peptide was dissolved in PBS to a concentration of 20 nM, and the pH was adjusted to 7-8. The resulting solution was called the dithiol peptide solution. The dithiol peptide sequence used in this embodiment is a peptide consisting of the amino acid sequence represented by Sequence ID No. 1 (Ac-GCRGGPAGMRGKGRCG-NH2: Sequence ID No. 1).

[0067] A gel precursor aqueous solution was obtained by mixing a polyether ester urethane (I) solution (polymer solution), a dithiol peptide solution, and a solvent so that the volume ratio of the polymer solution to the dithiol peptide solution was 1:1, 2:1, 3:1, or 4:1. At room temperature, a gel was formed via covalent bonding between maleimide groups and thiol groups. Figure 10 shows photographs of examples of gels. Figure 10(A) is a gel using a 15 w / v% polymer solution (from left to right, the volume ratios of the polymer solution to the dithiol peptide solution are 1:1, 2:1, 3:1, and 4:1), Figure 10(B) is a gel using a 20 w / v% polymer solution (from left to right, the volume ratios of the polymer solution to the dithiol peptide solution are 1:1, 2:1, 3:1, and 4:1), and Figure 10(C) is a gel using a 25 w / v% polymer solution (from left to right, the volume ratios of the polymer solution to the dithiol peptide solution are 1:1, 2:1, 3:1, and 4:1). In this example, the viscosity of the gel increased with increasing polymer solution concentration or with increasing volume proportion of polymer solution in the gel precursor aqueous solution. A more stable gel was formed under conditions where the polymer solution concentration was 20 w / v% or higher, and where the volume ratio of polymer solution to dithiol peptide solution was 3:1 or 4:1.

[0068] <Image of the internal structure of the gel> Images of polyether ester urethane (I) gels were acquired using a scanning electron microscope (SEM). Figure 11 shows the structure of the freeze-dried gels. Figure 11(A) shows gels prepared under conditions of a polymer solution concentration of 20 w / v% and a volume ratio of polymer solution to dithiol peptide solution of 3:1, and Figure 11(B) shows gels prepared under conditions of a polymer solution concentration of 25 w / v% and a volume ratio of polymer solution to dithiol peptide solution of 3:1, both images taken at 500x magnification. The gels were composed of fibers and numerous pores, and the numerous pores were interconnected.

[0069] <Cell culture evaluation - without gel formation> MSCs were seeded in 24-well plates (flat bottom, 15.7 mm diameter) so that approximately 10,000 cells were present per well. Each well was pre-filled with either an MSC-specific culture medium containing polyether ester urethane (I) at concentrations of 0.1 mg / 500 μl / well, 1 mg / 500 μl / well, or 10 mg / 500 μl / well, or an MSC-specific culture medium without polyether ester urethane (I). After continuous culture for 7 or 14 days using standard cell proliferation culture methods, the cytotoxicity of the polymer was evaluated by WST-1 cell viability assay and cell counting. On day 7 or 14 from the start of culture, the actin fibers of the MSCs were stained with phalloidin-actin, and the nuclei were stained with DAPI reagent, and images were acquired. The acquired images are shown in Figures 12(A) and 12(B). Figures 12(A) and (B) show cell nuclei and actin fibers stained with DAPI. The number of cells in the obtained images was measured using dedicated cell counting software. The results of this cell count measurement are shown as black triangles in the graph in Figure 12(C). In addition, on day 7 (Day-7) or day 14 (Day-14) from the start of culture, WST-1 reagent was added to the wells and the absorbance of the plate was measured at an observation wavelength of 438 nm. The measurement results are shown as a bar graph in Figure 12(C). In Figure 12(B), the vertical axis (left) shows absorbance, and the vertical axis (right) shows cell number. In Figure 12(C), the horizontal axis shows the content of polyether ester urethane (I) in the culture medium. From Figure 12(C), it was found that on day 14 from the start of culture, the absorbance was higher when the polyether ester urethane (I) content was 0.1 mg / 500 μl / well and 1 mg / 500 μl / well. This is thought to be because the gel according to this embodiment was able to effectively capture the cells.

[0070] <Cell culture evaluation - gel formation present> MSCs, the aforementioned polyether ester urethane (I) solution (polymer solution), and a culture medium specifically for MSCs were mixed with dithiol peptide (20 nM) (polymer solution concentration = 25 w / v%, volume ratio of polymer solution to dithiol peptide solution = 3:1), and a gel was formed on a low-adhesion 24-well plate (flat bottom, diameter 15.7 mm). The cell concentration of the MSCs initially mixed was adjusted so that the number of cells per well was 3000. After culturing the MSCs in the gel for 3 days, cell viability was evaluated by LIVE / DEAD staining. The results are shown in Figure 13(A). Figure 13(A) shows that when using the polyether ester urethane (I) of the present invention, almost no dead cells were observed.

[0071] A polyether ester urethane (I) gel (polymer solution concentration = 25 w / v%, volume ratio of polymer solution to dithiol peptide solution = 3:1) was formed on a low-adhesion 24-well plate (flat bottom, 15.7 mm diameter), and then MSCs were seeded on the gel surface (3000 cells / well). After culturing for 3 days, actin fibers were stained with phalloidin-actin and nuclei were stained with DAPI reagent, and images of the cells were obtained. The obtained images are shown in Figure 13(B). Figure 13(B) shows that good cell viability was observed when using the polyether ester urethane (I) of the present invention.

Claims

1. A polymer having a molecular weight of 5,000 to 30,000 g / mol and possessing 2 to 15 repeating units represented by the following formula (1). 【Chemistry 1】 [In the formula, There are multiple R １ Each of these is independently an alkylene group having 2 to 12 carbon atoms or a group represented by the following formula (2): 【Chemistry 2】 R ４ This is a polyoxyethylene group with a molecular weight of 176 to 5060 g / mol. There are multiple R ２ Each of these is independently a group represented by formula (2) or a group represented by formula (3) below, 【Transformation 3】 R ５ This is an alkylene group having 2 to 12 carbon atoms. R １ and R ２ At least one of them is a group represented by formula (2), There are multiple R ３ Each of these is independently a reactive side chain of an amino acid, and the reactive side chain is a side chain having at least one selected from the group consisting of a hydroxyl group, a carboxyl group, a thiol group, and an amino group. R in the repeating unit ３ The content is 1.5 to 1.9 equivalents. x + y is 1, [Both x and y are greater than 0]

2. R ３ The polymer according to claim 1, wherein the R is modified with an activating functional group.

3. The polymer according to claim 2, wherein the activating functional group is a maleimide group.

4. A bioconjugation product that is a complex of the polymer described in any one of claims 1 to 3 with at least one selected from the group consisting of peptides, antibodies, antigens, RNA, and DNA.

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