Polyrotaxane containing aldehyde group-added cyclic molecule, method for producing said polyrotaxane, stretchable biomaterial and method for producing said biomaterial

Abstract

[Problem] To develop an elastic biomaterial (in particular, elastic collagen). [Solution] A method for producing a polyrotaxane containing a cyclic molecule having an aldehyde group added thereto, said method being capable of specifically adding the aldehyde group to the cyclic molecule of the polyrotaxane. A method for producing an elastic biomaterial, said method including a cross-linking method that can suppress reductive amination and the formation of a free aldehyde. Further, it was confirmed that filamentous collagen has elasticity so that the present disclosure has been completed.

Description

[Technical Field]

[0001] The present disclosure relates to a polyrotaxane containing an aldehyde group-added cyclic molecule and a method for producing the polyrotaxane, as well as a stretchable biomaterial and a method for producing the biomaterial. This application claims priority from Japanese Application No. 2022-129674, which is incorporated herein by reference.

[0002] (macrocyclic compounds) Macrocyclic compounds are useful in the development of functional materials in the field of materials chemistry. In particular, cyclodextrins (α-, β-, and γ-CD) have attracted considerable attention due to their high water solubility, ease of mass production, and suitability for chemical modification (Non-Prior Patent Document 1). Furthermore, the internal cavity of CDs can encapsulate the amphiphilic and hydrophobic parts of molecules through inclusion phenomena, making them potentially useful as inclusion compounds for drug delivery systems, artificial enzymes, chemical sensors, and so on (Non-Prior Patent Document 2).

[0003] (Polyrotaxane) Polyrotaxanes (PRs), which consist of CDs and amphiphilic polymers such as polyethylene glycol (PEG) and polypropylene glycol (PPG), are promising soft materials. PEG and PPG penetrate into the cavities of CDs from both the primary and secondary hydroxyl groups. As a result, CDs are randomly oriented along the PR chain, with both the same and opposite orientations, allowing them to move and rotate freely along the chain, resulting in composite materials with the characteristic of slide-rings. These characteristics lead to unique properties such as improved mechanical properties, stimuli-responsiveness, and self-healing properties (Non-Patent Document 3).

[0004] (collagen) Collagen is a highly biocompatible protein that constitutes the majority of mammalian organs and tissues. Furthermore, atelocollagen, which does not contain N- and C-terminal telopeptides, exhibits relatively low immunogenicity compared to collagen. Currently, collagen and atelocollagen are considered promising for realizing human-friendly biomaterials that can be used both in vivo and in vitro. Specifically, atelocollagen is being developed as a biomaterial that can be applied to nucleic acid delivery, regenerative medicine, drug discovery, and other fields. Collagen and atelocollagen can be processed into various forms (gels, sponges, filaments, membranes, fibers, etc.) and can be used in basic and clinical research.

[0005] (thread-like collagen) Several thread-like collagens have been reported (Patent Documents 1 to 3). However, no filamentous collagen cross-linked with polyrotaxane has been disclosed. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Patent Publication No. 2005-314865 [Patent Document 2] Patent Publication No. 2003-193328 [Patent Document 3] Patent Publication No. 2017-086066 [Non-patent literature]

[0007] [Non-Patent Document 1] Chem. Soc. Rev. 2017, 46 (9), 2459-2478. [Non-patent document 2] Chem. Soc. Rev. 2005, 34 (2), 120-132. [Non-patent document 3] Chem. Commun. 2020, 56 (32), 4381-4395. Summary of the Invention [Problem to be solved by the invention]

[0008] The present inventors aimed to develop a stretchable biomaterial (especially stretchable collagen), and attempted to produce thread-like collagen cross-linked with polyrotaxane. However, using conventional methods, (1) it was confirmed that it was difficult to crosslink polyrotaxanes to biomaterials, and furthermore, (2) it was confirmed that the functional group formed by crosslinking (imine, also known as Schiff base) can undergo hydrolysis in vivo to produce free aldehydes. [Means for solving the problem]

[0009] The present inventors have discovered, in order to address the above-mentioned problem (1), a method for producing a polyrotaxane containing an aldehyde group-added cyclic molecule that can specifically add an aldehyde group to a cyclic molecule of the polyrotaxane, and, in order to address the above-mentioned problem (2), a method for producing a stretchable biomaterial that includes a crosslinking method that can suppress reductive amination and the generation of free aldehydes. They have also confirmed that thread-like collagen has stretchability, and have completed the present disclosure. In addition, the present inventors have confirmed that stronger collagen threads can be obtained by controlling the inclusion rate of cyclic molecules in polyrotaxanes containing aldehyde group-added cyclic molecules, thereby completing the present disclosure.

[0010] The present disclosure is as follows. 1. Polyrotaxanes containing aldehyde group-added cyclic molecules, including: Linear molecules: a blocking group (stopper molecule), wherein the blocking group is located at both ends of the linear molecule; and an aldehyde-group-added cyclic molecule, wherein the interior of the aldehyde-group-added cyclic molecule is threaded through the linear molecule; Polyrotaxane. 2. The polyrotaxane according to item 1 above, wherein the polyrotaxane is a water-soluble polyrotaxane. 3. The polyrotaxane according to item 1 or 2 above, wherein the aldehyde group-added cyclic molecule is substantially free of ketone groups. 4. A crosslinked composition comprising the polyrotaxane according to item 1 or 2 above. 5. The crosslinking composition according to the preceding item 4, which is used for crosslinking biomaterials. 6. The polyrotaxane according to item 1 or 2 above, wherein the linear molecule is a structural unit based on polyethylene glycol, and the aldehyde group-added cyclic molecule is a structural unit based on aldehyde group-added cyclodextrin. 7. The polyrotaxane according to item 1 or 2 above, wherein the linear molecule is a structural unit based on polypropylene glycol, and the aldehyde group-added cyclic molecule is a structural unit based on aldehyde group-added cyclodextrin. 8. The polyrotaxane according to item 1 or 2 above, wherein the linear molecule is a structural unit based on poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol), and the aldehyde group-added cyclic molecule is a structural unit based on aldehyde group-added cyclodextrin. 9. A method for producing a polyrotaxane containing an aldehyde group-added cyclic molecule, comprising the steps of: (1) a step of contacting a linear molecule with a cyclic molecule having a hydroxyl group to obtain a compound 3 in which the linear molecule is threaded through the interior of the cyclic molecule having a hydroxyl group: (2) contacting the compound 3 with a blocking group to obtain a compound 2 in which a linear molecule having blocking groups at both ends is threaded through the interior of a cyclic molecule having a hydroxyl group; and (3) A step of oxidizing the compound 2 in the presence of a TEMPO derivative and an iodobenzene derivative to obtain a polyrotaxane containing an aldehyde group-added cyclic molecule. 10. The production method according to the preceding item 9, wherein in the step (2), the compound 3 is dissolved in tetrahydrofuran. 11. The production method according to item 9 or 10 above, wherein in step (3), N,N-diisopropylethylamine is further present. 12. The production method according to the preceding item 9 or 10, wherein in step (3), the compound 2 is dissolved in hexamethylphosphoric triamide, N,N-dimethylformamide, or dimethyl sulfoxide. 13. The production method according to item 9 or 10 above, wherein in step (3), compound 2 is dissolved in hexamethylphosphoric triamide containing N,N-diisopropylethylamine. 14. The production method according to item 9 or 10 above, wherein the linear molecule and the cyclic molecule having a hydroxyl group are selected so that the inclusion rate, which is the molar fraction of aldehyde group-added cyclic molecules per number of repeating units in the linear molecule, is 1 to 40 mol%. 15. The production method according to item 9 or 10 above, wherein the inclusion rate is 2 to 15 mol%. 16. Biomaterials cross-linked with polyrotaxanes. wherein the polyrotaxane comprises: Linear molecules: a blocking group (stopper molecule), wherein the blocking group is located at both ends of the linear molecule; and a cyclic molecule, wherein the interior of the cyclic molecule is threaded through the linear molecule; Biomaterials. 17. The biomaterial according to item 16 above, wherein the polyrotaxane is a polyrotaxane containing an aldehyde group-added cyclic molecule. 18. The biomaterial according to item 17 above, wherein the aldehyde group-added cyclic molecule is substantially free of ketone groups. 19. The biomaterial according to item 17 or 18 above, wherein the biomaterial is collagen. 20. The biomaterial according to item 17 or 18 above, wherein the biomaterial is thread-like collagen. 21. The biomaterial described in the preceding paragraph 20, wherein the linear molecule is a structural unit based on polyethylene glycol, polypropylene glycol, or poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol), the aldehyde group-added cyclic molecule is a structural unit based on aldehyde group-added cyclodextrin, and the biomaterial is elastic thread-like collagen. 22. The biomaterial according to paragraph 21 above, having the following properties: (1) Breaking stress: 280 to 3200 kPa (2) Breaking strain is 40-70% (3) Elastic modulus: 18 to 220 kPa (4) Toughness of 83 to 350 kJ / m 3 23. The biomaterial according to paragraph 22, further having the following properties: (1) Stress is 10 kPa to 1000 kPa or less when strain is 30% to 40%. 24. A method for producing a biomaterial cross-linked with polyrotaxane, comprising the steps of: (1) A step of subjecting a biomaterial having a lysine residue to a reductive amination reaction in the presence of a polyrotaxane containing an aldehyde group-added cyclic molecule. 25. The method according to the preceding paragraph 24, wherein step (1) is a step of contacting a biomaterial having a lysine residue with a buffer solution containing a polyrotaxane containing an aldehyde group-added cyclic molecule, and subjecting the biomaterial to a reductive amination reaction. 26. The production method according to the preceding paragraph 25, wherein the buffer solution in step (1) contains a hydride reducing agent. [Effects of the Invention]

[0011] The present disclosure has one or more of the following advantages. (1) Providing a polyrotaxane containing an aldehyde group-added cyclic molecule (particularly, a polyrotaxane containing an aldehyde group-added cyclic molecule in which a ketone group is not substantially added to the cyclic molecule) (2) Provision of polyrotaxanes containing water-soluble aldehyde-group-added cyclic molecules (3) To provide a method for producing polyrotaxanes containing aldehyde group-added cyclic molecules, which can specifically add aldehyde groups to the cyclic molecules of polyrotaxanes. (4) To provide a method for producing a stretchable biomaterial, including a crosslinking method capable of suppressing the generation of free aldehydes. (5) Providing stretchable biomaterials (especially stretchable collagen) (6) To provide a polyrotaxane containing an aldehyde group-added cyclic molecule with a controlled inclusion rate of the cyclic molecule, and a method for producing the same. [Brief explanation of the drawings]

[0012] [Figure 1] Synthesis scheme of polyrotaxanes containing aldehyde-group-added cyclic molecules of the present disclosure [Figure 2] Schematic diagram of the preparation of atelocollagen threads of the present disclosure. For tensile testing, atelocollagen threads of the present disclosure were fixed to a 2.5 cm x 4.5 cm plastic sheet. [Figure 3] One- and two-dimensional NMR spectra (500 MHz, DO, 298 K) (R = H, D) of a polyrotaxane containing an aldehyde-group-added cyclic molecule of the present disclosure. (a) H NMR and (b) NOESY spectrum. Measurements were performed in DO (100% deuteration) containing 15 mM PRβCD1. Relaxation time: 2 seconds, mixing time: 0.68 seconds. [Figure 4]Mechanical properties of the prepared atelocollagen threads (Col-PRβCD1, Col-PRαCD1). (a) Representative stress-strain curves for Col alone, Col-PRβCD1, Col-PRαCD1, and Col-GA (glutaraldehyde), (b) breaking stress, (c) breaking strain, (d) Young's modulus, and (e) toughness. The breaking stress was calculated by dividing the breaking test force (N) by the cross-sectional area of ​​the thread (approximately 0.2 mm2). The breaking strain was calculated as the ratio of the length of the thread sample at break to its initial length (25 mm). Young's modulus was determined in the initial region of the stress-strain curve, where stress and strain are linearly correlated. Toughness was calculated as the area under the stress-strain curve. Experimental data are expressed as mean ± SD (n = 6). Tukey's test was used for statistical analysis (*p < 0.05, **p < 0.01). ns: no significant difference. Col-GA: atelocollagen-GA thread (glutaraldehyde has two cross-linking points, which bind to collagen), Col-PRβCD1:PRβCD1 cross-linked atelocollagen thread, Col-PRαCD1:PRαCD1 cross-linked atelocollagen thread, Col alone: ​​atelocollagen thread not containing GA, PRβCD1, or PRαCD1. [Figure 5] Stress during repeated strain loading of Col-PRβCD1 yarn. During 50 repeated measurements, the yarn was stretched in the range of 30% to 40% relative strain. [Figure 6] Synthesis scheme 2 of polyrotaxanes containing aldehyde-group-added cyclic molecules of the present disclosure [Figure 7] 1H NMR spectrum of PRαCD3 (DMSO-d6, 400 MHz, 298K) [Figure 8] 1H NMR spectrum of PRαCD2 (DMSO-d6, 400 MHz, 298K) [Figure 9] 1H NMR spectrum of PRαCD1 (DMSO-d6 / D2O, 400MHz, 298K) [Figure 10] Schematic diagram of stretchable collagen threads cross-linked by polyrotaxane containing aldehyde-group-added cyclic molecules [Figure 11]1H NMR spectrum of βCD (DO, 400 MHz, 298K) [Figure 12] 1H NMR spectrum of βCD after oxidation with DMP (DO, 400MHz, 298K) [Figure 13] 1H NMR spectrum of βCD after oxidation with TEMPO / PhI(OAc)2 redox couple (DO, 400 MHz, 298K) [Figure 14] 1H NMR spectrum of PluPRβCD3 (CDCl3, 400 MHz, 293K). Polyethylene glycol (x = 85), polypropylene glycol (y = 30) [Figure 15] 1H NMR spectrum of PluPRβCD2 (DMSO-d6, 400 MHz, 293K). Polyethylene glycol (x = 85), polypropylene glycol (y = 30) [Figure 16] 1H NMR spectra: (a) PegPRαCD1-DMHZ, (b) PluPRβCD1-DMHZ (DMSO-d6, 400 MHz, 318 K) [Figure 17] Mechanical properties evaluated by tensile testing: (a) Photographs during tensile testing, (b) Representative stress-strain curves, (c) Breaking stress, (d) Toughness, (e) Young's modulus, (f) Breaking elongation. Breaking stress was calculated by dividing the test force at break by the cross-sectional area. Young's modulus was calculated as the slope of the initial linear region (up to 6% elongation) of the stress-strain curve. Statistical analysis: *p < 0.01, **p < 0.05 (Tukey's test). DETAILED DESCRIPTION OF THE INVENTION

[0013] (Subject of this disclosure) The present disclosure relates to polyrotaxanes containing aldehyde-group-added cyclic molecules (particularly, polyrotaxanes containing aldehyde-group-added cyclic molecules in which ketone groups are not substantially added to the cyclic molecules), a method for producing polyrotaxanes containing aldehyde-group-added cyclic molecules that can specifically add aldehyde groups to the cyclic molecules of the polyrotaxane (hereinafter, sometimes abbreviated as "method for producing polyrotaxanes containing aldehyde-group-added cyclic molecules of the present disclosure"), stretchable biomaterials (particularly, stretchable collagen), a method for producing a stretchable biomaterial that includes a crosslinking method that can suppress the generation of free aldehyde (hereinafter, sometimes abbreviated as "method for producing a stretchable biomaterial of the present disclosure"), and polyrotaxanes containing aldehyde-group-added cyclic molecules in which the inclusion rate of cyclic molecules is controlled and a method for producing the same.

[0014] (Polyrotaxanes containing aldehyde-group-added cyclic molecules) The polyrotaxanes comprising aldehyde group-added cyclic molecules of the present disclosure include the following: (1) Linear molecule. (2) Blocking groups (stopper molecules). The blocking groups are located at both ends of the linear molecule. (3) Aldehyde-group-added cyclic molecules. The interior of the aldehyde-group-added cyclic molecules is penetrated by linear molecules. The polyrotaxanes containing the aldehyde group-added cyclic molecules of the present disclosure have been confirmed to be water-soluble by the following examples. Specifically, it is believed that the polyrotaxanes are water-soluble when one or more aldehydes are present in one cyclic molecule. In addition, the polyrotaxane containing an aldehyde group-added cyclic molecule obtained by the method for producing a polyrotaxane containing an aldehyde group-added cyclic molecule of the present disclosure contains an aldehyde group-added cyclic molecule that is substantially free of ketone groups. "Substantially no ketone groups added" not only means that no ketone groups are added to the cyclic molecule at all, but also includes cases where the number of added ketone groups is so low that there is no effect from the ketone groups. More specifically, according to the results of the Examples below, this means that 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more of the hydroxy groups of the cyclic molecule are substituted with ketone groups.

[0015] (linear molecule) The linear molecule is not limited as long as it is a linear molecule used in known polyrotaxanes, but preferably does not have an added, free, or non-added aldehyde group, hydroxyl group, or amino group. However, any linear molecule can be used as long as these groups are protected by some kind of protecting group. The linear molecule may also contain branched chains. For example, examples of linear molecules include structural units based on PEG (polyethylene glycol), PPG (polypropylene glycol), polyethylene glycol-polypropylene glycol copolymer, poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol), PES15 polyester polyol, viologen polymer, linear polyethyleneimine, Ionene-6.10, polylactic acid-polyethylene glycol-polylactic acid triblock copolymer, polylactic acid-polyethylene glycol block copolymer, polydimethylsiloxane, etc.

[0016] (blocking group) The blocking group (stopper molecule) is a structure of a size sufficient to prevent the aldehyde group-added cyclic molecule from being separated from the linear molecule. It is not limited to blocking groups used in known polyrotaxanes, but preferably does not have or contain an attached or free amino group. However, any blocking group protected by a protecting group can be used. Additionally, the blocking group using cyclodextrin may have an aldehyde group attached. Examples of structural units include triazine derivatives represented by the following general formula (I), α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, adamantane, om-dinitrobenzene, trityl glycine, adamantanecarboxylic acid, trityl amide, fluorescein isothiocyanate, trityl aniline, trityl phenol, and trityl chloride.

[0017] [ka]

[0018] In the formula, R 1 ~R 3 may be the same or different and represent a hydrogen atom, a hydroxymethylamino group, an amino group, a hydroxyl group, a halogen atom, an aryl group, a linear or branched alkyl or alkenyl group having from 1 to 6 carbon atoms, 4-(aminomethyl)-N-methylaniline, or a linear or branched alkoxy or alkenyloxy group having from 1 to 6 carbon atoms. Specific examples include N2,N4-bis(4-(aminomethyl)phenyl)-6-chloro-1,3,5-triazine-2,4-diamine, 2,4-diamino-1,3,5-triazine, 2-chloro-4,6-diamino-1,3,5-triazine, 2,4,6-triamino-1,3,5-triazine, 2,4,6-trihydroxy-1,3,5-triazine, and trichloro-1,3,5-triazine.

[0019] A preferred blocking group (stopper molecule) is represented by the following formula (1): If necessary, the amino group may be protected with a protecting group or the like.

[0020] [ka]

[0021] (cyclic molecule) The cyclic molecule is not limited as long as it is a cyclic molecule used in known polyrotaxanes, but it is necessary that an OH group is present. Examples include structural units based on α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, cycloawadolin, 2-hydroxymethyl-12-crown-4, 2-hydroxymethyl-15-crown-5, 2-hydroxymethyl-18-crown-6, and the like. Even if a functional group is added to the cyclic molecule, it is sufficient if it is protected with a protecting group.

[0022] (Embodiments of polyrotaxanes containing aldehyde group-added cyclic molecules) A preferred embodiment of the polyrotaxane containing the aldehyde group-added cyclic molecule of the present disclosure is as follows. The linear molecules are building blocks based on polyethylene glycol, polypropylene glycol or poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol). The blocking group (stopper molecule) is a structure that is large enough to prevent the aldehyde group-added cyclic molecule from being separated from the linear molecule. The aldehyde-group-added cyclic molecule is a building block based on an aldehyde-group-added cyclodextrin.

[0023] (Method for producing polyrotaxanes containing aldehyde group-added cyclic molecules according to the present disclosure) The method for producing a polyrotaxane containing an aldehyde group-added cyclic molecule of the present disclosure includes the following steps, as shown in FIG. (1) A step of contacting a linear molecule with a cyclic molecule having a hydroxyl group to obtain Compound 3 in which the linear molecule is threaded through the interior of the cyclic molecule having a hydroxyl group, with stirring as necessary. (2) A step of contacting the compound 3 with a blocking group to obtain a compound 2 in which a linear molecule having the blocking groups at both ends is threaded through the interior of a cyclic molecule having a hydroxyl group, with stirring as necessary. (3) A step of oxidizing the compound 2 in the presence of a TEMPO derivative and an iodobenzene derivative to obtain a polyrotaxane containing an aldehyde group-added cyclic molecule. The temperature, pressure, solvent, etc. used in the above steps may be those known per se.

[0024] In step (2), the present inventors found it difficult to produce the target compound in which linear molecules having blocking groups at both ends penetrate the interior of a cyclic molecule having a hydroxyl group under the conditions (oxidizing agent, solvent, and blocking group used) used for conventional polyrotaxanes. Therefore, in the production method of the present disclosure, unlike conventional methods for producing polyrotaxanes, a triazine derivative represented by general formula (I) (particularly, a compound represented by formula (1)), which is a previously unknown blocking group, is preferably used by dissolving it in a solvent such as hexamethylphosphoric triamide and / or tetrahydrofuran.

[0025] Regarding step (3), in the "Reaction of βCD with DMP" in the following example, when DMP (Dess-Martin Periodinane), an oxidizing agent used in conventional methods, and DMSO, a solvent, were used, both the primary and secondary alcohol moieties of CD were oxidized, resulting in a mixture of products that could not be isolated. On the other hand, in the example below, "Reaction of βCD with TEMPO / PhI(OAc)2 redox couple," βCD monoaldehyde was obtained as a white solid. Therefore, in the production method of the present disclosure, unlike conventional methods for producing polyrotaxanes, a compound in which a linear molecule having blocking groups at both ends penetrates a cyclic molecule having a hydroxyl group is oxidized, preferably in the presence of a TEMPO derivative and an iodobenzene derivative (and, if necessary, in the presence of N,N-diisopropylethylamine). Furthermore, the compound is dissolved in hexamethylphosphoric triamide, N,N-dimethylformamide, or dimethyl sulfoxide (more preferably, hexamethylphosphoric triamide containing N,N-diisopropylethylamine). This allows specific introduction of aldehyde groups into polyrotaxanes under mild conditions.

[0026] Commercially available TEMPO derivatives can be used. For example, 4-(2-Iodoacetamido)-2,2,6,6-tetramethylpiperidine 1-Oxyl Free Radical, 2,2,6,6-Tetramethylpiperidine 1-Oxyl Free Radical, 4-Amino-2,2,6,6-tetramethylpiperidine 1-Oxyl Free Radical, 4-Oxo-2,2,6,6-tetramethylpiperidine 1-Oxyl Free Radical, 4-Oxo-2,2,6,6-tetramethylpiperidine 1-Oxyl Free Radical, 4-Hydroxy-2,2,6,6-tetramethylpiperidine 1-Oxyl Free Radical, 4-Carboxy-2,2,6,6-tetramethylpiperidine 1-Oxyl Free Radical, 4-Methacryloyloxy-2,2,6,6-tetramethylpiperidine 1-Oxyl Free Radical, 4-Glycidyloxy-2,2,6,6-tetramethylpiperidine 1-Oxyl Free Radical, 4-Isothiocyanato-2,2,6,6-tetramethylpiperidine 1-Oxyl Free Radical, 4-Amino-2,2,6,6-tetramethylpiperidine 1-Oxyl Free Radical, 2,2,6,6-Tetramethylpiperidine 1-Oxyl Free Radical, 2-Hydroxy-2-azaadamantane, 4-Hydroxy-2,2,6,6-tetramethylpiperidine 1-Oxyl Benzoate Free Radical, 2,2,6,6-Tetramethyl-4-(2-propynyloxy)piperidine 1-Oxyl Free Radical, 4-Acetamido-2,2,6,6-tetramethylpiperidine 1-Oxyl Free Radical, 4-Cyano-2,2,6,Examples include 6-tetramethylpiperidine 1-oxyl free radical and 4-methoxy-2,2,6,6-tetramethylpiperidine 1-oxyl free radical. Commercially available iodobenzene derivatives can be used, such as iodobenzene diacetate and [bis(trifluoroacetoxy)iodo]benzene.

[0027] (Method for controlling the inclusion rate of cyclic molecules in polyrotaxanes containing aldehyde group-added cyclic molecules of the present disclosure) The inclusion rate of the cyclic molecules in the polyrotaxane containing the aldehyde group-added cyclic molecules can be controlled by appropriately selecting the types of the linear molecules and the cyclic molecules. The inclusion rate is defined as the molar fraction of aldehyde group-added cyclic molecules per number of repeating units in a linear molecule. The number of repeating units in a linear molecule can be calculated, for example, by dividing the molecular weight of the polymer by the molecular weight per repeating unit. The molar fraction of all cyclic molecules in a linear molecule can be calculated, for example, by dividing the signal intensity (e.g., NMR spectrum) of all cyclic molecules in the linear molecule (polyrotaxane) by the signal intensity (e.g., NMR spectrum) per cyclic molecule. For example, the following description will be given with reference to the following examples. The intensity of the signal derived from the cyclic molecules is 84H. If the intensity of the signal per cyclic molecule is 7H, it can be calculated that there are 12 cyclic molecules (84H÷7H=12). The entire linear molecule has 200 repeat units. The inclusion rate is the ratio of the number of cyclic molecules to the number of repeating units in the entire linear molecule, and can be calculated as (12 / 200) x 100 = 6 mol%.

[0028] The inclusion rate of the present disclosure is not particularly limited, but is 1-40 mol%, preferably 2-15 mol%, more preferably 2-10 mol%, according to the following examples. It has been confirmed in the examples below that an inclusion rate of 2-10 mol% results in increased toughness (particularly breaking stress and toughness) compared with other ranges of inclusion rates.

[0029] (Biomaterials cross-linked with polyrotaxane) The polyrotaxane-crosslinked biomaterial of the present disclosure includes the following polyrotaxanes: (1) Linear molecule. (2) Blocking groups (stopper molecules). The blocking groups are located at both ends of the linear molecule. (3) Cyclic molecules. The inside of the cyclic molecules is penetrated by linear molecules. Preferably, the cyclic molecule is an aldehyde group-added cyclic molecule. Furthermore, the polyrotaxane containing an aldehyde group-added cyclic molecule contains an aldehyde group-added cyclic molecule that is substantially free of a ketone group. "Substantially no ketone groups added" not only means that no ketone groups are added to the cyclic molecule at all, but also includes cases where the number of added ketone groups is so low that there is no effect from the ketone groups. More specifically, according to the results of the Examples below, this means that 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more of the hydroxy groups of the cyclic molecule are substituted with ketone groups. The biomaterials cross-linked with the polyrotaxane of the present disclosure (particularly, thread-like collagen cross-linked with the polyrotaxane) have been confirmed to have stretchability in the examples below.

[0030] (Biomaterials) The biomaterial of the present disclosure is not particularly limited as long as it has a lysine residue, and examples thereof include proteins (particularly collagen), enzymes, antibodies, peptides, etc., which have lysine residues. A preferred example of the biomaterial is collagen filaments (particularly atelocollagen filaments). The method for producing collagen filaments is not particularly limited, but for example, collagen filaments can be produced by discharging a collagen solution into a coagulation bath through an air gap in the form of filaments, thereby causing the collagen solution to elongate and flow just before spinning.

[0031] (Embodiments of biomaterials cross-linked with polyrotaxane) Preferred embodiments of the biomaterial cross-linked with the polyrotaxane of the present disclosure (particularly, the biomaterial cross-linked with the polyrotaxane containing an aldehyde group-added cyclic molecule) are as follows. The linear molecules are building blocks based on polyethylene glycol, polypropylene glycol or poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol). The blocking groups (stopper molecules) are building blocks based on triazine derivatives or adamantanecarboxylic acids. The aldehyde-group-added cyclic molecule is a building block based on an aldehyde-group-added cyclodextrin. The biomaterial is thread-like collagen.

[0032] The linear molecule is a structural unit based on polyethylene glycol, polypropylene glycol, or poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol), the blocking group (stopper molecule) is a structural unit based on a triazine derivative, the aldehyde group-added cyclic molecule is a structural unit based on an aldehyde group-added cyclodextrin, and the biomaterial is elastic thread-like collagen. The mechanical properties of the biomaterial are as follows: (1) Breaking stress is 280 to 1300, 280 to 360, 800 to 1300, or 2000 to 3200 kPa (2) Breaking strain is 40-70%, 62-70%, or 40-52% (3) an elastic modulus of 18 to 55, 18 to 34, 39 to 55, or 100 to 220 kPa; (4) Toughness of 83 to 240, 83 to 101, 100 to 240, or 200 to 350 kJ / m 3 (5) Stress is 10 kPa to 1000 kPa or less when strain is 30% to 40%. For each of the above measurement methods, the sample to be measured is placed in a commercially available microautograph (e.g., MST-X system (Shimadzu Corporation)), and one end is continuously pulled at a rate of 2 mm / min, while preventing drying if necessary, and the stress and strain of the sample are measured until the sample breaks. In addition, the stress is calculated by dividing the test force detected in the tensile test by the cross-sectional area of ​​the yarn (the cross-sectional area of ​​the yarn is calculated by measuring the diameter of the yarn under a microscope and calculating it as (radius) x (radius) x 3.14). The biomaterial has any one of properties 1, 2, 3, 4, or 5 of the above (1) to (5), and therefore has stretchability.

[0033] (Method for producing biomaterials cross-linked with polyrotaxanes of the present disclosure) The method for producing a biomaterial cross-linked with the polyrotaxane of the present disclosure (particularly, a biomaterial cross-linked with a polyrotaxane containing an aldehyde group-added cyclic molecule) preferably involves carrying out the following reductive amination reaction under mild conditions (see FIG. 2). Polyrotaxane - CHO + NH2 of Lysine Residues - Biomaterials ⇒Polyrotaxane - CH2-NH-Lys - Biomaterial In the above reaction, the aldehyde groups of the polyrotaxane react specifically and selectively with the lysine residues of the biomaterial, resulting in crosslinking (see Figure 10). On the other hand, when crosslinking is performed by an amide coupling reaction using a general carboxyl group instead of an aldehyde group, nonspecific crosslinking occurs by reacting with serine and tyrosine residues (commonly known as an esterification reaction). Furthermore, reductive amination can be performed under mild conditions, allowing the properties of the biomaterial to be maintained. The method for producing biomaterials cross-linked with polyrotaxanes will be outlined below using the example of thread-like collagen. The collagen solution is neutralized, degassed, and then extruded into a buffer solution. Next, the collagen thread is immersed in a weakly basic buffer solution, and polyrotaxane is added. The thread is then shaken in the presence of a reducing agent. Finally, the surface of the thread is washed, air-dried, and then immobilized on a frame to produce a collagen thread crosslinked with polyrotaxane.

[0034] The method for producing a biomaterial cross-linked with the polyrotaxane of the present disclosure (particularly, a biomaterial cross-linked with the polyrotaxane containing an aldehyde group-added cyclic molecule) preferably includes the following steps. (1) A step of subjecting a biomaterial having a lysine residue to a reductive amination reaction in the presence of a polyrotaxane containing a cyclic molecule (particularly, an aldehyde group-added cyclic molecule). Examples of the reducing agent used in the reductive amination reaction include known reducing agents such as sodium cyanoborohydride, sodium borohydride, lithium aluminum hydride, sodium triacetoxyborohydride, 2-picoline borane, and sodium dithionite. Furthermore, it is preferable to store and react the biomaterial (particularly, thread-like collagen) and / or polyrotaxane in a buffer solution at a pH of 5.0 to 10.0 (preferably, pH 7.5 to 9.5), which is a condition for maintaining the biomaterial in a stabilized state. Examples of the buffer solution include known buffer solutions such as acetic acid, phosphate, carbonate, boric acid, and HEPES. Additionally, in order to suppress the formation of free aldehydes in the reductive amination reaction step, a hydride reducing agent (sodium cyanoborohydride (NaBHCN), sodium borohydride, lithium aluminum hydride, sodium triacetoxyborohydride, 2-picoline borane, sodium dithionite) may be added to the reaction system. The temperature, pressure, solvent, etc. used in the above steps may be those known per se.

[0035] (Crosslinked composition) The crosslinking composition of the present disclosure contains, as an active ingredient, a polyrotaxane containing the aldehyde group-added cyclic molecule of the present disclosure. In particular, the crosslinking composition of the present disclosure is not particularly limited as long as it is a material having an amino group, and is preferably used for, for example, biomaterials, organic materials, or inorganic materials.

[0036] The present disclosure will be described in more detail below using experimental examples. The following experimental examples should be considered as an aid in gaining a concrete understanding of the present disclosure, and the scope of the present disclosure is not limited by the following experimental examples in any way. [Example]

[0037] Materials and Methods The materials and methods used in this example are as follows.

[0038] (Experimental Method) 〇Material The linear molecular building block, PPG-NH2 (Mn: 4,000), was purchased from Sigma-Aldrich. Sodium chloride (NaCl), glutaraldehyde (GA), 2,4-dinitrophenylhydrazine (DNPH), and βCD, the cyclic molecular building block, were purchased from Fujifilm Wako Pure Chemical Industries, Ltd. Absolute ethanol (EtOH, 99.5%), anhydrous tetrahydrofuran (THF), diethyl ether (Et2O), TEMPO (2,2,6,6-tetramethylpiperidine-1-oxyl), iodobenzene diacetate (PhI(OAc)2), disodium hydrogen phosphate decahydrate (Na2HPO4 12H2O), sodium dihydrogen phosphate dihydrate (NaH2PO4 2H2O), and sodium cyanoborohydride (NaBH3CN) were purchased from Kanto Chemical Co., Ltd. Hexamethylphosphoric triamide (HMPA), (bis(trifluoroacetoxy)iodo)benzene PhI(OAcTf)2, DMP (Dess-Martin Periodinane), and N,N-diisopropylethylamine (DIPEA) were purchased from Tokyo Chemical Industry Co., Ltd. All reagents were of special grade and were used as purchased without further purification. The compound of formula 1, which serves as the building block of the stopper molecule, was synthesized according to the method described in the literature: Org. Lett. 2000, 2 (6), 843-845. For the preparation of the thread-like atelocollagen of the present disclosure, a 3% aqueous solution (30 mg / mL) of medical type I atelocollagen manufactured and sold by the applicant was used.

[0039] (Synthesis of polyrotaxane containing aldehyde group-added cyclic molecule of the present disclosure 1) The polyrotaxane of the present disclosure was synthesized according to the synthesis scheme shown in Figure 1. The details are as follows. Synthesis of PRβCD3 PPG-NH2 (0.50 g, 0.125 mmol) was placed in a 1000 mL beaker. Subsequently, an aqueous solution containing water (600 mL) and βCD (6.6 g, 5.8 mmol) was added and stirred at 25 °C for 7 days, resulting in a white precipitate. This precipitate was centrifuged and lyophilized to give PRβCD3 (3.2 g, 0.11 mmol) as a white solid in 88% yield. δH (DMSO-d6): 0.85-1.04 (3H, -CH3, PPG), 3.55-3.57 (14H, βCD), 3.61-3.66 (28H, βCD), 4.46 (7H, βCD), 4.82 (7H, βCD), 5.70-5.76 (14H, βCD).

[0040] Synthesis of PRβCD2 The stopper molecule (2.0 g, 3.6 mmol) represented by the chemical formula (1) was dissolved in a solution of DIPEA (2.0 mL, 11.5 mmol) and dry THF (5.0 mL). PRβCD3 powder (1.0 g, 0.034 mmol) was added in small portions to the solution containing the stopper molecule, and the suspension was vigorously stirred at 25 °C for 4 days. The suspension was then thoroughly washed with cold THF and centrifuged until the supernatant became colorless. The resulting solid was dried under vacuum and then passed through a dialysis membrane (Spectra / Por (R) The mixture was dialyzed in water using a centrifuge tube (7, MWCO = 1 kDa) for 3 days. The inner dialysate was lyophilized to obtain PRβCD2 as a white solid (0.47 g, 47 wt%). Calculations based on the ratio of each characteristic signal confirmed that the number of βCD molecules constituting one PRβCD2 was 23 to 25. δH (DMSO-d6): 0.85-1.04 (3H, -CH3, PPG), 1.47 (9H, -C(CH3)3, Boc), 3.54-3.57 (14H, βCD), 3.61-3.65 (28H, βCD), 4.47 (7H, βCD), 4.83 (7H, βCD), 5.69-5.74 (14H, βCD), 7.32-7.48 (4H, benzene ring, stopper site), 7.64 (1H, -NH-Boc, stopper site), 9.30 (1H, Ar-NH-Trz, stopper site), 10.1 (1H, Ar-NH-Trz, stopper site). IR (KBr): v = 3383, 1155, 1080, 1032 cm- 1 .

[0041] Reaction of βCD and DMP The synthesis of βCD monoaldehyde was attempted according to a previously published method (see Tetrahedron Lett. 1995, 36 (46), 8371-8374). Specifically, βCD (1.0 g, 0.88 mmol) and two equivalents of freshly opened DMP (0.75 g, 1.76 mmol) were dissolved in DMSO (25 mL) and stirred at 25 °C for 1 h. The reaction mixture was then poured into cold acetone (150 mL) and cooled to -10 °C to precipitate the crude product. This precipitation process was repeated until the DMP by-product was completely removed. Subsequent extraction with water and lyophilization afforded oxidized βCD as a white powder (0.9 g, 90%). Because both the primary and secondary alcohol moieties of βCD were oxidized, the resulting product was a mixture and could not be isolated. 1 The peaks in the 1 H NMR spectra were assigned based on pure βCD. δH(D2O): 3.45-3.57(m, 14H), 3.73-3.89(m, 28H), 4.96-5.04(m, 7H), 5.31-5.43(m, acetal (-CH(OD)2, -CH(OH)(OD), -CH(OH)2)). δ 13 C(D2O): 60.3, 71.8, 72.1, 73.1, 81.1, 101.9.

[0042] Reaction of βCD with the TEMPO / PhI(OAc)2 redox couple βCD (1.0 g, 0.88 mmol), a catalytic amount of TEMPO (0.014 g, 0.09 mmol), and PhI(OAc) (0.28 g, 0.88 mmol) were dissolved in dry DMF (25 mL) and stirred at 25 °C for 24 h. The crude mixture was then added to EtO (500 mL) and cooled to 0 °C overnight. The precipitate was then filtered and washed thoroughly with acetonitrile. Extraction with water followed by lyophilization afforded βCD monoaldehyde as a white solid (0.85 g, 85%). δH(D2O): 3.47-3.58(m, 14H), 3.76-3.90(m, 28H), 4.98(d, 7H), 5.30-5.36(m, 1H, acetal (-CH(OD)2, -CH(OH)(OD), -CH(OH)2)). δ 13 C(D2O):60.3, 71.8, 72.1, 73.1, 101.9.

[0043] Synthesis of PRβCD1 Approximately 23 βCD molecules (7.6 × 10 -2 mmol) containing PRβCD2 (100 mg, 3.3 × 10 -3 The TEMPO (2.4 mg, 1.5 × 10 mmol) was dissolved in cold HMPA (5 mL) containing DIPEA (200 μL, 1.2 mmol) at 4 °C. -2 mmol) and PhI(OAcTf)2 (33 mg, 7.6 × 10 -2 The solution was then added (20 mmol) and stirred vigorously at 4 °C for 10 days. The crude product was then precipitated by adding this solution dropwise to an excess of cold EtO, which was then washed repeatedly by centrifugation (1500 × g) in cold acetonitrile. Finally, the resulting solid was analyzed by Spectra / Por (R) The mixture was dialyzed in water using a dialysis membrane (MWCO: 1 kDa) for 2 days, and the resulting solution was freeze-dried to give PRβCD1 as a white solid (40 mg, 40 wt%). δH(DO): 1.20 (3H, -CH3, PPG and Boc), 2.45-2.59 (3H, PPG), 3.08 (1H, α hydrogen, acetal), 3.49-3.59 (14H, βCD), 3.72-4.00 (28H, βCD), 4.94 (7H, βCD), 5.23-5.38 (1H, acetal (-CH(OD)2, -CH(OH)(OD), -CH(OH)2)). δ 13 C(D2O):12.2, 16.3, 17.8, 34.5, 42.6, 54.4, 60.4, 62.6, 71.6, 71.8, 71.8, 72.0, 72.1, 73.1, 76.5, 77.2, 78 .2, 78.3, 80.3, 80.7, 80.8, 80.9, 81.1, 81.3, 98.0, 98.1, 100.8, 101.3, 101.4, 101.5, 101.7, 101.8, 101.9. IR(KBr):v = 3383, 1209, 1155, 1080, 1032 cm -1 .

[0044] (Preparation of Thread-like Atelocollagen of the Present Disclosure) The atelocollagen thread (Col-PRβCD1 thread) of the present disclosure was produced according to the synthesis scheme shown in Figure 2. The details are as follows. Specifically, 15.0 g of 3% atelocollagen solution was mixed with 3.0 g of 600 mM phosphate buffer (containing 3.3 M NaCl, pH 7). The mixture was stirred in an ice bath and then degassed using a centrifuge (1500 × g, 4 °C, 20 min). The mixture was then degassed under reduced pressure in an ice bath. Degassing was continued until all air bubbles were eliminated. The resulting viscous solution was drawn up into an 18-gauge disposable syringe and extruded through 25 cm of 18-gauge flexible tubing into 200 mL of warmed (37 °C) 50 mM phosphate buffer (pH 7) containing 0.28 M NaCl over 15 min. A stepwise reductive amination method (i.e., imination followed by imine reduction) was employed to crosslink atelocollagen molecules. First, the threads to be crosslinked were immersed in a buffer solution (heated to 37 °C) containing PRβCD1, which contained a number of aldehyde groups equal to the total number of lysine residues in atelocollagen (i.e., 102 equivalents of lysine residues). The crosslinking reaction was continued in 100 mM borate buffer containing 0.18 M NaCl (pH 8.5), and quantitative imination was confirmed by colorimetric detection of aldehydes using DNPH. After imination, NaBH3CN was added to a final concentration of 0.1 M, and the resulting solution was kept at 37 °C for 3 days to allow for reductive amination to stabilize the crosslinks. As a control for the evaluation of mechanical properties, a general-purpose aldehyde cross-linking agent, glutaraldehyde (GA), was also used to cross-link atelocollagen.

[0045] (Tensile test) A sample of atelocollagen threads was immersed in 50 mM phosphate buffer (pH 7) containing 0.28 M NaCl for 2 minutes. The thread was clamped between the top and bottom of a microautograph fixture, and the red sections on the left and right sides of the plastic sheet were removed as shown in Figure 2 to allow for strength measurements of the fixed thread. For the tensile test, the top of the thread was continuously pulled at a rate of 2 mm / min while the sample was exposed to a water mist to prevent drying. The stress and strain of the sample were measured until it broke. Statistical significance of the experimental data was assessed using Tukey's test, with p < 0.01 considered significant.

[0046] (Device) The prepared compounds were purified using a high-speed refrigerated centrifuge (CR21GIII, Hitachi). The synthesized compounds were freeze-dried using an FDU-2200 freeze dryer (EYELA) equipped with a vacuum pump. A thermominder 50 (TAITEC) thermostatic chamber and an SDPC-1 syringe pump (AS ONE) were used to prepare thrombus-like collagen fibers. Crosslinking of the thrombus-like collagen fibers was performed at 37 °C using an FF-12 incubator (Fine). Mechanical properties were measured using a Micro Autograph MST-X system (Shimadzu Corporation). NMR measurements were performed using ECS-400, ECA-500, and ECZ600R NMR spectrometers (JEOL). [Example]

[0047] (Synthesis of polyrotaxanes containing aldehyde-group-added cyclic molecules) Synthesis of PRβCD2 The stopper molecule was synthesized in a yield comparable to that achieved in previous reports (i.e., approximately 95%). The remaining carbon-bonded chlorine atom in the stopper molecule was replaced with the terminal amino group of the pseudo-PR. By modifying the terminal with the stopper at room temperature, PRβCD2 was obtained in approximately 50% yield. Several previous studies have reported that a similar reaction can be achieved by heating the reaction mixture (at 60 °C or 80 °C) to efficiently synthesize triazine derivatives (https: / / doi.org / 10.1016 / B978-008096519-2.00042-4). However, in the case of pseudo-PR (PRβCD3), the synthesis yield significantly decreased (<1 wt%) when the reaction was performed at high temperatures.

[0048] (Synthesis of βCD monoaldehyde) Before introducing one aldehyde group per βCD of PR, we tested the oxidation reaction using DMP and βCD alone. To directly detect the aldehyde group, we first performed the oxidation test in DMSO-d6. 1H NMR measurements were performed. However, the intensity of the signal derived from the aldehyde group changed during several measurements. This was thought to be due to the reactivity of the aldehyde group (such as acetal formation with the remaining hydroxyl group of βCD) hindering quantitative analysis. Therefore, we performed measurements of oxidized βCD in DO. 1 Comparison of H NMR spectra revealed significant changes in the chemical structure of βCD (Figures 11 and 12). Specifically, signals attributable to C-H(Hi) were partially downfield shifted compared to those observed with βCD alone. Furthermore, new signals were detected at 5.31–5.43 ppm, which we attribute to the presence of ketones and aldehydes in the βCD structure. DMP is known to oxidize primary and secondary alcohols, so it can oxidize the Cd-OH, Cg-OH, and Ch-OH groups of βCD. From a general organic chemistry perspective, the formation of ketones from Cg or Ch results in a partial downfield shift of the nearby Hi signals. The three peaks at approximately 5.31–5.43 ppm can be assigned to three acetals (i.e., -CH(OD)2, -CH(OH)(OD), and -CH(OH)2) generated from aldehydes. The chemical shifts of the acetal-related peaks are within the range previously reported for synthetic acetals. On the other hand, oxidation using the TEMPO / PhI(OAc)2 redox couple did not result in any change in the Hi signal shape (Figures 11 and 13). Furthermore, the spectral shapes and intensities of other proton species were almost identical to those of the unoxidized raw material βCD (Figures 11 and 13). These results suggest that the Cg-OH and Ch-OH remain largely unreacted, suggesting that the primary alcohol moiety of βCD reacted selectively. Depending on the reaction conditions, both primary and secondary alcohols can be oxidized with TEMPO. However, due to the steric hindrance caused by the two dimethyl groups of TEMPO, the primary alcohol (i.e., the Cd-OH of βCD) was preferentially oxidized to form an aldehyde. Therefore, the TEMPO / PhI(OAc)2 redox couple is more suitable than DMP for the selective production of aldehydes in βCD.

[0049] (PRβCD1 synthesis) Based on the above results, we first selectively introduced an aldehyde group into PRβCD1 using the TEMPO / PhI(OAc)2 redox couple. When this reaction was performed in typical polar solvents (DMF and DMSO), the yield of PRβCD1 was low (<0.1 wt%). This low yield can be explained from a reaction mechanism perspective. More specifically, as the oxidation of the hydroxymethyl group progresses, the reaction solution becomes acidic. This acidification protonates the nitrogen atom of the stopper molecule, increasing the electrophilicity of the triazine ring and accelerating the nucleophilic substitution reaction with potential nucleophiles in the reaction solution (e.g., DMF, DMSO, acetate anion). As a result of this nucleophilic substitution reaction, the stopper molecule loses bulk, resulting in the release of βCD from PRβCD2, significantly reducing the synthetic yield. To avoid this problem, we used PhI(OAcTf)2 as an oxidant and performed the oxidation reaction in HMPA in the presence of a base (DIPEA). After stirring at 4 °C for 10 days, the yield of PRβCD1 increased to 40 wt%.

[0050] (NMR analysis of PRβCD1) PRβCD1 1The H NMR spectrum is shown in Figure 3a. The signal intensity at 2.45–2.59 ppm is almost equal to the signal intensity at 1.20 ppm assigned to the methyl groups of PPG and Boc. These signals were assigned to the Hb, Hb', Hc, and Hc' atoms of PPG. These signals were detected at a relatively high magnetic field compared to free PPG, which is attributed to the amphiphilic PPG contained within the βCD structure. Furthermore, the signals at 3.49–3.59 ppm and 3.72–4.00 ppm were assigned to βCD(Hd-h'). Based on the signal ratios for Ha,a' and Hd-h', PRβCD1 was determined to be composed of approximately 25 βCD molecules. The intensity ratios of Ha,a' and Hi,i' also indicated that the number of βCD molecules per PRβCD was the same. Interestingly, a new signal was detected at 3.08 ppm. The intensity ratio of this signal to the Ha and a' signals suggested the presence of a chemical species with an intensity corresponding to one H. Since Hd' could be attributed to the acetal species detected in βCD monoaldehyde (i.e., -CH(OD)2, -CH(OH)(OD)2, and -CH(OH)2), the signal at 3.08 ppm was considered to be derived from the α-hydrogen atom (He') attached to the acetal. Since acetal species are formed by hydration of aldehyde groups, the intensity of the He' peak, which is derived from the acetal, can be used to calculate the number of aldehyde groups contained in PRβCD1. The calculated number of aldehyde groups was estimated to be approximately 25, which is in good agreement with the number of βCD molecules contained in PRβCD1. In other words, one aldehyde group is added per βCD molecule. Furthermore, considering the detection sensitivity of NMR, it was revealed that the aldehyde group addition reaction described above (synthesis of PRβCD1) proceeded with an efficiency of over 95%. Furthermore, negative NOE correlations were observed for the combinations of Ha,a' and He' and Ha,a' and Hd-h' (Fig. 3b). From the above, the results of one-dimensional and two-dimensional NMR measurements confirmed that the PPG unit is enclosed within the βCD structure, and furthermore, the successful formation of the PR structure in aqueous solution confirmed water solubility. [Example]

[0051] (Confirmation of mechanical properties of thread-like atelocollagen of the present disclosure) After confirming the successful synthesis of PRβCD1, we prepared the presently disclosed atelocollagen filament (PRβCD1-reinforced atelocollagen thread (Col-PRβCD1)) by reductive amination. We also used GA, a conventional aldehyde-based crosslinker, to examine the effect of the PR structure on the mechanical properties of the resulting thread. As shown in Figure 4b, cross-linking the yarn with GA significantly increased the breaking stress from 60 (± 6) kPa to 480 (± 65) kPa. Meanwhile, the breaking strain decreased from 40% (± 5%) to 20% (± 3.5%) compared to Col alone (Figure 4c). The Young's modulus was determined from the initial region of the stress-strain curve, where stress and strain are linearly correlated. As shown in Figure 4d, the Young's modulus after cross-linking increased significantly, from 34 (± 3) kPa to 570 (± 45) kPa. The increase in breaking stress and Young's modulus and the decrease in breaking strain are typical behaviors of chemical cross-linking. The area under the stress-strain curve representing the toughness of Col-GA (i.e., 190 (± 60) μJ) increased 3.8-fold compared to Col alone (i.e., 50 (± 22) μJ) (Figure 4e). The stress-strain curve of the atelocollagen thread (Col-PRβCD1) was significantly different from that of Col-GA or Col alone. Specifically, Col-PRβCD1 yarn exhibited a J-shaped stress-strain curve, typical of PR-based cross-linked materials (Fig. 4a). Cross-linking with PRβCD1 increased both the breaking stress and breaking strain, with the breaking stress increasing 5.4-fold (320 (± 40) kPa) and the breaking strain increasing 1.6-fold (66% (± 4%)) (see Figs. 4a-4c). As a result, the toughness of Col-PRβCD1 was 92 (± 9) kJ / m 3 The Young's modulus of the PRβCD1-treated yarn was the highest among the yarns tested (Fig. 4e). Note that cross-linking with PRβCD1 had almost no effect on the Young's modulus (26 (± 8) kPa) (Fig. 4d). Overall, the mechanical properties observed for Col-PRβCD1 were characteristic of a PR sliding ring. [Example]

[0052] (Confirmation of stress of thread-like atelocollagen of the present disclosure) The stress in the atelocollagen thread of the present disclosure was measured when repeated strain loads were applied (FIG. 5). The strain was set to a relative value of 30 to 40%. As a result, it was found that the stress of the thread remained roughly constant in the range of 10 to 100 kPa. This result indicates that the atelocollagen thread of the present disclosure maintains its mechanical properties within the relative strain range where Col alone would break (Figure 4c). Therefore, the thread-like atelocollagen of the present disclosure has the property of being stretchable and can be employed as a biomaterial that can be used for a long period of time in the field of regenerative medicine, etc. [Example]

[0053] (Synthesis of polyrotaxane containing aldehyde group-added cyclic molecule of the present disclosure 2) In this example, a polyrotaxane containing an aldehyde-added cyclic molecule was synthesized based on the procedure described in Example 1, except that the linear molecular building block was PEG500k-NH2 instead of PPG-NH2 (Mn: 4,000) and α-cyclodextrin instead of β-cyclodextrin (Figure 6). PEG500k-NH2 was prepared by a known method (see Chem, 2016, 1, 766).

[0054] (Synthesis of PRαCD3) PEG 500k -NH2 (0.5 g, 0.001 mmol) and α-cyclodextrin (αCD) (5.0 g, 5.1 mmol) were stirred in 100 mL of pure water at 25°C for 7 days. After stirring, the resulting white precipitate was collected by centrifugation (1500 g, 25°C, 10 min). The supernatant was removed, and the white precipitate was freeze-dried to obtain 3.0 g of PEGαCD3. Synthesis was confirmed by 1 1 H NMR. PRαCD3 1 The H NMR spectrum is shown in Figure 7. The observation of the spectra of PEG and αCD confirmed the synthesis of PRαCD3. However, because PRαCD3 does not have stopper molecules at either end, the polyrotaxane structure was observed in a dissociated state in organic solvents such as DMSO.

[0055] (Synthesis of PRαCD2) PEGαCD3 (1.0 g, 4.3 × 10 -4 The following compounds were dissolved in 2.3 mL of DMF cooled to 4°C: 1H-benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate (BOP) (0.38 g, 0.27 mmol), adamantanecarboxylic acid (Ad-COOH) (0.16 g, 0.89 mmol), and 1-hydroxybenzotriazole monohydrate (HOBt·HO) (0.12 g, 0.89 mmol). The mixture was stirred for 3 days. After the reaction, the reaction mixture was added dropwise to diethyl ether, and the resulting precipitate was washed with acetonitrile and centrifuged (1500 g, 4°C, 10 min) to remove excess DIPEA, BOP, and Ad-COOH, yielding PRαCD2. Synthesis was confirmed by 1 1 H NMR. PRαCD2 1 The H NMR spectrum is shown in Figure 8. Peaks from polypropylene glycol and αCD were observed and broadened. In addition, peaks from the adamantyl group were observed. These results confirmed the synthesis of PRαCD2. Although several types of residual solvents were detected, they were used as they were, as this does not, in principle, affect the synthesis of PRαCD1.

[0056] (Synthesis of PRαCD1) PEGαCD2 (0.05 g, 2.2 × 10 -5 mmol), TEMPO(2.0 mg, 1.3 × 10 -2 mmol), PhI(OAcTf)2(18mg,4.2 × 10 -2(0.1 mL, 0.57 mmol) and DIPEA (0.1 mL, 0.57 mmol) were dissolved in 1.7 mL of HMPA cooled to 4°C and stirred for 10 days. After the reaction, the reaction solution was added dropwise to diethyl ether, and the resulting precipitate was washed with acetonitrile and centrifuged (1500 g, 4°C, 10 min) repeatedly, and then dried under reduced pressure to obtain 0.03 g of PRαCD1. Synthesis was confirmed by 1 1 H NMR. PRαCD1 1 The H NMR spectrum is shown in Figure 9. Peaks derived from PEG and αCD were observed, as well as peaks (d', e') derived from the acetal formed by hydration of the aldehyde. These results confirmed the synthesis of PRαCD1. Furthermore, based on the integral ratio, the αCD incorporation rate per PRαCD1 molecule was calculated to be 25 mol%. In other words, it was confirmed that approximately 3,000 αCD molecules were contained per single PRαCD1 chain. [Example]

[0057] Col-PRαCD1, a type of thread-like atelocollagen, was prepared using the PRαCD1 prepared in Example 5 by the manufacturing method described above in "Preparation of thread-like atelocollagen of the present disclosure." Furthermore, the mechanical properties of Col-PRαCD1 were confirmed by the method described above in "Tensile test." The results are shown in FIG. 4 and Table 1 below. Compared to Col-PRβCD1, Col-PRαCD1 had an increased breaking stress of 1050 ± 50 kPa, an increased modulus of elasticity of 47 ± 8 kPa, and a toughness of 170 ± 70 kJ / m 3 The strain at break increased to 46 ± 6%, which was similar to that of Col alone. These results confirmed that Col-PRαCD1 has elasticity similar to that of Col-PRβCD1.

[0058] [Table 1] [Example]

[0059] In this example, the polyrotaxane (Plu) of the present invention with a controlled inclusion rate was 15k βCD1, Plu 9k βCD1) was synthesized. Details are as follows.

[0060] Hydroxyl-terminated Pluronic (登録商標) Reagents (Plu 9k -OH and Plu 15k -OH) was purchased from Sigma-Aldrich. Carboxyl-terminated Pluronic (登録商標) Compound (Plu 9k -COOH and Plu 15k -COOH) was synthesized via bleach oxidation according to a known method ( Chem. Lett. 2016, 45, 991-993.). Tritylamine was purchased from Tokyo Chemical Industry Co., Ltd. Spectra / Por was used for purification at each step. (登録商標) A dialysis membrane (MWCO: 1 kDa) was used.

[0061] (Both terminal carboxylic acid Pluronic (登録商標) Pseudopolyrotaxane formation using (Synthesis of PluPRβCD3) Pluronic-COOH (1 g) and β-CD (1 g, 0.88 mmol) were stirred in 56 mL of purified water for 1 week. The resulting white precipitate was centrifuged (14,000 × g, 20 °C, 30 min), washed with cold water, and centrifuged again. Finally, the precipitate was freeze-dried to obtain a white solid. δH (DMSO-d6): 1.01-1.05 (m, 3H, C j -H), 3.30-3.38 (m, C b,d -H and C k,l -H), 3.41-3.67(m, 32H, C c,e,f -H and C m -H), 3.94 (br, 4H,C n -H), 4.37-4.40 (m, 7H, OHg), 4.83-4.84 (m, 7H, C a -H), 5.63-5.68 (m, 14H, OH h,i ) Figure 14 1 The H NMR spectrum showed peaks derived from Pluronic and β-cyclodextrin, confirming the formation of PluPRβCD3. The inclusion rate was calculated to be 6 mol% based on the intensity ratio of the Pluronic signal (j) to the β-cyclodextrin signal (a). The method for calculating the inclusion rate is as follows. The intensity of signal (j) is 90H per Pluronic chain, so this portion was normalized to 90H. In this case, the intensity of signal (a) derived from β-cyclodextrin was 84H. Since the intensity of signal (a) per β-cyclodextrin molecule is 7H, it was calculated that there were 12 β-cyclodextrin molecules (84H ÷ 7H = 12). Meanwhile, the entire Pluronic chain has 200 repeating units (85 × 2 + 30 = 200). The inclusion rate is the ratio of the number of β-cyclodextrin molecules to the number of repeating units in the entire Pluronic chain, so it was calculated as (12 / 200) × 100 = 6 mol%.

[0062] (Introduction of a trityl amide blocking group (synthesis of PluPRβCD2)) Tritylamine (0.2 g, 0.76 mmol), N,N-diisopropylethylamine (140 μL, 0.82 mmol), and BOP (0.34 g, 0.76 mmol) were mixed and stirred in 3 mL of dehydrated acetonitrile. Next, powdered PluPRβCD3 (0.5 g, 0.02 mmol) was gradually added and stirred at 25°C for 48 hours. After the reaction, the reaction solution was added dropwise to diethyl ether, and the resulting precipitate was washed with acetonitrile and centrifuged. The remaining white powder was dissolved in dichloromethane and filtered. The residue was then dissolved in water and dialyzed. Finally, the mixture was lyophilized to obtain PluPRβCD2 with a tritylamide blocking group. δ H (DMSO-d6):1.03-1.05 (br, 3H, C j -H), 3.28-3.38 (br, C b,d -Hand Ck,l -H), 3.42-3.66 (br, 32H, C c,e,f -H,C m,n -H), 4.45-4.47 (m, 7H, OH g ), 4.82-4.83 (m, 7H, C a -H), 5.68-5.75 (m, 14H, OH h,i ), 7.16-7.32 (m, trityl group) Figure 15 1 The H NMR spectrum showed a signal (o) derived from the blocking group, confirming the synthesis of PluPRβCD2. The inclusion rate was calculated to be 6 mol% based on the intensity ratio of the Pluronic signal (j) and the β-cyclodextrin signal (a). The method for calculating the inclusion rate is as follows. The intensity of signal (j) is 90H per Pluronic chain, so this portion was normalized to 90H. In this case, the intensity of signal (a) derived from β-cyclodextrin was 84H. Since the intensity of signal (a) per β-cyclodextrin molecule is 7H, it was calculated that there were 12 β-cyclodextrin molecules (84H ÷ 7H = 12). Meanwhile, the entire Pluronic chain has 200 repeating units (85 × 2 + 30 = 200). The inclusion rate is the ratio of the number of β-cyclodextrin molecules to the number of repeating units in the entire Pluronic chain, so it was calculated as (12 / 200) × 100 = 6 mol%.

[0063] (Aldehyde group introduction (synthesis of PluPRβCD1)) PluPRβCD2 (120 mg, equivalent to 0.065 mmol βCD), TEMPO (1.1 mg, 0.0065 mmol), and PhI(OAc)2 (21 mg, 0.065 mmol) were dissolved in 3 mL of hexamethylphosphoric triamide and stirred at 4 °C for 10 days. After the reaction, the mixture was added dropwise to diethyl ether, and the precipitate was washed with acetonitrile. The resulting precipitate was dissolved in water and dialyzed. Finally, the mixture was freeze-dried to obtain a white powder.

[0064] (Quantitative detection of aldehyde groups) PluPRβCD1 (10 mg) was dissolved in a 0.2 M DMHZ (1,1-dimethylhydrazine) aqueous solution (pH 8.2) and stirred at 37°C for 48 hours. NaBH3CN was then added to a final concentration of 0.2 M, and the reaction mixture was stirred at 37°C for an additional 48 hours. After the reaction, the mixture was dialyzed for 24 hours using a 1 kDa MWCO dialysis membrane and lyophilized. Finally, the resulting powder was washed with ethanol and dried under reduced pressure to obtain PluPRβCD1 labeled with DMHZ at the aldehyde group.

[0065] 1 H NMR spectral assignments PluPRβCD1-DMHZ δH (DMSO-d6): 1.04-1.05 (br,3H, C l -H), 2.54 (s, 3H, C k -H), 3.25 (br, N j -H, C b,b',c,c' -H and C m,n -H), 3.40-3.52 (m, 4H, C o -H), 3.64-3.82(br, 32H, C p -H, C c,e,f -H and C c',e',f' -H), 4.34 (br, 7H, OH g ), 4.83 (br, 7H, C a,a' -H), 5.59 (br, 14H, OH h,h',i,i' ) (Figure 16b) In Figure 16b, a broad spectrum associated with polyrotaxane formation was observed, particularly in signals (g) and (h, h', i, i'). The clear signal (l) derived from the axial polymer and the signals (a, a') derived from β-cyclodextrin led to a calculated inclusion rate of 6 mol%. Furthermore, by labeling the aldehyde group with DMHZ, a signal (k) derived from DMHZ appeared. The intensity ratio of signals (a, a') and (k) confirmed that one aldehyde group had been introduced per β-cyclodextrin. Calculation method for the inclusion rate: The intensity of signal (j) is 90H per Pluronic chain, so this portion was normalized to 90H. In this case, the intensity of signal (a, a') derived from β-cyclodextrin was 84H. Since the intensity of signal (a, a') per β-cyclodextrin molecule is 7H, it was calculated that there were 12 β-cyclodextrin molecules (84H ÷ 7H = 12). Meanwhile, the entire Pluronic chain has 200 repeating units (85 × 2 + 30 = 200). The inclusion rate is the ratio of the number of β-cyclodextrin molecules to the number of repeating units in the entire Pluronic chain, so it was calculated as (12 / 200) × 100 = 6 mol%. Furthermore, when signals (a, a') were normalized to 7H, the intensity of signal (k) derived from DMHZ was 6H. Since the intensity of signal (k) per molecule is 6H, one molecule of DMHZ exists per molecule of β-cyclodextrin. Since the equivalent of DMHZ is equal to the equivalent of the aldehyde group, it was concluded that one aldehyde group exists per molecule of β-cyclodextrin. [Example]

[0066] In this example, the polyrotaxane (PEG) of the present invention with a controlled inclusion rate was 20k PRαCD1, Peg 10k PRαCD1) was synthesized. The structural unit of the linear molecule is PEG 10k -OH or Peg 20k The synthesis method of Example 5 was used except that Peg 10k -OH and Peg 20k -OH was purchased from FUJIFILM Wako PureChemical Corporation.

[0067] (Quantitative detection of aldehyde groups) PegPRαCD1 was dissolved in a DMHZ aqueous solution (0.2 M, pH = 8.2) and stirred at 37°C for 48 hours. Next, NaBH3CN was added to a final concentration of 0.2 M, and the reaction mixture was stirred at 37°C for an additional 48 hours. After the reaction, the mixture was dialyzed for 24 hours using a 1 kDa MWCO dialysis membrane and lyophilized. Finally, the resulting powder was washed with ethanol and dried under reduced pressure to obtain PegPRαCD1 labeled with DMHZ at the aldehyde group.

[0068] 1 H NMR spectral assignments PegPRαCD1-DMHZ δH (DMSO-d6):2.54 (s, 3H, C k -H), 3.24 (br, N j -H, C b,b',d,d'- H), 3.40-3.52 (m, 4H, C l,m -H), 3.59-3.81 (br, 28H, C n -H,C c,e,f -H and C c',e',f' -H), 4.31(br, 6H, OH g ), 4.80-4.81 (br, 6H, C a,a' -H), 5.51 (br, 6H, OH h,h',i,i' ) (Figure 16a) In Figure 16a, a broad spectrum associated with polyrotaxane formation was observed, particularly in signals (g) and (h, h', i, i'). The inclusion rate was calculated to be 20 mol% from the signals (l, m) derived from the axial polymer and the signals (n, c, e, f, c', e', f') derived from α-cyclodextrin. Furthermore, by labeling the aldehyde group with DMHZ, a signal (k) derived from DMHZ appeared. The intensity ratio of signals (a, a') and (k) confirmed that one aldehyde group was introduced per α-cyclodextrin. Calculation method for the inclusion rate: The signals (a, a') derived from α-cyclodextrin were normalized to 6H, which is the intensity per molecule. The intensity of the signals (l, m) derived from polyethylene glycol was 20H. Since the intensity per repeating unit of polyethylene glycol is 4H, 20H corresponds to five repeating units. Since there is one molecule of α-cyclodextrin for every five repeating units of polyethylene glycol, the inclusion rate was calculated as (1 / 5) × 100 = 20 mol%. Furthermore, when the signals (a, a') were normalized to 6H, the intensity of the signal (k) derived from DMHZ was 6H. Since the intensity of signal (k) per molecule is 6H, it was calculated that there is one molecule of DMHZ per molecule of α-cyclodextrin. Since the equivalent weight of DMHZ and the equivalent weight of aldehyde groups are equal, it was concluded that there is one aldehyde group per molecule of α-cyclodextrin. [Example]

[0069] (Preparation of thrombus-like atelocollagen of the present invention and confirmation of its mechanical properties 2) The preparation and mechanical properties of the atelocollagen threads of the present invention were confirmed in the same manner as in the above examples. Briefly, an atelocollagen solution (25 mg / mL) in 0.1 M phosphate buffer (pH = 7.0) was injected into 0.05 M phosphate buffer at 37 °C through an 18 G plastic tube. The resulting atelocollagen threads were subjected to stepwise reductive amination in 0.1 M borate buffer (pH = 8.5) to synthesize PEG-1. 20k PRαCD1, Peg 10k PRαCD1, Plu 15k βCD1 or Plu 9k The crosslinked yarn was then crosslinked with βCD1. The crosslinked yarn was washed with an aqueous ethanol solution and dried at room temperature. Finally, the yarn was fixed to a flexible polypropylene sheet for tensile testing. The fixed sample was immersed in 50 mM phosphate buffer (pH = 7) for 3 minutes, and then a tensile test was performed in the wet state. A Shimadzu Micro Autograph MST-X HS / HR was used for the tensile test.

[0070] The results of the tensile test are shown in Figure 17 (Figure 17a is a representative photograph taken during the tensile test). 10k PRαCD1 and Peg 20k Cross-linking with PRαCD1 significantly increased the breaking stress and breaking elongation of atelocollagen thread (AtCol) (Fig. 17b, c, f). This was accompanied by a five-fold increase in toughness (Fig. 17d). In the early stage of the stress-strain curve, stress and elongation were linearly related, and Young's modulus was calculated from this region. As a result, Peg 10k PRαCD1 and Peg 20k Cross-linking with PRαCD1 increased the Young's modulus by 4-fold (Fig. 17e). On the other hand, changing the molecular weight of the PEG chains did not significantly affect the mechanical properties of the atelocollagen threads. Plu 9k PRβCD1 and Plu 15k Cross-linking with PegPRβCD1 further improved the mechanical properties of atelocollagen threads. Specifically, the breaking stress increased by 2.3-fold compared to PegPRαCD1 (Fig. 17b, c). Concomitantly, the toughness increased by 1.6-fold (Fig. 17c and 17d). 9k PRβCD1 and Plu 15k (There was no statistically significant difference between the PEG-PRβCD1 and PEG-PRβCD1 crosslinks.) On the other hand, the Young's modulus and elongation at break remained unchanged (Figures 17e and 17f). Furthermore, the fact that the molecular weight of the axial polymer does not affect the mechanical properties of the atelocollagen threads is consistent with the results obtained with PEG-PRβCD1 crosslinks. That is, it was confirmed that in order to strengthen the thread-like atelocollagen of the present invention, it is more important to reduce the inclusion rate of cyclic molecules than to increase the molecular weight of PR (especially linear molecules).

[0071] From the results of Table 1 and FIG. 17 above, it is believed that stretchable biomaterials (especially stretchable collagen) have one or more of the following mechanical properties. (1) Breaking stress: 280 to 3200 kPa (2) Breaking strain is 40-70% (3) Elastic modulus: 18 to 220 kPa (4) Toughness of 83 to 350 kJ / m3 (5) Stress is 10 kPa to 1000 kPa or less when strain is 30% to 40%.

[0072] (General remarks) To develop stretchable biomaterials, we synthesized novel aldehyde-containing polyrotaxanes and crosslinked the biomaterials by reductive amination. Using a redox pair consisting of the catalytic oxidant 2,2,6,6-tetramethylpiperidine 1-oxyl (TEMPO) and the reductant (bis(trifluoroacetoxy)iodo)benzene (PhI(OAcTf)2), we were able to selectively generate aldehyde groups on the cyclodextrins (βCD, αCD) in PRβCD1 and PRαCD1. Comparative studies using Dess-Martin periodinane confirmed that the redox pairs in both βCD and PRβCD1 were suitable for generating aldehyde groups. Crosslinking of biomaterials with PRβCD1 and PRαCD1 significantly improved the mechanical strength and flexibility of the biomaterials, as evidenced by stress-strain curves and repeated strain loading experiments. Furthermore, by changing the types of linear molecules, blocking groups (stopper molecules), and cyclic molecules, not only the stretchability but also the mechanical properties can be controlled. By controlling the inclusion rate, it is possible to achieve toughening of the biomaterial. [Industrial Applicability]

[0073] It is possible to provide a polyrotaxane containing an aldehyde group-added cyclic molecule and a stretchable biomaterial (particularly, a stretchable collagen).

Claims

1. A method for producing a polyrotaxane containing an aldehyde group-added cyclic molecule, comprising the following steps: (1) a step of contacting a linear molecule with a cyclic molecule having a hydroxyl group to obtain a compound 3 in which the linear molecule is threaded through the interior of the cyclic molecule having a hydroxyl group: (2) contacting the compound 3 with a blocking group to obtain a compound 2 in which a linear molecule having blocking groups at both ends is threaded through the interior of a cyclic molecule having a hydroxyl group; and (3) A step of oxidizing the compound 2 in the presence of a TEMPO derivative and an iodobenzene derivative to obtain a polyrotaxane containing an aldehyde group-added cyclic molecule.

2. The manufacturing method described in claim 1, wherein in step (2), compound 3 is dissolved in tetrahydrofuran.

3. A manufacturing method described in claim 1 or 2, wherein N,N-diisopropylethylamine is further present in step (3).

4. A manufacturing method described in claim 1 or 2, wherein in step (3), compound 2 is dissolved in hexamethylphosphoric triamide, N,N-dimethylformamide or dimethyl sulfoxide.

5. A manufacturing method described in claim 1 or 2, wherein in step (3), compound 2 is dissolved in hexamethylphosphoric triamide containing N,N-diisopropylethylamine.

6. A manufacturing method described in claim 1 or 2, wherein the linear molecule and the cyclic molecule having a hydroxyl group are selected so that the inclusion rate, which is the molar fraction of aldehyde group-added cyclic molecules per number of repeating units in the linear molecule, is 1-40 mol%.

7. The manufacturing method described in claim 1 or 2, wherein the inclusion rate is 2-15 mol%.

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