Conjugate, method for producing the conjugate, and compound

Abstract

The purpose of the present disclosure is to provide: a complex of a cell and a material; a method for producing a complex of a cell and a material; and a novel compound that can be used to obtain said complex.　In a complex according to this embodiment, a cell having an azido group on the cell surface and a material having an azido group (excluding a fluorescent dye having an azido group) are bonded to each other by means of dibenzocyclooctadiene having a polar functional group.

Description

[Technical Field]

[0001] The present disclosure relates to conjugates, methods for making the conjugates, and compounds. [Background technology]

[0002] There is a demand for technologies to elucidate and control cellular processes (eg, proliferation, differentiation, morphogenesis, and cell death).

[0003] Dibenzocyclooctadiyne (5,6,11,12-Tetradehydrodibenzo[a,e]cyclooctene) is a strained cyclic molecule containing two alkynes with carbon-carbon triple bonds within the molecule, and is known to be capable of undergoing strain-promoted azide-alkyne cycloaddition (SPAAC) reactions twice.

[0004] Using the SPAAC reaction, dibenzocyclooctadiyne was bound to cells having azide groups on the cell surface, and then another alkyne carried by the dibenzocyclooctadiyne was bound to a fluorescent dye having an azide group, and the fluorescently modified cells were observed by this procedure (see, for example, Patent Document 1, Example 22). [Prior art documents] [Patent documents]

[0005] [Patent Document 1] International Publication No. 2011 / 118394 Summary of the Invention [Problem to be solved by the invention]

[0006] Patent Document 1 discloses that cells can be bound to fluorescent dyes by using dibenzocyclooctadiyne. However, the inventors have found that the invention disclosed in Patent Document 1 may not provide a sufficient reaction rate when binding to cells or when binding to fluorescent dyes, or may not allow sufficient amounts of fluorescent dye to bind to cells.

[0007] As a result of extensive research, the present inventors have found that if the solubility of dibenzocyclooctadiyne used in the SPAAC reaction can be increased, the reactivity when binding to cells etc. can be improved and the amount of fluorescent dye that binds to cells can be increased.

[0008] The inventors further developed this finding and thought that if the solubility of dibenzocyclooctadiyne could be increased, the reactivity could be improved and it might be possible to bind materials other than fluorescent dyes, such as materials with large molecular weights that are generally difficult to bind to cells, or even between cells.

[0009] In other words, the present disclosure aims to provide a cell-material complex, which has been difficult to provide using conventional techniques, a method for producing a cell-material complex, and a novel compound that can be used to obtain the complex. [Means for solving the problem]

[0010] The present inventors have found that a composite of cells and a material can be obtained by using a dibenzocyclooctadiyne having a polar functional group. Furthermore, in the course of investigating dibenzocyclooctadiynes having a polar functional group, the present inventors have also found a novel compound.

[0011] An example aspect of this embodiment is described as follows.

[0012] (1) A complex in which cells having azide groups on their surface and materials having azide groups (excluding fluorescent dyes having azide groups) are bound together by dibenzocyclooctadiyne, which has a polar functional group. (2) The composite according to (1), wherein the material is at least one material selected from cells having azide groups on the cell surface and azide-modified inorganic materials. (3) The composite according to (2), wherein the inorganic material is at least one inorganic material selected from a culture substrate, a glass substrate, an AFM cantilever, and a metal. (4) The composite according to any one of (1) to (3), wherein the dibenzocyclooctadiyne having a polar functional group is a compound represented by the following general formula (I): [ka] (In general formula (I), each X is independently a group having a polar functional group, each A is independently a hydrocarbon group, n is an integer of 1 to 4, m is an integer of 0 to 3, n+m is an integer of 1 to 4, n' is an integer of 1 to 4, m' is an integer of 0 to 3, and n'+m' is an integer of 1 to 4.) (5) The composite according to any one of (1) to (3), wherein the dibenzocyclooctadiyne having a polar functional group is a compound represented by the following general formula (II): [ka] (In general formula (II), each X is independently a group having a polar functional group, and each n is independently an integer of 1 to 4.) (6) The composite according to any one of (1) to (3), wherein the dibenzocyclooctadiyne having a polar functional group is a compound represented by the following general formula (III): [ka] (In general formula (III), each R is independently a group selected from a group having a polar functional group and an alkyl group, and each n is independently an integer of 1 to 4.) (7) The composite according to any one of (1) to (3), wherein the dibenzocyclooctadiyne having a polar functional group is a compound represented by the following general formula (IV): [ka] (In general formula (IV), each R is independently a group selected from a group having a polar functional group and an alkyl group.) (8) The composite according to any one of (1) to (3), wherein the dibenzocyclooctadiyne having a polar functional group is at least one compound selected from the following formulae 1a to 1j: [ka] [ka] (In formula 1g, RGD is a peptide chain of three residues consisting of arginine-glycine-aspartic acid, and in formula 1j, Y is each independently a group selected from a group having a polar functional group and an alkyl group.) (9) The complex according to any one of (1) to (8), wherein the cells having an azide group on the cell surface are cells in which an azide group has been introduced into the sialic acid moiety at the terminal of the sugar chain on the cell surface through the sialic acid metabolic pathway of the cells by adding acetylated azidomannosamine (Ac4ManNAz) to a cell culture medium.

[0013] (10) A step of binding cells having azide groups on the cell surface to a dibenzocyclooctadiyne having a polar functional group to obtain a conjugate (A) of the dibenzocyclooctadiyne having a polar functional group and the cell; and A method for producing a complex, comprising the steps of: combining a conjugate (A) with a material having an azide group (excluding fluorescent dyes having an azide group) to obtain a complex in which the cell and the material are bound by a dibenzocyclooctadiyne having a polar functional group. (11) a step of bonding a material having an azide group (excluding fluorescent dyes having an azide group) with a dibenzocyclooctadiyne having a polar functional group to obtain a bond (B) of the dibenzocyclooctadiyne having a polar functional group and the material; and A method for producing a complex, comprising the steps of binding a conjugate (B) to a cell having an azide group on the cell surface, and obtaining a complex in which the cell and the material are bound by a dibenzocyclooctadiyne having a polar functional group. (12) The method for producing a composite according to (10) or (11), wherein the material is at least one material selected from cells having azide groups on the cell surface and azide-modified inorganic materials. (13) The method for producing a composite according to (12), wherein the inorganic material is at least one inorganic material selected from a culture substrate, a glass substrate, an AFM cantilever, and a metal. (14) The method for producing a composite according to any one of (10) to (13), wherein the dibenzocyclooctadiyne having a polar functional group is a compound represented by the following general formula (I): [ka] (In general formula (I), each X is independently a group having a polar functional group, each A is independently a hydrocarbon group, n is an integer of 1 to 4, m is an integer of 0 to 3, n+m is an integer of 1 to 4, n' is an integer of 1 to 4, m' is an integer of 0 to 3, and n'+m' is an integer of 1 to 4.) (15) The method for producing a composite according to any one of (10) to (13), wherein the dibenzocyclooctadiyne having a polar functional group is a compound represented by the following general formula (II): [ka] (In general formula (II), each X is independently a group having a polar functional group, and each n is independently an integer of 1 to 4.) (16) The method for producing a composite according to any one of (10) to (13), wherein the dibenzocyclooctadiyne having a polar functional group is a compound represented by the following general formula (III): [ka] (In general formula (III), each R is independently a group selected from a group having a polar functional group and an alkyl group, and each n is independently an integer of 1 to 4.) (17) The method for producing a composite according to any one of (10) to (13), wherein the dibenzocyclooctadiyne having a polar functional group is a compound represented by the following general formula (IV): [ka] (In general formula (IV), each R is independently a group selected from a group having a polar functional group and an alkyl group.) (18) The method for producing a composite according to any one of (10) to (13), wherein the dibenzocyclooctadiyne having a polar functional group is at least one compound selected from the following formulae 1a to 1j: [ka] [ka] (In formula 1g, RGD is a peptide chain of three residues consisting of arginine-glycine-aspartic acid, and in formula 1j, Y is each independently a group selected from a group having a polar functional group and an alkyl group.) (19) At least one compound selected from the following formulae 1b to 1j: [ka] [ka] (In formula 1g, RGD is a peptide chain of three residues consisting of arginine-glycine-aspartic acid, and in formula 1j, Y is each independently a group selected from a group having a polar functional group and an alkyl group.) This specification includes the disclosure of Japanese Patent Application No. 2021-028795, from which this application claims priority. [Effects of the Invention]

[0014] The present disclosure provides cell-material complexes, methods for producing cell-material complexes, and novel compounds that can be used to obtain the complexes. [Brief explanation of the drawings]

[0015] [Figure 1] Figure 1 shows a schematic diagram of the process by which azidomannosamine (Ac4ManNAz) is added to the cell culture medium, and an azide group is introduced into the sialic acid moiety at the terminal of the glycan on the cell surface via the sialic acid metabolic pathway of the cell. [Figure 2A] FIG. 2A is a schematic diagram of cell attachment (adhesion) to a glass surface (Example 5). [Figure 2B] FIG. 2B shows a photomicrograph of 1b-bound cells seeded on a glass plate modified with azide by methods A1 and B1, analyzed by a reflected interference microscope, and the results of an analysis of the adhesion area to the substrate. [Figure 2C] FIG. 2C shows photomicrographs analyzed by reflected interference microscopy of the time course of azide-modified glass plates seeded with cells bound to 1b by methods A1 and B1. [Figure 3] Figure 3 shows a photomicrograph (right) of Example 5, taken using a reflected interference microscope, of the time course of cells bound to 1a, 1c, or 1d seeded on a glass plate modified with azide by methods A1 and B2, and the analyzed area of ​​strong adhesion (distance from the substrate h ≦40 nm) (left) of Figure 3. [Figure 4A] FIG. 4A is a schematic diagram of cell attachment (adhesion) to a glass surface (Example 5). [Figure 4B] FIG. 4B is a micrograph showing the time course of cells adhered to glass using 1a-1d in Example 5. [Figure 5A] FIG. 5A is a schematic diagram of cell-cell binding (crosslinking) (Example 6). [Figure 5B] Figure 5B shows a micrograph of cell clusters bound (adhered) with 1a. The control in Figure 5B is a micrograph of cells not treated with 1a. [Figure 6]Figure 6A shows a schematic diagram of cantilever bridging to a cell using 1b (Example 7). Figure 6B is a photograph showing the state of a cell bound to the cantilever in (1) of Figure 6A. In Figure 6C, the horizontal axis shows the distance between the cell and the cantilever, and the vertical axis shows the magnitude of cantilever deflection. [Figure 7A] FIG. 7A is a schematic diagram showing the scheme of Reference Example 1. [Figure 7B] FIG. 7B shows the results of measuring the fluorescence derived from FITC in Example 7 for 1a, 1b, 1c, and 1d. [Figure 7C] The results of Figure 7B are quantified and shown in Figure 7C. [Figure 7D] FIG. 7D shows the results of measuring FITC-derived fluorescence in Example 7 for 1a, unsubstituted CODY. [Figure 7E] The results of Figure 7D are quantified and shown in Figure 7E. [Figure 8] FIG. 8 shows the cell engraftment rate calculated in Example 8. [Figure 9A] FIG. 9A is a schematic diagram showing the scheme of Example 9. [Figure 9B] FIG. 9B shows the results of Example 9 in the molecular function category. DETAILED DESCRIPTION OF THE INVENTION

[0016] The present invention will be described in detail below.

[0017] <Complex> The complex of this embodiment is a complex in which cells having azide groups on their cell surface and materials having azide groups (excluding fluorescent dyes having azide groups) are bonded via dibenzocyclooctadiyne having polar functional groups. In this complex, the cells and materials are bonded via dibenzocyclooctadiyne having polar functional groups, rather than via dibenzocyclooctadiyne (5,6,11,12-Tetradehydrodibenzo[a,e]cyclooctene) (hereinafter also referred to as unsubstituted CODY), allowing for rapid cell-to-material bonding. The inventors speculate that this is due to the superior solubility of dibenzocyclooctadiyne having polar functional groups compared to unsubstituted CODY. Furthermore, due to its superior solubility, dibenzocyclooctadiyne having more polar functional groups can bond with azide groups on the cell surface compared to unsubstituted CODY, resulting in stronger bonding between the cells and materials.

[0018] (cell) The cells having an azide group on the cell surface may be any cells having an azide group on the cell surface. Preferably, the cells having an azide group on the cell surface are cells in which an azide group has been introduced into the sialic acid moiety at the terminal sugar chain on the cell surface through the sialic acid metabolic pathway of the cells by adding acetylated azidomannosamine (Ac4ManNAz) to the cell culture medium. Alternatively, the cells may be cells in which an azide group has been introduced onto the cell surface through another metabolic pathway. Note that the cells in which an azide group has been introduced onto the cell surface may also have an azide group introduced onto a site other than the cell surface. The structure of Ac4ManNAz is shown below.

[0019] [ka]

[0020] Figure 1 shows a schematic diagram of the process in which azide groups are introduced into the sialic acid moieties at the terminals of glycans on the cell surface via the sialic acid metabolic pathway of the cells by adding Ac4ManNAz to the cell culture medium.

[0021] Cultured cells are usually used as the cells into which the azide group is introduced. The cells into which the azide group is introduced may be cells of human origin or cells of non-human origin. Non-human cells include cells of mammalian origin (excluding humans) and cells of organisms other than mammals, such as birds, fish, insects, plants, algae, and fungi. Mammals (excluding humans) include, for example, mice, rats, horses, sheep, pigs, goats, and cows.

[0022] The cells are not particularly limited, and examples thereof include PC-9, PC-14, PC-1, PC-3, PC-6, PC-7, PC-10, PC-13, PC-17a, PC-17b, PC-19, PC-20, (L)PC3, (L)PC6, (L)PC10, A549, ABC-1, EBC-1, FT821, FM205, GLS, GLL-1, H-69, KTA7, KTA9, and K TZ6, L-1-5, L-2-3, L-8-1, L-27, L-58-1, L-62-2, L-63-5, LC-3-JCK, LC-4-JCK, LC-6-JCK, LC-7-JC K, LC-8-JCK, LC-9-JCK, LC-10-JCK, LC-11-JCK, LC-12-JCK, LC-13-JCK, LC-14-JCK, LC-15-JCK, LC- 16-JCK, LC-17-JCK, LC-18-JCK, LC-19-JCK, LC-20-JCK, LC-21-JCK, LC-22-JCK, LC-23-JCK, LC-24 -JCK, LC-25-JCK, LC-26-JCK, LC-27-JCK, LCT-1, LCT-2, LCT-6, Lu-24, Lu-61, Lu-65, Lu-99, Lu-116 , Lu-130, Lu-135, LX-1, Mgnu1, Msnu1, N-231, OS2-RA, OTUK, QG-56, QG-90, RERF-LC-AI, RERF-LC-K J, SBC-1, SBC-2, SBC-3, SBC-4, SBC-5, SK-AK-LCL, SK-MES-1, B16F10, Colon-26, MCF7, HepG2, Lewis Examples include lung cancer, ISO-HAS-B, NIH / 3T3, TFK-1, A549, Jurkat, SW1990, MDA-MB-231, PANC-1, CHO, HEK-293T, HUVEC, MSC, MIN6, etc.

[0023] (material) The material having an azide group (excluding fluorescent dyes having an azide group) is not particularly limited as long as it has an azide group. The azide group may be present in a portion of the material that is desired to bind to cells via dibenzocyclooctadiyne having a polar functional group, and the azide group may be present on a part of the surface of the material or on the entire surface of the material.

[0024] Examples of the material include cells having azide groups on their surface, azide-modified inorganic materials, and organic materials having azide groups, and it is preferable that the material be at least one material selected from cells having azide groups on their surface and azide-modified inorganic materials. The composite of this embodiment can easily bond cells (cells having azide groups on their surface) that are usually difficult to bond with each other, or cells and inorganic materials that are usually difficult to bond with, and therefore can be used in various research projects that were previously difficult to do.

[0025] The inorganic material is preferably at least one inorganic material selected from a culture substrate, a glass substrate, an AFM cantilever, and a metal.

[0026] The material used in this embodiment may be one type alone or two or more types. When two or more types are used, for example, after a composite is formed, another material may be bonded to the composite.

[0027] There are no particular limitations on the method for introducing azide groups into a material. For example, if the material is a cell having azide groups on its surface, the cells described in the above section (Cells) can be used. If the material is an organic material having azide groups, the azide groups can be introduced, for example, according to a conventional organic synthesis method. If the material is an inorganic material having azide groups, the azide groups can be introduced by, for example, binding organic molecules or organic-inorganic hybrid molecules to the surface using a compound having an azide group as the organic molecule or organic-inorganic hybrid molecule, as described in the Examples. Alternatively, if the organic molecule or organic-inorganic hybrid molecule does not have an azide group, the azide groups can be introduced by further modifying or denaturing the organic molecule or organic-inorganic hybrid molecule.

[0028] (Dibenzocyclooctadiyne with polar functional groups) The dibenzocyclooctadiyne having a polar functional group includes a molecule in which a hydrogen atom on the benzene ring of unsubstituted CODY is substituted with a polar functional group.

[0029] The dibenzocyclooctadiyne having a polar functional group is preferably a compound represented by the following general formula (I), more preferably a compound represented by the following general formula (II), particularly preferably a compound represented by the following general formula (III), and most preferably a compound represented by the following general formula (IV).

[0030] [ka] (In general formula (I), each X is independently a group having a polar functional group, each A is independently a hydrocarbon group, n is an integer of 1 to 4, m is an integer of 0 to 3, n+m is an integer of 1 to 4, n' is an integer of 1 to 4, m' is an integer of 0 to 3, and n'+m' is an integer of 1 to 4.)

[0031] [ka] (In general formula (II), each X is independently a group having a polar functional group, and each n is independently an integer of 1 to 4.)

[0032] [ka] (In general formula (III), each R is independently a group selected from a group having a polar functional group and an alkyl group, and each n is independently an integer of 1 to 4.)

[0033] [ka] (In general formula (IV), each R is independently a group selected from a group having a polar functional group and an alkyl group.)

[0034] In general formulas (I) and (II), X's each independently represent a group having a polar functional group, and in general formulas (III) and (IV), R's each independently represent a group selected from a group having a polar functional group and an alkyl group.

[0035] The polar functional group may be at least one polar functional group selected from an ionic functional group and a nonionic polar functional group. The group having a polar functional group may have at least one polar functional group, and may have multiple polar functional groups. There is no particular upper limit on the number of polar functional groups. For example, X may have 20 or less polar functional groups per group having a polar functional group, and R may have 19 or less polar functional groups per group having a polar functional group.

[0036] The ionic functional group may be, for example, a cation (e.g., -NH3 + , -NH2Z + , -NHZ2 + , -NZ3 + ) and a counter anion (e.g., a halide ion (F - , Cl - , Br - , I - )) functional groups, anions (e.g., -SO3 - , -O - , -COO - , -PO3 - ) and a counter cation (e.g., an alkali metal ion (e.g., Li + , Na + , K. + )) and functional groups having zwitterions (for example, the functional groups having the above-mentioned cations and anions). When the group having a polar functional group has an ionic functional group, it may have one ionic functional group or multiple ionic functional groups. When it has multiple ionic functional groups, it may have the same type of ionic functional groups or different types of ionic functional groups.

[0037] Examples of nonionic polar functional groups include -NH, -NHZ, -NZ, -SOH, -OH, -COOH, -NH-CONH, -NH-CONHZ, -NH-CONZ, -NZ-CONH, -NZ-CONHZ, -NZ-CONZ, -CO- (carbonyl group), -COO- (ester group), and -O- (ether group). When the group having a polar functional group has a nonionic polar functional group, it may have one or more nonionic polar functional groups. When it has multiple nonionic polar functional groups, it may have the same type of nonionic polar functional group or different types of nonionic polar functional groups.

[0038] Z is a hydrocarbon group having 1 to 18 carbon atoms, and preferably an alkyl group having 1 to 6 carbon atoms. When there are multiple Z's, they may be the same group or different groups.

[0039] The structure of the group having a polar functional group other than the polar functional group is not particularly limited, but is usually a saturated or unsaturated hydrocarbon group, preferably a saturated hydrocarbon group. The saturated or unsaturated hydrocarbon group may be linear, branched, or have a ring structure.

[0040] X preferably has a molecular weight of 16 to 1,100, more preferably 16 to 200.

[0041] R preferably has a molecular weight of 15 to 584, more preferably 15 to 184. As described above, R are each independently a group selected from a group having a polar functional group and an alkyl group, and it is preferable that at least one of R in one molecule is a group having a polar functional group. When R is an alkyl group, examples of the alkyl group include alkyl groups having 1 to 18 carbon atoms, and preferred examples include alkyl groups having 1 to 6 carbon atoms. One preferred embodiment of the alkyl group is a methyl group.

[0042] X and R preferably have at least one structure selected from a peptide chain, a sugar chain, a urea group (e.g., -NH-CONH2, -NH-CONHZ, -NH-CONZ2, -NZ-CONH2, -NZ-CONHZ, -NZ-CONZ2), a carbonyl group, an ester bond, and an ether group. Examples of the peptide chain include peptide chains of 1 to 5 residues.

[0043] In one preferred embodiment, X is —OR in general formula (III), that is, in one preferred embodiment, the bonding site to the benzene ring of X is —O— (ether group).

[0044] In general formula (I), each A independently represents a hydrocarbon group, which is preferably a hydrocarbon group having 1 to 18 carbon atoms, and more preferably an alkyl group having 1 to 6 carbon atoms.

[0045] In general formulas (I), (II), and (III), n is each independently an integer of 1 to 4, preferably an integer of 1 to 3, more preferably 1 or 2, and particularly preferably 1. In general formula (I), n' is an integer of 1 to 4, preferably an integer of 1 to 3, more preferably 1 or 2, and particularly preferably 1.

[0046] In general formula (I), m is an integer of 0 to 3, preferably 0 or 1, and more preferably 0. In general formula (I), m' is an integer of 0 to 3, preferably 0 or 1, and more preferably 0.

[0047] In general formula (I), n+m is an integer of 1 to 4, preferably an integer of 1 to 3, more preferably 1 or 2, and particularly preferably 1. Furthermore, n'+m' is an integer of 1 to 4, preferably an integer of 1 to 3, more preferably 1 or 2, and particularly preferably 1.

[0048] In one preferred embodiment, the group having the polar functional group has at least one functional group selected from a functional group having at least a cation and a counter anion, a functional group having an anion and a counter cation, and a functional group having a zwitterion. When the group having the polar functional group has these polar functional groups, the dibenzocyclooctadiynes having the polar functional groups tend to have particularly excellent solubility, which is preferred. Furthermore, since cell surfaces tend to be anionic, it is more preferred that the polar functional group has a cation and a counter anion in terms of the reactivity between the azide groups on the cell surface and the dibenzocyclooctadiynes having the polar functional groups.

[0049] Specific examples of dibenzocyclooctadiynes having a polar functional group include at least one compound selected from the following formulae 1a to 1j, and these compounds are particularly preferred. As the at least one compound selected from the formulae 1a to 1j, 1a is preferred. The compounds of formulae 1b to 1j are novel compounds. In the present disclosure, "compound 1a" may be simply written as "1a," "compound 1b" may be simply written as "1b," "compound 1c" may be simply written as "1c," "compound 1d" may be simply written as "1d," "compound 1e" may be simply written as "1e," "compound 1f" may be simply written as "1f," "compound 1g" may be simply written as "1g," "compound 1h" may be simply written as "1h," "compound 1i" may be simply written as "1i," and "compound 1j" may be simply written as "1j."

[0050] [ka] [ka]

[0051] In formula 1g, RGD is a three-residue peptide chain consisting of arginine-glycine-aspartic acid.

[0052] In formula 1j, each Y is independently a group selected from a group having a polar functional group and an alkyl group.

[0053] When Y is a group having a polar functional group, examples thereof include the groups exemplified above for X. However, the upper limit of the molecular weight of Y is 28 molecular weights lower than the upper limit of X. When Y is an alkyl group, examples thereof include alkyl groups having 1 to 18 carbon atoms, preferably alkyl groups having 1 to 6 carbon atoms. As the alkyl group, a methyl group is one of the preferred embodiments.

[0054] There are no particular limitations on the method for synthesizing dibenzocyclooctadiynes having polar functional groups, but examples include a method in which dibenzocyclooctadiynes having a hydroxy group (-OH) represented by S5 as described in the Examples are synthesized, and then the hydroxy group is etherified by a conventionally known organic synthesis method or the like to convert the hydroxy group to -OR. Another method includes a method in which dibenzocyclooctadiynes having a hydroxy group (-OH) represented by S5 as described in the Examples are synthesized, and then the hydroxy group is esterified by a conventionally known organic synthesis method or the like. Yet another method includes a method in which dibenzocyclooctadiynes having a carboxy group (-COOH) are synthesized, and then the carboxy group is esterified.

[0055] (complex) As described above, the complex of this embodiment is a complex in which cells having azide groups on the cell surface and a material having azide groups (excluding fluorescent dyes having azide groups) are bound together by dibenzocyclooctadiyne having a polar functional group.

[0056] Dibenzocyclooctadiyne, which has a polar functional group, has two alkynes in its molecule. One of them undergoes an SPAAC reaction with the azide group of a cell that has an azide group on its surface, and the other undergoes an SPAAC reaction with the azide group of a material that also has an azide group, thereby obtaining a complex in which the cell and material are bound by the dibenzocyclooctadiyne, which has a polar functional group.

[0057] The composite of this embodiment can easily bond cells that are normally difficult to bond (cells with azide groups on their surface) with each other, or cells with inorganic materials that are normally difficult to bond with, enabling applications in a variety of research fields that were previously difficult to achieve. For example, when the material is cells with azide groups on their surface, spheroids can be easily produced, and even if the cells are non-adherent, cell aggregates can be easily obtained, allowing the cultivation of non-adherent cells. Furthermore, by using a material with microchannels as the inorganic material and modifying desired positions of the microchannels with azide groups, cells can be bonded to desired positions. Another potential application is to layer cells on a cell sheet to form a multilayered tissue.

[0058] Taking the case where dibenzocyclooctadiyne having a polar functional group is a compound represented by the above general formula (IV) as an example, the structure of the complex can be represented by the following general formulas (2-1) to (2-4).

[0059] [ka] In the general formulas (2-1) to (2-4), R is independently a group selected from a group having a polar functional group and an alkyl group, and Z 1 is a bound cell derived from a cell with azide groups on the cell surface, and Z 2 is a bound material derived from a material having an azide group.

[0060] The structure of the complex is such that the dibenzocyclooctadiyne having a polar functional group is a compound represented by the above general formula (IV), and the two R in the general formula (IV) are different groups, R 1 and R 2 For example, the case can be represented by the following compound group (general formula (3)).

[0061] [ka] (In the general formula (3), R 1and R 2 has the same meaning as R in general formula (IV), and R 1 and R 2 is a group different from Z 1 is a bound cell derived from a cell with azide groups on the cell surface, and Z 2 is a bound material derived from a material having an azide group.

[0062] The complex according to this embodiment may have different structures as shown in the above formulas (2-1) to (2-4) and formula (3) even when the same type of cells, the same type of materials, and dibenzocyclooctadiynes having the same type of polar functional groups are used. However, unless otherwise specified in this disclosure, the structures represented by the above formulas (2-1) to (2-4) and the group of compounds represented by formula (3) are not particularly distinguished from one another.

[0063] <Method of manufacturing the composite> The method for producing a composite according to this embodiment is a method for producing the composite according to the embodiment described above. The method for producing a composite according to this embodiment can be broadly divided into two modes.

[0064] The first aspect of the method for producing a complex comprises the steps of: binding cells having azide groups on their surface to a dibenzocyclooctadiyne having a polar functional group to obtain a conjugate (A) of the dibenzocyclooctadiyne having the polar functional group and the cells; and binding the conjugate (A) to a material having an azide group (excluding fluorescent dyes having an azide group) to obtain a complex in which the cells and the material are bound by the dibenzocyclooctadiyne having the polar functional group.

[0065] The second embodiment of the method for producing a complex comprises the steps of: bonding a material having an azide group (excluding fluorescent dyes having an azide group) to a dibenzocyclooctadiyne having a polar functional group to obtain a conjugate (B) of the dibenzocyclooctadiyne having a polar functional group and the material; and bonding the conjugate (B) to a cell having an azide group on its surface to obtain a complex in which the cell and the material are bonded via the dibenzocyclooctadiyne having a polar functional group.

[0066] The biggest difference between the first and second embodiments is which is first bound to the dibenzocyclooctadiyne having a polar functional group: cells or a material. In the first embodiment, the dibenzocyclooctadiyne is bound to cells first, and then bound to the material after obtaining conjugate (A). On the other hand, in the second embodiment, the dibenzocyclooctadiyne is bound to the material first, and then bound to cells after obtaining conjugate (B). In either the first or second embodiment, the reaction proceeds rapidly, making it possible to produce a complex.

[0067] The method for obtaining conjugate (A) is not particularly limited as long as it allows contact between cells having azide groups on the cell surface and dibenzocyclooctadiyne having a polar functional group, but an example is a method in which a cell suspension containing cells having azide groups on the cell surface is prepared, and dibenzocyclooctadiyne having a polar functional group is added to the cell suspension and incubated to obtain conjugate (A). The dibenzocyclooctadiyne having a polar functional group to be added to the cell suspension is preferably added as a solution containing dibenzocyclooctadiyne having a polar functional group.

[0068] The method for obtaining the conjugate (B) is not particularly limited as long as it can bring a material having an azide group into contact with a dibenzocyclooctadiyne having a polar functional group. An example of the method is to bring a material having an azide group into contact with a solution containing a dibenzocyclooctadiyne having a polar functional group to obtain the conjugate (B).

[0069] The method for obtaining a complex from the conjugate (A) is not particularly limited, and examples thereof include a method for obtaining a complex by applying a suspension containing the conjugate (A) onto a material having an azide group, and a method for obtaining a complex by contacting or immersing a material having an azide group in a suspension containing the conjugate (A).

[0070] The method for obtaining a complex from the conjugate (B) is not particularly limited, and examples thereof include a method for obtaining a complex by applying a cell suspension containing cells having azide groups on the cell surface onto the conjugate (B), and a method for obtaining a complex by contacting or immersing the conjugate (B) in a cell suspension containing cells having azide groups on the cell surface.

[0071] The method for producing a composite according to this embodiment can usually be carried out at 4 to 40° C., and more preferably at 15 to 37° C. The examples described below were carried out at room temperature (rt) (20 to 30° C.) unless otherwise specified.

[0072] The method for producing a composite according to this embodiment may be carried out in air or in an inert gas (e.g., nitrogen, argon) atmosphere. It may also be carried out under pressure or under reduced pressure. The examples described below were carried out in air unless otherwise specified. [Example]

[0073] The present embodiment will be described below with reference to examples, but the present disclosure is not limited to these examples.

[0074] In the examples, Silica gel 60 (spherical, particle size 40 to 100 μm, Kanto Chemical) was used in flash chromatography.

[0075] In the examples, 1 H and 13 C NMR spectra were measured using a JEOL JNM-AL 300, JNM-ECX 400, or JNM-ECA 500. The solvent was CDCl3 ( 1 H NMR; δ=7.26 ppm,13 C NMR; δ = 77.0 ppm), DMSO-d6 ( 1 H NMR; δ=2.50 ppm, 13 C NMR; δ=39.5 ppm), CD3CN ( 1 H NMR; δ=1.94 ppm, 13 C NMR; δ=118.3 ppm) or DO ( 1 1 H NMR; δ=4.79 ppm) was used, and the signals shown in parentheses were used as the reference.

[0076] 1 The H NMR data are reported in terms of chemical shift (δ, ppm), multiplicity (s, singlet; d, doublet; t, triplet; m, multiplet; br, broad), coupling constant (Hz), and integral. 13 C NMR data are reported in terms of chemical shifts (δ, ppm).

[0077] Mass spectra were measured using a JEOL JMS-T100LC mass spectrometer in ESI-MS mode using methanol as a solvent.

[0078] The synthesis scheme of dibenzocyclooctadiynes having polar functional groups is shown below.

[0079] [ka]

[0080] In Scheme 1, a) boron tribromide, dichloromethane, 0°C → rt; b) tert-butyldimethylchlorosilane, imidazole, dimethylformamide, rt; c) lithium diisopropylamide, tetrahydrofuran, -78°C; d) potassium bifluoride, tetrahydrofuran / methanol, 0°C.

[0081] [ka]

[0082] In Scheme 2, a) triphenylphosphine, N,N-dimethylaminoethanol, diisopropyl azodicarboxylate, 0°C → rt; b) iodomethane, rt; c) 1,3-propane sultone, rt; d) 1,3-propane sultone, cesium carbonate, tetrahydrofuran, 0°C.

[0083] [ka]

[0084] In Scheme 3, a) 2,3,4,6-Tetra-O-acetyl-β-D-galactopyranosyl 2,2,2-Trichloroacetimidate, CHCl, TMSOTf, 0°C; b) potassium bifluoride, tetrahydrofuran / methanol, 0°C; then potassium carbonate, iodomethane, dimethylformamide; c) potassium carbonate, tetrahydrofuran / methanol.

[0085] [Synthesis Example 1] The compound S2 shown in Scheme 1 was synthesized.

[0086] [ka]

[0087] To a solution of S1 (160 mg, 293 μmol) in dichloromethane, boron tribromide (2.06 mmol, 1.0 mol / L in dichloromethane) was added at 0°C and stirred at room temperature under an argon atmosphere for 3 h. Methanol (1 mL) and saturated aqueous sodium bicarbonate solution were added dropwise to the reaction mixture at 0°C. The mixture was extracted with dichloromethane and dried over anhydrous magnesium sulfate. The residue was recrystallized in dichloromethane to give S2 (129 mg, 85%) as a white solid.

[0088] 1H-NMR (301 MHz, DMSO-D6) δ 10.05 (s, 2H), 7.77 (t, J = 7.1 Hz, 2H), 7.63 (t, J = 7.7 Hz, 4H), 7.32 (d, J = 8.3 Hz, 4H), 7.15 (d, J = 8.6 Hz, 2H), 7.07 (s, 2H), 6.71 (dd, J = 8.6, 2.1 Hz, 2H), 6.47 (d, J = 2.1 Hz, 2H); 13 HRMS (ESI-TOF) 539.0599 (M + Na) + calcd for C 28 H 20 O6S2Na 539.0599.

[0089] [Synthesis Example 2] The compound S3 shown in Scheme 1 was synthesized.

[0090] [ka]

[0091] S2 (124 mg, 240 μmol) and imidazole (130 mg, 1.91 mmol) were dissolved in dimethylformamide (3 mL), tert-butyldimethylchlorosilane (199 mg, 1.32 mmol) was added, and the mixture was stirred for 5 hours under an argon atmosphere. Ethyl acetate was added to the reaction mixture, which was washed with water and dried over anhydrous magnesium sulfate. The residue was recrystallized in ethyl acetate to give S3 (114 mg, 64%) as a white solid. Note that TBS stands for tert-butyldimethylsilyl group.

[0092] 1H-NMR (301 MHz, CHLOROFORM-D) δ 7.60-7.65 (m, 2H), 7.36-7.48 (m, 10H), 7.19 (s, 2H), 6.73 (dd, J = 8.6, 2.8 Hz, 2H), 6.42 (d, J = 2.4 Hz, 2H), 0.93 (s, 18H), 0.15 (s, 12H); 13 C-NMR (76 MHz, CHLOROFORM-D) δ 156.75, 144.73, 139.17, 138.36, 137.50, 133.95, 132.43, 129.04, 128.25, 121.62, 120.52, 118.41, 25.74, 18.41, -4.28; HRMS (ESI-TOF) 767.2327 (M + Na) + calcd for C 40 H 48 O6S2Si2Na 767.2329.

[0093] [Synthesis Example 3] The compound S4 shown in Scheme 1 was synthesized.

[0094] [ka]

[0095] S3 (114 mg, 153 μmol) was dissolved in tetrahydrofuran (4.1 mL), and lithium diisopropylamide (780 μmol, 1 mol / L hexane / tetrahydrofuran solution) was added dropwise at -78°C, followed by stirring for 40 minutes. Saturated aqueous ammonium chloride solution was added to the reaction mixture at 0°C, and the mixture was extracted with ethyl acetate and dried over anhydrous magnesium sulfate. The residue was purified by silica gel column chromatography [hexane / ethyl acetate (15:1)] to give S4 (56.5 mg, 82%) as an orange solid.

[0096] 1 H-NMR (500 MHz, CDCl3). 1H-NMR (400 MHz, CDCl3) δ 6.60 (d, J = 8.2 Hz, 2H), 6.34 (dd, J = 8.2, 2.3 Hz, H), 6.25 (d, J = 2.3 Hz, 2H), 0.94 (s, 18H), 0.17 (s, 12H); 13 HRMS (ESI-TOF) 483.2153 (M + Na) + calcd for C 28 H 36 O2Si2Na 483.2152.

[0097] [Synthesis Example 4] Compounds S5 and S7 shown in Scheme 1 above were synthesized.

[0098] [ka]

[0099] S4 (57 mg, 123 μmol) was dissolved in THF-MeOH (3:1, 2.0 mL), and aqueous potassium bifluoride (60 μmol, 1 M) was added at 0 °C. After stirring for 30 min, hydrochloric acid (1.2 M, 60 μL) and saturated brine were added. The organic phase was extracted with ethyl acetate, dried over anhydrous magnesium sulfate, and the residue was purified on a silica gel column [hexane / ethyl acetate (5:1)] to give S5 (12 mg, 39%) and S7 (25.1 mg, 59%) as yellow powders. Because S5 was unstable in the concentrated state, it was dissolved in ethyl acetate and stored at −30 °C.

[0100] S5: 1H-NMR (400 MHz, CD3CN) δ 7.29 (s, 2H), 6.65 (d, J = 8.7 Hz, 2H), 6.39 (dd, J = 8.5, 2.5 Hz, 2H), 6.30 (d, J = 2.7 Hz, 2H); 13 C-NMR (126 MHz, CHLOROFORM-D) δ 159.00, 135.63, 129.23, 123.38, 116.23, 115.16, 110.89, 107.68; HRMS (ESI-TOF) 345.1304 (M - H) - calcd for C 16 H7O2231.0449; S7: 1 H-NMR (301 MHz, CDCl3) δ 6.61 (dd, J = 8.3, 2.0 Hz, 3H), 6.34 (td, J = 5.4, 2.7 Hz, 2H), 6.26 (dd, J = 12.9, 2.6 Hz, 1H), 5.11 (s, 1H), 0.94 (s, 9H), 0.17 (s, 6H); 13 C-NMR (126 MHz, CHLOROFORM-D) δ156.84, 156.49, 135.32, 135.01, 128.14, 128.12, 124.73, 124.41, 119.99, 119.32, 115.48, 114.36, 110.57, 110.07, 107.53, 107.42, 77.45, 77.20, 76.94, 25.73, 18.34, -4.28; HRMS (ESI-TOF) 345.1304 (M + Na)+ calcd for C 22 H 21 O2SiNa 345.1311.

[0101] [Synthesis example 5] The above description shows the synthesis of the S6 compound.

[0102]

change

[0103] Under an argon atmosphere, S5 (40 mg, 172 μmol) and triphenylphosphine (181 mg, 689 μmol) were dissolved in dry THF (2.0 mL), and N,N-dimethylaminoethanol (69 μL, 689 μmol) and diisopropyl azodicarboxylate (135 μL, 689 μmol) were added at 0 °C. After stirring at room temperature under an argon atmosphere for 4 h, the solvent was removed from the reaction mixture by evaporation under reduced pressure. The residue was purified using a silica gel column [chloroform / methanol (1:1)] to give S6 (41 mg, 64%) as a yellow powder.

[0104] [Synthesis Example 6] The compound 1a shown in Scheme 2 above was synthesized.

[0105] [ka]

[0106] Hydrochloric acid (1.2 M, 40 μL) was added to S6, and the mixture was evaporated under reduced pressure to obtain a yellow powder 1a.

[0107] 1 H-NMR (301 MHz, D2O) δ 6.81 (d, J = 8.3 Hz, 2H), 6.60 (dd, J = 8.6, 2.8 Hz, 2H), 6.52 (d, J = 2.8 Hz, 2H), 4.28 (t, J = 5.0 Hz, 4H), 3.55 (t, J = 5.0 Hz, 4H), 2.93 (s, 12H); 13 C-NMR (126 MHz, D2O) δ158.28, 133.97, 128.50, 124.03, 115.18, 113.41, 109.97, 107.26, 61.63, 56.05, 42.79; HRMS (ESI-TOF) 375.2069 (M+H) + calcd for C 24 H 27 N2O2375.2075.

[0108] [Example 1] Compound 1b shown in Scheme 2 above was synthesized.

[0109] [ka]

[0110] Iodomethane (500 μL) was added to S6 (17 mg, 45.4 μmol) and the mixture was stirred and heated under reflux for 8 hours. The reaction solution was concentrated and then purified on an ODS column (Sep-Pak C18, Waters) [saturated brine → HO:MeOH = 9:1] to obtain 1b as a yellow powder (3.7 mg, 17%).

[0111] 1 H-NMR (500 MHz, D2O) δ 6.79 (d, J = 8.5 Hz, 2H), 6.58 (dd, J = 9, 3.0 Hz, 2H), 6.50 (d, J = 3.0 Hz, 2H), 4.40 (s, 4H), 3.76 (m, 4H), 3.19 (s, 18H); 13 C-NMR (126 MHz, D2O) δ 158.13, 133.96, 128.53, 123.96, 115.31, 113.42, 109.94, 107.23, 64.91, 61.89, 53.92; HRMS (ESI-TOF) 404.2461 calcd for C 26 H 32 N2O2404.2464.

[0112] [Example 2] Compound 1c shown in Scheme 2 above was synthesized.

[0113] [ka]

[0114] 1,3-Propanesultone (500 μL) was added to S6 (22 mg, 58.7 μmol) and stirred at 40° C. for 7 hours. The reaction solution was purified on an ODS column (Sep-Pak C18, Waters) [HO:MeOH = 1:1] to obtain 1c as a yellow powder (5.5 mg, 15%).

[0115] 1 H-NMR (500 MHz, D2O) δ 6.71 (d, J = 9.0 Hz, 2H), 6.57 (dd, J = 8.5, 2.5 Hz, 2H), 6.43 (d, J = 2.0 Hz, 2H), 4.37 (s, 4H), 3.78 (s, 4H), 3.52 (m, 4H), 3.17 (s, 12H), 2.84 (t, J = 7.5 Hz, 4H), 2.22 (m, 4H); 13 C-NMR (126 MHz, D2O) δ 158.30, 134.04, 128.59, 123.98, 115.56, 115.44, 113.28, 110.39, 107.82, 103.41, 63.44, 62.29, 61.91, 51.94, 47.42, 18.53; HRMS (ESI-TOF) 641.1968 (M + Na) + calcd for C 30 H 38 N2NaO8S2641.1967.

[0116] [Example 3] Compound 1d shown in Scheme 2 above was synthesized.

[0117] [ka]

[0118] S5 (3.6 mg, 15.5 μmol) was dissolved in dry tetrahydrofuran (500 μL), and cesium carbonate (25.2 mg, 77.3 μmol) and 1,3-propane sultone (6.8 μL, 77.4 μmol) were added at 0°C. After stirring at 0°C for 18 hours, the resulting solid was collected and purified on an ODS column [50% saturated brine → water / methanol (4:1)] to give 1d (3.4 mg, 22%).

[0119] 1 H-NMR (500 MHz, D2O) δ 6.69 (d, J = 8.5 Hz, 1H), 6.49 (dd, J = 8.5, 3 Hz, 1H), 6.40 (d, J = 2.0 Hz, 1H), 4.00 (t, J = 6.0 Hz, 2H), 3.00 (t, J = 7.0 Hz, 2H), 2.09-2.14 (m, 2H); 13 C-NMR (126 MHz, D2O) δ159.12, 133.99, 128.39, 123.35, 115.10, 113.41, 110.17, 107.50, 66.57, 47.71, 24.12; HRMS (ESI-TOF) 474.0427 M - calcd for C 22 H 18 O8S2474.0443.

[0120] [Synthesis Example 7] The compound S8 shown in Scheme 3 was synthesized.

[0121] [ka]

[0122] To a solution of S7 (23.1 mg, 66.7 μmol) and 2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl 2,2,2-trichloroacetimidate (49.3 mg, 0.10 mmol) in CHCl (2 mL) was added TMSOTf (trimethylsilyl trifluoromethanesulfonate) (1.21 μL, 6.67 μmol) at 0 °C under an Ar atmosphere. After stirring at 0 °C for 1.5 h, the reaction mixture was quenched with EtN (50 μL) and concentrated in vacuo. The residue was purified by silica gel column chromatography (hexane / EtOAc = 4:1) to give S8 (15.8 mg, 0.023 mmol, 35%).

[0123] 1 H-NMR (301 MHz, CDCl3) δ 6.64 (d, J = 8.4 Hz, 1H), 6.61 (d, J = 8.1 Hz, 1H), 6.49 (dd, J = 8.4, 2.6 Hz, 1H), 6.39 (d, J = 2.7 Hz, 1H), 6.36 (dd, J = 8.4, 2.4 Hz, 1H), 6.26 (d, J = 2.4 Hz, 1H), 5.27 (t, J = 9.6 Hz, 1H), 5.21 (dd, J = 10.8, 3.9 Hz, 1H), 5.12 (t, J = 9.5 Hz, 1H), 4.97 (d, J = 7.6 Hz, 1H), 4.26 (dd, J = 12.3, 5.4 Hz, 1H), 4.14 (dd, J = 12.4, 2.4 Hz, 1H), 3.80-3.85 (m, 1H), 2.10 (s, 3H), 2.05 (s, 3H), 2.04 (s, 3H), 2.03 (s, 3H), 0.94 (s, 9H), 0.17 (s, 6H); 13C NMR (101 MHz, CDCl3) δ 170.73, 170.33, 169.52, 169.37, 157.31, 156.91, 135.36, 134.68, 128.17, 127.80, 127.21, 124.70, 120.18, 119.54, 116.34, 115.95, 110.87, 109.41, 108.35, 98.56, 72.71, 72.30, 71.08, 68.28, 62.02, 25.69, 20.89, 20.74, 18.30. -4.31; HRMS (ESI-TOF) 699.22170 (M + H) + calcd for C 36 H 40 O 11 SiNa 699.22376.

[0124] [Synthesis Example 8] The compound S9 shown in Scheme 3 was synthesized.

[0125] [ka]

[0126] To a solution of S8 (27.5 mg, 40.6 μmol) in MeOH (200 μL) and THF (600 μL) was added KHF (40.6 μmol) at 0 °C. After stirring for 5 min, the reaction mixture was quenched with saturated aqueous solution of HCl at 0 °C and extracted with EtOAc. The organic layer was washed with brine, dried over MgSO, filtered, and concentrated in vacuo to give the alcohol. To a solution of the alcohol and KCO (14.2 mg, 103 μmol) in DMF (1 mL) was added MeI (4.31 μL, 68.4 μmol) at room temperature and stirred for 70 min. The reaction mixture was added EtOAc, washed with water, and dried over MgSO. After washing with water, the solvent was removed in vacuo. The residue was purified by silica gel column chromatography (hexane / EtOAc = 2:3) to give S9 (12.1 mg, 52%, 2 steps).

[0127] 1H-NMR (500 MHz, CDCl3) δ 6.66 (t, J = 8.6 Hz, 2H), 6.49 (dd, J = 8.3, 2.6 Hz, 1H), 6.40 (dd, J = 10.0, 2.6 Hz, 2H), 6.34 (d, J = 2.9 Hz, 1H), 5.27 (dd, J = 13.8, 4.5 Hz, 1H), 5.20 (t, J = 8.6 Hz, 1H), 5.12 (t, J = 9.7 Hz, 1H), 4.97 (d, J = 7.4 Hz, 1H), 4.25 (dd, J = 7.8, 3.3 Hz, 1H), 4.14 (dd, J = 12.3, 2.6 Hz, 1H), 3.80-3.84 (m, 1H), 3.72 (s, 3H), 2.10 (s, 3H), 2.05 (s, 3H), 2.04 (s, 3H), 2.02 (s, 3H); 13 C NMR (126 MHz, CDCl3) δ 170.72, 170.33, 169.52, 169.36, 160.49, 157.33, 135.37, 134.67, 128.10, 127.83, 127.17, 123.88, 116.28, 115.89, 114.99, 112.27, 110.86, 109.53, 98.54, 72.68, 72.28, 71.05, 68.25,61.99, 55.52, 20.88, 20.76, 20.73

[0128] [Example 4] Compound 1e shown in Scheme 3 above was synthesized.

[0129] [ka]

[0130] To a solution of S9 (3.4 mg, 5.90 μmol) in MeOH (500 μL) and THF (500 μL) was added K2CO3 (4.08 mg, 29.5 μmol) at room temperature. After stirring for 55 min, the reaction mixture was transferred to a cation exchange resin (DOWEX TM50Wx8 100-200 mesh (H) cation exchange resin) was added, filtered, and concentrated in vacuo to give 1e (3.0 mg, quantitative).

[0131] 1 H-NMR (500 MHz, DMSO) δ 6.82 (d, J = 3.4 Hz, 1H), 6.81 (d, J = 3.4,Hz, 1H), 6.65 (dd, J = 8.6, 2.9 Hz, 1H), 6.58 (dd, J = 2.3, 8.0 Hz, 1H), 6.55 (d, J = 2.3 Hz, 1H), 6.52 (d, J = 2.3 Hz, 1H), 5.36 (br, 1H), 5.18 (br, 1H), 5.11 (br, 1H), 4.82 (d, J = 8.0 Hz, 1H), 4. 60 (m, 1H), 3.70 (s, 3H), 3.65 (m, 1H), 3.42 (m, 1H), 3.27 (m, 1H), 3.24 (m, 1H), 3.13 (m, 2H)

[0132] (Solubility test) The synthesized compounds 1a-1d and dibenzocyclooctadiyne (unsubstituted CODY) were dissolved in pure water and the absorbance at 350 nm was measured. From the absorbance, the solubilities of 1a-1d and dibenzocyclooctadiyne were calculated to be 6.8 mM, 3.7 mM, 7.4 mM, 2.0 mM, and 0.002 mM, respectively. Dibenzocyclooctadiyne was obtained from Tokyo Chemical Industry Co., Ltd. (product code: T3241).

[0133] PC-9 cells (a lung cancer-derived suspension cell line) used in the following examples were cultured in RPMI (containing 10% FBS, 1% glutamine, 0.75 mg / L sodium bicarbonate, and 5000 units / L penicillin / streptomycin) at 37°C and 5% CO2. PC-9 cells were cultured at a concentration of 1 × 10 5 The cells were cultured until the total number of cells reached 10 ...

[0134] The inverted microscope systems used in the following examples were the A1R MP (Nikon) and ECLIPS Ts2 (Nikon). 13 mm diameter and 12 mm diameter circular cover glasses were purchased from Matsunami Glass Industry. A Min-Eximer (Ushio Inc.) UV irradiator was used.

[0135] [Example 5] (Hydrophilic glass surface) Method A1: A 12 mm diameter circular cover glass (Iwaki) was placed in a beaker, immersed in acetone, and treated in an ultrasonic cleaner for 3 minutes. Ethanol, methanol, and MilliQ water were also treated in the same manner. The glass was then immersed in MilliQ water: 28 v / v % NH3 aq.: 30 v / v % HO2 aq. = 5:1:1, heated to 60°C, and held for 30 minutes. It was then thoroughly washed with MilliQ water. Method A2: A 13 mm diameter circular cover glass was irradiated with UV (172 nm) in air for 15 minutes.

[0136] (Azide modification of glass surface) Method B1: Hydrophilic glass plates were ultrasonically cleaned in methanol, purged with toluene, and then treated with 3-aminopropyltriethoxysilane (APTES) (1.0 v / v% in toluene, 10 mL) for 15 minutes. Then, ultrasonically cleaned with methanol and MilliQ water. Furthermore, the plates were incubated with 200 μL of sulfo-SANPAH (sulfosuccinimidyl 6-(4'-azido-2'-nitrophenylamino)hexanoate) (2.0 mg / mL in 20 mM HEPES buffer, pH 8.5) per glass plate in the dark for 30 minutes, followed by rinsing with RPMI (-) FBS.

[0137] Method B2: A hydrophilically treated glass plate was treated with 1-azido-11-(triethoxysilyl)undecane solution (0.50 v / v% toluene solution, 7 mL) on a 7 cm diameter Teflon dish for 15 minutes. The glass plate was washed with toluene and methanol in that order and dried under reduced pressure for 2 hours.

[0138] (Bonding of 1a, 1b, 1c or 1d to a glass surface) Method C: Azide-modified glass plates prepared by Method B were treated with 500 μL of a 50 μM solution of 1a, 1b, 1c, or 1d in water in a 24-well plate for 5 minutes, then washed three times with 500 μL of MilliQ water and used immediately in the next experiment.

[0139] (Binding (adhesion) of cells to glass surfaces and quantification of adhesion) 5.0×10 4 Cells at 1.0 × 10 cells / mL were cultured in 10 mL of medium containing 100 μM Ac4ManNAz and 0.1% DMSO in a 10 cm diameter circular dish. After 24 hours, the cells were washed with RPMI(-)FBS (10 mL) and then resuspended in RPMI(-)FBS at a cell concentration of 1.0 × 10 cells / mL. 5 A cell suspension of 100 cells / mL was prepared.

[0140] 5 μL of 1a, 1b, 1c, 1d, or unsubstituted CODY (Dibenzocyclooctadiyne: 5,6,11,12-Tetradehydrodibenzo[a,e]cyclooctene) (10 mM aq.) was added to 1 mL of this cell suspension (final concentration: 50 μM) and incubated for 5 min. The medium was removed, and the cells were suspended in 500 μL of RPMI(-)FBS. They were then plated on glass plates modified with azide by methods A1 and B1 and analyzed (see Figure 2B, C). Similarly, glass plates modified with azide by methods A1 and B2 were plated and analyzed (see Figure 3). Negative controls were treated in the same manner except that 1a-1d were not included.

[0141] (Cell adhesion to glass surfaces, changes in adhesion over time, and cell viability) 5.0×10 4Cells at 1.0 × 10 cells / mL were cultured in 10 mL of medium containing 100 μM Ac4ManNAz and 0.1% DMSO in a 10 cm diameter circular dish. After 24 hours, the cells were washed with RPMI(-)FBS (10 mL) and then resuspended in RPMI(-)FBS at a cell concentration of 1.0 × 10 cells / mL. 5 A cell suspension of 100 cells / mL was prepared.

[0142] 500 μL of the cell suspension was added to glass plates with 1a, 1b, 1c, 1d, or unsubstituted CODY conjugated, which were prepared by applying method C to azide-modified glass plates prepared by methods A2 and B2. After 15 minutes of incubation, the medium was removed and replaced with 500 μL of RPMI containing FBS. Images were taken immediately and 6 hours later. Viability was determined by adding trypan blue solution (0.4% in PBS(-)) or 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) solution (5 mg / mL in PBS(-)) to the medium.

[0143] Figure 2A shows a schematic diagram of cell attachment (adhesion) to a glass surface. Figure 2B shows micrographs of 1b-conjugated cells seeded on azide-modified glass plates by methods A1 and B1, analyzed by reflected light interference microscopy, along with the analyzed adhesion area. The black contrast areas in Figure 2B indicate the adhesion site between the cell and the substrate. Because the interference intensity can be converted to the distance h between the cell and the substrate, we defined the strong adhesion contact area (h < 50 nm) and the weak adhesion contact area (h < 100 nm) based on the height at which biochemical adhesion occurs (h ~ 40 nm). The addition of 1b significantly increased the strong adhesion area compared to the weak adhesion area, indicating that 1b strongly adhered to the substrate. Figure 2C shows micrographs of 1b-conjugated cells seeded on azide-modified glass plates by methods A1 and B1, analyzed by reflected light interference microscopy, over time. The white arrows in Figure 2C indicate the actual formation of a black contrast region, indicating cytoplasmic extension. Figure 3 shows the time course of cells bound to 1a, 1c, or 1d seeded on azide-modified glass plates (methods A1 and B2) analyzed with a reflected light interference microscope (right) and the analyzed area of ​​strong adhesion (distance from the substrate h ≦40 nm) (left) (Figure 3). In Figure 3, t indicates the incubation time (min).

[0144] Figure 4A shows a schematic diagram of cell binding (adhesion) to a glass surface. Figure 4B shows the time course of cells adhered to glass using 1a-1d. The negative control in Figure 4B shows the experimental results of a similar treatment except that 1a-1d was not used. Figure 4B shows photographs taken immediately after cell seeding and 6 hours later. For 1a, viability was assessed using MTT and trypan blue 6 hours later.

[0145] [Example 6] (Cell and cell-cell bonds (bridges)) 5.0×10 4Cells were cultured in a 10-cm diameter circular dish at 100 cells / mL in 10 mL of medium containing 100 μM Ac4ManNAz and 0.1% DMSO. After 24 hours, the cells were washed with RPMI(-)FBS (10 mL) and then cultured at a cell concentration of 2.0 × 10 5 A cell suspension of 100 cells / mL (cell A suspension) was prepared.

[0146] To 500 μL of cell A suspension, 500 μL of 1a solution (100 μM in RPMI(-)FBS, 500 μL; final concentration 50 μM) was added, incubated for 5 minutes, washed with RPMI(-)FBS, centrifuged, and the supernatant discarded to obtain a pellet of cell B. The same procedure was performed as for the control, except that 1a was not added.

[0147] The cell B pellet was mixed with 500 μL of the cell A suspension and centrifuged at 1500 G for 30 minutes. The formed pellet was pipetted, and the cell aggregates that remained aggregated were photographed under a microscope.

[0148] In Example 6, cell A is a cell into which azide (-N3) has been introduced, and cell B is a cell into which azide has been introduced and then 1a has been bound via the azide.

[0149] Figure 5A shows a schematic diagram of cell-cell binding (crosslinking), and Figure 5B shows a micrograph of the bound (adhered) cell cluster.

[0150] [Example 7] (Cell adhesion to AFM cantilever) An AFM cantilever (OMCL-TR400-PSA, Olympus) was irradiated with UV ozone cleaner (ProCleaner, Bioforce) for 15 minutes, immersed in APTES (3-aminopropyltriethoxysilane) (200 μL, 1.0 v / v% in toluene), washed with methanol, and dried.

[0151] The cantilever was further treated with the following solution (50 μL, 2.0 mg / mL in 20 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) buffer, pH 8.5) for 30 minutes, and then washed with RPMI(-)FBS.

[0152] [ka]

[0153] 5.0×10 4 Cells at 100 cells / mL were cultured in 10 mL of medium containing 100 μM Ac4ManNAz and 0.1% DMSO in a 10 cm diameter circular dish. After 24 hours, the cells were washed with 10 mL of RPMI(-)FBS and then suspended in 10 mL of RPMI(-)FBS. Meanwhile, the cantilever was incubated with 1b (50 μL, 50 μM in RPMI(-)FBS) for 15 minutes and then washed with RPMI(-)FBS. The cantilever surface was then contacted with the cells for 5 minutes using an atomic force microscope (AFM, NanoWizard, JPK).

[0154] To confirm that cells had adhered to the cantilever surface, we observed the transmission image of the cells when the cantilever was moved away from the substrate surface (see Figure 6b). Next, we contacted the cantilever with the cells attached to it with glass modified with 1b using methods A1, B1, and C described in Example 5, and then measured the force acting on the cantilever when it was peeled off, obtaining a force-distance curve (see Figure 6c).

[0155] Figure 6A shows a schematic diagram of cantilever crosslinking to cells using 1b. Figure 6A(1): When an azide-modified cantilever reacted with 1b was contacted with cells previously treated with Ac4ManNAz for 24 hours, the cells and the cantilever immediately adhered to each other. (2): When the cantilever with azide-modified cells was contacted with glass modified with 1b by method C, the cells immediately adhered to the glass. (3) and (4): The attached cells were lifted from the glass by pulling up the cantilever. Figure 6B is a photograph showing the state of cells binding to the cantilever in (1). Figure 6C shows the distance between the cell and the cantilever (unit: μm) on the horizontal axis and the magnitude of the cantilever deflection (i.e., a measure of the force acting on the cantilever) (unit: au) on the vertical axis. The cantilever with the azide-modified cell was approached to the glass modified with 1b along the dotted line (Approach) (Figure 6C(2)), and the cantilever was pulled up along the solid line (Retract) (Figure 6C(3) and (4)). Because the binding between the cell and the substrate was strong, the deflection of the cantilever during pulling up exceeded the detection limit.

[0156] [Reference example 1] 5.0×10 4 Cells (100 μM / mL) were cultured in 10 mL of medium containing 100 μM Ac4ManNAz and 0.1% DMSO in a 10 cm diameter circular dish. After 24 hours, the cells were washed with RPMI(-)FBS (10 mL).

[0157] 1.0 × 10 5 Cells were treated with 50 μM of 1a, 1b, 1c, 1d, or unsubstituted CODY (Dibenzocyclooctadiyne: 5,6,11,12-Tetradehydrodibenzo[a,e]cyclooctene) in 500 μL of RPMI(-)FBS for 5 minutes, and then washed with 500 μL of RPMI(-)FBS.

[0158] Next, the cells were treated with FITC-PEG3-N3 solution (10 μM in RPMI(-)FBS, 0.1% DMSO, 500 μL) for 5 minutes, then washed with RPMI(-)FBS (1 mL) and PBS(-) (1 mL). The cells were fixed with paraformaldehyde solution (4% in PBS(-)) for 30 minutes, washed with PBS(-) and MilliQ water (500 μL each), and suspended in MilliQ water (25 μL). The entire volume was placed on a glass slide and air-dried for 1 hour. 10 μL of mounting medium (Fluorescence Mounting Medium, DAKO) was added, and the cells were sealed with a cover glass.

[0159] The fluorescence intensity of FITC-derived fluorescence was measured using a fluorescence microscope. Because unsubstituted CODY has low solubility in water, 0.1% DMSO was added during the 5-minute treatment with cells.

[0160] The experimental scheme is shown in Figure 7A. This figure illustrates the following steps: (i) introduction of azide groups onto the cell surface by incubation with Ac4ManNAz (100 μM, 24 hours), (ii) binding of cells with CODY derivatives (1a, 1b, 1c, 1d, or unsubstituted CODY) (50 μM, 5 min), and (iii) staining of the cell surface with fluorescent dyes via two strain-promoted azide-alkyne cycloaddition reactions with FITC-PEG3-N3 (50 μM, 5 min).

[0161] The FITC-derived fluorescence measurement results from the above experiment are shown in Figures 7B and 7D. Figure 7B shows the results for 1a, 1b, 1c, and 1d, and Figure 7D shows the results for 1a and unsubstituted CODY. Quantitative results from Figure 7B are shown in Figure 7C, and quantitative results from Figure 7D are shown in Figure 7E.

[0162] [Example 8] (Binding (adhesion) of cells to glass surfaces and quantification of adhesion) 5.0×10 4Cells at 100 cells / mL were cultured in 5 mL of medium containing 100 μM Ac4ManNAz and 0.1% DMSO in a 6 cm diameter circular dish. After 24 hours, 1 mL of the culture medium was dispensed, and the cells were washed with 1 mL of RPMI (-) FBS, and the medium was removed.

[0163] 500 μL of 50 μM RPMI (-) FBS solution of 1a, 1b, 1c, or unsubstituted CODY (dibenzocyclooctadiyne) was added to the cells and incubated for 5 minutes. The medium was removed, the cells were washed with 500 μL of RPMI (-) FBS, and then suspended in 300 μL of RPMI (-) FBS. Azide-modified glass plates prepared using Method B2 were placed in a 24-well plate and 500 μL of RPMI (-) FBS was added. 300 μL of the compound-treated cell suspension was added to each well. After 30 minutes, the medium was removed, 1 mL of RPMI 10% FBS was added, and the plate was incubated at 37°C in 5% CO2 for 6 hours. The medium was then changed three times to remove floating cells. Adherent cells were collected with a cell scraper, stained with trypan blue, and the number of viable cells was counted. The cell engraftment rate (engrafted cells / seeded cells) (%) was calculated by dividing the number of cells seeded (Figure 8).

[0164] As a negative control, the cell engraftment rate of 1a was calculated by carrying out the same procedure except that Ac4ManNAz was not used.

[0165] [Example 9] (Binding (adhesion) of cells to glass surfaces and quantification of adhesion) 5.0×10 4 Cells at 100 cells / mL were cultured in 5 mL of medium containing 100 μM Ac4ManNAz and 0.1% DMSO in a 6 cm diameter circular dish. After 24 hours, 1 mL of the culture medium was dispensed, and the cells were washed with 1 mL of RPMI (-) FBS, and the medium was removed.

[0166] 500 μL of 50 μM RPMI (-) FBS solution (1a) was added to the cells and incubated for 5 minutes. The medium was removed, the cells were washed with 500 μL of RPMI (-) FBS, and then suspended in 300 μL of RPMI (-) FBS. Azide-modified glass plates prepared using Method B2 were placed in a 24-well plate, and 500 μL of RPMI (-) FBS was added. 300 μL of the compound-treated cell suspension was added to each well. After 30 minutes, the medium was removed, 1 mL of RPMI 10% FBS was added, and the plates were incubated at 37°C in 5% CO2 for 6 hours. The medium was then changed three times to remove floating cells. Total RNA was extracted from cells 24 hours after adhesion. As a control, RNA from cells treated with Ac4ManNAz but not seeded on glass plates was used (Figure 9A). The 241 genes whose gene expression levels were increased by 1.5-fold or more in the cells after 24 hours compared to the control were analyzed using DAVID. 1), 2) GO enrichment analysis was performed using the molecular function category. The results are shown in Figure 9B in descending order of -log10(P-value). 1) Huang DW, Sherman BT, Lempicki RA. Systematic and integrative analysis of large gene lists using DAVID Bioinformatics Resources. Nature Protoc. 2009;4(1):44-57. 2) Huang DW, Sherman BT, Lempicki RA. Bioinformatics enrichment tools: paths toward the comprehensive functional analysis of large gene lists. Nucleic Acids Res. 2009;37(1):1-13.

[0167] The examples demonstrate that various materials can be used to form composites in which materials and cells are bound. These results could not have been predicted from the prior art, which used unsubstituted CODY. In Figures 2 to 7, the polar functional groups of dibenzocyclooctadiynes having polar functional groups are sometimes omitted, and sometimes written as -OR.

[0168] All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety.

[0169] The upper and / or lower limit values ​​of the numerical ranges described herein can be arbitrarily combined to define a preferred range. For example, the upper and lower limit values ​​of the numerical ranges can be arbitrarily combined to define a preferred range, the upper limit values ​​of the numerical ranges can be arbitrarily combined to define a preferred range, and the lower limit values ​​of the numerical ranges can be arbitrarily combined to define a preferred range.

[0170] Throughout this specification, singular terms should be understood to include the plural concept unless otherwise stated. Thus, singular articles (e.g., "a," "an," "the," etc. in English) should be understood to include the plural concept unless otherwise stated.

[0171] Although the present embodiment has been described in detail above, the specific configuration is not limited to this embodiment, and even if there are design changes within the scope that do not deviate from the gist of this disclosure, they are included in this disclosure.

Claims

[Claim 1] At least one compound selected from the following formulae 1b to 1j: 【Chemistry 1】 【Chemistry 2】 (In formula 1g, RGD is a peptide chain of three residues consisting of arginine-glycine-aspartic acid, In formula 1j, each Y is independently a group selected from a group having a polar functional group and an alkyl group, the polar functional group is at least one selected from an ionic functional group and a nonionic polar functional group, the ionic functional group is selected from functional groups having a cation selected from —NH 3 + , —NH 2 Z + , —NHZ 2 + and —NZ 3 + and a counter anion selected from a halide ion, functional groups having an anion selected from —SO 3 − , —O − , —COO − and —PO 3 − and a counter cation selected from an alkali metal ion, and functional groups having the above cation and the above anion; the nonionic polar functional group is selected from —NH 2 , —NHZ, —NZ 2 , —SO 3 H, —OH, —COOH, —NH—CONH 2 , —NH—CONHZ, —NH—CONZ 2 , —NZ—CONH 2 , —NZ—CONHZ, —NZ—CONZ 2 , —CO—, —COO—, and —O—; Z is a hydrocarbon group having 1 to 18 carbon atoms.

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