WO2022181695A1 - 複合体、複合体の製造方法及び化合物 - Google Patents

複合体、複合体の製造方法及び化合物 Download PDF

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WO2022181695A1
WO2022181695A1 PCT/JP2022/007624 JP2022007624W WO2022181695A1 WO 2022181695 A1 WO2022181695 A1 WO 2022181695A1 JP 2022007624 W JP2022007624 W JP 2022007624W WO 2022181695 A1 WO2022181695 A1 WO 2022181695A1
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group
polar functional
functional group
dibenzocyclooctadiyne
azide
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French (fr)
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Inventor
正行 寺
浩平 北川
萌華 吉永
賢寿 松崎
菜穂 大熊
洋史 吉川
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Tokyo University of Agriculture and Technology NUC
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Tokyo University of Agriculture and Technology NUC
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    • C07C217/00—Compounds containing amino and etherified hydroxy groups bound to the same carbon skeleton
    • C07C217/02—Compounds containing amino and etherified hydroxy groups bound to the same carbon skeleton having etherified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton
    • C07C217/04—Compounds containing amino and etherified hydroxy groups bound to the same carbon skeleton having etherified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated
    • C07C217/06—Compounds containing amino and etherified hydroxy groups bound to the same carbon skeleton having etherified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one etherified hydroxy group and one amino group bound to the carbon skeleton, which is not further substituted
    • C07C217/14—Compounds containing amino and etherified hydroxy groups bound to the same carbon skeleton having etherified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one etherified hydroxy group and one amino group bound to the carbon skeleton, which is not further substituted the oxygen atom of the etherified hydroxy group being further bound to a carbon atom of a six-membered aromatic ring
    • C07C217/24—Compounds containing amino and etherified hydroxy groups bound to the same carbon skeleton having etherified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one etherified hydroxy group and one amino group bound to the carbon skeleton, which is not further substituted the oxygen atom of the etherified hydroxy group being further bound to a carbon atom of a six-membered aromatic ring the six-membered aromatic ring being part of a condensed ring system containing rings other than six-membered aromatic rings
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    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
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    • C07C275/04—Derivatives of urea, i.e. compounds containing any of the groups, the nitrogen atoms not being part of nitro or nitroso groups having nitrogen atoms of urea groups bound to acyclic carbon atoms
    • C07C275/06—Derivatives of urea, i.e. compounds containing any of the groups, the nitrogen atoms not being part of nitro or nitroso groups having nitrogen atoms of urea groups bound to acyclic carbon atoms of an acyclic and saturated carbon skeleton
    • C07C275/10—Derivatives of urea, i.e. compounds containing any of the groups, the nitrogen atoms not being part of nitro or nitroso groups having nitrogen atoms of urea groups bound to acyclic carbon atoms of an acyclic and saturated carbon skeleton being further substituted by singly-bound oxygen atoms
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    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C279/00—Derivatives of guanidine, i.e. compounds containing the group, the singly-bound nitrogen atoms not being part of nitro or nitroso groups
    • C07C279/04—Derivatives of guanidine, i.e. compounds containing the group, the singly-bound nitrogen atoms not being part of nitro or nitroso groups having nitrogen atoms of guanidine groups bound to acyclic carbon atoms of a carbon skeleton
    • C07C279/14—Derivatives of guanidine, i.e. compounds containing the group, the singly-bound nitrogen atoms not being part of nitro or nitroso groups having nitrogen atoms of guanidine groups bound to acyclic carbon atoms of a carbon skeleton being further substituted by carboxyl groups
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C309/00—Sulfonic acids; Halides, esters, or anhydrides thereof
    • C07C309/01—Sulfonic acids
    • C07C309/02—Sulfonic acids having sulfo groups bound to acyclic carbon atoms
    • C07C309/03—Sulfonic acids having sulfo groups bound to acyclic carbon atoms of an acyclic saturated carbon skeleton
    • C07C309/07—Sulfonic acids having sulfo groups bound to acyclic carbon atoms of an acyclic saturated carbon skeleton containing oxygen atoms bound to the carbon skeleton
    • C07C309/09—Sulfonic acids having sulfo groups bound to acyclic carbon atoms of an acyclic saturated carbon skeleton containing oxygen atoms bound to the carbon skeleton containing etherified hydroxy groups bound to the carbon skeleton
    • C07C309/11—Sulfonic acids having sulfo groups bound to acyclic carbon atoms of an acyclic saturated carbon skeleton containing oxygen atoms bound to the carbon skeleton containing etherified hydroxy groups bound to the carbon skeleton with the oxygen atom of at least one of the etherified hydroxy groups further bound to a carbon atom of a six-membered aromatic ring
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    • C07—ORGANIC CHEMISTRY
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    • C07C309/00—Sulfonic acids; Halides, esters, or anhydrides thereof
    • C07C309/01—Sulfonic acids
    • C07C309/02—Sulfonic acids having sulfo groups bound to acyclic carbon atoms
    • C07C309/03—Sulfonic acids having sulfo groups bound to acyclic carbon atoms of an acyclic saturated carbon skeleton
    • C07C309/13—Sulfonic acids having sulfo groups bound to acyclic carbon atoms of an acyclic saturated carbon skeleton containing nitrogen atoms, not being part of nitro or nitroso groups, bound to the carbon skeleton
    • C07C309/14—Sulfonic acids having sulfo groups bound to acyclic carbon atoms of an acyclic saturated carbon skeleton containing nitrogen atoms, not being part of nitro or nitroso groups, bound to the carbon skeleton containing amino groups bound to the carbon skeleton
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    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/06—Phosphorus compounds without P—C bonds
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    • C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
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    • C12N1/00—Microorganisms; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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    • C12N5/06—Animal cells or tissues; Human cells or tissues

Definitions

  • the present disclosure relates to a complex, a method for producing the complex, and a compound.
  • dibenzocyclooctadiyne (5,6,11,12-Tetradehydrodibenzo[a,e]cyclooctene) is a strained cyclic molecule with two carbon-carbon triple bond alkynes in the molecule. It is known that the strain-promoted azide-alkyne cycloaddition reaction (SPAAC reaction) can occur twice.
  • SPAAC reaction strain-promoted azide-alkyne cycloaddition reaction
  • Patent Document 1 discloses that cells can be bound to a fluorescent dye by using dibenzocyclooctadiyne. According to the studies of the present inventors, the invention disclosed in Patent Document 1 They have found that in some cases the reaction rate is not sufficient when binding to cells or binding to a fluorescent dye, and in some cases the amount of fluorescent dye that binds to cells is insufficient.
  • 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 or the like can be improved. It was found that the amount of fluorescent dye that
  • the present inventors further developed the above findings, and found that if the solubility of dibenzocyclooctadiyne can be increased, the reactivity can be improved, and materials other than fluorescent dyes, for example, generally can bind to cells. I thought that it would be possible to bind even difficult materials with large molecular weights and cells.
  • the present disclosure provides complexes of cells and materials, methods for producing complexes of cells and materials, and novel compounds that can be used to obtain the complexes, which have been difficult to provide with conventional techniques. intended to provide
  • the present inventors have found that by using dibenzocyclooctadiyne having a polar functional group, it is possible to obtain a cell-material complex. In addition, the present inventors also discovered a new compound in the process of studying dibenzocyclooctadiyne having a polar functional group.
  • the material is at least one material selected from cells having an azide group on the cell surface and an azide-modified inorganic material.
  • the inorganic material is at least one inorganic material selected from culture substrates, glass substrates, AFM cantilevers, and metals.
  • 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
  • n'+m' is an integer of 1 to 4.
  • 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.
  • the complex 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).
  • each R is independently a group selected from a group having a polar functional group and an alkyl group.
  • the complex according to any one of (1) to (3), wherein the dibenzocyclooctadiyne having a polar functional group is at least one compound selected from formulas 1a to 1j below.
  • RGD is a three-residue peptide chain consisting of arginine-glycine-aspartic acid, and in formula 1j, each Y is a group independently selected from a group having a polar functional group and an alkyl group.
  • acetylated azidomannosamine Ac 4 ManNAz
  • cells having an azide group on the cell surface convert sialic acid at the end of the sugar chain on the cell surface through the sialic acid metabolic pathway of the cell.
  • the conjugate according to any one of (1) to (8), which is a cell having an azide group introduced at the site.
  • (10) a step of combining cells having an azide group on the cell surface with dibenzocyclooctadiyne having a polar functional group to obtain a conjugate (A) of dibenzocyclooctadiyne having a polar functional group and cells; , The conjugate (A) is combined with a material having an azide group (excluding a fluorescent dye having an azide group) to obtain a complex in which the cell and the material are bonded by dibenzocyclooctadiyne having a polar functional group.
  • a method for producing a composite comprising steps.
  • 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
  • n'+m' is an integer of 1 to 4.
  • 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 complex 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).
  • 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.
  • the method for producing a conjugate 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).
  • each R is independently a group selected from a group having a polar functional group and an alkyl group.
  • RGD is a three-residue peptide chain consisting of arginine-glycine-aspartic acid
  • each Y is a group independently selected from a group having a polar functional group and an alkyl group.
  • RGD is a three-residue peptide chain consisting of arginine-glycine-aspartic acid
  • each Y is a group independently selected from a group having a polar functional group and an alkyl group.
  • a cell-material composite a method for producing a cell-material composite, and a novel compound that can be used to obtain the composite are provided.
  • FIG. 1 shows the process by which an azide group is introduced to the sialic acid site at the end of a sugar chain on the cell surface through the sialic acid metabolic pathway of cells by adding acetylated azidomannosamine (Ac 4 ManNAz) to the cell culture medium.
  • FIG. 2A is a schematic representation of cell attachment (adhesion) to a glass surface (Example 5).
  • FIG. 2B shows micrographs of 1b-bound cells seeded on azide-modified glass plates by methods A1 and B1, analyzed by reflection interference microscopy, and analysis results of adhesion regions to substrates.
  • FIG. 1 is a schematic representation of cell attachment (adhesion) to a glass surface (Example 5).
  • FIG. 2B shows micrographs of 1b-bound cells seeded on azide-modified glass plates by methods A1 and B1, analyzed by reflection interference microscopy, and analysis results of adhesion regions to substrates.
  • FIG. 2C is a photomicrograph obtained by analyzing changes over time in a glass plate modified with azido by methods A1 and B1 with 1b-bound cells seeded thereon, using a reflection interference microscope.
  • Fig. 3 is a photomicrograph obtained by analyzing changes over time in a glass plate modified with azido by methods A1 and B2 in Example 5 with cells bound with 1a, 1c or 1d, and analyzed with a reflection interference microscope (Fig. 3, right). and the analyzed area of strong adhesion (distance h ⁇ 40 nm from the substrate) (Fig. 3, left).
  • FIG. 4A is a schematic representation of cell attachment (adhesion) to a glass surface (Example 5).
  • FIG. 4B is a photomicrograph showing time course of cells adhered to glass using 1a-1d in Example 5.
  • FIG. FIG. 5A is a schematic diagram of cells and intercellular binding (crosslinking) (Example 6).
  • FIG. 5B shows a photomicrograph of cell clusters bound (adhered) in 1a.
  • Control in FIG. 5B is a photomicrograph of cells not treated with 1a.
  • FIG. 6A shows a schematic of cantilever cross-linking to cells using 1b (Example 7).
  • FIG. 6B is a photograph showing a state in which cells are bound to the cantilever in (1) of FIG. 6A.
  • FIG. 7A is a schematic diagram showing the scheme of Reference Example 1.
  • FIG. 7B shows measurement results of FITC-derived fluorescence of Example 7 for 1a, 1b, 1c, and 1d.
  • FIG. 7C shows a quantification of the results of FIG. 7B.
  • FIG. 7D is a measurement result of FITC-derived fluorescence of Example 7 for 1a, unsubstituted CODY. A quantification of the results of FIG. 7D is shown in FIG. 7E. 8 shows the cell engraftment rate calculated in Example 8.
  • FIG. 9A is a schematic diagram showing the scheme of Example 9.
  • FIG. 9B shows the results of the molecular function category of Example 9.
  • a cell having an azide group on the cell surface and a material having an azide group are bound by dibenzocyclooctadiyne having a polar functional group.
  • the complex according to the present embodiment is dibenzocyclooctadiyne (5,6,11,12-Tetradehydrodibenzo[a,e]cyclooctene) (hereinafter also referred to as unsubstituted CODY), but has a polar functional group. Since the cells and the material are bound by dibenzocyclooctadiyne, the cells and the material can be rapidly bound together.
  • dibenzocyclooctadiyne having a polar functional group is superior in solubility to unsubstituted CODY.
  • dibenzocyclooctadiyne which has many polar functional groups, can bind to the azide groups on the cell surface compared to binding unsubstituted CODY, and as a result, cells and materials It was speculated that they could be strongly combined with
  • the cell having an azide group on the cell surface may have an azide group on the cell surface.
  • acetylated azidomannosamine Ac 4 ManNAz
  • the sialic acid site at the end of the sugar chain on the cell surface is treated via the sialic acid metabolic pathway of the cell.
  • Cells into which an azide group has been introduced are preferred.
  • the cell may be a cell in which an azide group has been introduced to the cell surface through another metabolic pathway.
  • an azide group may be introduced on a surface other than the cell surface.
  • the structure of Ac4ManNAz is shown below.
  • FIG. 1 shows a conceptual diagram of the process of introducing an azide group to the sialic acid site of the sugar chain terminal on the cell surface through the sialic acid metabolic pathway of the cell by adding Ac 4 ManNAz to the cell culture medium.
  • Cultured cells are usually used as cells into which an azide group is introduced.
  • Cells into which an azide group is introduced may be human-derived cells or non-human-derived cells.
  • Non-human derived cells include cells derived from mammals (excluding humans), and cells derived from non-mammalian organisms such as birds, fish, insects, plants, algae, and fungi. Mammals (other than humans) include, for example, mice, rats, horses, sheep, pigs, goats, and cows.
  • Cells are not particularly limited, but for example 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 , KTZ6, 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-JCK, 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
  • the material having an azide group (excluding a fluorescent dye 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 in a portion of the surface of the material. may be present on the entire surface of the material.
  • Examples of materials include cells having an azide group on the cell surface, azide-modified inorganic materials, and organic materials having an azide group, and are selected from cells having an azide group on the cell surface and azide-modified inorganic materials.
  • the inorganic material is preferably at least one inorganic material selected from culture substrates, glass substrates, AFM cantilevers, and metals.
  • the materials used in this embodiment may be of one type or two or more types. When two or more materials are used, for example, once a composite is formed, another material may be further bonded to the composite.
  • an azide group into a material.
  • the material when the material is cells having an azide group on the cell surface, the cells described in the above section (Cells) can be used.
  • the azide group can be introduced, for example, according to conventionally known organic synthesis methods.
  • an organic molecule or an organic-inorganic hybrid molecule is bonded to the surface by, for example, the method described in the Examples, and a compound having an azide group as the organic molecule or organic-inorganic hybrid molecule is prepared.
  • the organic molecule or the organic-inorganic hybrid molecule is further denatured or modified to introduce an azide group.
  • a method of introducing a group can be mentioned.
  • dibenzocyclooctadiyne with polar functional group examples include molecules in which the hydrogen atom of the benzene ring of unsubstituted CODY is substituted with a polar functional group.
  • 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).
  • a compound represented by (III) is particularly preferable, and a compound represented by the following general formula (IV) is most preferable.
  • 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
  • n'+m' is an integer of 1 to 4.
  • each X is independently a group having a polar functional group, and each n is independently an integer of 1 to 4.
  • 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.
  • each R is independently a group selected from a group having a polar functional group and an alkyl group.
  • X is each independently a group having a polar functional group.
  • each R is independently a group selected from a group having a polar functional group and an alkyl group.
  • the polar functional group includes at least one polar functional group selected from ionic functional groups and nonionic polar functional groups.
  • the group having a polar functional group may have at least one polar functional group, and may have a plurality of polar functional groups. There is no particular upper limit to the number of polar functional groups.
  • X may have 20 or less polar functional groups per group having a polar functional group, for example, R may have 19 or less per group having a polar functional group. may have a polar functional group of
  • ionic functional groups include cations (eg, -NH 3 + , -NH 2 Z + , -NHZ 2 + , -NZ 3 + ) and counter anions (eg, halide ions (F - , Cl - , Br ⁇ , I ⁇ )), anions (eg —SO 3 ⁇ , —O ⁇ , —COO ⁇ , —PO 3 ⁇ ) and countercations (eg alkali metal ions (eg Li + , Na + , K + )), as well as functional groups with zwitterions (eg, functional groups with cations and anions as described above).
  • cations eg, -NH 3 + , -NH 2 Z + , -NHZ 2 + , -NZ 3 +
  • counter anions eg, halide ions (F - , Cl - , Br ⁇ , I ⁇ )
  • anions eg —
  • the group having a polar functional group When the group having a polar functional group has an ionic functional group, it may have one or a plurality of ionic functional groups. When it has a plurality of ionic functional groups, it may have the same kind of ionic functional groups or different kinds of ionic functional groups.
  • nonionic polar functional groups include -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- (carbonyl group), -COO- (ester group), and -O- (ether group).
  • the group having a polar functional group has a nonionic polar functional group, it may have one or a plurality of nonionic polar functional groups. When it has a plurality of nonionic polar functional groups, it may have the same type of nonionic polar functional group, or may have a different type of nonionic polar functional group.
  • Z is a hydrocarbon group having 1 to 18 carbon atoms, preferably an alkyl group having 1 to 6 carbon atoms. When there are a plurality of Z's, they may be the same group or different groups.
  • the structure of the group having a polar functional group other than the polar functional group is not particularly limited, but it 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.
  • X preferably has a molecular weight of 16-1100, more preferably 16-200.
  • the molecular weight is preferably 15-584, more preferably 15-184.
  • each R is a group independently selected from a group having a polar functional group and an alkyl group, but at least one of R in one molecule may be a group having a polar functional group. preferable.
  • R is an alkyl group, it includes an alkyl group having 1 to 18 carbon atoms, preferably an alkyl group having 1 to 6 carbon atoms.
  • a preferred embodiment of the alkyl group is a methyl group.
  • X and R include peptide chains, sugar chains, and urea groups (eg, -NH-CONH 2 , -NH-CONHZ, -NH-CONZ 2 , -NZ-CONH 2 , -NZ-CONHZ, -NZ-CONZ 2 ). , a carbonyl group, an ester bond, and an ether group.
  • Peptide chains include, for example, peptide chains of 1 to 5 residues.
  • X is -OR in general formula (III). That is, it is one of preferred embodiments that the bonding site of X to the benzene ring is —O— (ether group).
  • each A independently represents a hydrocarbon group.
  • the hydrocarbon group is preferably a hydrocarbon group having 1 to 18 carbon atoms, more preferably an alkyl group having 1 to 6 carbon atoms.
  • n is each independently an integer of 1 to 4, preferably an integer of 1 to 3, more preferably 1 or 2, particularly preferably 1.
  • n' is an integer of 1 to 4, preferably an integer of 1 to 3, more preferably 1 or 2, and particularly preferably 1.
  • m is an integer of 0 to 3, preferably 0 or 1, more preferably 0.
  • m' is an integer of 0 to 3, preferably 0 or 1, more preferably 0.
  • 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.
  • 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.
  • the group having a polar functional group at least one selected from functional groups 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 as the polar functional group.
  • Having a functional group is one of preferred embodiments.
  • dibenzocyclooctadiyne having a polar functional group tends to be particularly excellent in solubility, which is preferable.
  • the cell surface tends to have anionic properties
  • having a cation and a counter anion as polar functional groups is preferable from the viewpoint of reactivity between the azide group on the cell surface and dibenzocyclooctadiyne having a polar functional group. more preferred.
  • dibenzocyclooctadiyne having a polar functional group include at least one compound selected from the following formulas 1a to 1j, and these compounds are particularly preferred. As at least one compound selected from formulas 1a to 1j, 1a is preferred.
  • the compounds of formulas 1b to 1j are novel compounds. In the present disclosure, the "compound of 1a” is also simply referred to as “1a”, the “compound of 1b” is also simply referred to as “1b”, the “compound of 1c” is also simply referred to as “1c”, and the “compound of 1d” is also referred to simply as "1c”.
  • RGD is a three-residue peptide chain consisting of arginine-glycine-aspartic acid.
  • each Y is a group independently selected from a group having a polar functional group and an alkyl group.
  • Y When Y is a group having a polar functional group, it includes the groups exemplified for X above. However, the upper limit of the molecular weight of Y is lower than the upper limit of X by 28 molecular weights.
  • Y When Y is an alkyl group, it includes an alkyl group having 1 to 18 carbon atoms, preferably an alkyl group having 1 to 6 carbon atoms. A preferred embodiment of the alkyl group is a methyl group.
  • the method for synthesizing dibenzocyclooctadiyne having a polar functional group is not particularly limited.
  • a method of etherifying a hydroxy group by a conventionally known organic synthesis method or the like to convert the hydroxy group to —OR can be mentioned.
  • the hydroxy group is esterified by a conventionally known organic synthesis method or the like.
  • Still another method includes a method of synthesizing dibenzocyclooctadiyne having a carboxy group (--COOH) and then esterifying the carboxy group.
  • the complex according to the present embodiment is composed of a cell having an azide group on the cell surface and a material having an azide group (excluding a fluorescent dye having an azide group), dibenzocyclooctate having a polar functional group. It is a conjugate bound by a diyne.
  • Dibenzocyclooctadiyne having a polar functional group has two alkynes in the molecule, one of which undergoes a SPAAC reaction with the azide group of cells having an azide group on the cell surface, and the other of which is a material having an azide group.
  • SPAAC reaction with an azide group can yield a complex in which cells and materials are bound by dibenzocyclooctadiyne having a polar functional group.
  • the complex according to the present embodiment can easily bind cells (cells having an azide group on the cell surface), which are usually difficult to bind together, or cells and inorganic materials, which are usually difficult to bind. Therefore, it can be used for various researches that were difficult in the past.
  • the material is cells having an azide group on the cell surface
  • spheroids can be easily produced, and cell aggregates can be easily obtained even if the cells are difficult-to-adhere cells. Therefore, difficult-to-adhere cells can be cultured.
  • a material having a microchannel as the inorganic material and modifying a desired position of the microchannel with an azide group cells can be bound to the desired position.
  • a method of use in which cells are further layered on the cell sheet to form a multi-layered tissue is conceivable.
  • each R is independently a group selected from a group having a polar functional group and an alkyl group
  • Z 1 is a cell having an azide group on the cell surface
  • Z2 is the bound material derived from the material with an azide group.
  • Dibenzocyclooctadiyne having a polar functional group is the compound represented by the general formula (IV), and the two R in the general formula (IV) are different groups, R 1 and R 2 . Taking a certain case as an example, it can be represented by the following compound group (general formula (3)).
  • R 1 and R 2 have the same definitions as R in general formula (IV), are groups different from R 1 and R 2 , and Z 1 has an azide group on the cell surface. Bound cell derived from cells and Z2 is bound material derived from materials with azide groups.
  • the complex according to the present embodiment can be obtained by the above formulas (2-1) to (2-4) and formula ( As shown in 3), the structure of the complex may have a different structure, but unless otherwise specified in the present disclosure, it is represented by the above formulas (2-1) to (2-4)
  • the compound group represented by the structure or formula (3) shall not be particularly distinguished.
  • the method for manufacturing a composite according to this embodiment is a method for manufacturing the above-described composite according to this embodiment.
  • the method for manufacturing a composite according to this embodiment can be roughly divided into two aspects.
  • the method for producing a complex according to the first aspect comprises binding a cell having an azide group on the cell surface to dibenzocyclooctadiyne having a polar functional group, and binding dibenzocyclooctadiyne having a polar functional group to the cell.
  • a method for producing a conjugate, comprising the step of obtaining a conjugate bound by cyclooctadiyne.
  • the method for producing a composite according to the second aspect comprises combining a material having an azide group (excluding fluorescent dyes having an azide group) with dibenzocyclooctadiyne having a polar functional group, a step of obtaining a conjugate (B) of dibenzocyclooctadiyne and a material;
  • a method for producing a conjugate comprising the step of obtaining a conjugate bound by cyclooctadiyne.
  • the biggest difference between the first embodiment and the second embodiment is which of the cells and the material is bound first to the dibenzocyclooctadiyne having a polar functional group.
  • the conjugate is first bound to the cell to obtain the conjugate (A), and then bound to the material.
  • the material is first bound to obtain the conjugate (B), and then the cell is bound.
  • the reaction proceeds rapidly and it is possible to produce a complex.
  • the method for obtaining the conjugate (A) is not particularly limited as long as the cell having an azide group on the cell surface can be brought into contact with dibenzocyclooctadiyne having a polar functional group.
  • a method of obtaining the conjugate (A) includes preparing a cell suspension containing cells having an azide group, adding dibenzocyclooctadiyne having a polar functional group to the cell suspension, and incubating. 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.
  • the method for obtaining the conjugate (B) is not particularly limited as long as the material having an azide group and the dibenzocyclooctadiyne having a polar functional group can be brought into contact with each other.
  • a method of obtaining the conjugate (B) by contacting with a solution containing dibenzocyclooctadiyne having a functional group can be mentioned.
  • the method of obtaining the conjugate from the conjugate (A) is not particularly limited, but for example, a method of obtaining the conjugate by applying a suspension containing the conjugate (A) onto a material having an azide group. , a method of obtaining a composite by contacting and immersing a material having an azide group in a suspension containing the conjugate (A).
  • the method for obtaining the conjugate from the conjugate (B) is not particularly limited. and a method of obtaining a complex by contacting and immersing the conjugate (B) in a cell suspension containing cells having an azide group on the cell surface.
  • the method for producing a composite according to the present embodiment can usually be carried out at 4-40°C, more preferably at 15-37°C.
  • the examples described below were carried out at room temperature (r.t.) (20 to 30° C.) unless otherwise specified.
  • the method for producing a composite according to this embodiment may be performed in the atmosphere or in an inert gas (eg, nitrogen, argon) atmosphere. Moreover, it may be carried out under pressure or under reduced pressure. The examples below were carried out in the atmosphere unless otherwise specified.
  • an inert gas eg, nitrogen, argon
  • silica gel 60 (spherical, particle size 40-100 ⁇ m, Kanto Kagaku) was used in flash chromatography.
  • 1 H and 13 C NMR spectra were measured using JEOL JNM-AL 300, JNM-ECX 400 or JNM-ECA 500.
  • Mass spectra were measured using a JEOL JMS-T100LC mass spectrometer in ESI-MS mode with a methanol solvent.
  • 1,3-Propanesultone 500 ⁇ L was added to S6 (22 mg, 58.7 ⁇ mol) and stirred at 40° C. for 7 hours.
  • PC-9 cells lung cancer-derived suspension cell line
  • RPMI RPMI (10% FBS, 1 % glutamine, 0.75 mg/L sodium bicarbonate, 5000 units/L penicillin/ containing streptomycin).
  • PC-9 cells were cultured to 1 ⁇ 10 5 cells/mL and passaged every 3-4 days. In the following description, PC-9 cells are also simply referred to as cells.
  • Inverted microscope systems A1R MP (Nikon) and ECLIPS Ts2 (Nikon) were used for the fluorescence microscopes used in the following examples. 13 mm ⁇ and 12 mm ⁇ circular cover glasses were purchased from Matsunami Glass Industry. A Min-Eximer (Ushio Inc.) was used as the UV irradiator.
  • Method A2 A circular cover glass of 13 mm ⁇ was irradiated with UV (172 nm) in air for 15 minutes.
  • Method B1 A hydrophilically treated glass plate was ultrasonically washed with methanol, substituted with toluene, and then treated with 3-aminopropyltriethoxysilane (APTES) (1.0 v/v% in toluene, 10 mL) for 15 minutes. After that, ultrasonic cleaning was performed with methanol and MilliQ water.
  • APTES 3-aminopropyltriethoxysilane
  • sulfo-SANPAH sulfosuccinimidyl 6-(4'-azido-2'-nitrophenylamino) hexanoate
  • Method B2 A hydrophilically treated glass plate was treated with a 1-azido-11-(triethoxysilyl)undecane solution (0.50 v/v% toluene solution, 7 mL) on a 7 cm diameter Teflon (registered trademark) petri dish for 15 minutes. . The glass was washed with toluene and methanol in order and dried under reduced pressure for 2 hours.
  • Method C Azide-modified glass plates using Method B were treated with a solution containing 50 ⁇ M of 1a, 1b, 1c or 1d (50 ⁇ M in water, 500 ⁇ L) in a 24-well plate for 5 minutes. After that, it was washed three times with MilliQ water (500 ⁇ L) and immediately used for the next experiment.
  • 500 ⁇ L of the cell suspension was added to 1a, 1b, 1c, 1d prepared by performing Method C on the azide-modified glass plates by Methods A2 and B2, or to unsubstituted CODY-bound glass plates. After allowing to stand for 15 minutes, the medium was removed and replaced with 500 ⁇ L of RPMI containing FBS. For viability determination, 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(-)) was added to the medium.
  • trypan blue solution (0.4% in PBS(-)
  • MTT 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide
  • FIG. 2A shows a schematic diagram of cell binding (adhesion) to the glass surface.
  • FIG. 2B shows micrographs and analyzed adhesion areas of 1b-bound cells seeded on azide-modified glass plates by methods A1 and B1 analyzed by reflection interference microscopy. Areas showing black contrast in the brightness of the interference light in FIG. 2B indicate adhesion sites between the cells and the substrate. Since the intensity of the interference can be converted to the distance h between the cell and the substrate, the strong adhesion region Contact area (h ⁇ 50 nm) is based on the height at which biochemical adhesion occurs (h ⁇ 40 nm). and the weak adhesion region Contact area (h ⁇ 100 nm).
  • FIG. 2C shows micrographs of the change over time of azide-modified glass plate seeded with 1b-conjugated cells by methods A1 and B1, analyzed by reflection interference microscopy.
  • White arrows in FIG. 2C indicate the actual formation of dark contrast areas, indicating cytoplasmic extension.
  • Fig. 3 is a photomicrograph (Fig. 3, right) of a glass plate modified with azide by methods A1 and B2, and a micrograph (Fig.
  • FIG. 4A shows a schematic diagram of cell binding (adhesion) to the glass surface.
  • FIG. 4B shows the time course of cells adhered to glass using 1a-1d. Negative control in FIG. 4B shows experimental results when treated in the same manner except that 1a-1d was not used.
  • FIG. 4B shows photographs immediately after cell seeding and 6 hours after seeding. In 1a, viability determination was performed after 6 hours using MTT and trypan blue.
  • Example 6 (cells, bonds between cells (crosslinks)) Cells at 5.0 ⁇ 10 4 cells/mL were cultured in 10 mL medium containing 100 ⁇ M Ac 4 ManNAz and 0.1% DMSO in a 10 cm ⁇ circular dish. 24 hours later, after washing the cells with RPMI(-)FBS (10 mL), a cell suspension (cell A suspension) with a cell concentration of 2.0 ⁇ 10 5 cells/mL in RPMI(-)FBS was added. prepared.
  • the cell B pellet and 500 ⁇ L of the cell A suspension were mixed and centrifuged at 1500 G for 30 minutes.
  • the formed pellet was pipetted and the cell clumps remaining aggregated were photographed microscopically.
  • Example 6 cell A is a cell introduced with azide (-N 3 ), and cell B is a cell bound with 1a via azide after introduction of azide.
  • FIG. 5A shows a schematic diagram of cells and bonds (crosslinks) between cells.
  • FIG. 5B shows a micrograph of the bound (adhered) cell mass.
  • Example 7 Boding (adhesion) of cells to the AFM cantilever
  • An AFM cantilever atomic force microscope cantilever (OMCL-TR400-PSA, Olympus) was irradiated with a UV ozone cleaner (ProCleaner, Bioforce) for 15 minutes.
  • This cantilever was immersed in APTES (3-aminopropyltriethoxysilane) (200 ⁇ L, 1.0 v/v% in toluene), washed with methanol and dried.
  • APTES 3-aminopropyltriethoxysilane
  • This cantilever was further treated with the following s10 solution (50 ⁇ L, 2.0 mg/mL in 20 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) buffer, pH 8.5) for 30 minutes, then RPMI(-) Washed with FBS.
  • s10 solution 50 ⁇ L, 2.0 mg/mL in 20 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) buffer, pH 8.5
  • Cells at 5.0 ⁇ 10 4 cells/mL were cultured in 10 mL medium containing 100 ⁇ M Ac 4 ManNAz and 0.1% DMSO in a 10 cm ⁇ circular dish. After 24 hours, the cells were washed with RPMI(-)FBS (10 mL) and then suspended in RPMI(-)FBS (10 mL). On the other hand, the cantilever was reacted with 1b (50 ⁇ L, 50 ⁇ M in RPMI(-)FBS) for 15 minutes and washed with RPMI(-)FBS. The surface of the cantilever was brought into contact with the cells for 5 minutes using an atomic force microscope (AFM, NanoWizard, JPK).
  • AFM atomic force microscope
  • FIG. 6A shows a schematic diagram of cantilever cross-linking to cells using 1b.
  • Fig. 6A (1): When the azide-modified cantilever reacted with 1b was brought into contact with cells previously treated with Ac4ManNAz for 24 hours, the cells and cantilever immediately adhered. (2): When the cantilever with azidated cells was brought into contact with the glass modified with 1b by method C, the glass and the cells immediately adhered. (3) and (4): The attached cells were lifted from the glass by pulling up the cantilever.
  • FIG. 6B is a photograph showing a state in which cells are bound to the cantilever in (1) above. In FIG.
  • the horizontal axis indicates the distance between the cell and the cantilever (unit: ⁇ m), and the vertical axis indicates the magnitude of cantilever deflection (that is, a measure of the force applied to the cantilever) (unit: au).
  • the cantilever with azide cells was approached along the dotted line (Approach) to the glass modified with 1b (Fig. 6C (2)), and the cantilever was pulled up along the solid line (Retract) (Fig. 6C (3), (4). ). Since the bond between the cell and the substrate was strong, the deflection of the cantilever exceeds the upper limit of detection when it is pulled up.
  • 1.0 ⁇ 10 5 cells were treated with 1a, 1b, 1c, 1d, or unsubstituted CODY (Dibenzocyclooctadiyne: 5,6,11,12-Tetradehydrodibenzo[a,e]cyclooctene) in a solution containing 50 ⁇ M (50 ⁇ M in RPMI (- )FBS, 500 ⁇ L) for 5 minutes, followed by washing with RPMI( ⁇ )FBS (500 ⁇ L).
  • CODY Dibenzocyclooctadiyne: 5,6,11,12-Tetradehydrodibenzo[a,e]cyclooctene
  • the cells were treated with FITC-PEG 3 -N 3 solution (10 ⁇ M in RPMI(-)FBS, 0.1% DMSO, 500 ⁇ L) for 5 minutes, then RPMI(-)FBS (1 mL) and PBS(-) (1 mL ).
  • Cells were fixed with a paraformaldehyde solution (4% in PBS( ⁇ )) for 30 minutes, washed with PBS( ⁇ ) and MilliQ water (500 ⁇ L each), and suspended in MilliQ water (25 ⁇ L). The whole amount was placed on a slide glass and air-dried for 1 hour.
  • a mounting agent Fluorescence Mounting Medium, DAKO; 10 ⁇ L
  • DAKO Fluorescence Mounting Medium
  • the FITC-derived fluorescence was measured using a fluorescence microscope, and the fluorescence intensity was quantified. Since unsubstituted CODY has low solubility in water, 0.1% DMSO was added in the treatment with cells for 5 minutes.
  • FIG. 7A The scheme of the experiment is shown in Figure 7A.
  • a scheme for staining the cell surface with dyes is illustrated.
  • FIG. 7B and 7D show the measurement results of FITC-derived fluorescence in the above experiment.
  • FIG. 7B shows the results for 1a, 1b, 1c, 1d and
  • FIG. 7D shows the results for 1a, unsubstituted CODY.
  • FIG. 7C shows quantification of the results of FIG. 7B
  • FIG. 7E shows quantification of the results of FIG. 7D.
  • Example 8 Boding (adhesion) of cells to glass surface and quantification of adhesion) Cells at 5.0 ⁇ 10 4 cells/mL were cultured in 5 mL medium containing 100 ⁇ M Ac 4 ManNAz and 0.1% DMSO in a 6 cm ⁇ circular dish. After 24 hours, 1 mL of the culture solution was dispensed, the cells were washed with RPMI (-) FBS (1 mL), and the medium was removed.
  • RPMI ( ⁇ ) FBS solution of 1a, 1b, 1c or unsubstituted CODY Dibenzocyclooctadiyne
  • the medium was removed, washed with 500 ⁇ L of RPMI (-) FBS, and then suspended in 300 ⁇ L of RPMI (-) FBS.
  • Azide-modified glass plates were placed in a 24-well plate using method B2, and 500 ⁇ L RPMI ( ⁇ ) FBS was added. 300 ⁇ L of compound-treated cell suspension was added to the wells.
  • the cell engraftment rate (engrafted cells/seeded cells) (%) was calculated by dividing by the number of seeded cells (Fig. 8).
  • the cell engraftment rate of 1a was calculated in the same manner except that Ac 4 ManNAz was not used.
  • Example 9 Boding (adhesion) of cells to glass surface and quantification of adhesion)
  • Cells at 5.0 ⁇ 10 4 cells/mL were cultured in 5 mL medium containing 100 ⁇ M Ac 4 ManNAz and 0.1% DMSO in a 6 cm ⁇ circular dish. After 24 hours, 1 mL of the culture solution was dispensed, the cells were washed with RPMI (-) FBS (1 mL), and the medium was removed.
  • RPMI ( ⁇ ) FBS solution of 1a 500 ⁇ L was added to the cells and incubated for 5 minutes. The medium was removed, washed with 500 ⁇ L of RPMI (-) FBS, and then suspended in 300 ⁇ L of RPMI (-) FBS. Azide-modified glass plates were placed in a 24-well plate using method B2, and 500 ⁇ L RPMI ( ⁇ ) FBS was added. 300 ⁇ L of compound-treated cell suspension was added to the wells. After 30 minutes, the medium was removed, 1 mL of RPMI 10% FBS was added, and the cells were incubated at 37°C in 5% CO 2 for 6 hours. After that, floating cells were removed by exchanging the medium three times.
  • RNA from cells that had not been plated on glass after treatment with Ac 4 ManNAz was used (FIG. 9A).
  • the results of the molecular function category are shown in FIG. 9B in descending order of -log10(P-value).
  • 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.
  • the polar functional group of dibenzocyclooctadiyne having a polar functional group may be omitted, or may be indicated as -OR.
  • a preferred range can be defined by arbitrarily combining the upper and lower limits of the numerical range
  • a preferred range can be defined by arbitrarily combining the upper limits of the numerical range
  • the lower limit of the numerical range Any combination of values can be used to define a preferred range.

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