WO2022181695A1 - Complex, method for producing complex, and compound - Google Patents
Complex, method for producing complex, and compound Download PDFInfo
- Publication number
- 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
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- group
- polar functional
- functional group
- dibenzocyclooctadiyne
- azide
- Prior art date
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- 150000001875 compounds Chemical class 0.000 title claims abstract description 65
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 32
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- 125000000524 functional group Chemical group 0.000 claims abstract description 141
- 239000000463 material Substances 0.000 claims abstract description 70
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- IVRMZWNICZWHMI-UHFFFAOYSA-N azide group Chemical group [N-]=[N+]=[N-] IVRMZWNICZWHMI-UHFFFAOYSA-N 0.000 claims description 87
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- 125000000217 alkyl group Chemical group 0.000 claims description 30
- 239000002131 composite material Substances 0.000 claims description 24
- 229910010272 inorganic material Inorganic materials 0.000 claims description 20
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- 239000000758 substrate Substances 0.000 claims description 18
- IYMAXBFPHPZYIK-BQBZGAKWSA-N Arg-Gly-Asp Chemical compound NC(N)=NCCC[C@H](N)C(=O)NCC(=O)N[C@@H](CC(O)=O)C(O)=O IYMAXBFPHPZYIK-BQBZGAKWSA-N 0.000 claims description 14
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- PAERYIZRYCNIKO-QTVWNMPRSA-N (3S,4R,5S,6R)-3-amino-2-azido-6-(hydroxymethyl)oxane-2,4,5-triol Chemical compound N(=[N+]=[N-])C1(O)[C@@H](N)[C@@H](O)[C@H](O)[C@H](O1)CO PAERYIZRYCNIKO-QTVWNMPRSA-N 0.000 claims description 4
- 125000001183 hydrocarbyl group Chemical group 0.000 claims 2
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- VBKNTGMWIPUCRF-UHFFFAOYSA-M potassium;fluoride;hydrofluoride Chemical compound F.[F-].[K+] VBKNTGMWIPUCRF-UHFFFAOYSA-M 0.000 description 1
- FWZMWMSAGOVWEZ-UHFFFAOYSA-N potassium;hydrofluoride Chemical compound F.[K] FWZMWMSAGOVWEZ-UHFFFAOYSA-N 0.000 description 1
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- 238000000425 proton nuclear magnetic resonance spectrum Methods 0.000 description 1
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- BOLDJAUMGUJJKM-LSDHHAIUSA-N renifolin D Natural products CC(=C)[C@@H]1Cc2c(O)c(O)ccc2[C@H]1CC(=O)c3ccc(O)cc3O BOLDJAUMGUJJKM-LSDHHAIUSA-N 0.000 description 1
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 1
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- 238000010186 staining Methods 0.000 description 1
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- 125000001981 tert-butyldimethylsilyl group Chemical group [H]C([H])([H])[Si]([H])(C([H])([H])[H])[*]C(C([H])([H])[H])(C([H])([H])[H])C([H])([H])[H] 0.000 description 1
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Images
Classifications
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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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- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C275/00—Derivatives of urea, i.e. compounds containing any of the groups, the nitrogen atoms not being part of nitro or nitroso groups
- 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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- 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
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- 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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- 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
- C07F9/08—Esters of oxyacids of phosphorus
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- C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
- C07H15/20—Carbocyclic rings
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- C12N1/00—Microorganisms, e.g. protozoa; 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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- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
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- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- 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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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
本開示は、複合体、複合体の製造方法及び化合物に関する。
The present disclosure relates to a complex, a method for producing the complex, and a compound.
細胞プロセス（例えば、増殖・分化・形態形成・細胞死）の解明や制御を行うための技術が求められている。
There is a need for technologies to elucidate and control cellular processes (eg, proliferation, differentiation, morphogenesis, and cell death).
ところで、ジベンゾシクロオクタジイン（Dibenzocyclooctadiyne：5,6,11,12-Tetradehydrodibenzo[a,e]cyclooctene）は、分子内に炭素炭素三重結合であるアルキンを二つ有する歪みを有する環状分子であるため、歪み促進型アジド－アルキン付加環化反応（ＳＰＡＡＣ反応）を二度起こすことが可能であることが知られている。
By the way, 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反応を利用し、細胞表面にアジド基を有する細胞にジベンゾシクロオクタジインを結合し、更にジベンゾシクロオクタジインが有するもう一つのアルキンと、アジド基を有する蛍光色素とを結合すること、該操作によって蛍光修飾された細胞を蛍光観察することが既に行われていた（例えば、特許文献１、実施例２２参照）。
Using the SPAAC reaction, binding dibenzocyclooctadiyne to cells having an azide group on the cell surface, and further binding another alkyne possessed by dibenzocyclooctadiyne with a fluorescent dye having an azide group, said operation Fluorescence observation of cells fluorescently modified by has already been performed (see, for example, Patent Document 1, Example 22).
特許文献１には、ジベンゾシクロオクタジインを用いることにより、細胞と、蛍光色素とを結合できることが開示されているが、本発明者らの検討によると、特許文献１に開示された発明では、細胞と結合する際や蛍光色素と結合する際の反応速度が十分でない場合や、細胞に結合する蛍光色素の量が十分でない場合があることを見出した。
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.
本発明者らは、鋭意検討を重ねた結果、ＳＰＡＡＣ反応を行う際に用いるジベンゾシクロオクタジインの溶解性を高めることができれば、細胞等と結合する際の反応性が向上すること、細胞に結合する蛍光色素の量を増やせることを見出した。
As a result of intensive studies, 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
本発明者らは、前記知見を更に発展させ、ジベンゾシクロオクタジインの溶解性を高めることができれば、反応性を向上させることができ、蛍光色素以外の材料、例えば、一般に細胞と結合することが困難な分子量の大きな材料や、細胞同士であっても、結合することができるのではないかと考えた。
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.
すなわち、本開示は、従来の技術では提供することが困難であった、細胞と材料との複合体、細胞と材料との複合体の製造方法、該複合体を得るために使用可能な新規化合物を提供することを目的とする。
That is, 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
本発明者らは、極性官能基を有するジベンゾシクロオクタジインを用いることにより、細胞と材料との複合体を得ることができることを見出した。また、本発明者らは、極性官能基を有するジベンゾシクロオクタジインの検討の過程で、新規化合物についても見出した。
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.
本実施形態の態様例は、以下の通りに記載される。
Aspect examples of the present embodiment are described as follows.
（１） 細胞表面にアジド基を有する細胞とアジド基を有する材料（但し、アジド基を有する蛍光色素を除く）とが、極性官能基を有するジベンゾシクロオクタジインによって結合した複合体。
（２） 材料が、細胞表面にアジド基を有する細胞及びアジド修飾された無機材料から選択される少なくとも１種の材料である、（１）に記載の複合体。
（３） 無機材料が培養基板、ガラス基板、ＡＦＭカンチレバー、金属から選択される少なくとも１種の無機材料である、（２）に記載の複合体。
（４） 極性官能基を有するジベンゾシクロオクタジインが、下記一般式（Ｉ）で表される化合物である、（１）～（３）のいずれかに記載の複合体。
（一般式（Ｉ）において、Ｘはそれぞれ独立に極性官能基を有する基であり、Ａはそれぞれ独立に炭化水素基であり、ｎは１～４の整数であり、ｍは０～３の整数であり、ｎ＋ｍは１～４の整数であり、ｎ’は１～４の整数であり、ｍ’は０～３の整数であり、ｎ’＋ｍ’は１～４の整数である。）
（５） 極性官能基を有するジベンゾシクロオクタジインが、下記一般式（ＩＩ）で表される化合物である、（１）～（３）のいずれかに記載の複合体。
（一般式（ＩＩ）において、Ｘはそれぞれ独立に極性官能基を有する基であり、ｎはそれぞれ独立に１～４の整数である。）
（６） 極性官能基を有するジベンゾシクロオクタジインが、下記一般式（ＩＩＩ）で表される化合物である、（１）～（３）のいずれかに記載の複合体。
（一般式（ＩＩＩ）において、Ｒはそれぞれ独立に極性官能基を有する基及びアルキル基から選択される基であり、ｎはそれぞれ独立に１～４の整数である。）
（７） 極性官能基を有するジベンゾシクロオクタジインが、下記一般式（ＩＶ）で表される化合物である、（１）～（３）のいずれかに記載の複合体。
（一般式（ＩＶ）において、Ｒはそれぞれ独立に極性官能基を有する基及びアルキル基から選択される基である。）
（８） 極性官能基を有するジベンゾシクロオクタジインが、下記式１ａ～１ｊから選択される少なくとも１種の化合物である、（１）～（３）のいずれかに記載の複合体。
（式１ｇにおいて、ＲＧＤはアルギニン－グリシン－アスパラギン酸からなる３残基のペプチド鎖であり、式１ｊにおいて、Ｙはそれぞれ独立に極性官能基を有する基及びアルキル基から選択される基である。）
（９） 細胞表面にアジド基を有する細胞が、細胞培養培地にアセチル化アジドマンノサミン（Ac4ManNAz）を加えることにより、細胞のシアル酸代謝経路を通じて、細胞表面の糖鎖末端のシアル酸部位にアジド基が導入された細胞である、（１）～（８）のいずれかに記載の複合体。 (1) A complex in which a cell having an azide group on the cell surface and a material having an azide group (excluding fluorescent dyes having an azide group) are bound by dibenzocyclooctadiyne having a polar functional group.
(2) The composite according to (1), wherein the material is at least one material selected from cells having an azide group on the cell surface and an azide-modified inorganic material.
(3) The composite according to (2), wherein the inorganic material is at least one inorganic material selected from culture substrates, glass substrates, AFM cantilevers, and metals.
(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 (I).
(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, and 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 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 (II).
(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 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 (III).
(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 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).
(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 complex according to any one of (1) to (3), wherein the dibenzocyclooctadiyne having a polar functional group is at least one compound selected fromformulas 1a to 1j below.
(In formula 1g, 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. )
(9) By adding acetylated azidomannosamine (Ac 4 ManNAz) to the cell culture medium, 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.
（２） 材料が、細胞表面にアジド基を有する細胞及びアジド修飾された無機材料から選択される少なくとも１種の材料である、（１）に記載の複合体。
（３） 無機材料が培養基板、ガラス基板、ＡＦＭカンチレバー、金属から選択される少なくとも１種の無機材料である、（２）に記載の複合体。
（４） 極性官能基を有するジベンゾシクロオクタジインが、下記一般式（Ｉ）で表される化合物である、（１）～（３）のいずれかに記載の複合体。
（５） 極性官能基を有するジベンゾシクロオクタジインが、下記一般式（ＩＩ）で表される化合物である、（１）～（３）のいずれかに記載の複合体。
（６） 極性官能基を有するジベンゾシクロオクタジインが、下記一般式（ＩＩＩ）で表される化合物である、（１）～（３）のいずれかに記載の複合体。
（７） 極性官能基を有するジベンゾシクロオクタジインが、下記一般式（ＩＶ）で表される化合物である、（１）～（３）のいずれかに記載の複合体。
（８） 極性官能基を有するジベンゾシクロオクタジインが、下記式１ａ～１ｊから選択される少なくとも１種の化合物である、（１）～（３）のいずれかに記載の複合体。
（９） 細胞表面にアジド基を有する細胞が、細胞培養培地にアセチル化アジドマンノサミン（Ac4ManNAz）を加えることにより、細胞のシアル酸代謝経路を通じて、細胞表面の糖鎖末端のシアル酸部位にアジド基が導入された細胞である、（１）～（８）のいずれかに記載の複合体。 (1) A complex in which a cell having an azide group on the cell surface and a material having an azide group (excluding fluorescent dyes having an azide group) are bound by dibenzocyclooctadiyne having a polar functional group.
(2) The composite according to (1), wherein the material is at least one material selected from cells having an azide group on the cell surface and an azide-modified inorganic material.
(3) The composite according to (2), wherein the inorganic material is at least one inorganic material selected from culture substrates, glass substrates, AFM cantilevers, and metals.
(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 (I).
(5) 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 (II).
(6) 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 (III).
(7) 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).
(8) 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
(9) By adding acetylated azidomannosamine (Ac 4 ManNAz) to the cell culture medium, 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） 細胞表面にアジド基を有する細胞と、極性官能基を有するジベンゾシクロオクタジインとを結合し、極性官能基を有するジベンゾシクロオクタジインと細胞との結合体（Ａ）を得る工程、及び、
結合体（Ａ）と、アジド基を有する材料（但し、アジド基を有する蛍光色素を除く）とを結合し、細胞と材料とが極性官能基を有するジベンゾシクロオクタジインによって結合した複合体を得る工程を有する、複合体の製造方法。
（１１） アジド基を有する材料（但し、アジド基を有する蛍光色素を除く）と、極性官能基を有するジベンゾシクロオクタジインとを結合し、極性官能基を有するジベンゾシクロオクタジインと材料との結合体（Ｂ）を得る工程、及び、
結合体（Ｂ）と、細胞表面にアジド基を有する細胞とを結合し、細胞と材料とが極性官能基を有するジベンゾシクロオクタジインによって結合した複合体を得る工程を有する、複合体の製造方法。
（１２） 材料が、細胞表面にアジド基を有する細胞及びアジド修飾された無機材料から選択される少なくとも１種の材料である、（１０）又は（１１）に記載の複合体の製造方法。
（１３） 無機材料が培養基板、ガラス基板、ＡＦＭカンチレバー、金属から選択される少なくとも１種の無機材料である、（１２）に記載の複合体の製造方法。
（１４） 極性官能基を有するジベンゾシクロオクタジインが、下記一般式（Ｉ）で表される化合物である、（１０）～（１３）のいずれかに記載の複合体の製造方法。
（一般式（Ｉ）において、Ｘはそれぞれ独立に極性官能基を有する基であり、Ａはそれぞれ独立に炭化水素基であり、ｎは１～４の整数であり、ｍは０～３の整数であり、ｎ＋ｍは１～４の整数であり、ｎ’は１～４の整数であり、ｍ’は０～３の整数であり、ｎ’＋ｍ’は１～４の整数である。）
（１５） 極性官能基を有するジベンゾシクロオクタジインが、下記一般式（ＩＩ）で表される化合物である、（１０）～（１３）のいずれかに記載の複合体の製造方法。
（一般式（ＩＩ）において、Ｘはそれぞれ独立に極性官能基を有する基であり、ｎはそれぞれ独立に１～４の整数である。）
（１６） 極性官能基を有するジベンゾシクロオクタジインが、下記一般式（ＩＩＩ）で表される化合物である、（１０）～（１３）のいずれかに記載の複合体の製造方法。
（一般式（ＩＩＩ）において、Ｒはそれぞれ独立に極性官能基を有する基及びアルキル基から選択される基であり、ｎはそれぞれ独立に１～４の整数である。）
（１７） 極性官能基を有するジベンゾシクロオクタジインが、下記一般式（ＩＶ）で表される化合物である、（１０）～（１３）のいずれかに記載の複合体の製造方法。
（一般式（ＩＶ）において、Ｒはそれぞれ独立に極性官能基を有する基及びアルキル基から選択される基である。）
（１８） 極性官能基を有するジベンゾシクロオクタジインが、下記式１ａ～１ｊから選択される少なくとも１種の化合物である、（１０）～（１３）のいずれかに記載の複合体の製造方法。
（式１ｇにおいて、ＲＧＤはアルギニン－グリシン－アスパラギン酸からなる３残基のペプチド鎖であり、式１ｊにおいて、Ｙはそれぞれ独立に極性官能基を有する基及びアルキル基から選択される基である。）
（１９） 下記式１ｂ～１ｊから選択される少なくとも１種の化合物。
（式１ｇにおいて、ＲＧＤはアルギニン－グリシン－アスパラギン酸からなる３残基のペプチド鎖であり、式１ｊにおいて、Ｙはそれぞれ独立に極性官能基を有する基及びアルキル基から選択される基である。）
本明細書は本願の優先権の基礎となる日本国特許出願番号2021-028795号の開示内容を包含する。 (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.
(11) Bonding a material having an azide group (excluding fluorescent dyes having an azide group) with dibenzocyclooctadiyne having a polar functional group, and bonding the dibenzocyclooctadiyne having a polar functional group to the material obtaining the body (B); and
A method for producing a conjugate, comprising the step of binding the conjugate (B) to a cell having an azide group on the cell surface to obtain a conjugate in which the cell and the material are bound by 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 an azide group on the cell surface and an azide-modified inorganic material.
(13) The method for producing a composite according to (12), wherein the inorganic material is at least one inorganic material selected from culture substrates, glass substrates, AFM cantilevers, and metals.
(14) 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 (I).
(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, and 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 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 (II).
(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 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).
(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 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).
(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 complex according to any one of (10) to (13), wherein the dibenzocyclooctadiyne having a polar functional group is at least one compound selected from the followingformulas 1a to 1j.
(In formula 1g, 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. )
(19) At least one compound selected from the followingformulas 1b to 1j.
(In formula 1g, 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. )
This specification includes the disclosure content of Japanese Patent Application No. 2021-028795, which is the basis of priority of this application.
結合体（Ａ）と、アジド基を有する材料（但し、アジド基を有する蛍光色素を除く）とを結合し、細胞と材料とが極性官能基を有するジベンゾシクロオクタジインによって結合した複合体を得る工程を有する、複合体の製造方法。
（１１） アジド基を有する材料（但し、アジド基を有する蛍光色素を除く）と、極性官能基を有するジベンゾシクロオクタジインとを結合し、極性官能基を有するジベンゾシクロオクタジインと材料との結合体（Ｂ）を得る工程、及び、
結合体（Ｂ）と、細胞表面にアジド基を有する細胞とを結合し、細胞と材料とが極性官能基を有するジベンゾシクロオクタジインによって結合した複合体を得る工程を有する、複合体の製造方法。
（１２） 材料が、細胞表面にアジド基を有する細胞及びアジド修飾された無機材料から選択される少なくとも１種の材料である、（１０）又は（１１）に記載の複合体の製造方法。
（１３） 無機材料が培養基板、ガラス基板、ＡＦＭカンチレバー、金属から選択される少なくとも１種の無機材料である、（１２）に記載の複合体の製造方法。
（１４） 極性官能基を有するジベンゾシクロオクタジインが、下記一般式（Ｉ）で表される化合物である、（１０）～（１３）のいずれかに記載の複合体の製造方法。
（１５） 極性官能基を有するジベンゾシクロオクタジインが、下記一般式（ＩＩ）で表される化合物である、（１０）～（１３）のいずれかに記載の複合体の製造方法。
（１６） 極性官能基を有するジベンゾシクロオクタジインが、下記一般式（ＩＩＩ）で表される化合物である、（１０）～（１３）のいずれかに記載の複合体の製造方法。
（１７） 極性官能基を有するジベンゾシクロオクタジインが、下記一般式（ＩＶ）で表される化合物である、（１０）～（１３）のいずれかに記載の複合体の製造方法。
（１８） 極性官能基を有するジベンゾシクロオクタジインが、下記式１ａ～１ｊから選択される少なくとも１種の化合物である、（１０）～（１３）のいずれかに記載の複合体の製造方法。
（１９） 下記式１ｂ～１ｊから選択される少なくとも１種の化合物。
本明細書は本願の優先権の基礎となる日本国特許出願番号2021-028795号の開示内容を包含する。 (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.
(11) Bonding a material having an azide group (excluding fluorescent dyes having an azide group) with dibenzocyclooctadiyne having a polar functional group, and bonding the dibenzocyclooctadiyne having a polar functional group to the material obtaining the body (B); and
A method for producing a conjugate, comprising the step of binding the conjugate (B) to a cell having an azide group on the cell surface to obtain a conjugate in which the cell and the material are bound by 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 an azide group on the cell surface and an azide-modified inorganic material.
(13) The method for producing a composite according to (12), wherein the inorganic material is at least one inorganic material selected from culture substrates, glass substrates, AFM cantilevers, and metals.
(14) 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 (I).
(15) 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 (II).
(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).
(17) 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).
(18) The method for producing a complex 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
(19) At least one compound selected from the following
This specification includes the disclosure content of Japanese Patent Application No. 2021-028795, which is the basis of priority of this application.
本開示によれば、細胞と材料との複合体、細胞と材料との複合体の製造方法、該複合体を得るために使用可能な新規化合物が提供される。
According to the present disclosure, 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.
以下、本発明を詳細に説明する。
The present invention will be described in detail below.
＜複合体＞
本実施形態に係る複合体は、細胞表面にアジド基を有する細胞とアジド基を有する材料（但し、アジド基を有する蛍光色素を除く）とが、極性官能基を有するジベンゾシクロオクタジインによって結合した複合体である。本実施形態に係る複合体は、ジベンゾシクロオクタジイン（Dibenzocyclooctadiyne：5,6,11,12-Tetradehydrodibenzo[a,e]cyclooctene）（以下、無置換ＣＯＤＹとも記す。）ではなく、極性官能基を有するジベンゾシクロオクタジインによって、細胞と材料とが結合されているため、速やかに細胞と材料とを結合することができる。この理由を本発明者らは、極性官能基を有するジベンゾシクロオクタジインは、無置換ＣＯＤＹと比べて溶解性に優れるためであると推測した。また、溶解性に優れるため、無置換ＣＯＤＹを結合する場合と比べて、多くの極性官能基を有するジベンゾシクロオクタジインが細胞表面のアジド基と結合することが可能であり、結果として細胞と材料とを強固に結合できると推測した。 <Complex>
In the complex according to the present embodiment, a cell having an azide group on the cell surface and a material having an azide group (excluding fluorescent dyes having an azide group) are bound by dibenzocyclooctadiyne having a polar functional group. Complex. 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. The present inventors speculated that the reason for this is that dibenzocyclooctadiyne having a polar functional group is superior in solubility to unsubstituted CODY. In addition, due to its excellent solubility, 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
本実施形態に係る複合体は、細胞表面にアジド基を有する細胞とアジド基を有する材料（但し、アジド基を有する蛍光色素を除く）とが、極性官能基を有するジベンゾシクロオクタジインによって結合した複合体である。本実施形態に係る複合体は、ジベンゾシクロオクタジイン（Dibenzocyclooctadiyne：5,6,11,12-Tetradehydrodibenzo[a,e]cyclooctene）（以下、無置換ＣＯＤＹとも記す。）ではなく、極性官能基を有するジベンゾシクロオクタジインによって、細胞と材料とが結合されているため、速やかに細胞と材料とを結合することができる。この理由を本発明者らは、極性官能基を有するジベンゾシクロオクタジインは、無置換ＣＯＤＹと比べて溶解性に優れるためであると推測した。また、溶解性に優れるため、無置換ＣＯＤＹを結合する場合と比べて、多くの極性官能基を有するジベンゾシクロオクタジインが細胞表面のアジド基と結合することが可能であり、結果として細胞と材料とを強固に結合できると推測した。 <Complex>
In the complex according to the present embodiment, a cell having an azide group on the cell surface and a material having an azide group (excluding fluorescent dyes having an azide group) are bound by dibenzocyclooctadiyne having a polar functional group. Complex. 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. The present inventors speculated that the reason for this is that dibenzocyclooctadiyne having a polar functional group is superior in solubility to unsubstituted CODY. In addition, due to its excellent solubility, 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
（細胞）
前記細胞表面にアジド基を有する細胞としては、細胞表面にアジド基を有していればよい。細胞表面にアジド基を有する細胞としては、細胞培養培地にアセチル化アジドマンノサミン（Ac4ManNAz）を加えることにより、細胞のシアル酸代謝経路を通じて、細胞表面の糖鎖末端のシアル酸部位にアジド基が導入された細胞であることが好ましい。また、別の代謝経路を通じて、細胞表面にアジド基が導入された細胞であってもよい。なお、細胞表面にアジド基が導入された細胞としては、細胞表面以外にもアジド基が導入されていてもよい。Ac4ManNAzの構造を以下に示す。 (cell)
The cell having an azide group on the cell surface may have an azide group on the cell surface. By adding acetylated azidomannosamine (Ac 4 ManNAz) to the cell culture medium for cells with azide groups on the cell surface, 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. Alternatively, the cell may be a cell in which an azide group has been introduced to the cell surface through another metabolic pathway. In addition, as a cell in which an azide group is introduced on the cell surface, an azide group may be introduced on a surface other than the cell surface. The structure of Ac4ManNAz is shown below.
前記細胞表面にアジド基を有する細胞としては、細胞表面にアジド基を有していればよい。細胞表面にアジド基を有する細胞としては、細胞培養培地にアセチル化アジドマンノサミン（Ac4ManNAz）を加えることにより、細胞のシアル酸代謝経路を通じて、細胞表面の糖鎖末端のシアル酸部位にアジド基が導入された細胞であることが好ましい。また、別の代謝経路を通じて、細胞表面にアジド基が導入された細胞であってもよい。なお、細胞表面にアジド基が導入された細胞としては、細胞表面以外にもアジド基が導入されていてもよい。Ac4ManNAzの構造を以下に示す。 (cell)
The cell having an azide group on the cell surface may have an azide group on the cell surface. By adding acetylated azidomannosamine (Ac 4 ManNAz) to the cell culture medium for cells with azide groups on the cell surface, 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. Alternatively, the cell may be a cell in which an azide group has been introduced to the cell surface through another metabolic pathway. In addition, as a cell in which an azide group is introduced on the cell surface, an azide group may be introduced on a surface other than the cell surface. The structure of Ac4ManNAz is shown below.
細胞培養培地にAc4ManNAzを加えることにより、細胞のシアル酸代謝経路を通じて、細胞表面の糖鎖末端のシアル酸部位にアジド基が導入される過程の概念図を図１に示す。
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.
アジド基が導入される細胞としては、通常は培養細胞が用いられる。また、アジド基が導入される細胞としては、ヒト由来の細胞であっても、非ヒト由来の細胞であってもよい。非ヒト由来の細胞としては、哺乳類（ヒトを除く）由来の細胞、哺乳類以外の生物、例えば鳥類、魚類、昆虫、植物、藻類、菌類由来の細胞が挙げられる。哺乳類（ヒトを除く）としては、例えばマウス、ラット、ウマ、ヒツジ、ブタ、ヤギ、ウシが挙げられる。
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.
細胞としては特に制限はないが、例えば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、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－ＫＪ、ＳＢＣ－１、ＳＢＣ－２、ＳＢＣ－３、ＳＢＣ－４、ＳＢＣ－５、ＳＫ－ＡＫ－ＬＣＬ、ＳＫ－ＭＥＳ－１、Ｂ１６Ｆ１０、Ｃｏｌｏｎ－２６、ＭＣＦ７、ＨｅｐＧ２、Ｌｅｗｉｓ ｌｕｎｇ ｃａｎｃｅｒ、ＩＳＯ－ＨＡＳ－Ｂ、ＮＩＨ／３Ｔ３、ＴＦＫ－１、Ａ５４９、Ｊｕｒｋａｔ、ＳＷ１９９０、ＭＤＡ－ＭＢ－２３１、ＰＡＮＣ－１、ＣＨＯ、ＨＥＫ－２９３Ｔ、ＨＵＶＥＣ、ＭＳＣ、ＭＩＮ６等が挙げられる。
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, 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-KJ, SBC-1, SBC-2, SBC-3, SBC-4, SBC-5, SK-AK-LCL, SK-MES-1, B16F10, Colon -26, MCF7, HepG2, Lewis lung cancer, ISO-HAS-B, NIH/3T3, TFK-1, A549, Jurkat, SW1990, MDA-MB-231, PANC-1, CHO, HEK-293T, HUVEC, MSC , MIN6, and the like.
（材料）
前記アジド基を有する材料（但し、アジド基を有する蛍光色素を除く）としては、アジド基を有していればよく、特に制限はない。アジド基としては材料の、極性官能基を有するジベンゾシクロオクタジインを介して細胞と結合することが望まれる部分にアジド基が存在すればよく、アジド基は材料の表面の一部に存在してもよく、材料の表面全部に存在してもよい。 (material)
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.
前記アジド基を有する材料（但し、アジド基を有する蛍光色素を除く）としては、アジド基を有していればよく、特に制限はない。アジド基としては材料の、極性官能基を有するジベンゾシクロオクタジインを介して細胞と結合することが望まれる部分にアジド基が存在すればよく、アジド基は材料の表面の一部に存在してもよく、材料の表面全部に存在してもよい。 (material)
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.
材料としては例えば、細胞表面にアジド基を有する細胞、アジド修飾された無機材料、及びアジド基を有する有機材料が挙げられ、細胞表面にアジド基を有する細胞及びアジド修飾された無機材料から選択される少なくとも１種の材料であることが好ましい。本実施形態に係る複合体は、通常結合することが困難な細胞（細胞表面にアジド基を有する細胞）同士や、細胞と、通常結合することが困難な無機材料とを容易に結合することができるため、従来困難であった様々な研究への利用が可能である。
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. is preferably at least one material that 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.
無機材料が培養基板、ガラス基板、ＡＦＭカンチレバー、金属から選択される少なくとも１種の無機材料であることが好ましい。
The inorganic material is preferably at least one inorganic material selected from culture substrates, glass substrates, AFM cantilevers, and metals.
本実施形態に用いる材料としては一種単独でも、二種以上であってもよい。二種以上を用いる場合には、例えば、一度、複合体を形成した後に、更に別の材料を複合体に結合してもよい。
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.
材料にアジド基を導入する方法としては、特に制限はない。例えば、材料が細胞表面にアジド基を有する細胞である場合には、前述の（細胞）の項で記載した細胞を用いることができる。材料がアジド基を有する有機材料である場合には、例えば従来公知の有機合成方法にしたがって、アジド基を導入することが可能である。材料がアジド基を有する無機材料である場合には、例えば実施例に記載の方法により、表面に有機分子又は有機無機ハイブリッド分子を結合させ、該有機分子又は有機無機ハイブリッド分子としてアジド基を有する化合物を用いることによりアジド基を導入する方法や、該有機分子又は有機無機ハイブリッド分子がアジド基を有さない場合には、該有機分子又は有機無機ハイブリッド分子を更に変性、修飾等を行うことによりアジド基を導入する方法が挙げられる。
There are no particular restrictions on the method of introducing an azide group into a material. For example, when the material is cells having an azide group on the cell surface, the cells described in the above section (Cells) can be used. When the material is an organic material having an azide group, the azide group can be introduced, for example, according to conventionally known organic synthesis methods. When the material is an inorganic material having an azide group, 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. or when the organic molecule or the organic-inorganic hybrid molecule does not have an azide group, 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.
（極性官能基を有するジベンゾシクロオクタジイン）
前記極性官能基を有するジベンゾシクロオクタジインとしては、無置換ＣＯＤＹが有するベンゼン環の水素原子を極性官能基で置換した分子が挙げられる。 (Dibenzocyclooctadiyne with polar functional group)
Examples of the dibenzocyclooctadiyne having a polar functional group include molecules in which the hydrogen atom of the benzene ring of unsubstituted CODY is substituted with a polar functional group.
前記極性官能基を有するジベンゾシクロオクタジインとしては、無置換ＣＯＤＹが有するベンゼン環の水素原子を極性官能基で置換した分子が挙げられる。 (Dibenzocyclooctadiyne with polar functional group)
Examples of the dibenzocyclooctadiyne having a polar functional group include molecules in which the hydrogen atom of the benzene ring of unsubstituted CODY is substituted with a polar functional group.
極性官能基を有するジベンゾシクロオクタジインとしては、下記一般式（Ｉ）で表される化合物であることが好ましく、下記一般式（ＩＩ）で表される化合物であることがより好ましく、下記一般式（ＩＩＩ）で表される化合物であることが特に好ましく、下記一般式（ＩＶ）で表される化合物であることが最も好ましい。
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.
一般式（Ｉ）及び（ＩＩ）において、Ｘはそれぞれ独立に極性官能基を有する基である。また一般式（ＩＩＩ）及び（ＩＶ）において、Ｒはそれぞれ独立に極性官能基を有する基及びアルキル基から選択される基である。
In general formulas (I) and (II), X is each independently a group having a polar functional group. In general formulas (III) and (IV), each R is independently a group selected from a group having a polar functional group and an alkyl group.
極性官能基としては、イオン性官能基及び非イオン性極性官能基から選択される少なくとも１種の極性官能基が挙げられる。極性官能基を有する基としては、極性官能基を少なくとも１つ有していればよく、複数の極性官能基を有していてもよい。極性官能基の数の上限としては特に制限はない。例えば、Ｘにおいては、極性官能基を有する基１つあたり、２０個以下の極性官能基を有していてもよく、例えば、Ｒにおいては、極性官能基を有する基１つあたり、１９個以下の極性官能基を有していてもよい。
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. For example, 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
イオン性官能基としては、例えば、カチオン（例えば、－ＮＨ３
＋、－ＮＨ２Ｚ＋、－ＮＨＺ２
＋、－ＮＺ３
＋）及びカウンターアニオン（例えば、ハロゲン化物イオン（Ｆ－、Ｃｌ－、Ｂｒ－、Ｉ－））を有する官能基、アニオン（例えば、－ＳＯ３
－、－Ｏ－、－ＣＯＯ－、－ＰＯ３
－）及びカウンターカチオン（例えば、アルカリ金属イオン（例えばＬｉ＋、Ｎａ＋、Ｋ＋））を有する官能基、並びに双性イオン（zwitterion）を有する官能基（例えば、前述のカチオン及びアニオンを有する官能基）が挙げられる。極性官能基を有する基が、イオン性官能基を有する場合には、１つ有していても、複数のイオン性官能基を有していてもよい。複数のイオン性官能基を有する場合には、同種のイオン性官能基を有していてもよく、別種のイオン性官能基を有していてもよい。
Examples 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). 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.
非イオン性極性官能基としては、例えば、－ＮＨ２、－ＮＨＺ、－ＮＺ２、－ＳＯ３Ｈ、－ＯＨ、－ＣＯＯＨ、－ＮＨ－ＣＯＮＨ２、－ＮＨ－ＣＯＮＨＺ、－ＮＨ－ＣＯＮＺ２、－ＮＺ－ＣＯＮＨ２、－ＮＺ－ＣＯＮＨＺ、－ＮＺ－ＣＯＮＺ２、－ＣＯ－（カルボニル基）、－ＣＯＯ－（エステル基）、及び－Ｏ－（エーテル基）が挙げられる。極性官能基を有する基が、非イオン性極性官能基を有する場合には、１つ有していても、複数の非イオン性極性官能基を有していてもよい。複数の非イオン性極性官能基を有する場合には、同種の非イオン性極性官能基を有していてもよく、別種の非イオン性極性官能基を有していてもよい。
Examples of 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). When 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は、炭素数１～１８の炭化水素基であり、好ましくは炭素数１～６のアルキル基である。Ｚが複数存在する場合には、同一の基でも異なる基でもよい。
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.
極性官能基を有する基の、極性官能基以外の構造としては、特に制限はないが、通常は、飽和又は不飽和の炭化水素基であり、好ましくは飽和炭化水素基である。飽和又は不飽和の炭化水素基としては、直鎖状であってもよく、分岐を有する鎖状であってもよく、環構造を有していてもよい。
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としては、分子量が１６～１１００であることが好ましく、１６～２００であることがより好ましい。
X preferably has a molecular weight of 16-1100, more preferably 16-200.
Ｒとしては、分子量が１５～５８４であることが好ましく、１５～１８４であることがより好ましい。Ｒは前述のように、それぞれ独立に極性官能基を有する基及びアルキル基から選択される基であるが、一分子内のＲの内、少なくとも一つは極性官能基を有する基であることが好ましい。Ｒがアルキル基である場合には、炭素数１～１８のアルキル基が挙げられ、好ましくは炭素数１～６のアルキル基が挙げられる。アルキル基としては、メチル基が好ましい態様の一つである。
As for R, the molecular weight is preferably 15-584, more preferably 15-184. As described above, 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. When 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及びＲとしては、ペプチド鎖、糖鎖、ウレア基（例えば－ＮＨ－ＣＯＮＨ２、－ＮＨ－ＣＯＮＨＺ、－ＮＨ－ＣＯＮＺ２、－ＮＺ－ＣＯＮＨ２、－ＮＺ－ＣＯＮＨＺ、－ＮＺ－ＣＯＮＺ２）、カルボニル基、エステル結合、及びエーテル基から選択される少なくとも１種の構造を有することが好ましい。ペプチド鎖としては、例えば１～５残基のペプチド鎖が挙げられる。
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としては、一般式（ＩＩＩ）における‐ＯＲであることが好ましい態様の一つである。すなわち、Ｘとしてはベンゼン環との結合部位が－Ｏ－（エーテル基）であることが好ましい態様の一つである。
In one preferred embodiment, 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).
一般式（Ｉ）において、Ａはそれぞれ独立に炭化水素基を示す。炭化水素基としては、好ましくは炭素数１～１８の炭化水素基であり、より好ましくは炭素数１～６のアルキル基である。
In general formula (I), 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.
一般式（Ｉ）、（ＩＩ）及び（ＩＩＩ）において、ｎはそれぞれ独立に１～４の整数であり、好ましくは１～３の整数であり、より好ましくは１又は２であり、特に好ましくは１である。また、一般式（Ｉ）において、ｎ’は１～４の整数であり、好ましくは１～３の整数であり、より好ましくは１又は２であり、特に好ましくは１である。
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, 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.
一般式（Ｉ）において、ｍは０～３の整数であり、好ましくは０又は１であり、より好ましくは０である。また、一般式（Ｉ）において、ｍ’は０～３の整数であり、好ましくは０又は１であり、より好ましくは０である。
In general formula (I), m is an integer of 0 to 3, preferably 0 or 1, more preferably 0. In general formula (I), m' is an integer of 0 to 3, preferably 0 or 1, more preferably 0.
一般式（Ｉ）において、ｎ＋ｍは１～４の整数であり、好ましくは１～３の整数であり、より好ましくは１又は２であり、特に好ましくは１である。また、ｎ’＋ｍ’は１～４の整数であり、好ましくは１～３の整数であり、より好ましくは１又は２であり、特に好ましくは１である。
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. Also, 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.
前述の極性官能基を有する基としては、極性官能基として、少なくともカチオン及びカウンターアニオンを有する官能基、アニオン及びカウンターカチオンを有する官能基、並びに双性イオンを有する官能基から選択される少なくとも１つの官能基を有することが好ましい態様の一つである。極性官能基を有する基が、これらの極性官能基を有すると、極性官能基を有するジベンゾシクロオクタジインは、特に溶解性に優れる傾向があるため好ましい。また、細胞表面はアニオン性を有する傾向にあるため、極性官能基として、カチオン及びカウンターアニオンを有することが、細胞表面のアジド基と極性官能基を有するジベンゾシクロオクタジインとの反応性の観点からより好ましい。
As 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. When the group having a polar functional group has these polar functional groups, dibenzocyclooctadiyne having a polar functional group tends to be particularly excellent in solubility, which is preferable. In addition, since 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.
極性官能基を有するジベンゾシクロオクタジインとしては、具体例としては下記式１ａ～１ｊから選択される少なくとも１種の化合物が挙げられ、これらの化合物は特に好ましい。式１ａ～１ｊから選択される少なくとも１種の化合物としては、１aが好ましい。なお、式１ｂ～１ｊの化合物は新規化合物である。本開示において、「１ａの化合物」を、単に「１ａ」とも記し、「１ｂの化合物」を、単に「１ｂ」とも記し、「１ｃの化合物」を、単に「１ｃ」とも記し、「１ｄの化合物」を、単に「１ｄ」とも記し、「１ｅの化合物」を、単に「１ｅ」とも記し、「１ｆの化合物」を、単に「１ｆ」とも記し、「１ｇの化合物」を、単に「１ｇ」とも記し、「１ｈの化合物」を、単に「１ｈ」とも記し、「１ｉの化合物」を、単に「１ｉ」とも記し、「１ｊの化合物」を、単に「１ｊ」とも記す。
Specific examples of the 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". " is also simply written as "1d", "1e compound" is also simply written as "1e", "1f compound" is also simply written as "1f", and "1g compound" is also simply written as "1g". , "compound of 1h" is also simply written as "1h", "compound of 1i" is also simply written as "1i", and "compound of 1j" is also simply written as "1j".
式１ｇにおいて、ＲＧＤはアルギニン－グリシン－アスパラギン酸からなる３残基のペプチド鎖である。
In formula 1g, RGD is a three-residue peptide chain consisting of arginine-glycine-aspartic acid.
式１ｊにおいて、Ｙはそれぞれ独立に極性官能基を有する基及びアルキル基から選択される基である。
In Formula 1j, each Y is a group independently selected from a group having a polar functional group and an alkyl group.
Ｙが極性官能基を有する基である場合には、前記Ｘで例示した基が挙げられる。但し、Ｙの分子量の上限はＸの上限よりも２８分子量が低い。Ｙがアルキル基である場合には、炭素数１～１８のアルキル基が挙げられ、好ましくは炭素数１～６のアルキル基が挙げられる。アルキル基としては、メチル基が好ましい態様の一つである。
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. 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.
極性官能基を有するジベンゾシクロオクタジインの合成方法としては特に制限はないが、例えば、実施例で記載したＳ５で表されるヒドロキシ基（－ＯＨ）を有するジベンゾシクロオクタジインを合成した後に、該ヒドロキシ基を、従来公知の有機合成法等により、エーテル化し、ヒドロキシ基を、－ＯＲへ変換する方法が挙げられる。別の方法としては実施例で記載したＳ５で表されるヒドロキシ基（－ＯＨ）を有するジベンゾシクロオクタジインを合成した後に、該ヒドロキシ基を、従来公知の有機合成法等により、エステル化する方法が挙げられる。さらに別の方法としては、カルボキシ基（－ＣＯＯＨ）を有するジベンゾシクロオクタジインを合成した後に、該カルボキシ基をエステル化する方法が挙げられる。
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. As another method, after synthesizing dibenzocyclooctadiyne having a hydroxy group (—OH) represented by S5 described in Examples, the hydroxy group is esterified by a conventionally known organic synthesis method or the like. is mentioned. Still another method includes a method of synthesizing dibenzocyclooctadiyne having a carboxy group (--COOH) and then esterifying the carboxy group.
（複合体）
本実施形態に係る複合体は、前述のように細胞表面にアジド基を有する細胞とアジド基を有する材料（但し、アジド基を有する蛍光色素を除く）とが、極性官能基を有するジベンゾシクロオクタジインによって結合した複合体である。 (complex)
As described above, 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.
本実施形態に係る複合体は、前述のように細胞表面にアジド基を有する細胞とアジド基を有する材料（但し、アジド基を有する蛍光色素を除く）とが、極性官能基を有するジベンゾシクロオクタジインによって結合した複合体である。 (complex)
As described above, 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.
極性官能基を有するジベンゾシクロオクタジインは、分子内に二つのアルキンを有しており、片方が細胞表面にアジド基を有する細胞のアジド基と、ＳＰＡＡＣ反応し、他方がアジド基を有する材料のアジド基と、ＳＰＡＡＣ反応することにより、細胞と材料とが、極性官能基を有するジベンゾシクロオクタジインによって結合した複合体を得ることができる。
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.
本実施形態に係る複合体は、通常結合することが困難な細胞（細胞表面にアジド基を有する細胞）同士や、細胞と、通常結合することが困難な無機材料とを容易に結合することができるため、従来困難であった様々な研究への利用が可能である。例えば、材料が、細胞表面にアジド基を有する細胞である場合には、スフェロイドを製造することが容易に可能であり、細胞が難接着性細胞であっても、細胞の凝集体を容易に得ることができるため、難接着性細胞の培養を行うことができる。また、無機材料として、マイクロ流路を備える材料を用い、マイクロ流路の所望の位置をアジド基で修飾することにより、所望の位置に細胞を結合することができる。また、細胞シート上にさらに細胞を積層し複層組織を形成する利用法が考えられる。
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. For example, when 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. In addition, by using 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. In addition, a method of use in which cells are further layered on the cell sheet to form a multi-layered tissue is conceivable.
複合体の構造を、極性官能基を有するジベンゾシクロオクタジインが、上記一般式（ＩＶ）で表される化合物である場合を例にすると、以下の一般式（２－１）～（２－４）で表すことができる。
Taking the structure of the complex as an example where dibenzocyclooctadiyne having a polar functional group is a compound represented by the general formula (IV), the following general formulas (2-1) to (2-4) ).
複合体の構造を、極性官能基を有するジベンゾシクロオクタジインが、上記一般式（ＩＶ）で表される化合物であり、一般式（ＩＶ）における二つのＲが異なる基、Ｒ１及びＲ２である場合を例にすると、以下の化合物群（一般式（３））で表すことができる。
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)).
本実施形態に係る複合体は、同種の細胞、同種の材料、同種の極性官能基を有するジベンゾシクロオクタジインを用いた場合でも、上記式（２－１）～（２－４）や式（３）で示したように、複合体の構造として、異なる構造となることがあるが、本開示において特に言及した場合を除き、上記式（２－１）～（２－４）で表される構造や式（３）で表される化合物群は特に区別しないものとする。
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.
＜複合体の製造方法＞
本実施形態に係る複合体の製造方法は、前述の本実施形態に係る複合体を製造するための方法である。本実施形態に係る複合体の製造方法は、大きく二つの態様に分けられる。 <Method for producing composite>
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.
本実施形態に係る複合体の製造方法は、前述の本実施形態に係る複合体を製造するための方法である。本実施形態に係る複合体の製造方法は、大きく二つの態様に分けられる。 <Method for producing composite>
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.
第一の態様の複合体の製造方法は、細胞表面にアジド基を有する細胞と、極性官能基を有するジベンゾシクロオクタジインとを結合し、極性官能基を有するジベンゾシクロオクタジインと細胞との結合体（Ａ）を得る工程、及び、結合体（Ａ）と、アジド基を有する材料（但し、アジド基を有する蛍光色素を除く）とを結合し、細胞と材料とが極性官能基を有するジベンゾシクロオクタジインによって結合した複合体を得る工程を有する、複合体の製造方法である。
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 step of obtaining the body (A), and binding the conjugate (A) with a material having an azide group (excluding a fluorescent dye having an azide group), and the cell and the material are dibenzos having a polar functional group A method for producing a conjugate, comprising the step of obtaining a conjugate bound by cyclooctadiyne.
第二の態様の複合体の製造方法は、アジド基を有する材料（但し、アジド基を有する蛍光色素を除く）と、極性官能基を有するジベンゾシクロオクタジインとを結合し、極性官能基を有するジベンゾシクロオクタジインと材料との結合体（Ｂ）を得る工程、及び、結合体（Ｂ）と、細胞表面にアジド基を有する細胞とを結合し、細胞と材料とが極性官能基を有するジベンゾシクロオクタジインによって結合した複合体を得る工程を有する、複合体の製造方法である。
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.
第一の態様と、第二の態様の最も大きな違いは、細胞と、材料とのどちらを先に極性官能基を有するジベンゾシクロオクタジインと結合させるかである。第一の態様では、先に細胞と結合させ、結合体（Ａ）を得た後に、材料と結合させる。一方で、第二の態様では、先に材料と結合させ、結合体（Ｂ）を得た後に、細胞と結合させる。第一の態様、第二の態様のいずれであっても、反応は速やかに進行し、複合体を製造することが可能である。
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. In the first aspect, the conjugate is first bound to the cell to obtain the conjugate (A), and then bound to the material. On the other hand, in the second aspect, the material is first bound to obtain the conjugate (B), and then the cell is bound. In either of the first aspect and the second aspect, the reaction proceeds rapidly and it is possible to produce a complex.
結合体（Ａ）を得る方法としては、細胞表面にアジド基を有する細胞と、極性官能基を有するジベンゾシクロオクタジインとを接触させることができればよく、特に制限はないが、例えば、細胞表面にアジド基を有する細胞を含む細胞懸濁液を調製し、細胞懸濁液に極性官能基を有するジベンゾシクロオクタジインを添加し、インキュベートすることにより、結合体（Ａ）を得る方法が挙げられる。細胞懸濁液に添加する極性官能基を有するジベンゾシクロオクタジインは、極性官能基を有するジベンゾシクロオクタジインを含む溶液として添加することが好ましい。
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.
結合体（Ｂ）を得る方法としては、アジド基を有する材料と、極性官能基を有するジベンゾシクロオクタジインとを接触させることができればよく、特に制限はないが、アジド基を有する材料に、極性官能基を有するジベンゾシクロオクタジインを含む溶液を接触させ、結合体（Ｂ）を得る方法が挙げられる。
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.
結合体（Ａ）から複合体を得る方法としては、特に制限はないが、例えば、結合体（Ａ）を含む懸濁液を、アジド基を有する材料上に塗布することにより複合体を得る方法、結合体（Ａ）を含む懸濁液に、アジド基を有する材料を接触、浸漬することにより複合体を得る方法が挙げられる。
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).
結合体（Ｂ）から複合体を得る方法としては、特に制限はないが、例えば、細胞表面にアジド基を有する細胞を含む細胞懸濁液を、結合体（Ｂ）上に塗布することにより複合体を得る方法、細胞表面にアジド基を有する細胞を含む細胞懸濁液に、結合体（Ｂ）を、接触、浸漬することにより複合体を得る方法が挙げられる。
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.
本実施形態に係る複合体の製造方法は、通常は４～４０℃で行うことができ、１５～３７℃で行うことがより好ましい。なお、後述の実施例は特に記載した場合を除き、室温（r.t.）（２０～３０℃）で実施した。
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.
本実施形態に係る複合体の製造方法は、大気下で行っても、不活性ガス（例えば、窒素、アルゴン）雰囲気下で行ってもよい。また、加圧下で行っても、減圧下で行ってもよい。後述の実施例は特に記載した場合を除き、大気下で行った。
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.
以下、実施例を挙げて本実施形態を説明するが、本開示はこれらの例によって限定されるものではない。
The present embodiment will be described below with examples, but the present disclosure is not limited by these examples.
実施例において、フラッシュクロマトグラフィーでは、Silica gel 60 （球形、粒径４０～１００μｍ、関東化学）を使用した。
In the examples, silica gel 60 (spherical, particle size 40-100 μm, Kanto Kagaku) was used in flash chromatography.
実施例において、1H及び13C NMRスペクトルは、JEOL JNM-AL 300、JNM-ECX 400又はJNM-ECA 500を用いて測定した。溶媒としては、CDCl3 (1H NMR; δ=7.26ppm, 13C NMR; δ=77.0ppm)、DMSO-d6 (1H NMR; δ=2.50ppm, 13C NMR; δ=39.5ppm)、CD3CN (1H NMR; δ=1.94ppm, 13C NMR; δ=118.3ppm) 又はD2O (1H NMR; δ=4.79ppm)を用い、括弧内に記載のシグナルを基準とした。
In the examples, 1 H and 13 C NMR spectra were measured using JEOL JNM-AL 300, JNM-ECX 400 or JNM-ECA 500. As a solvent, CDCl 3 ( 1 H NMR; δ=7.26 ppm, 13 C NMR; δ=77.0 ppm), DMSO-d 6 ( 1 H NMR; δ=2.50 ppm, 13 C NMR; δ=39.5 ppm), Signals in brackets were referenced using CD3CN ( 1H NMR; ?=1.94 ppm, 13C NMR; ?=118.3 ppm) or D2O ( 1H NMR; ?=4.79 ppm).
1H NMRのデータは、化学シフト (δ，ppm)、多重度(s, 一重線; d, 二重線; t, 三重線; m, 多重線; br, ブロード)、カップリング定数 (Hz)、積分値について記載した。
13C NMRのデータは、化学シフト (δ，ppm) に関して記載した。 1 H NMR data are given as chemical shift (δ, ppm), multiplicity (s, singlet; d, doublet; t, triplet; m, multiplet; br, broad), coupling constant (Hz) , described the integral value.
13 C NMR data are reported in terms of chemical shifts (δ, ppm).
13C NMRのデータは、化学シフト (δ，ppm) に関して記載した。 1 H NMR data are given as chemical shift (δ, ppm), multiplicity (s, singlet; d, doublet; t, triplet; m, multiplet; br, broad), coupling constant (Hz) , described the integral value.
13 C NMR data are reported in terms of chemical shifts (δ, ppm).
質量スペクトルは、JEOL JMS-T100LC質量分析計を用い、ESI-MSモードにてメタノール溶媒で測定した。
Mass spectra were measured using a JEOL JMS-T100LC mass spectrometer in ESI-MS mode with a methanol solvent.
極性官能基を有するジベンゾシクロオクタジインの合成スキームを以下に示す。
A synthetic scheme for dibenzocyclooctadiyne with a polar functional group is shown below.
スキーム１において、a) 三臭化ホウ素, ジクロロメタン, ０℃→r.t.; b) tert-ブチルジメチルクロロシラン, イミダゾール, ジメチルホルムアミド, r.t.; c) リチウムジイソプロピルアミド, テトラヒドロフラン, －７８℃; d) フッ化水素カリウム, テトラヒドロフラン/メタノール, ０℃である。
In Scheme 1, a) boron tribromide, dichloromethane, 0°C → r.t.; b) tert-butyldimethylchlorosilane, imidazole, dimethylformamide, r.t.; c) lithium diisopropylamide, tetrahydrofuran, -78°C; d) hydrogen fluoride Potassium, tetrahydrofuran/methanol, 0°C.
スキーム２において、a) トリフェニルホスフィン, N,N-ジメチルアミノエタノール,アゾジカルボン酸ジイソプロピル, ０℃→r.t.; b) ヨードメタン, r.t.; c) 1,3-プロパンスルトン, r.t.; d) 1,3-プロパンスルトン, 炭酸セシウム, テトラヒドロフラン, ０℃である。
In Scheme 2, a) triphenylphosphine, N,N-dimethylaminoethanol, diisopropyl azodicarboxylate, 0°C → r.t.; b) iodomethane, r.t.; c) 1,3-propanesultone, r.t.; d) 1,3 -propane sultone, cesium carbonate, tetrahydrofuran, 0°C.
スキーム３において、a) 2,3,4,6-Tetra-O-acetyl-β-D-galactopyranosyl 2,2,2-Trichloroacetimidate、CH2Cl2, TMSOTf, ０℃； b) フッ化水素カリウム, テトラヒドロフラン/メタノール, ０℃； その後炭酸カリウム、ヨードメタン、ジメチルホルムアミド c） 炭酸カリウム, テトラヒドロフラン/メタノールである。
In Scheme 3, a) 2,3,4,6-Tetra-O-acetyl-β-D- galactopyranosyl 2,2,2 - Trichloroacetimidate, CH2Cl2, TMSOTf, 0 °C; b) potassium hydrogen fluoride, Tetrahydrofuran/methanol, 0°C; then potassium carbonate, iodomethane, dimethylformamide c) Potassium carbonate, tetrahydrofuran/methanol.
［合成例１］
前記スキーム１に示したＳ２の化合物を合成した。 [Synthesis Example 1]
A compound of S2 shown inScheme 1 was synthesized.
前記スキーム１に示したＳ２の化合物を合成した。 [Synthesis Example 1]
A compound of S2 shown in
ジクロロメタン中のS1(160mg, 293μmol)の溶液に、三臭化ホウ素(2.06mmol,ジクロロメタン中濃度1.0mol/L)を0℃で添加し、室温・アルゴン雰囲気下で3時間撹拌した。反応混合物にメタノール (1mL)及び飽和炭酸水素ナトリウム水溶液を0℃で滴下した。混合物をジクロロメタンで抽出し、無水硫酸マグネシウムで乾燥させた。残渣をジクロロメタン中で再結晶し、S2 (129mg, 85%)を白色の固体として得た。
Boron tribromide (2.06 mmol, concentration 1.0 mol/L in dichloromethane) was added to a solution of S1 (160 mg, 293 μmol) in dichloromethane at 0°C and stirred for 3 hours at room temperature under an argon atmosphere. Methanol (1 mL) and saturated aqueous sodium hydrogen carbonate 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 (129mg, 85%) as a white solid.
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); 13C-NMR (76 MHz, DMSO-D6) δ 158.25, 143.91, 138.68, 138.35, 137.10, 134.20, 131.80, 129.48, 127.65, 118.66, 115.77, 113.56; HRMS (ESI-TOF) 539.0599 (M + Na)+calcd for C28H20O6S2Na 539.0599.
1 H-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 C-NMR (76 MHz, DMSO-D6) δ 158.25, 143.91, 138.68, 138.35, 137.10, 134.20, 131.80, 129.48, 127.65, 118.66, 115.77, 113.56; + calcd for C28H20O6S2Na 539.0599 .
［合成例２］
前記スキーム１に示したＳ３の化合物を合成した。 [Synthesis Example 2]
A compound of S3 shown inScheme 1 was synthesized.
前記スキーム１に示したＳ３の化合物を合成した。 [Synthesis Example 2]
A compound of S3 shown in
S2(124mg, 240μmol)とイミダゾール (130mg, 1.91mmol)をジメチルホルムアミド(3mL) に溶解し、tert-ブチルジメチルクロロシラン(199mg, 1.32mmol)を加え、アルゴン雰囲気下で5時間撹拌した。反応混合物に酢酸エチルを加え、水で洗浄し、無水硫酸マグネシウムで乾燥させた。残渣を酢酸エチル中で再結晶して、S3 (114mg, 64%) を白色の固体として得た。なお、ＴＢＳは、tert-ブチルジメチルシリル基を意味する。
Dissolve S2 (124 mg, 240 μmol) and imidazole (130 mg, 1.91 mmol) in dimethylformamide (3 mL), add tert-butyldimethylchlorosilane (199 mg, 1.32 mmol), and stir for 5 hours under an argon atmosphere. Ethyl acetate was added to the reaction mixture, washed with water, and dried over anhydrous magnesium sulfate. The residue was recrystallized in ethyl acetate to give S3 (114mg, 64%) as a white solid. Incidentally, TBS means a tert-butyldimethylsilyl group.
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); 13C-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 C40H48O6S2Si2Na 767.2329.
1 H-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); , 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 C40H48O6S27Si2Na296
［合成例３］
前記スキーム１に示したＳ４の化合物を合成した。 [Synthesis Example 3]
A compound of S4 shown inScheme 1 was synthesized.
前記スキーム１に示したＳ４の化合物を合成した。 [Synthesis Example 3]
A compound of S4 shown in
S3(114mg, 153μmol)をテトラヒドロフラン(4.1mL)に溶解し、リチウムジイソプロピルアミド(780μmol、ヘキサン/テトラヒドロフラン溶液1mol/L)を－78℃で滴下し、40分間撹拌した。反応混合物に、飽和塩化アンモニウム水溶液を0℃で加え、酢酸エチルで抽出し、無水硫酸マグネシウムで乾燥させた。残渣をシリカゲルカラムクロマトグラフィー[ヘキサン/酢酸エチル(15:1)]で精製して、S4(56.5mg,82%)をオレンジ色の固体として得た。
S3 (114 mg, 153 μmol) was dissolved in tetrahydrofuran (4.1 mL), lithium diisopropylamide (780 μmol, hexane/tetrahydrofuran solution 1 mol/L) was added dropwise at -78°C, and stirred for 40 minutes. A saturated aqueous ammonium chloride solution was added to the reaction mixture at 0° C., 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.
1H-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); 13C-NMR (101 MHz, CHLOROFORM-D) δ 156.76, 134.99, 128.04, 124.88, 119.92, 119.26, 110.20, 107.68, 25.74, 18.34, -4.27; HRMS (ESI-TOF) 483.2153 (M + Na)+ calcd for C28H36O2Si2Na 483.2152.
1 H-NMR (500 MHz, CDCl 3 ). 1 H-NMR (400 MHz, CDCl 3 ) 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); , 110.20, 107.68, 25.74, 18.34, -4.27; HRMS (ESI-TOF) 483.2153 (M + Na) + calcd for C28H36O2Si2Na 483.2152 .
［合成例４］
前記スキーム１に示したＳ５の化合物及びＳ７の化合物を合成した。 [Synthesis Example 4]
A compound of S5 and a compound of S7 shown inScheme 1 were synthesized.
前記スキーム１に示したＳ５の化合物及びＳ７の化合物を合成した。 [Synthesis Example 4]
A compound of S5 and a compound of S7 shown in
S4(57mg, 123μmol)をTHF－MeOH (3:1, 2.0mL)に溶解し、フッ化水素カリウム水溶液(60μmol, 1M) を0℃で加えた。混合物を30分間撹拌した後、塩酸(1.2M, 60μL)及び飽和食塩水を混合物に加えた。有機相を酢酸エチルで抽出し、無水硫酸マグネシウムで乾燥させた残渣をシリカゲルカラム[ヘキサン/酢酸エチル(5:1)]で精製し、S5(12mg, 39%)、S7(25.1mg, 59%)を黄色の粉末として得た。S5は濃縮状態で不安定なため、酢酸エチルに溶解し－30 ℃で保存した。
S4 (57 mg, 123 μmol) was dissolved in THF-MeOH (3:1, 2.0 mL), and an aqueous potassium hydrogen fluoride solution (60 μmol, 1 M) was added at 0°C. After stirring the mixture for 30 minutes, hydrochloric acid (1.2 M, 60 μL) and saturated brine were added to the mixture. The organic phase was extracted with ethyl acetate, and the residue dried over anhydrous magnesium sulfate was purified with a silica gel column [hexane/ethyl acetate (5:1)] to obtain S5 (12 mg, 39%), S7 (25.1 mg, 59%). ) was obtained as a yellow powder. Since S5 is unstable in a concentrated state, it was dissolved in ethyl acetate and stored at -30°C.
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); 13C-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 C16H7O2231.0449;
S7: 1H-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); 13C-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 C22H21O2SiNa 345.1311. S5: 1 H-NMR (400 MHz, CD 3 CN) 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 C16H7O2 231.0449 ;
S7: 1 H-NMR (301 MHz, CDCl 3 ) 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); , 128.12, 124.73, 124.41, 119.99, 119.32, 115.48, 114.36, 110.57, 110.07, 107.42, 107.42, 77.42, 77.42, 77.42, 76.94, 25.34, -14.28; HRMS (ESI -TOF) 345.104 for C22H21O2SiNa 345.1311 .
S7: 1H-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); 13C-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 C22H21O2SiNa 345.1311. S5: 1 H-NMR (400 MHz, CD 3 CN) 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 C16H7O2 231.0449 ;
S7: 1 H-NMR (301 MHz, CDCl 3 ) 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); , 128.12, 124.73, 124.41, 119.99, 119.32, 115.48, 114.36, 110.57, 110.07, 107.42, 107.42, 77.42, 77.42, 77.42, 76.94, 25.34, -14.28; HRMS (ESI -TOF) 345.104 for C22H21O2SiNa 345.1311 .
［合成例５］
前記スキーム２に示したＳ６の化合物を合成した。 [Synthesis Example 5]
A compound of S6 shown inScheme 2 was synthesized.
前記スキーム２に示したＳ６の化合物を合成した。 [Synthesis Example 5]
A compound of S6 shown in
アルゴン雰囲気下、S5(40mg, 172μmol) 及びトリフェニルホスフィン(181mg, 689μmol)を乾燥THF(2.0mL)に溶解し、N,N-ジメチルアミノエタノール(69μL, 689μmol)及びアゾジカルボン酸ジイソプロピル(135 μL, 689μmol)を0℃で加えた。室温・アルゴン雰囲気下で４時間攪拌した後、反応混合物から溶媒を減圧留去した。残渣をシリカゲルカラム[クロロホルム/メタノール (1:1)]で精製し、黄色の粉末S6 (41mg,64%)を得た。
Under an argon atmosphere, S5 (40 mg, 172 μmol) and triphenylphosphine (181 mg, 689 μmol) were dissolved in dry THF (2.0 mL), N,N-dimethylaminoethanol (69 μL, 689 μmol) and diisopropyl azodicarboxylate (135 μL). , 689 μmol) was added at 0°C. After stirring for 4 hours at room temperature under an argon atmosphere, the solvent was distilled off from the reaction mixture under reduced pressure. The residue was purified with a silica gel column [chloroform/methanol (1:1)] to obtain yellow powder S6 (41 mg, 64%).
［合成例６］
前記スキーム２に示した１ａの化合物を合成した。 [Synthesis Example 6]
A compound of 1a shown inScheme 2 above was synthesized.
前記スキーム２に示した１ａの化合物を合成した。 [Synthesis Example 6]
A compound of 1a shown in
S6に塩酸(1.2M, 40μL)を加え、減圧留去して、黄色の粉末1aを得た。
Hydrochloric acid (1.2 M, 40 μL) was added to S6 and evaporated under reduced pressure to obtain yellow powder 1a.
1H-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); 13C-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 C24H27N2O2375.2075.
1 H-NMR (301 MHz, D 2 O) 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, D 2 O) 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 C24H27N2O2 375.2075 .
［実施例１］
前記スキーム２に示した１ｂの化合物を合成した。 [Example 1]
Compound 1b shown in Scheme 2 above was synthesized.
前記スキーム２に示した１ｂの化合物を合成した。 [Example 1]
S6(17mg,45.4μmol)に、ヨードメタン(500μL)を加え8時間攪拌・加熱還流した。反応溶液を濃縮後、ODSカラム (Sep-Pak C18, Waters)[飽和食塩水→H2O:MeOH = 9:1]で精製し、1bを黄色の粉末 (3.7 mg, 17%)として得た。
Iodomethane (500 μL) was added to S6 (17 mg, 45.4 μmol), and the mixture was stirred and heated under reflux for 8 hours. After concentrating the reaction solution, it was purified with an ODS column (Sep-Pak C18, Waters) [saturated saline → H 2 O:MeOH = 9:1] to obtain 1b as a yellow powder (3.7 mg, 17%). .
1H-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); 13C-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 C26H32N2O2404.2464.
1 H-NMR (500 MHz, D 2 O) 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); , 64.91, 61.89, 53.92; HRMS (ESI - TOF) 404.2461 calcd for C26H32N2O2 404.2464 .
［実施例２］
前記スキーム２に示した１ｃの化合物を合成した。 [Example 2]
A compound of 1c shown inScheme 2 above was synthesized.
前記スキーム２に示した１ｃの化合物を合成した。 [Example 2]
A compound of 1c shown in
S6(22mg, 58.7μmol)に、1,3-プロパンスルトン(500μL)を加え、40℃で7時間攪拌した。反応溶液をODSカラム (Sep-Pak C18, Waters) [H2O:MeOH = 1:1]で精製し、1cを黄色の粉末 (5.5mg, 15%)として得た。
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 with an ODS column (Sep-Pak C18, Waters) [H 2 O:MeOH = 1:1] to obtain 1c as a yellow powder (5.5mg, 15%).
1H-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); 13C-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 C30H38N2NaO8S2641.1967.
1 H-NMR (500 MHz, D 2 O) 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); -Nmr (126 mhz, d 2 o) δ 158.30, 134.04, 128.59, 123.98, 115.56, 115.44, 113.39, 107.82, 103.41, 63.44, 62.91, 61.91 ( M + Na) + calcd for C30H38N2NaO8S2 641.1967 .
［実施例３］
前記スキーム２に示した１ｄの化合物を合成した。 [Example 3]
Compound 1d shown in Scheme 2 above was synthesized.
前記スキーム２に示した１ｄの化合物を合成した。 [Example 3]
S5(3.6mg, 15.5μmol)を乾燥テトラヒドロフラン(500μL)に溶解し、炭酸セシウム(25.2mg, 77.3μmol) 及び1,3-プロパンスルトン(6.8μL, 77.4μmol)を０℃で加えた。０℃で18時間撹拌した後、得られた固体を収集し、ODSカラム[50% 飽和食塩水→水/メタノール (4:1)]で精製し1d(3.4 mg, 22％)を得た。
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-propanesultone (6.8 μL, 77.4 μmol) were added at 0°C. After stirring for 18 h at 0° C., the resulting solid was collected and purified by ODS column [50% saturated saline→water/methanol (4:1)] to give 1d (3.4 mg, 22%).
1H-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); 13C-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 C22H18O8S2474.0443.
1 H-NMR (500 MHz, D 2 O) 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, D 2 O) δ159.12, 133.99, 128.39, 123.35, 115.10 , 113.41 , 110.17, 107.50, 66.57, 47.71 , 24.12 ;
［合成例７］
前記スキーム３に示したＳ８の化合物を合成した。 [Synthesis Example 7]
A compound of S8 shown inScheme 3 above was synthesized.
前記スキーム３に示したＳ８の化合物を合成した。 [Synthesis Example 7]
A compound of S8 shown in
CH2Cl2（2mL）中のS7（23.1mg、66.7μmol）および2,3,4,6-テトラ-O-アセチル-β-D-ガラクトピラノシル2,2,2-トリクロロアセトイミデート（49.3mg、0.10mmol）の溶液にTMSOTf（トリフルオロメタンスルホン酸トリメチルシリル）（1.21μL、6.67μmol）をAr雰囲気下0℃で添加した。0℃で1.5時間撹拌した後、反応混合物をEt3N（50μL）でクエンチし、真空で濃縮した。残渣をシリカゲルカラムクロマトグラフィー（ヘキサン/ EtOAc = 4：1）で精製して、S8（15.8mg、0.023mmol、35％）を得た。
S7 (23.1 mg, 66.7 μmol) and 2,3,4,6-tetra-O-acetyl-β-D- galactopyranosyl 2,2,2-trichloroacetimidate in CH2Cl2 ( 2 mL) To a solution of (49.3 mg, 0.10 mmol) was added TMSOTf (trimethylsilyl trifluoromethanesulfonate) (1.21 μL, 6.67 μmol) at 0° C. under Ar atmosphere. After stirring for 1.5 h at 0° C., the reaction mixture was quenched with Et3N (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%).
1H-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 C36H40O11SiNa 699.22376.
1 H-NMR (301 MHz, CDCl 3 ) 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); 13 C NMR (101 MHz, CDCl3) δ 170.73, 170.33, 169.52, 169.37, 157.31, 156.91, 135.36, 134.68, 128.17, 127.74, 127.8 , 120.18, 119.54, 116.34, 115.95, 110.87, 109.41, 108.35, 98.56, 98.56, 72.30, 72.08, 68.02, 62.02, 25.69 + calcd for C36H40O11SiNa 699.22376 .
［合成例８］
前記スキーム３に示したＳ９の化合物を合成した。 [Synthesis Example 8]
A compound of S9 shown inScheme 3 above was synthesized.
前記スキーム３に示したＳ９の化合物を合成した。 [Synthesis Example 8]
A compound of S9 shown in
S8（27.5mg、40.6μmol）のMeOH（200μL）とTHF（600μL）の溶液に、KHF2（40.6μmol）を0℃で加えた。５分間撹拌した後、反応混合物を飽和水溶液でクエンチした。0℃でNH4Clを加え、EtOAcで抽出した。有機層をブラインで洗浄し、MgSO4で乾燥し、濾過し、真空で濃縮してアルコールを得た。そのアルコールとK2CO3（14.2mg、103μmol）のDMF（1 mL）溶液に、MeI（4.31μL、68.4μmol）を室温で加え、70分間撹拌した。反応混合物に、ＥｔＯＡｃを加え、水で洗浄し、MgSO4で乾燥させた。水で洗浄し、溶媒を真空で除去した。残渣をシリカゲルカラムクロマトグラフィー（ヘキサン/ EtOAc = 2：3）で精製して、S9（12.1 mg、52％、2ステップ）を得た。
To a solution of S8 (27.5 mg, 40.6 μmol) in MeOH (200 μL) and THF (600 μL) was added KHF2 (40.6 μmol) at 0°C. After stirring for 5 minutes, the reaction mixture was quenched with saturated aqueous solution. NH 4 Cl was added at 0° C. and extracted with EtOAc. The organic layer was washed with brine, dried over MgSO4 , filtered and concentrated in vacuo to give the alcohol. MeI (4.31 μL, 68.4 μmol) was added to a solution of the alcohol and K 2 CO 3 (14.2 mg, 103 μmol) in DMF (1 mL) at room temperature and stirred for 70 minutes. To the reaction mixture was added EtOAc, washed with water and dried over MgSO4 . Washed with water and solvent 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).
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); 13C 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
1 H-NMR (500 MHz, CDCl 3 ) 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, CDCl 3 ) 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
［実施例４］
前記スキーム３に示した１ｅの化合物を合成した。 [Example 4]
A compound of 1e shown inScheme 3 above was synthesized.
前記スキーム３に示した１ｅの化合物を合成した。 [Example 4]
A compound of 1e shown in
S9（3.4mg、5.90μmol）のMeOH（500μL）およびTHF（500μL）溶液に、K2CO3（4.08mg、29.5μmol）を室温で加えた。55分間撹拌した後、反応混合物に陽イオン交換樹脂（DOWEXTM50Wx8 100-200メッシュ（H）陽イオン交換樹脂）を加え、濾過し、真空で濃縮して、1e（3.0mg、定量的）を得た。
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, cation exchange resin (DOWEX TM 50Wx8 100-200 mesh (H) cation exchange resin) was added to the reaction mixture, filtered and concentrated in vacuo to give 1e (3.0 mg, quantitative). Obtained.
1H-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)
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)
（溶解性試験）
合成した、1a-1d及び、ジベンゾシクロオクタジイン（無置換ＣＯＤＹ）を純水に溶解し３５０ｎｍにおける吸光度を測定した。吸光度から1a-1d、ジベンゾシクロオクタジインの溶解度はそれぞれ、6.8 ｍＭ、3.7 ｍＭ、7.4 ｍＭ、2.0 ｍＭ、0.002 ｍＭと算出された。なお、ジベンゾシクロオクタジインは東京化成工業（製品コード：T3241）より入手した。 (Solubility test)
Synthesized 1a-1d and dibenzocyclooctadiyne (unsubstituted CODY) were dissolved in pure water and the absorbance at 350 nm was measured. The solubilities of 1a-1d and dibenzocyclooctadiyne were calculated from the absorbance to be 6.8 mM, 3.7 mM, 7.4 mM, 2.0 mM and 0.002 mM, respectively. Dibenzocyclooctadiyne was obtained from Tokyo Kasei Kogyo (product code: T3241).
合成した、1a-1d及び、ジベンゾシクロオクタジイン（無置換ＣＯＤＹ）を純水に溶解し３５０ｎｍにおける吸光度を測定した。吸光度から1a-1d、ジベンゾシクロオクタジインの溶解度はそれぞれ、6.8 ｍＭ、3.7 ｍＭ、7.4 ｍＭ、2.0 ｍＭ、0.002 ｍＭと算出された。なお、ジベンゾシクロオクタジインは東京化成工業（製品コード：T3241）より入手した。 (Solubility test)
Synthesized 1a-1d and dibenzocyclooctadiyne (unsubstituted CODY) were dissolved in pure water and the absorbance at 350 nm was measured. The solubilities of 1a-1d and dibenzocyclooctadiyne were calculated from the absorbance to be 6.8 mM, 3.7 mM, 7.4 mM, 2.0 mM and 0.002 mM, respectively. Dibenzocyclooctadiyne was obtained from Tokyo Kasei Kogyo (product code: T3241).
以下の実施例で使用したＰＣ－９細胞(肺がん由来浮遊細胞株)は37℃、5%CO2においてRPMI(10%FBS、1%グルタミン、0.75mg/L炭酸水素ナトリウム、５０００units/L ペニシリン/ストレプトマイシンを含む)中で培養した。ＰＣ－９細胞は1 × 105 cells/mLになるまで培養し3－4日毎に継代した。なお、以下の記載において、ＰＣ－９細胞を、単に細胞とも記す。
PC-9 cells (lung cancer-derived suspension cell line) used in the following examples are 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.
以下の実施例で使用した蛍光顕微鏡には倒立顕微鏡システムであるA1R MP(Nikon)及びECLIPS Ts2(Nikon)を用いた。13mmΦ及び12mmΦ円形カバーガラスは松浪硝子工業から購入した。UV照射器にはMin-Eximer(ウシオ電機)を用いた。
Inverted microscope systems A1R MP (Nikon) and ECLIPS Ts2 (Nikon) were used for the fluorescence microscopes used in the following examples. 13 mmΦ and 12 mmΦ circular cover glasses were purchased from Matsunami Glass Industry. A Min-Eximer (Ushio Inc.) was used as the UV irradiator.
［実施例５］
（ガラス表面の親水化）
方法A1: ビーカーに12 mmΦ円形カバーガラス（Iwaki）を静置し、アセトンに浸漬し超音波洗浄機で3分間処理した。エタノール、メタノール、MilliQ水でも同様に処理した。次に、MilliQ水 : 28 v/v % NH3 aq. : 30 v/v % H2O2aq. = 5:1:1に浸漬し、60℃に昇温した後30分間保持した。次にMilliQ水で十分に洗浄した。
方法A2: 13 mmΦの円形カバーガラスに対し、UV (172 nm) を大気中で15分間照射した。 [Example 5]
(Hydrophilization of glass surface)
Method A1: A 12 mmΦ circular cover glass (Iwaki) was placed in a beaker, immersed in acetone, and treated with an ultrasonic cleaner for 3 minutes. Ethanol, methanol, and MilliQ water were treated in the same manner. Next, it was immersed in MilliQ water: 28 v/v % NH 3 aq.: 30 v/v % H 2 O 2 aq. = 5:1:1, heated to 60°C and held for 30 minutes. It was then thoroughly washed with MilliQ water.
Method A2: A circular cover glass of 13 mmΦ was irradiated with UV (172 nm) in air for 15 minutes.
（ガラス表面の親水化）
方法A1: ビーカーに12 mmΦ円形カバーガラス（Iwaki）を静置し、アセトンに浸漬し超音波洗浄機で3分間処理した。エタノール、メタノール、MilliQ水でも同様に処理した。次に、MilliQ水 : 28 v/v % NH3 aq. : 30 v/v % H2O2aq. = 5:1:1に浸漬し、60℃に昇温した後30分間保持した。次にMilliQ水で十分に洗浄した。
方法A2: 13 mmΦの円形カバーガラスに対し、UV (172 nm) を大気中で15分間照射した。 [Example 5]
(Hydrophilization of glass surface)
Method A1: A 12 mmΦ circular cover glass (Iwaki) was placed in a beaker, immersed in acetone, and treated with an ultrasonic cleaner for 3 minutes. Ethanol, methanol, and MilliQ water were treated in the same manner. Next, it was immersed in MilliQ water: 28 v/v % NH 3 aq.: 30 v/v % H 2 O 2 aq. = 5:1:1, heated to 60°C and held for 30 minutes. It was then thoroughly washed with MilliQ water.
Method A2: A circular cover glass of 13 mmΦ was irradiated with UV (172 nm) in air for 15 minutes.
（ガラス表面のアジド修飾）
方法B1: 親水処理したガラス板をメタノールで超音波洗浄し、トルエンで置換したのち、3-アミノプロピルトリエトキシシラン(APTES) (1.0v/v% in toluene, 10mL) で15分間処理した。その後、メタノール、MilliQ水で超音波洗浄した。さらに、ガラス一枚につき200μLのsulfo-SANPAH（スルフォスクシニミジル 6-（4'-アジド-2'-ニトロフェニルアミノ）ヘキサノアート） (2.0mg/mL in 20mM HEPES buffer, pH8.5) と暗所で30分間反応させた後、RPMI (－) FBSで洗浄した。 (Azide modification of glass surface)
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. In addition, 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 and reacted in the dark for 30 minutes, then washed with RPMI (-) FBS.
方法B1: 親水処理したガラス板をメタノールで超音波洗浄し、トルエンで置換したのち、3-アミノプロピルトリエトキシシラン(APTES) (1.0v/v% in toluene, 10mL) で15分間処理した。その後、メタノール、MilliQ水で超音波洗浄した。さらに、ガラス一枚につき200μLのsulfo-SANPAH（スルフォスクシニミジル 6-（4'-アジド-2'-ニトロフェニルアミノ）ヘキサノアート） (2.0mg/mL in 20mM HEPES buffer, pH8.5) と暗所で30分間反応させた後、RPMI (－) FBSで洗浄した。 (Azide modification of glass surface)
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. In addition, 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 and reacted in the dark for 30 minutes, then washed with RPMI (-) FBS.
方法B2: 親水処理したガラス板を、1-アジド-11-(トリエトキシシリル)ウンデカン溶液 (0.50v/v%トルエン溶液, 7mL)で、直径7cmテフロン（登録商標）シャーレ上で15分間処理した。ガラスをトルエン、メタノールで順に洗浄し、減圧条件下で2時間乾燥させた。
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.
（ガラス表面への1a、1b、1c又は1dの結合）
方法C: 方法Bを用いてアジド修飾したガラス板を、24ウェルプレート中、1a、1b、1c又は1dを50μM含む溶液(50μM in water, 500μL)で5分間処理した。その後、MilliQ水(500μL)で3回洗浄し、時間を置かずに次の実験に用いた。 (Binding of 1a, 1b, 1c or 1d to the glass surface)
Method C: Azide-modified glass plates using Method B were treated with a solution containing 50 μM of 1a, 1b, 1c or 1d (50 μM in water, 500 μL) in a 24-well plate for 5 minutes. After that, it was washed three times with MilliQ water (500 μL) and immediately used for the next experiment.
方法C: 方法Bを用いてアジド修飾したガラス板を、24ウェルプレート中、1a、1b、1c又は1dを50μM含む溶液(50μM in water, 500μL)で5分間処理した。その後、MilliQ水(500μL)で3回洗浄し、時間を置かずに次の実験に用いた。 (Binding of 1a, 1b, 1c or 1d to the glass surface)
Method C: Azide-modified glass plates using Method B were treated with a solution containing 50 μM of 1a, 1b, 1c or 1d (50 μM in water, 500 μL) in a 24-well plate for 5 minutes. After that, it was washed three times with MilliQ water (500 μL) and immediately used for the next experiment.
（ガラス表面への細胞の結合（接着）及び、接着の定量）
5.0×104cells/mLの細胞を、10cmΦの円形ディッシュ中で、100μMのAc4ManNAzと0.1%DMSOを含む10 mLの培地で培養した。24時間後、細胞をRPMI(－)FBS (10 mL) で洗浄した後のち、RPMI(－)FBS中、細胞濃度が1.0×105cells/mLの細胞懸濁液を調製した。 (Binding (adhesion) of cells to glass surface and quantification of adhesion)
Cells at 5.0×10 4 cells/mL were cultured in 10 mL medium containing 100 μM Ac 4 ManNAz and 0.1% DMSO in a 10 cmΦ circular dish. After 24 hours, the cells were washed with RPMI(-)FBS (10 mL), and a cell suspension with a cell concentration of 1.0×10 5 cells/mL was prepared in RPMI(-)FBS.
5.0×104cells/mLの細胞を、10cmΦの円形ディッシュ中で、100μMのAc4ManNAzと0.1%DMSOを含む10 mLの培地で培養した。24時間後、細胞をRPMI(－)FBS (10 mL) で洗浄した後のち、RPMI(－)FBS中、細胞濃度が1.0×105cells/mLの細胞懸濁液を調製した。 (Binding (adhesion) of cells to glass surface and quantification of adhesion)
Cells at 5.0×10 4 cells/mL were cultured in 10 mL medium containing 100 μM Ac 4 ManNAz and 0.1% DMSO in a 10 cmΦ circular dish. After 24 hours, the cells were washed with RPMI(-)FBS (10 mL), and a cell suspension with a cell concentration of 1.0×10 5 cells/mL was prepared in RPMI(-)FBS.
この細胞懸濁液1mLに1a、1b、1c、1d、又は無置換CODY（Dibenzocyclooctadiyne：5,6,11,12-Tetradehydrodibenzo[a,e]cyclooctene）(10mM aq.)を5μL加え(終濃度50μM)、5分間インキュベートした。培地を除き、500μLのRPMI(－)FBSに懸濁した後、方法A1及びB1によってアジド修飾したガラス板に播種し解析した(図2B, C参照)。同様に、方法A1及びB2によってアジド修飾したガラス板に対しても播種し解析した(図3参照)。ネガティブコントロールにおいては、1a-1dを含まない以外は同様に処理した。
To 1 mL of this cell suspension, add 5 μL of 1a, 1b, 1c, 1d, or unsubstituted CODY (Dibenzocyclooctadiyne: 5,6,11,12-Tetradehydrodibenzo[a,e]cyclooctene) (10 mM aq.) (final concentration 50 μM ), incubated for 5 minutes. The medium was removed, suspended in 500 μL of RPMI(−) FBS, seeded on an azide-modified glass plate by methods A1 and B1, and analyzed (see FIGS. 2B and 2C). Similarly, azide-modified glass plates by methods A1 and B2 were seeded and analyzed (see FIG. 3). Negative controls were treated in the same manner except that 1a-1d was not included.
（ガラス表面への細胞の結合（接着）、接着の経時変化及び細胞生存率）
5.0×104cells/mLの細胞を、10cmΦの円形ディッシュ中で、100μMのAc4ManNAzと0.1%DMSOを含む10 mLの培地で培養した。24時間後、細胞をRPMI(－)FBS (10 mL) で洗浄した後のち、RPMI(－)FBS中、細胞濃度が1.0×105cells/mLの細胞懸濁液を調製した。 (Binding (Adhesion) of Cells to Glass Surface, Changes in Adhesion over Time and Cell Viability)
Cells at 5.0×10 4 cells/mL were cultured in 10 mL medium containing 100 μM Ac 4 ManNAz and 0.1% DMSO in a 10 cmΦ circular dish. After 24 hours, the cells were washed with RPMI(-)FBS (10 mL), and a cell suspension with a cell concentration of 1.0×10 5 cells/mL was prepared in RPMI(-)FBS.
5.0×104cells/mLの細胞を、10cmΦの円形ディッシュ中で、100μMのAc4ManNAzと0.1%DMSOを含む10 mLの培地で培養した。24時間後、細胞をRPMI(－)FBS (10 mL) で洗浄した後のち、RPMI(－)FBS中、細胞濃度が1.0×105cells/mLの細胞懸濁液を調製した。 (Binding (Adhesion) of Cells to Glass Surface, Changes in Adhesion over Time and Cell Viability)
Cells at 5.0×10 4 cells/mL were cultured in 10 mL medium containing 100 μM Ac 4 ManNAz and 0.1% DMSO in a 10 cmΦ circular dish. After 24 hours, the cells were washed with RPMI(-)FBS (10 mL), and a cell suspension with a cell concentration of 1.0×10 5 cells/mL was prepared in RPMI(-)FBS.
細胞懸濁液500μLを、方法A2及びB2によってアジド修飾したガラス板に方法Cを行うことにより調製した1a、1b、1c、1d、又は無置換CODYが結合したガラス板に加えた。15分間静置した後、培地を除き、FBS入りRPMI 500μLに入れ替え、直後と6時間後に顕微鏡撮影した。生死判別では、トリパンブルー溶液 (0.4% in PBS(－))又は3-(4,5-ジメチル-チアゾール-2-イル)-2, 5-ジフェニルテトラゾリウムブロマイド(MTT)溶液 (5 mg/mL in PBS(－))を培地に添加することにより行った。
500 μL 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 µL 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.
図2Aに、ガラス表面への細胞の結合（接着）の模式図を示す。図2Bに方法A1及びB1によってアジド修飾したガラス板に1bを結合した細胞を播種したものを、反射干渉顕微鏡で解析した顕微鏡写真及び解析した接着面積を示す。図2Bの干渉光の輝度で黒いコントラストを示すエリアが細胞と基板の接着部位を示している。干渉の輝度が細胞と基板との距離hに変換することができるため、生化学的な接着が行われる高さ（h ～ 40 nm）を基準に、強接着領域Contact area（h ＜50 nm）と弱接着領域Contact area（h ＜100 nm）を規定した。1bの添加によって弱接着領域の変化よりも、強接着領域の顕著な増大が確認できたことから、1bによって細胞が基板に強く接着したことを示している。図2Cに方法A1及びB1によってアジド修飾したガラス板に1bを結合した細胞を播種したものの経時変化を反射干渉顕微鏡で解析した顕微鏡写真を示す。図2Cに記した白い矢印は、実際に黒いコントラストの領域が形成される様子を示しており、細胞質の伸展を示している。図3に方法A1及びB2によってアジド修飾したガラス板に、1a、1c又は1dを結合した細胞を播種したものの経時変化を反射干渉顕微鏡で解析した顕微鏡写真（図３右）及び解析した強接着面積（基板との距離h ≦40 nm）（図３左）を示す。図3におけるｔは培養時間（分）を示す。
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). Addition of 1b resulted in a marked increase in the strong adhesion area rather than a change in the weak adhesion area, indicating that cells strongly adhered to the substrate due to 1b. 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. 3, right) of analysis of changes over time of cells bound with 1a, 1c, or 1d that were seeded and analyzed with a reflection interference microscope. (distance h ≤ 40 nm from the substrate) (Fig. 3 left). t in FIG. 3 indicates the culture time (minutes).
図４Aに、ガラス表面への細胞の結合（接着）の模式図を示す。図4Bに、1a-1dを用いてガラスに接着した細胞の経時変化を示す。図4Bにおけるnegative controlでは、1a-1dを用いていない以外は同様に処理した場合の実験結果を示す。図4Bでは、細胞播種直後及び6時間後の写真を示している。1aにおいて6時間後の生死判定をMTT及びトリパンブルーを用いて行った。
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.
［実施例６］
（細胞、細胞間の結合（架橋））
5.0×104cells/mLの細胞を、10cmΦの円形ディッシュ中で、100μMのAc4ManNAzと0.1%DMSOを含む10 mLの培地で培養した。24時間後、細胞をRPMI(－)FBS (10 mL) で洗浄した後のち、RPMI(－)FBS中細胞濃度が2.0×105cells/mLの細胞懸濁液(細胞A懸濁液)を調製した。 [Example 6]
(cells, bonds between cells (crosslinks))
Cells at 5.0×10 4 cells/mL were cultured in 10 mL medium containing 100 μM Ac 4 ManNAz and 0.1% DMSO in a 10 cmΦ 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.
（細胞、細胞間の結合（架橋））
5.0×104cells/mLの細胞を、10cmΦの円形ディッシュ中で、100μMのAc4ManNAzと0.1%DMSOを含む10 mLの培地で培養した。24時間後、細胞をRPMI(－)FBS (10 mL) で洗浄した後のち、RPMI(－)FBS中細胞濃度が2.0×105cells/mLの細胞懸濁液(細胞A懸濁液)を調製した。 [Example 6]
(cells, bonds between cells (crosslinks))
Cells at 5.0×10 4 cells/mL were cultured in 10 mL medium containing 100 μM Ac 4 ManNAz and 0.1% DMSO in a 10 cmΦ 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.
細胞A懸濁液500μLに対し、1aの溶液(100μM in RPMI(－)FBS, 500μL; 終濃度50μM)を加え、5分間インキュベートした後、RPMI(－)FBSで洗浄し、遠心して上清を捨て細胞Bのペレットとした。Controlでは、1aを加えていない以外は同じ操作を行った。
Add solution 1a (100 μM in RPMI(-) FBS, 500 μL; final concentration 50 μM) to 500 μL of cell A suspension, incubate for 5 minutes, wash with RPMI(-) FBS, centrifuge and remove the supernatant. A discarded cell B was used as a pellet. In Control, the same operation was performed except that 1a was not added.
細胞Bのペレットと、細胞A懸濁液500μLを混合し、1500Gで30分間遠心した。形成されたペレットをピペッティングし、凝集を保っている細胞塊を顕微鏡撮影した。
The cell B pellet and 500 μL 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.
なお、実施例６において細胞Aは、アジド（－N3）が導入された細胞であり、細胞Bはアジドを導入した後に、該アジドを介して1aが結合された細胞である。
In 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.
図５Aに、細胞、細胞間の結合（架橋）の模式図を示す。図５Bに結合（接着）した細胞塊の顕微鏡写真を示す。
Fig. 5A shows a schematic diagram of cells and bonds (crosslinks) between cells. FIG. 5B shows a micrograph of the bound (adhered) cell mass.
［実施例７］
（AFMカンチレバーへの細胞の結合（接着））
AFMカンチレバー（原子間力顕微鏡カンチレバー）（OMCL-TR400-PSA、Olympus）に対し、UVオゾンクリーナー（ProCleaner、Bioforce）を15分間照射した。このカンチレバーをAPTES（3-アミノプロピルトリエトキシシラン）(200μL, 1.0v/v% in toluene)に浸漬したのち、メタノールで洗浄し乾燥させた。 [Example 7]
(Binding (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 μL, 1.0 v/v% in toluene), washed with methanol and dried.
（AFMカンチレバーへの細胞の結合（接着））
AFMカンチレバー（原子間力顕微鏡カンチレバー）（OMCL-TR400-PSA、Olympus）に対し、UVオゾンクリーナー（ProCleaner、Bioforce）を15分間照射した。このカンチレバーをAPTES（3-アミノプロピルトリエトキシシラン）(200μL, 1.0v/v% in toluene)に浸漬したのち、メタノールで洗浄し乾燥させた。 [Example 7]
(Binding (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 μL, 1.0 v/v% in toluene), washed with methanol and dried.
このカンチレバーをさらに下記ｓ１０の溶液(50μL, 2.0mg/mL in 20 mM HEPES（4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid） buffer, pH8.5) で30分間処理したのち、RPMI(－)FBSで洗浄した。
This cantilever was further treated with the following s10 solution (50 μL, 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.
5.0×104cells/mLの細胞を、10cmΦの円形ディッシュ中で、100μMのAc4ManNAzと0.1%DMSOを含む10 mLの培地で培養した。24時間後、細胞をRPMI(－)FBS (10 mL) で洗浄した後のち細胞をRPMI(－)FBS (10 mL)に懸濁した。一方、カンチレバーを1b (50μL, 50μM in RPMI(－)FBS) と15分間反応させ、RPMI(－)FBSで洗浄した。原子間力顕微鏡(AFM, NanoWizard, JPK)を用いてカンチレバーの表面を細胞に5分間接触させた。
Cells at 5.0×10 4 cells/mL were cultured in 10 mL medium containing 100 μM Ac 4 ManNAz and 0.1% DMSO in a 10 cmΦ 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 µL, 50 µM 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).
カンチレバー表面に細胞が接着したことを確認するため、カンチレバーを基板表面から遠ざけたときの細胞の透過像を観察した(図６b参照)。次に、この細胞が接着したカンチレバーを、実施例５に記載の方法A1、B1及びCを用いて1bで修飾したガラスに対して接触させ、その後引き剥がした時にカンチレバーにかかる力を計測し、フォース－ディスタンスカーブを取得した(図６c参照)。
In order to confirm that the cells adhered to the cantilever surface, a transmission image of the cells was observed when the cantilever was moved away from the substrate surface (see Fig. 6b). Next, the cantilever to which the cells adhered was brought into contact with the glass modified with 1b using methods A1, B1 and C described in Example 5, and then the force applied to the cantilever when it was peeled off was measured, A force-distance curve was obtained (see Figure 6c).
図６Aに、1bを用いた細胞へのカンチレバー架橋の模式図を示す。図６A（１）：1bと反応させたアジド修飾カンチレバーを、あらかじめAc4ManNAzで24時間処理した細胞に接触させたところ、細胞とカンチレバーが即座に接着された。（２）：アジド化細胞付きカンチレバーを方法Cにより1bで修飾したガラスに接触させたところ、ガラスと細胞が即座に接着された。（３）及び（４）：カンチレバーを引き上げることで、接着した細胞をガラスから持ちあげた。図６Bは前記（１）においてカンチレバーに細胞が結合している状態を示す写真である。図６Cは、横軸が細胞とカンチレバーの距離（単位μｍ）、縦軸がカンチレバーのたわみの大きさ（すなわち、カンチレバーにかかっている力の尺度となる）（単位a.u.）を示す。点線（Approach）に沿ってアジド化細胞付きカンチレバーを1bで修飾したガラスにアプローチし(図６C（２）)、実線（Retract）に沿ってカンチレバーを引き上げた (図６C（３）、（４）)。細胞と基板との結合が強固であったため、引き上げる際にカンチレバーのたわみが検出上限を超えている。
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. 6C, the horizontal axis indicates the distance between the cell and the cantilever (unit: μm), 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.
［参考例１］
5.0×104cells/mLの細胞を、10cmΦの円形ディッシュ中で、100μMのAc4ManNAzと0.1%DMSOを含む10 mLの培地で培養した。24時間後、細胞をRPMI(－)FBS (10 mL)で洗浄した。 [Reference example 1]
Cells at 5.0×10 4 cells/mL were cultured in 10 mL medium containing 100 μM Ac 4 ManNAz and 0.1% DMSO in a 10 cmΦ circular dish. After 24 hours, cells were washed with RPMI(-)FBS (10 mL).
5.0×104cells/mLの細胞を、10cmΦの円形ディッシュ中で、100μMのAc4ManNAzと0.1%DMSOを含む10 mLの培地で培養した。24時間後、細胞をRPMI(－)FBS (10 mL)で洗浄した。 [Reference example 1]
Cells at 5.0×10 4 cells/mL were cultured in 10 mL medium containing 100 μM Ac 4 ManNAz and 0.1% DMSO in a 10 cmΦ circular dish. After 24 hours, cells were washed with RPMI(-)FBS (10 mL).
1.0 × 105個の細胞を、1a、1b、1c、1d、又は無置換CODY（Dibenzocyclooctadiyne：5,6,11,12-Tetradehydrodibenzo[a,e]cyclooctene）を50μM含む溶液(50μM in RPMI(－)FBS, 500μL)で5分間処理した後、RPMI(－)FBS (500μL)で洗浄した。
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 µM (50 µM in RPMI (－ )FBS, 500 μL) for 5 minutes, followed by washing with RPMI(−)FBS (500 μL).
次に、細胞をFITC-PEG3-N3溶液 (10μM in RPMI(－)FBS, 0.1%DMSO, 500μL)で5分間処理したのち、RPMI(－)FBS (1mL)及びPBS(－)(1mL)で洗浄した。細胞をパラホルムアルデヒド溶液 (4% in PBS(－))で30分間固定した後、PBS(－)、MilliQ水(各500μL)で洗浄し、MilliQ水(25μL)に懸濁した。全量をスライドガラスに乗せ、1時間風乾した。マウント剤(Fluorescence Mounting Medium, DAKO; 10μL)を滴下し、カバーガラスで封入した。
Next, the cells were treated with FITC-PEG 3 -N 3 solution (10 μM in RPMI(-)FBS, 0.1% DMSO, 500 μL) 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 μL each), and suspended in MilliQ water (25 μL). The whole amount was placed on a slide glass and air-dried for 1 hour. A mounting agent (Fluorescence Mounting Medium, DAKO; 10 µL) was dropped, and the plate was sealed with a cover glass.
蛍光顕微鏡を用いてFITC由来の蛍光を測定し、蛍光強度の定量を行った。なお、無置換CODYは、水への溶解性が低いため、細胞との５分間の処理において、0.1%DMSOを添加した。
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.
前記実験のスキームを図７Aに示す。図７Aでは、(i)培養培地にAc4ManNAzを加えインキュベーション(100μM, 24 hours) することで細胞表面にアジド基を導入し、(ii) CODY誘導体（1a、1b、1c、1d、又は無置換CODY）(50μM, 5 min)と細胞を結合し、(iii) FITC-PEG3-N3(50 μM, 5 min) 処理により、二度の歪み促進型アジド－アルキン付加環化反応を通じて蛍光色素で細胞表面を染色するスキームを図示した。
The scheme of the experiment is shown in Figure 7A. In FIG. 7A, (i) Ac 4 ManNAz was added to the culture medium and incubated (100 μM, 24 hours) to introduce an azide group to the cell surface, and (ii) a CODY derivative (1a, 1b, 1c, 1d, or no Substituted CODY) (50 μM, 5 min) bound cells and (iii) treated with FITC-PEG 3 -N 3 (50 μM, 5 min) to induce fluorescence through a double strain-promoted azide-alkyne cycloaddition reaction. A scheme for staining the cell surface with dyes is illustrated.
前記実験のFITC由来の蛍光の測定結果を図７B、図７Dに示す。図７Bは1a、1b、1c、1dの結果を示し、図７Dは1a、無置換CODYの結果を示す。前記図７Bの結果を定量したものを図７C、図７Dの結果を定量したものを図７Eに示す。
Figures 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, and FIG. 7E shows quantification of the results of FIG. 7D.
［実施例８］
（ガラス表面への細胞の結合（接着）及び、接着の定量）
5.0×104cells/mLの細胞を、6cmΦの円形ディッシュ中で、100μMのAc4ManNAzと0.1%DMSOを含む5mLの培地で培養した。24時間後、培養液を1mL分注し、細胞をRPMI (-) FBS(1mL)で洗浄し、培地を除いた。 [Example 8]
(Binding (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 μM Ac 4 ManNAz and 0.1% DMSO in a 6 cmΦ 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.
（ガラス表面への細胞の結合（接着）及び、接着の定量）
5.0×104cells/mLの細胞を、6cmΦの円形ディッシュ中で、100μMのAc4ManNAzと0.1%DMSOを含む5mLの培地で培養した。24時間後、培養液を1mL分注し、細胞をRPMI (-) FBS(1mL)で洗浄し、培地を除いた。 [Example 8]
(Binding (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 μM Ac 4 ManNAz and 0.1% DMSO in a 6 cmΦ 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.
この細胞に1a、1b、1c又は無置換CODY（Dibenzocyclooctadiyne）の50μM RPMI (-) FBS溶液500μLを加え、5分間インキュベートした。培地を除き、500μLのRPMI (-) FBSで洗浄した後、RPMI (-) FBS 300μLに懸濁した。方法B2を用いてアジド修飾したガラス板を24ウェルプレートに静置し、500μL RPMI (-) FBSを加えた。化合物処理した細胞懸濁液300μLをウェルに加えた。30分後、培地を除き、RPMI 10% FBSを1mL加え、5% CO2、37℃で6時間インキュベートした。その後、培地を3回交換することで浮遊している細胞を取り除いた。接着している細胞をセルスクレイパーで回収し、トリパンブルーで染色して生細胞数をカウントした。播種した細胞数で除することで細胞生着率（生着した細胞/播種した細胞）（％）を算出した（図８）。
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, washed with 500 µL of RPMI (-) FBS, and then suspended in 300 µL of RPMI (-) FBS. Azide-modified glass plates were placed in a 24-well plate using method B2, and 500 μL RPMI (−) FBS was added. 300 μL 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. Adhering cells were collected with a cell scraper, stained with trypan blue, and viable cells were counted. The cell engraftment rate (engrafted cells/seeded cells) (%) was calculated by dividing by the number of seeded cells (Fig. 8).
ネガティブコントロールとして、Ac4ManNAzを使用しないこと以外は同様に行った1aの細胞生着率を算出した。
As a negative control, the cell engraftment rate of 1a was calculated in the same manner except that Ac 4 ManNAz was not used.
［実施例９］
（ガラス表面への細胞の結合（接着）及び、接着の定量）
5.0×104cells/mLの細胞を、6cmΦの円形ディッシュ中で、100μMのAc4ManNAzと0.1%DMSOを含む5mLの培地で培養した。24時間後、培養液を1mL分注し、細胞をRPMI (-) FBS(1mL)で洗浄し、培地を除いた。 [Example 9]
(Binding (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 μM Ac 4 ManNAz and 0.1% DMSO in a 6 cmΦ 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.
（ガラス表面への細胞の結合（接着）及び、接着の定量）
5.0×104cells/mLの細胞を、6cmΦの円形ディッシュ中で、100μMのAc4ManNAzと0.1%DMSOを含む5mLの培地で培養した。24時間後、培養液を1mL分注し、細胞をRPMI (-) FBS(1mL)で洗浄し、培地を除いた。 [Example 9]
(Binding (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 μM Ac 4 ManNAz and 0.1% DMSO in a 6 cmΦ 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.
この細胞に1aの50μM RPMI (-) FBS溶液500μLを加え、5分間インキュベートした。培地を除き、500μLのRPMI (-) FBSで洗浄した後、RPMI (-) FBS 300μLに懸濁した。方法B2を用いてアジド修飾したガラス板を24ウェルプレートに静置し、500μL RPMI (-) FBSを加えた。化合物処理した細胞懸濁液300μLをウェルに加えた。30分後、培地を除き、RPMI 10% FBSを1mL加え、5% CO2、37℃で6時間インキュベートした。その後、培地を3回交換することで浮遊している細胞を取り除いた。接着24時間後の細胞からtotal RNAを抽出した。コントロールとして、Ac4ManNAzで処理したのちガラスに播種はしていない細胞のRNAを用いた (図９A)。コントロールに対して24時間後の細胞で1.5倍以上遺伝子発現が上昇していた241遺伝子について、DAVID1), 2)を用いたGOエンリッチメント解析を行った。molecular function カテゴリーの結果を-log10(P-value)の降順に図９Bに示した。
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. 500 μL of 50 μM RPMI (−) FBS solution of 1a was added to the cells and incubated for 5 minutes. The medium was removed, washed with 500 µL of RPMI (-) FBS, and then suspended in 300 µL of RPMI (-) FBS. Azide-modified glass plates were placed in a 24-well plate using method B2, and 500 μL RPMI (−) FBS was added. 300 μL 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. Total RNA was extracted from cells 24 hours after adhesion. As a control, RNA from cells that had not been plated on glass after treatment with Ac 4 ManNAz was used (FIG. 9A). GO enrichment analysis using DAVID 1), 2) was performed on 241 genes whose gene expression was increased 1.5-fold or more in cells after 24 hours compared to the control. The results of the molecular function category are shown in FIG. 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.
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. 500 μL of 50 μM RPMI (−) FBS solution of 1a was added to the cells and incubated for 5 minutes. The medium was removed, washed with 500 µL of RPMI (-) FBS, and then suspended in 300 µL of RPMI (-) FBS. Azide-modified glass plates were placed in a 24-well plate using method B2, and 500 μL RPMI (−) FBS was added. 300 μL 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. Total RNA was extracted from cells 24 hours after adhesion. As a control, RNA from cells that had not been plated on glass after treatment with Ac 4 ManNAz was used (FIG. 9A). GO enrichment analysis using DAVID 1), 2) was performed on 241 genes whose gene expression was increased 1.5-fold or more in cells after 24 hours compared to the control. The results of the molecular function category are shown in FIG. 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.
実施例より、材料と細胞とが結合した複合体を構成する材料として、様々なものが使用できることが、明らかとなった。このような結果は、無置換ＣＯＤＹを用いた従来技術からは、到底推測されるものではなかった。なお、実施例に関する図２～７においては、極性官能基を有するジベンゾシクロオクタジインの極性官能基の記載を省略して記す場合、及び‐ＯＲと記す場合がある。
From the examples, it was clarified that various materials can be used as the materials that form the composites in which the materials and cells are bound. Such a result could not have been inferred from the prior art using unsubstituted CODY. 2 to 7 relating to the examples, the polar functional group of dibenzocyclooctadiyne having a polar functional group may be omitted, or may be indicated as -OR.
本明細書で引用した全ての刊行物、特許及び特許出願はそのまま引用により本明細書に組み入れられるものとする。
All publications, patents and patent applications cited herein are hereby incorporated by reference as is.
本明細書中に記載した数値範囲の上限値及び／又は下限値は、それぞれ任意に組み合わせて好ましい範囲を規定することができる。例えば、数値範囲の上限値及び下限値を任意に組み合わせて好ましい範囲を規定することができ、数値範囲の上限値同士を任意に組み合わせて好ましい範囲を規定することができ、また、数値範囲の下限値同士を任意に組み合わせて好ましい範囲を規定することができる。
The upper and/or lower limits of the numerical ranges described herein can be combined arbitrarily to define a preferred range. For example, 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, and the lower limit of the numerical range Any combination of values can be used to define a preferred range.
本明細書の全体にわたり、単数形の表現は、特に言及しない限り、その複数形の概念をも含むことが理解されるべきである。したがって、単数形の冠詞（例えば、英語の場合は「ａ」、「ａｎ」、「ｔｈｅ」等）は、特に言及しない限り、その複数形の概念をも含むことが理解されるべきである。
Throughout this specification, it should be understood that singular expressions also include their plural concepts unless otherwise specified. Thus, articles in the singular (eg, "a," "an," "the," etc. in the English language) should be understood to include their plural concepts as well, unless specifically stated otherwise.
以上、本実施形態を詳述したが、具体的な構成はこの実施形態に限定されるものではなく、本開示の要旨を逸脱しない範囲における設計変更があっても、それらは本開示に含まれるものである。
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 of the present disclosure, they are included in the present disclosure. It is.
Claims (19)
- 細胞表面にアジド基を有する細胞とアジド基を有する材料（但し、アジド基を有する蛍光色素を除く）とが、極性官能基を有するジベンゾシクロオクタジインによって結合した複合体。 A complex in which a cell with an azide group on the cell surface and a material with an azide group (excluding fluorescent dyes with an azide group) are bonded by dibenzocyclooctadiyne with a polar functional group.
- 材料が、細胞表面にアジド基を有する細胞及びアジド修飾された無機材料から選択される少なくとも１種の材料である、請求項１に記載の複合体。 The composite according to claim 1, wherein the material is at least one material selected from cells having an azide group on the cell surface and an azide-modified inorganic material.
- 無機材料が培養基板、ガラス基板、ＡＦＭカンチレバー、金属から選択される少なくとも１種の無機材料である、請求項２に記載の複合体。 The composite according to claim 2, wherein the inorganic material is at least one inorganic material selected from culture substrates, glass substrates, AFM cantilevers, and metals.
- 極性官能基を有するジベンゾシクロオクタジインが、下記一般式（Ｉ）で表される化合物である、請求項１～３のいずれか１項に記載の複合体。
- 極性官能基を有するジベンゾシクロオクタジインが、下記一般式（ＩＩ）で表される化合物である、請求項１～３のいずれか１項に記載の複合体。
- 極性官能基を有するジベンゾシクロオクタジインが、下記一般式（ＩＩＩ）で表される化合物である、請求項１～３のいずれか１項に記載の複合体。
- 極性官能基を有するジベンゾシクロオクタジインが、下記一般式（ＩＶ）で表される化合物である、請求項１～３のいずれか１項に記載の複合体。
- 極性官能基を有するジベンゾシクロオクタジインが、下記式１ａ～１ｊから選択される少なくとも１種の化合物である、請求項１～３のいずれか１項に記載の複合体。
- 細胞表面にアジド基を有する細胞が、細胞培養培地にアセチル化アジドマンノサミン（Ac4ManNAz）を加えることにより、細胞のシアル酸代謝経路を通じて、細胞表面の糖鎖末端のシアル酸部位にアジド基が導入された細胞である、請求項１～８のいずれか１項に記載の複合体。 By adding acetylated azidomannosamine (Ac 4 ManNAz) to the cell culture medium, cells with an azide group on the cell surface can transfer azide to the sialic acid site at the end of the sugar chain on the cell surface through the cellular sialic acid metabolism pathway. The complex according to any one of claims 1 to 8, which is a group-introduced cell.
- 細胞表面にアジド基を有する細胞と、極性官能基を有するジベンゾシクロオクタジインとを結合し、極性官能基を有するジベンゾシクロオクタジインと細胞との結合体（Ａ）を得る工程、及び、
結合体（Ａ）と、アジド基を有する材料（但し、アジド基を有する蛍光色素を除く）とを結合し、細胞と材料とが極性官能基を有するジベンゾシクロオクタジインによって結合した複合体を得る工程を有する、複合体の製造方法。 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. - アジド基を有する材料（但し、アジド基を有する蛍光色素を除く）と、極性官能基を有するジベンゾシクロオクタジインとを結合し、極性官能基を有するジベンゾシクロオクタジインと材料との結合体（Ｂ）を得る工程、及び、
結合体（Ｂ）と、細胞表面にアジド基を有する細胞とを結合し、細胞と材料とが極性官能基を有するジベンゾシクロオクタジインによって結合した複合体を得る工程を有する、複合体の製造方法。 A material having an azide group (excluding fluorescent dyes having an azide group) and dibenzocyclooctadiyne having a polar functional group are combined to form a conjugate of dibenzocyclooctadiyne having a polar functional group and the material (B ), and
A method for producing a conjugate, comprising the step of binding the conjugate (B) to a cell having an azide group on the cell surface to obtain a conjugate in which the cell and the material are bound by dibenzocyclooctadiyne having a polar functional group. . - 材料が、細胞表面にアジド基を有する細胞及びアジド修飾された無機材料から選択される少なくとも１種の材料である、請求項１０又は１１に記載の複合体の製造方法。 The method for producing a composite according to claim 10 or 11, wherein the material is at least one material selected from cells having an azide group on the cell surface and an azide-modified inorganic material.
- 無機材料が培養基板、ガラス基板、ＡＦＭカンチレバー、金属から選択される少なくとも１種の無機材料である、請求項１２に記載の複合体の製造方法。 The method for producing a composite according to claim 12, wherein the inorganic material is at least one inorganic material selected from culture substrates, glass substrates, AFM cantilevers, and metals.
- 極性官能基を有するジベンゾシクロオクタジインが、下記一般式（Ｉ）で表される化合物である、請求項１０～１３のいずれか１項に記載の複合体の製造方法。
- 極性官能基を有するジベンゾシクロオクタジインが、下記一般式（ＩＩ）で表される化合物である、請求項１０～１３のいずれか１項に記載の複合体の製造方法。
- 極性官能基を有するジベンゾシクロオクタジインが、下記一般式（ＩＩＩ）で表される化合物である、請求項１０～１３のいずれか１項に記載の複合体の製造方法。
- 極性官能基を有するジベンゾシクロオクタジインが、下記一般式（ＩＶ）で表される化合物である、請求項１０～１３のいずれか１項に記載の複合体の製造方法。
- 極性官能基を有するジベンゾシクロオクタジインが、下記式１ａ～１ｊから選択される少なくとも１種の化合物である、請求項１０～１３のいずれか１項に記載の複合体の製造方法。
- 下記式１ｂ～１ｊから選択される少なくとも１種の化合物。
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| Title |
|---|
| DEWEY A. SUTTON, SEOK-HO YU, RICHARD STEET, VLADIMIR V. POPIK: "Cyclopropenone-caged Sondheimer diyne (dibenzo[a,e]cyclooctadiyne): a photoactivatable linchpin for efficient SPAAC crosslinking", CHEMICAL COMMUNICATIONS, ROYAL SOCIETY OF CHEMISTRY, UK, vol. 52, no. 3, 1 January 2016 (2016-01-01), UK , pages 553 - 556, XP055589528, ISSN: 1359-7345, DOI: 10.1039/C5CC08106H * |
| KOHEI KITAGAWA, NAHO OKUMA, KENJU MATSUZAKI, HIROSHI YOSHIKAWA, MASAMI SUGANUMA, MASAYUKI TERA: "A24-3am-08 Cell surface modification using water-soluble strained diyne", BOOK OF ABSTRACTS, THE 101ST CSJ ANNUAL MEETING (2021); MARCH 19-22, 2021, CHEMICAL SOCIETY OF JAPAN, JP, 4 March 2021 (2021-03-04) - 22 March 2021 (2021-03-22), JP, pages A24 - 3am, XP009539388 * |
| MASAYUKI TERA, ZAHRA HARATI TAJI, NATHAN W. LUEDTKE: "Intercalation-enhanced "Click" Crosslinking of DNA", ANGEWANDTE CHEMIE INTERNATIONAL EDITION, VERLAG CHEMIE, vol. 57, no. 47, 19 November 2018 (2018-11-19), pages 15405 - 15409, XP055589511, ISSN: 1433-7851, DOI: 10.1002/anie.201808054 * |
| SHARMA KRISHNA, STRIZHAK ALEXANDER V., FOWLER ELAINE, WANG XUELU, XU WENSHU, HATT JENSEN CLAUS, WU YUTENG, SORE HANNAH F., LAU YU : "Water-soluble, stable and azide-reactive strained dialkynes for biocompatible double strain-promoted click chemistry", ORGANIC & BIOMOLECULAR CHEMISTRY, ROYAL SOCIETY OF CHEMISTRY, vol. 17, no. 34, 28 August 2019 (2019-08-28), pages 8014 - 8018, XP055961525, ISSN: 1477-0520, DOI: 10.1039/C9OB01745C * |
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