Compound Localized in Golgi Apparatus and Its Use

Abstract

To provide a compound specifically localized in a Golgi body.SOLUTION: A compound expressed by the following formula (1) [in formula (1), Z denotes a group expressed by the following formula (2) or (3), L denotes a divalent hydrocarbon group having the carbon number 1 to 50 which may be interrupted by substitution of -CH2- into -O-, -CO-, -NH- or a group expressed by the following formula (4), R1 denotes a group in which photodegradable protective group is further bound to a group induced from a labeling substance or a group induced from a medicine, a group which functions as a ligand of tag protein or a group which functions as a ligand of the tag protein, and in the following formula (2) and (3), R2 denotes a saturated or unsaturated straight-chain hydrocarbon group having the carbon number 9 or more, R3 severally independently denotes a hydrogen atom or a methyl group, and * denotes an asymmetric carbon atom]. [formula 1]SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a compound localized in the Golgi apparatus and its use. More specifically, the present invention relates to a novel compound, a Golgi apparatus labeling agent, a drug delivery system targeting the Golgi apparatus, an agent for localizing a tagged protein to the Golgi apparatus, a method for controlling the localization of a target protein in a cell, and a kit for controlling the localization of a tagged protein in a cell.

Background Art

[0002] The Golgi apparatus is an organelle present in eukaryotic cells. Most secreted proteins and membrane proteins are modified by glycosylation and cleavage in the Golgi apparatus and then sorted and delivered to organelles of the secretory pathway, the cell membrane, or the extracellular space. Dysfunctions in the structure and function of the Golgi apparatus are being revealed to cause abnormalities in the structure, function, and delivery of these proteins, and are associated with neurodegenerative diseases, glycosylation disorders, carcinogenesis, and the like.

[0003] As Golgi apparatus-labeling fluorescent probes, ceramide derivatives such as NBD-C6-Ceramide, BODIPY FL C5-Ceramide, and BODIPY TR Ceramide are known. When these compounds are added to cells, they are taken up into the cells and used as reagents for selectively staining the Golgi apparatus (see, for example, Non-Patent Documents 1 to 3).

[0004] The inventors have previously synthesized a compound (mgcTMP) in which trimethoprim (TMP), a ligand of Escherichia coli-derived dihydrofolate reductase (eDHFR), is bound to a localization motif for the cell membrane or the Golgi apparatus (see, for example, Non-Patent Document 4). By introducing mgcTMP into cells, a fusion protein of Escherichia coli-derived dihydrofolate reductase (eDHFR) expressed in the cytoplasm can be localized to the cell membrane or the Golgi apparatus. The chemical formula of trimethoprim is shown in the following formula (A1).

[0005]

Chemical Formula

[0006] Further, the chemical formula of mgcTMP is shown by the following formula (A2). Among mgcTMP, the portion consisting of a myristoyl group - glycine residue - cysteine residue functions as a localization motif to the cell membrane or Golgi apparatus.

[0007]

Chemical formula

Prior art documents

Non-patent documents

[0008]

Non-patent document 1

Non-patent document 2

Non-patent document 3

Non-patent document 4

Summary of the Invention

Problems to be Solved by the Invention

[0009] However, there is room for improvement in the specificity of conventional ceramide derivatives for the Golgi apparatus. In addition, mgcTMP is a compound that also localizes to the cell membrane and is not a compound that specifically localizes only to the Golgi apparatus. Therefore, an object of the present invention is to provide a compound that specifically localizes to the Golgi apparatus.

Means for Solving the Problems

[0010] The present invention includes the following aspects. [1] A compound represented by the following formula (1).

Chemical formula

Chemical formula

[10] After the step (a2), the method according to [9], further comprising a step (b) of introducing a ligand of the tag protein into the cell, wherein in the step (b), a group that functions as the ligand competes with the ligand, and the group that functions as the ligand and the ligand are substituted in at least a part of the tag protein localized in the Golgi apparatus, and as a result, the target protein moves from the Golgi apparatus.

[11] The method according to any one of [7] to

[10] , wherein the target protein is a signal transduction protein.

[12] A kit for controlling the localization of a tag protein in a cell, comprising the localization agent according to [6].

[13] The kit according to

[12] , further comprising a cell that expresses a fusion protein of the target protein and the tag protein, an expression vector of the fusion protein of the target protein and the tag protein, or a vector for producing the fusion protein of the target protein and the tag protein. The kit according to

[12] or

[13] , further comprising a ligand for the tag protein.

Advantages of the Invention

[0011] According to the present invention, a compound specifically localized in the Golgi apparatus can be provided.

Brief Description of the Drawings

[0012]

Figure 1

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Figure 27

Mode for Carrying Out the Invention

[0013] [Novel Compound] In one embodiment, the present invention provides a compound represented by the following formula (1).

Chemical formula

[0014] In the above formulas (2) and (3), at least one R 3 is preferably a methyl group. As will be described later in the examples, the compound of this embodiment localizes in the Golgi apparatus. More specifically, the compound of this embodiment localizes on the cytoplasmic side of the membrane of the Golgi apparatus. Further, by using the compound of this embodiment, a tag protein can be localized in the Golgi apparatus. In addition, by expressing the tag protein as a fusion protein with a target protein in cells, any target protein can be localized in the Golgi apparatus together with the tag protein.

[0015] Also, as will be described later in the examples, in order for the compound of this embodiment to localize in the Golgi apparatus, the structure of the group represented by the above formula (2) or (3) is particularly important. The group represented by the above formula (2) or (3) is a structure in which the portion consisting of a myristoyl group - glycine residue - cysteine residue among the above-described mgcTMP is modified.

[0016] Also, as will be described later in the examples, the inventors have clarified that if the carbon number of the group represented by R 2 is 9 or more, it can localize in the Golgi apparatus. The group represented by R 2 is preferably a saturated linear hydrocarbon group having 9 or more carbon atoms.

[0017] ​In the compound represented by the above formula (1), L is not particularly limited as long as it functions as a linker that links a group represented by the above formula (2) or (3) with a group derived from a labeling substance, a group derived from a drug, a group that functions as a ligand for a tag protein, or a group in which a photodegradable protecting group is further bonded to a group that functions as a ligand for the tag protein. More specifically, L represents a divalent hydrocarbon group having 1 to 50 carbon atoms, which may be interrupted by -CH2- being substituted with -O-, -CO-, -NH-, or a group represented by the above formula (4).

[0018] In the above formula (2), * indicates an asymmetric carbon atom. That is, the group represented by (2) may be a group represented by the following formula (2-1) or a group represented by the following formula (2-2). In the following formulas (2-1) and (2-2), R 2 and R 3 The groups represented by are the same as those in the above formula (2).

[0019]

Chemical formula

[0020] The SH group in the above formula (2-1) can also be said to be the side chain of the L-cysteine residue. Also, the SH group in the above formula (2-2) can also be said to be the side chain of the D-cysteine residue.

[0021] Similarly, in the above formula (3), * indicates an asymmetric carbon atom. That is, the group represented by (3) may be a group represented by the following formula (3-1) or a group represented by the following formula (3-2). In the following formulas (3-1) and (3-2), R 2 and R 3 The groups represented by are the same as those in the above formula (3).

[0022]

Chemical formula

[0023] The SH group in the above formula (3-1) can also be said to be the side chain of the L-cysteine residue. Further, the SH group in the above formula (3-2) can also be said to be the side chain of the D-cysteine residue.

[0024] When the group represented by the above formula (2) or (3) contains a D-cysteine residue, the localization of the compound represented by the above formula (1) to the Golgi apparatus tends to be maintained for a longer time.

[0025] In the compound of this embodiment, the combination of the tag protein and the group that functions as the ligand of the tag protein is not particularly limited, and any combination can be used.

[0026] Specific examples of the combination of the tag protein and the group that functions as the ligand of the tag protein include the combination of Escherichia coli dihydrofolate reductase (eDHFR) protein and the group represented by the following formula (5), the combination of FK506 binding protein (FKBP) and the group represented by the following formula (6), the combination of carbonic anhydrase I protein or carbonic anhydrase II protein and the group represented by the following formula (7), the combination of SNAP-tag (registered trademark) protein and the group represented by the following formula (8) or the following formula (9), the combination of HaloTag (registered trademark) protein and the group represented by the following formula (10), or the combination of photoactive yellow protein and the group represented by the following formula (11), etc.

[0027]

Chemical formula

[0028] The tag protein and the group that functions as a ligand for the tag protein may be modified as long as they can bind to each other. For example, the tag protein may have an amino acid sequence in which one or several amino acids are deleted, substituted, or added in the amino acid sequence of the tag protein described above. Here, one or several means, for example, it may be 1 to 10, for example, it may be 1 to 5, and for example, it may be 1 to 3. Further, the group that functions as a ligand for the tag protein may have a substituent in the group described above.

[0029] In the compound of this embodiment, a photocleavable protecting group may be further bound to the group that functions as a ligand for the tag protein. Such a compound is called a caged compound.

[0030] As will be described later in the examples, in the compound of this embodiment, when a photocleavable protecting group is further bound to the group that functions as a ligand for the tag protein, it is considered that its function can be controlled by light irradiation or the like.

[0031] In a state where a photocleavable protecting group is further bound to the group that functions as a ligand for the tag protein, the tag protein and the group that functions as a ligand for the tag protein do not bind. However, when the photocleavable protecting group is deprotected by irradiating light of a predetermined wavelength or the like, the tag protein and the group that functions as a ligand for the tag protein can bind.

[0032] The photocleavable protecting group is not particularly limited, and any one can be used. Specific examples of the photocleavable protecting group include, for example, a 6-nitroveratryloxycarbonyl (NVOC) group represented by the following formula (12), a 4,5-dimethoxy-2-nitrobenzyl (DMNB) group represented by the following formula (13), an α-methyl-6-nitropiperonyloxymethyl (MNPOM) group represented by the following formula (14), a (7-diethylaminocoumarin-4-yl)methyloxycarbonyl (DEACM) group represented by the following formula (15), a (7-diethylaminothiocoumarin-4-yl)methyloxycarbonyl (DEATCOC) group represented by the following formula (16), a (6-bromo-7-hydroxycoumarin-4-yl)methyloxycarbonyl (BHCOC) group represented by the following formula (17), an (8-bromo-7-hydroxyquinolin-2-yl)methyloxycarbonyl (BHQOC) group represented by the following formula (18), a (3-nitrodibenzofuran-2-yl)methyloxycarbonyl (NDBFOC) group represented by the following formula (19), a 3-nitrodibenzofuran-2-ylmethyl (NDBF) group represented by the following formula (20), and the like.

[0033] [Chemical formula]

[0034] [Golgi body labeling agent] In one embodiment, the present invention provides a Golgi body labeling agent comprising a compound in which R in the above formula (1) is a group derived from a labeling substance. 1 Examples of the labeling substance include a fluorescent substance, a Raman tag, a peptide tag, biotin, and the like.

[0035] Examples of the fluorescent substance include low molecular weight compounds such as fluorescein, coumarin, rhodamine, cyanine dye, BODIPY, and derivatives thereof.

[0036] Examples of the Raman tag include alkyne compounds and nitrile compounds.

[0037] Examples of the Raman tag include alkyne compounds and nitrile compounds.

[0038] Examples of the peptide tag include an HA tag, a myc tag, a His tag, and the like.

[0039] Examples of the group derived from the labeling substance include a monovalent group formed by removing a hydrogen atom, a hydroxyl group, or the like from the above-described labeling substance.

[0040] As will be described later in the examples, according to the Golgi body labeling agent of the present embodiment, the Golgi body can be specifically labeled.

[0041] [Drug Delivery System Targeting the Golgi Body] In one embodiment, the present invention provides a drug delivery system targeting the Golgi body, which comprises a compound in which R in the above formula (1) is a group derived from a drug. 1

[0042] The drug is not particularly limited, and examples thereof include an anticancer agent and a vesicular transport inhibitor. Examples of the anticancer agent include a compound that exhibits anticancer activity when delivered to the Golgi body. Examples of the vesicular transport inhibitor include (+)-brefeldin A and its derivatives.

[0043] Examples of the group derived from the drug include a monovalent group formed by removing a hydrogen atom, a hydroxyl group, or the like from the above-described drug.

[0044] According to the drug delivery system of the present embodiment, a drug can be specifically delivered to the Golgi body.

[0045] [Localizing Agent for Tagged Protein to the Golgi Body] In one embodiment, the present invention provides a localizing agent for a tagged protein to the Golgi body, which comprises a compound in which R in the above formula (1) is a group that functions as a ligand for the tagged protein. 1

[0046] The tag protein and the group that functions as a ligand for the tag protein are the same as those described above. As will be described later in the examples, the localization agent of the present embodiment can be used for the purpose of localizing the tag protein to the Golgi apparatus.

[0047] [Method for Controlling the Localization of a Target Protein in a Cell] (First Embodiment) The method of the first embodiment is a method for controlling the localization of a target protein in a cell, and inside the cell, a localization agent described above, wherein R 1 is a group that functions as a ligand for the tag protein, is introduced. As a result, the group that functions as the ligand of the localization agent binds to the tag protein, and the tag protein localizes to the Golgi apparatus together with the target protein in step (a). The cell is a cell that expresses a fusion protein of the target protein and the tag protein. The method of the first embodiment is a method for controlling the localization of a target protein in a cell using a localization agent that does not have a photodegradable protecting group.

[0048] In the method of the first embodiment, as the cell, a cell that expresses a fusion protein of a target protein and a tag protein is used. The cell is preferably a eukaryotic cell. Examples of eukaryotic cells include animal cells, insect cells, plant cells, yeast cells, and the like.

[0049] The number of target proteins contained in the fusion protein may be one or two or more. Also, the number of tag proteins contained in the fusion protein may be one or two or more. Here, when there are a plurality of target proteins, those target proteins may be of one type or two or more types. Also, when there are a plurality of tag proteins, those tag proteins may be of one type or two or more types.

[0050] In the fusion protein, the order of arrangement of the target protein and the tag protein is not particularly limited, and either may be present on the N-terminal side. For example, they may be arranged in the order of the target protein and the tag protein from the N-terminal side, or in the order of the tag protein and the target protein from the N-terminal side. Further, when there are a plurality of target proteins and tag proteins respectively, the order of their arrangement in the fusion protein is not particularly limited.

[0051] For example, as described later in the examples, the fusion protein may have one tag protein and two target proteins. The target protein is not particularly limited, and can be a fluorescent protein, a signal transduction protein, an enzyme, etc.

[0052] In the method of the first embodiment, in step (a), the above-described localizing agent is introduced into the cell in which the fusion protein of the target protein and the tag protein is expressed. The introduction of the localizing agent can be carried out, for example, by adding the localizing agent to the culture medium of the cells.

[0053] As described later in the examples, as a result of introducing the localizing agent into the cell, the group that functions as the ligand of the localizing agent binds to the tag protein, and the tag protein specifically localizes to the Golgi apparatus together with the target protein.

[0054] The method of the first embodiment may further include step (b) of introducing the ligand of the tag protein into the cell after step (a). In this case, in step (b), the group that functions as the ligand competes with the ligand, and the group that functions as the ligand and the ligand are substituted in at least a part of the tag protein localized in the Golgi apparatus, and as a result, the target protein moves from the Golgi apparatus. That is, the localization of the target protein is eliminated. By step (b), the localization of the localized substance can be controlled and eliminated.

[0055] (Second Embodiment) The method of the second embodiment is a method for controlling the localization of a target protein in a cell, and inside the cell, there is a localization agent described above, where the R 1 is a group in which a photodegradable protecting group is further bonded to a group that functions as a ligand for the tag protein. The method includes a step (a1) of introducing the localization agent into the cell, and after the step (a1), irradiating the cell with light. As a result, the photodegradable protecting group detaches from the localization agent, the group that functions as the ligand of the localization agent binds to the tag protein, and the tag protein localizes to the Golgi apparatus together with the target protein. The cell is a cell that expresses a fusion protein of the target protein and the tag protein. The method of the second embodiment mainly differs from the method of the first embodiment in that a caged compound is used as the localization agent.

[0056] In the method of the second embodiment, in step (a1), a caged compound as a localization agent is introduced into the cell of a cell that expresses a fusion protein of a target protein and a tag protein. In the method of the second embodiment, the method of introducing the localization agent into the cell is the same as that of the first embodiment.

[0057] Subsequently, after step (a1), a step (a2) of irradiating the cell with light is performed. The light irradiated in step (a2) may be light under conditions that can deprotect the photodegradable protecting group of the caged compound, and may be appropriately adjusted according to the used photodegradable protecting group. For example, as described later in the examples, when an NVOC group is used as the photodegradable protecting group, light with a wavelength of 405 nm may be irradiated. Also, when a DEACM group is used as the photodegradable protecting group, light with a wavelength of 405 nm, light with a wavelength of 445 nm, etc. may be irradiated.

[0058] As a result of irradiating with light, the photodegradable protecting group detaches from the localization agent. Subsequently, the group that functions as the ligand of the localization agent binds to the tag protein, and the tag protein localizes to the Golgi apparatus together with the target protein.

[0059] As will be described later in the examples, by the method of the second embodiment, the localization of the tag protein can be controlled at the single cell level or in a specific region on a single cell.

[0060] The method of the second embodiment may further include a step (b) of introducing a ligand of the tag protein into the cell after the step (a2). The step (b) in the method of the second embodiment is the same as the step (b) in the method of the first embodiment.

[0061] In this case, in the step (b), the group functioning as the ligand competes with the ligand, and the group functioning as the ligand substitutes for at least a part of the tag protein localized in the Golgi apparatus, and as a result, the target protein moves from the Golgi apparatus. That is, the localization of the target protein is eliminated. By the step (b), the localization of the localized target substance can be controlled and eliminated.

[0062] [Kit] In one embodiment, the present invention provides a kit for controlling the localization of a tag protein in a cell, which includes the localization agent described above. By using the kit of the present embodiment, the localization of the tag protein in the cell can be controlled.

[0063] In the kit of the present embodiment, the localization agent may or may not have a photodegradable protecting group.

[0064] The kit of the present embodiment may further include a cell expressing a fusion protein of a target protein and a tag protein, an expression vector of a fusion protein of a target protein and a tag protein, or a vector for producing a fusion protein of a target protein and a tag protein.

[0065] In the kit of this embodiment, the same cells as those described above can be used. When the kit of this embodiment contains cells expressing a fusion protein of a target protein and a tag protein, the localization of the tag protein in the cell can be controlled by introducing the above-described localization agent into the cell.

[0066] When the kit of this embodiment contains an expression vector for a fusion protein of a target protein and a tag protein, a cell expressing a fusion protein of a target protein and a tag protein can be prepared by introducing the expression vector into a desired cell and expressing it. By introducing the above-described localization agent into this cell, the localization of the tag protein in the cell can be controlled.

[0067] Examples of the vector for producing a fusion protein of a target protein and a tag protein include, for example, a vector containing a gene encoding a tag protein and a multiple cloning site downstream of a promoter. Either the gene encoding the tag protein or the multiple cloning site may be arranged on the promoter side. By cloning a gene encoding a desired target protein into the multiple cloning site of the vector for producing a fusion protein, an expression vector for a fusion protein of a target protein and a tag protein can be prepared.

[0068] Subsequently, a cell expressing a fusion protein of a target protein and a tag protein can be prepared by introducing the expression vector into a desired cell and expressing it. By introducing the above-described localization agent into this cell, the localization of the tag protein in the cell can be controlled.

[0069] The kit of this embodiment may further contain a ligand for the tag protein. As will be described later in the examples, after localizing the tag protein, the localization of the tag protein can be eliminated by introducing a ligand for the tag protein into the cell.

Examples

[0070] Next, examples will be shown to explain the present invention in more detail. However, the present invention is not limited to the following examples.

[0071] [Experimental Example 1] (Synthesis of Compounds) mgc(1Me)TMP with a chemical formula shown in the following formula (21), mgc(2Me)TMP with a chemical formula shown in the following formula (22), mgc(3Me)TMP with a chemical formula shown in the following formula (23), and mc(2Me)TMP with a chemical formula shown in the following formula (24) were each synthesized.

[0072]

Chem.

[0073]

Chem.

[0074]

Chem.

[0075]

Chem.

[0076] 《Synthesis of mgc(1Me)TMP》 mgc(1Me)TMP was synthesized according to the following scheme (1).

[0077]

Chem.

[0078] Specifically, mgc(1Me)TMP was synthesized on Rink amide resin according to standard solid-phase peptide synthesis using the 9-fluorenylmethyloxycarbonyl (Fmoc) protecting group and the N-methylation protocol.

[0079] Fmoc deprotection was carried out using 20% piperidine in N,N-dimethylformamide (DMF) at room temperature for 15 minutes. The amino acid coupling reaction for standard (non-methylated) amino acids was carried out at room temperature using a mixture of Fmoc-protected amino acid (3.1 equivalents), 1-[bis(dimethylamino)methylene]-1H-benzotriazolium 3-oxide hexafluorophosphate (HBTU, 3.0 equivalents), 1-hydroxybenzotriazole (HOBt, 3.0 equivalents), and N,N-diisopropylethylamine (DIPEA, 6.0 equivalents) in DMF. Also, the amino acid coupling reaction for N-methylated amino acids was carried out at room temperature using a mixture of Fmoc-protected amino acid (3.1 equivalents), 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazol[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU, 3.0 equivalents), 1-hydroxy-7-azabenzotriazole (HOAt, 3.0 equivalents), and N,N-diisopropylethylamine (DIPEA, 6.0 equivalents) in NMP. All Fmoc deprotection and coupling steps were monitored by Kaiser test and chloranil test. Also, all washing procedures were carried out using DMF or NMP.

[0080] First, Rink amide resin (0.55 mmol / g) (36.3 mg, 20 μmol) was Fmoc-deprotected and washed. Subsequently, Fmoc-Lys(Aloc)-OH was coupled to the resin and washed with DMF.

[0081] Subsequently, using Fmoc-Adox-OH as a building block, the Fmoc deprotection and coupling reactions were repeated three times.

[0082] Fmoc deprotection of the Fmoc-Adox terminus on the resin was performed, followed by N-methylation of the terminal amino group. For N-methylation, first, (1) the resin was treated with 2-nitrobenzenesulfonyl chloride (4.0 equivalents) and 2,4,6-collidine (10.0 equivalents) in NMP at room temperature for 15 minutes to effect 2-nitrobenzenesulfonylation. (2) After washing with NMP, the resin was treated with DBU (3.0 equivalents) in NMP for 3 minutes. Then, (3) dimethyl sulfate (10 equivalents) was added and the treatment was continued for another 2 minutes. N-methylation was carried out by repeating the operations of (2) to (3) once more. Next, the resin was treated with 2-mercaptoethanol (10.0 equivalents) and DBU (5.0 equivalents) in NMP for 5 minutes. By repeating this operation once more, deprotection of the 2-nitrobenzenesulfonyl group was achieved.

[0083] Subsequently, using Fmoc-Cys(Trt)-OH and Fmoc-Gly-OH as building blocks, Fmoc deprotection and coupling reactions were repeated.

[0084] The N-terminus was myristoylated using a mixture of myristic acid (4.1 equivalents), HBTU (4.0 equivalents), HOBt (4.0 equivalents), and DIPEA (8.0 equivalents) in DMF / CH2Cl2 (1:1).

[0085] Subsequently, the resin was treated with chloroform containing Pd(PPh3)4 (3.0 equivalents), acetic acid (5%), and 4-methylmorpholine (2.5%) to selectively deprotect the Aloc group. The resin was washed with DMF containing DIPEA (0.5%), and further washed with DMF containing sodium diethyldithiocarbamate (0.5%).

[0086] Subsequently, a mixture of compound X1 (3.1 equivalents), HBTU (3.0 equivalents), HOBt (3.0 equivalents), and DIPEA (8.0 equivalents) in DMF was reacted to attach compound X1 to the side chain of the lysine residue.

[0087] Trt deprotection and cleavage from the resin were carried out using trifluoroacetic acid (TFA) containing 2.5% triisopropylsilane (TIPS). Subsequently, the crude product was precipitated with diethyl ether and purified by reverse-phase HPLC on a semi-preparative C18 column using a linear gradient of acetonitrile containing 0.1% TFA and 0.1% hydrated TFA to obtain mgc(1Me)TMP as a white solid.

[0088] 1 1H NMR (400 MHz, CD3OD): δ 7.21 (1H, s), 6.56 (2H, s), 5.12 - 4.96 (1H, m), 4.42 (1H, m), 4.03 (2H, s), 4.00 (4H, s), 3.91, (2H, t), 3.86 (2H, s, s), 3.80 (6H, s), 3.66 (14H, m), 3.60 (6H, m), 3.46 (6H, m), 3.23 - 2.96 (5H, m), 2.88 - 2.72 (2H, m), 2.25 (4H, m), 1.80 (2H, m), 1.72 (2H, m), 1.61 (2H, m), 1.53 (2H, m), 1.40 (2H, m), 1.28 (22H, m), 0.89 (3H, t).

[0089] 《Synthesis of mgc(2Me)TMP》 According to the following Scheme (2), mgc(2Me)TMP was synthesized.

[0090]

Chemical formula

[0091] In Scheme (2), in addition to the N-methylation of Adox in Scheme (1), N-methylation of Cys was also carried out to synthesize mgc(2Me)TMP.

[0092] 11H NMR (400 MHz, CD3OD): δ 7.21 (1H, s), 6.56 (2H, s), 5.62 - 5.40 (1H, m), 4.41 (1H, m), 4.03 (2H, s), 4.00 (4H, s), 3.91 (2H, t), 3.80 (6H, s), 3.66 (16H, m), 3.60 (6H, m), 3.46 (6H, m), 3.17 (2H, t), 3.04 - 2.90 (6H, m), 2.68 - 2.56 (2H, m), 2.27 (4H, m), 1.80 (2H, m), 1.72 (2H, m), 1.62 (2H, m), 1.53 (2H, m), 1.40 (2H, m), 1.28 (22H, m), 0.90 (3H, t).

[0093] Synthesis of mgc(3Me)TMP mgc(3Me)TMP was synthesized according to the following Scheme (3).

[0094]

Chemical Structure

[0095] In Scheme (3), in addition to the N-methylation of Adox in Scheme (1), the N-methylation of Cys and the N-methylation of Gly were also carried out to synthesize mgc(3Me)TMP. Also, instead of the Rink amide resin in Scheme (1), Siber amide resin was used, instead of Fmoc-Cys(Trt)-OH, Fmoc-Cys(Mmt)-OH was used, and instead of DMF, NMP was used as the condensation solvent for myristic acid. Mmt deprotection and cleavage from the resin were carried out using CH2Cl2 containing 2.5% triisopropylsilane (TIPS) and 5% TFA.

[0096] 11H NMR (400 MHz, CD3OD): δ 7.21 (1H, s), 6.56 (2H, s), 5.66 - 5.44 (1H, m), 4.41 (1H, m), 4.03 (2H, s), 3.99 (4H, s), 3.91 (2H, t), 3.80 (6H, s), 3.66 (16H, m), 3.60 (6H, m), 3.45 (6H, m), 3.17 (2H, t), 3.10 - 2.89 (9H, m), 2.76 - 2.60 (2H, m), 2.44 (2H, m), 2.25 (2H, m), 1.80 (2H, m), 1.70 (2H, m), 1.61 (2H, m), 1.53 (2H, m), 1.38 (2H, m), 1.28 (22H, m), 0.90 (3H, t).

[0097] Synthesis of mc(2Me)TMP According to the following Scheme (4), mc(2Me)TMP was synthesized.

[0098]

Chemical Structure

[0099] In Scheme (4), mc(2Me)TMP was synthesized by excluding the coupling of Gly in Scheme (3).

[0100] 1 1H NMR (400 MHz, CD3OD): δ 7.22 (1H, s), 6.56 (2H, s), 5.66 - 5.44 (1H, m), 4.41 (1H, m), 4.03 (2H, s), 3.99 (4H, s), 3.93 (2H, t), 3.80 (6H, s), 3.66 (16H, m), 3.60 (6H, m), 3.46 (6H, m), 3.36 - 2.90 (6H, m), 2.68 - 2.54 (2H, m), 2.42 (2H, m), 2.25 (2H, m), 1.80 (2H, m), 1.72 (2H, m), 1.61 (2H, m), 1.53 (2H, m), 1.38 (2H, m), 1.28 (22H, m), 0.90 (3H, t).

[0101] [Experimental Example 2] (Examination of Localization) Using mgcTMP, mgc(1Me)TMP, mgc(2Me)TMP, mgc(3Me)TMP, and mc(2Me)TMP, the localization of the target protein in cells was examined. Specifically, first, a fusion protein of the target protein and a tag protein was expressed in HeLa cells, a cell line derived from human cervical cancer. EGFP was used as the target protein, and eDHFR was used as the tag protein. eDHFR was placed on the N-terminal side of the fusion protein, and EGFP was placed on the C-terminal side. Hereinafter, this fusion protein may be referred to as "DG". The amino acid sequence of DG is shown in SEQ ID NO: 1.

[0102] Subsequently, mgcTMP, mgc(1Me)TMP, mgc(2Me)TMP, mgc(3Me)TMP, and mc(2Me)TMP were each added to the medium of HeLa cells expressing the fusion protein (hereinafter sometimes referred to as "DG-expressing cells") to a final concentration of 10 μM. Subsequently, the fluorescence of EGFP was observed with a confocal laser microscope.

[0103] Figure 1(a) is a confocal laser micrograph. In Figure 1(a), "mgc" indicates the result of adding mgcTMP, "mgc(1Me)" indicates the result of adding mgc(1Me)TMP, "mgc(2Me)" indicates the result of adding mgc(2Me)TMP, "mgc(3Me)" indicates the result of adding mgc(3Me)TMP, "mc(2Me)" indicates the result of adding mc(2Me)TMP, "-" indicates the result before the addition of each compound, "+5min" indicates the result 5 minutes after the addition of each compound, "+30min" indicates the result 30 minutes after the addition of each compound, and "+60min" indicates the result 60 minutes after the addition of each compound.

[0104] Figure 1(b) is a graph that numerically represents over time the ratio (F / F0) of the fluorescence intensity F in the cytoplasm after the addition of the compound to the fluorescence intensity F0 in the cytoplasm before the addition of the compound. When EGFP localizes to the Golgi apparatus, the fluorescence intensity in the cytoplasm decreases. In Figure 1(b), the vertical axis represents the normalized fluorescence intensity (relative value) in the cytoplasm, and the horizontal axis represents the time (minutes) after the addition of the compound.

[0105] As a result, when mgcTMP was added, it became clear that EGFP localized to the cell membrane and the Golgi apparatus 5 minutes after the addition. Also, it became clear that the localization of EGFP spontaneously disappeared 40 minutes after the addition of mgcTMP.

[0106] Also, when mgc(1Me)TMP was added, it became clear that EGFP localized to the Golgi apparatus 5 minutes after the addition. Also, it became clear that the localization of EGFP spontaneously disappeared 60 minutes after the addition of mgc(1Me)TMP.

[0107] Also, when mgc(2Me)TMP, mgc(3Me)TMP, or mc(2Me)TMP was added, it became clear that EGFP localized to the Golgi apparatus 5 minutes after the addition. Also, when these compounds were added, it became clear that the localization of EGFP to the Golgi apparatus was maintained even after 60 minutes. Among them, mgc(3Me)TMP had high specificity for the Golgi apparatus, and a tendency for long-term localization to the Golgi apparatus was observed.

[0108] Figures 2(a) and (b) are confocal laser microscope photographs taken 180 minutes after the addition of mgc(3Me)TMP. The scale bar is 20 μm. Also, Figure 2(c) is a graph plotting the fluorescence intensity along the straight line shown in Figure 2(b).

[0109] Figures 2(d) and (e) are confocal laser microscope photographs taken 180 minutes after the addition of mc(2Me)TMP. The scale bar is 20 μm. Also, Figure 2(f) is a graph plotting the fluorescence intensity along the straight line shown in Figure 2(e).

[0110] As a result, it was confirmed that by adding these compounds, the fusion protein could be localized to the Golgi apparatus.

[0111] [Experimental Example 3] (Synthesis of Golgi apparatus labeling agent) mgc(3Me)FDA with the chemical formula shown in the following formula (25) and mgc(3Me)DEAC with the chemical formula shown in the following formula (26) were each synthesized.

[0112] [Chemical formula]

[0113] [Chemical formula]

[0114] 《Synthesis of mgc(3Me)FDA》 mgc(3Me)FDA was synthesized according to the following scheme (5).

[0115] [Chemical formula]

[0116] Specifically, mgc(Trt)(3Me)K was first synthesized on Sieber amide resin according to the same protocol as the synthesis of mgc(3Me)TMP.

[0117] First, Sieber amide resin (0.79 mmol / g) (50.6 mg, 40 μmol) was Fmoc-deprotected and washed. Subsequently, it was coupled to the resin in the order of Fmoc-Lys(Aloc)-OH and Fmoc-Adox-OH, and washed with DMF.

[0118] After the Fmoc deprotection of the Fmoc-Adox terminus on the resin, N-methylation was carried out in the same manner as in Scheme (3). Subsequently, using Fmoc-Cys(Trt)-OH and Fmoc-Gly-OH as building blocks, the coupling reaction, Fmoc deprotection, and N-methylation were repeated. Thereafter, N-terminal myristoylation was performed.

[0119] Subsequently, the Aloc group was selectively deprotected in the same manner as in Scheme (1).

[0120] Subsequently, the resin was treated in CH2Cl2 containing 5% TFA for 5 minutes, and the operation of recovering the solution was repeated 5 times. Toluene was added to the recovered solution and concentrated to obtain mgc(Trt)(3Me)K. Subsequently, the crude product was purified by reverse-phase HPLC using a linear gradient of acetonitrile containing 0.1% TFA and 0.1% hydrated TFA on a semi-preparative C4 column to obtain mgc(Trt)(3Me)K as a white solid.

[0121] Subsequently, in DMF, mgc(Trt)(3Me)K was reacted with 5-carboxyfluorescein diacetate (1.0 equivalent), HBTU (1.0 equivalent), and DIPEA (3.0 equivalents) to introduce 5-carboxyfluorescein diacetate (FDA) into the side chain of the lysine residue. After distilling off the solvent, Trt deprotection was carried out with CH2Cl2 containing 25% TFA and 2.5% TIPS. The crude product was purified by reverse-phase HPLC using a linear gradient of acetonitrile containing 0.1% TFA and 0.1% hydrated TFA on a semi-preparative C4 column to obtain mgc(Trt)(3Me)FDA as a white solid.

[0122] ESI-MS [M+Na] + Theoretical value: 1167.5295 Measured value: 1167.4677 [M+K] + Theoretical value: 1183.5034 Measured value: 1183.4409

[0123] 《Synthesis of mgc(3Me)DEAC》 mgc(3Me)DEAC was synthesized according to the following scheme (6). In scheme (6), mgc(3Me)DEAC was synthesized by reacting mgc(Trt)(3Me)K synthesized in scheme (5) with 7-(diethylamino)coumarin-3-carboxylic acid.

[0124] [Chemical formula]

[0125] ESI-MS [M+Na] + Theoretical value: 968.5501 Measured value: 968.6678 [M+K] + Theoretical value: 984.5241 Measured value: 984.6440

[0126] [Experimental Example 4] (Staining of Golgi apparatus) The Golgi apparatus of HeLa cells was stained using mgc(3Me)FDA, mgc(3Me)DEAC, and BODIPY FL C5-Ceramide (product number "D3521", Thermo Fisher Scientific), a conventional Golgi apparatus staining reagent.

[0127] 《Staining of Golgi apparatus 1 by mgc(3Me)FDA》 mgc(3Me)FDA was added to the medium of HeLa cells to a final concentration of 1 μM and incubated at room temperature for 5 minutes. Subsequently, it was washed with a medium containing 3 mg / mL bovine serum albumin (BSA) and observed under a microscope.

[0128] Figure 3(a) is a fluorescence micrograph. It was irradiated with excitation light of wavelength 488 nm, and fluorescence with a wavelength of 500 - 540 nm was observed. The scale bar is 20 μm. Figure 3(b) is an image obtained by synthesizing a fluorescence micrograph and a differential interference contrast micrograph in the same field of view as Figure 3(a). Also, Figure 3(c) is a graph plotting the fluorescence intensity along the straight line shown in Figure 3(b).

[0129] As a result, it was revealed that mgc(3Me)FDA can specifically stain the Golgi apparatus.

[0130] 《Staining of the Golgi Apparatus with mgc(3Me)FDA 2》 HeLa cells expressing the fusion protein mCherry-Giantin, which is a fusion protein of the fluorescent protein mCherry and the Golgi marker protein Giantin, were prepared. The amino acid sequence of mCherry-Giantin is shown in SEQ ID NO: 2. mgc(3Me)FDA was added to the medium of these cells to a final concentration of 1 μM and incubated at room temperature for 5 minutes. Subsequently, it was washed with a medium containing 3 mg / mL BSA and observed under a microscope.

[0131] Figure 4(a) is a fluorescence micrograph. Excitation light with a wavelength of 488 nm was irradiated, and fluorescence with a wavelength of 500 - 540 nm was observed. The scale bar is 20 μm. Figure 4(b) is a fluorescence micrograph of the same field of view as Figure 4(a), observing the fluorescence of mCherry. Figure 4(c) is an image synthesized from Figure 4(a) and Figure 4(b).

[0132] As a result, it was revealed that the fluorescence of mgc(3Me)FDA coincides with the fluorescence of mCherry. This result further supports the fact that mgc(3Me)FDA can specifically stain the Golgi apparatus.

[0133] 《Staining of the Golgi Apparatus with mgc(3Me)DEAC 1》 mgc(3Me)DEAC was added to the medium of HeLa cells to a final concentration of 1 μM and incubated at room temperature for 5 minutes. Subsequently, it was washed with a medium containing 3 mg / mL BSA and observed under a microscope.

[0134] Figure 5(a) is a fluorescence micrograph. The sample was irradiated with excitation light of wavelength 405 nm, and fluorescence with wavelengths of 430 - 470 nm was observed. The scale bar is 20 μm. Figure 5(b) is an image obtained by synthesizing a fluorescence micrograph and a differential interference contrast micrograph in the same field of view as in Figure 5(a). Further, Figure 5(c) is a graph plotting the fluorescence intensity along the straight line shown in Figure 5(b).

[0135] As a result, it was revealed that mgc(3Me)DEAC can specifically stain the Golgi apparatus.

[0136] 《Staining of Golgi Apparatus with mgc(3Me)DEAC 2》 HeLa cells expressing the fusion protein mCherry - Giantin, which is a fusion protein of the fluorescent protein mCherry and the Golgi marker protein Giantin, were prepared. mgc(3Me)DEAC was added to the medium of these cells to a final concentration of 1 μM, and the cells were incubated at room temperature for 5 minutes. Subsequently, the cells were washed with a medium containing 3 mg / mL BSA and observed under a microscope.

[0137] Figure 6(a) is a fluorescence micrograph. The sample was irradiated with excitation light of wavelength 405 nm, and fluorescence with wavelengths of 430 - 470 nm was observed. The scale bar is 20 μm. Figure 6(b) is a fluorescence micrograph of the fluorescence of mCherry observed in the same field of view as in Figure 6(a). Figure 6(c) is an image obtained by synthesizing Figure 6(a) and Figure 6(b).

[0138] As a result, it was revealed that the fluorescence of mgc(3Me)DEAC coincides with the fluorescence of mCherry. This result further supports the fact that mgc(3Me)DEAC can specifically stain the Golgi apparatus.

[0139] 《Staining of Golgi Apparatus with BODIPY FL C5 - Ceramide 1》 To the medium of HeLa cells, a complex of BODIPY FL C5-Ceramide (product number "D3521", Thermo Fisher Scientific) and BSA was added to a final concentration of 5 μM, and the mixture was incubated at 4 °C for 30 minutes. Subsequently, it was washed with buffer, further incubated at 37 °C for 30 minutes, and then observed under a microscope.

[0140] Figure 7(a) is a fluorescence micrograph. It was irradiated with excitation light of wavelength 488 nm, and fluorescence with wavelengths of 500 - 540 nm was observed. The scale bar is 20 μm. Figure 7(b) is an image obtained by synthesizing a fluorescence micrograph and a differential interference contrast micrograph in the same field of view as Figure 7(a). Also, Figure 7(c) is a graph plotting the fluorescence intensity along the straight line shown in Figure 7(b).

[0141] As a result, it became clear that it is difficult for BODIPY FL C5-Ceramide to specifically stain only the Golgi apparatus.

[0142] 《Staining of Golgi Apparatus with BODIPY FL C5-Ceramide 2》 HeLa cells expressing the fusion protein mCherry-Giantin, which is a fusion protein of the fluorescent protein mCherry and the Golgi marker protein Giantin, were prepared. To the medium of these cells, a complex of BODIPY FL C5-Ceramide (product number "D3521", Thermo Fisher Scientific) and BSA was added to a final concentration of 5 μM, and the mixture was incubated at 4 °C for 30 minutes. Subsequently, it was washed with buffer, further incubated at 37 °C for 30 minutes, and then observed under a microscope.

[0143] Figure 8(a) is a fluorescence micrograph. It was irradiated with excitation light of wavelength 488 nm, and fluorescence with wavelengths of 500 - 540 nm was observed. The scale bar is 20 μm. Figure 8(b) is a fluorescence micrograph observing the fluorescence of mCherry in the same field of view as Figure 8(a). Figure 8(c) is an image synthesized from Figure 8(a) and Figure 8(b).

[0144] As a result, it was revealed that the fluorescence of BODIPY FL C5-Ceramide did not coincide with the fluorescence of mCherry. This result further supports the fact that it is difficult to specifically stain only the Golgi apparatus with BODIPY FL C5-Ceramide.

[0145] [Experimental Example 5] (mg D Examination of localization by c(3Me)TMP) A localization agent in which the cysteine residue of mgc(3Me)TMP was replaced with the D-form (hereinafter sometimes referred to as "mg D c(3Me)TMP") was synthesized, and the localization ability of the tagged protein was examined.

[0146] 《Synthesis of mg D c(3Me)TMP》 First, mg D c(3Me)TMP was synthesized according to the following Scheme (7).

[0147]

Chemical formula

[0148] mg D c(3Me)TMP was synthesized according to the following Scheme (7). Specifically, the same reaction as in Scheme (3) was carried out, except that Fmoc-D-Cys(Mmt)-OH was used instead of Fmoc-Cys(Mmt)-OH in Scheme (3) of Experimental Example 1. Subsequently, the product was purified by reverse-phase HPLC to obtain mg D c(3Me)TMP as a white solid.

[0149] 11H NMR (400 MHz, CD3OD): δ 7.22 (1H, s), 6.56 (2H, s), 5.61 - 5.39 (1H, m), 4.41 (1H, m), 4.03 (2H, s), 3.99 (4H, s), 3.92 (2H, t), 3.80 (6H, s), 3.66 (16H, m), 3.60 (6H, m), 3.46 (6H, m), 3.18 (2H, t), 3.10 - 2.90 (9H, m), 2.76 - 2.60 (2H, m), 2.44 (2H, m), 2.25 (2H, m), 1.78 (2H, m), 1.72 (2H, m), 1.71 (2H, m), 1.57 (2H, m), 1.36 (2H, m), 1.29 (22H, m), 0.89 (3H, t).

[0150] 《mg D Examination of Localization by c(3Me)TMP》 mg was added to the medium of DG-expressing cells to a final concentration of 10 μM, and the fluorescence of EGFP was observed with a confocal laser microscope. D c(3Me)TMP was added, and the fluorescence of EGFP was observed with a confocal laser microscope.

[0151] Figures 9(a) to (d) are confocal laser micrographs. The scale bar is 20 μm. Figure 9(a) is a photograph of DG-expressing cells before the addition of mg D c(3Me)TMP. Also, Figure 9(b) is a photograph of DG-expressing cells taken 5 minutes after the addition of mg D c(3Me)TMP. Also, Figure 9(c) is a photograph of DG-expressing cells taken 30 minutes after the addition of mg D c(3Me)TMP. Also, Figure 9(d) is a photograph of DG-expressing cells taken 60 minutes after the addition of mg D c(3Me)TMP.

[0152] As a result, mg D it was revealed that EGFP was localized to the Golgi apparatus 5 minutes after the addition of c(3Me)TMP. Also, mg D it was revealed that the localization of EGFP to the Golgi apparatus was maintained even 60 minutes after the addition of c(3Me)TMP.

[0153] [Experimental Example 6] (Examination of the Structure of the Myristoyl Group Portion of mgc(3Me)TMP) The number of carbon atoms in the myristoyl group portion of mgc(3Me)TMP was modified, and the localization ability of the tagged protein was examined. First, in the compound represented by the following formula (27), R 2 The group represented by is the octyl group shown in the following formula (28) (hereinafter, may be referred to as "cap-gc(3Me)TMP"), and R in the compound represented by the following formula (27) 2 The group represented by is the nonyl group shown in the following formula (29) (hereinafter, may be referred to as "pel-gc(3Me)TMP") was synthesized.

[0154] [Chemical formula] [In formula (27), R 2 represents a linear hydrocarbon group having 9 or more carbon atoms and being saturated or unsaturated.]

[0155] [Chemical formula]

[0156] 《Synthesis of cap-gc(3Me)TMP》 First, cap-gc(3Me)TMP was synthesized according to the following scheme (8). Specifically, the same reaction as in scheme (3) was carried out except that capric acid was used instead of myristic acid in scheme (3) of Experimental Example 1. Subsequently, the product was purified by reverse-phase HPLC, and cap-gc(3Me)TMP was obtained as a white solid.

[0157] [Chemical formula]

[0158] 11H NMR (400 MHz, CD3OD): δ 7.22 (1H, s), 6.56 (2H, s), 5.61 - 5.39 (1H, m), 4.41 (1H, m), 4.03 (2H, s), 4.00 (4H, s), 3.92 (2H, t), 3.80 (6H, s), 3.66 (16H, m), 3.60 (6H, m), 3.46 (6H, m), 3.18 (2H, t), 3.19 - 2.90 (9H, m), 2.76 - 2.60 (2H, m), 2.44 (2H, m), 2.25 (2H, m), 1.80 (2H, m), 1.72 (2H, m), 1.60 (2H, m), 1.53 (2H, m), 1.38 (2H, m), 1.29 (16H, m), 0.89 (3H, t).

[0159] Synthesis of pel - gc(3Me)TMP Subsequently, pel - gc(3Me)TMP was synthesized according to the following Scheme (9). Specifically, the same reaction as in Scheme (3) was carried out, except that pelargonic acid was used instead of myristic acid in Scheme (3) of Experimental Example 1. Subsequently, the product was purified by reverse - phase HPLC to obtain pel - gc(3Me)TMP as a white solid.

[0160] [Chemical formula]

[0161] 1 1H NMR (400 MHz, CD3OD): δ 7.22 (1H, s), 6.56 (2H, s), 5.61 - 5.39 (1H, m), 4.41 (1H, m), 4.03 (2H, s), 4.00 (4H, s), 3.92 (2H, t), 3.80 (6H, s), 3.66 (16H, m), 3.60 (6H, m), 3.46 (6H, m), 3.18 (2H, t), 3.22 - 2.90 (9H, m), 2.76 - 2.60 (2H, m), 2.44 (2H, m), 2.25 (2H, m), 1.80 (2H, m), 1.72 (2H, m), 1.59 (2H, m), 1.53 (2H, m), 1.40 (2H, m), 1.29 (14H, m), 0.89 (3H, t).

[0162] "Examination of Localization by cap-gc(3Me)TMP and pel-gc(3Me)TMP" cap-gc(3Me)TMP was added to the medium of DG-expressing cells to a final concentration of 30 μM, and the fluorescence of EGFP was observed with a confocal laser microscope. Also, pel-gc(3Me)TMP was added to the medium of DG-expressing cells to a final concentration of 60 μM, and the fluorescence of EGFP was observed with a confocal laser microscope.

[0163] Figures 10(a) to (h) are confocal laser microscope photographs. The scale bar is 20 μm. Figures 10(a) to (d) show the results of adding cap-gc(3Me)TMP, and Figures 10(e) to (h) show the results of adding pel-gc(3Me)TMP. Figures 10(a) to (d) are photographs of DG-expressing cells taken before the addition of cap-gc(3Me)TMP, 5 minutes after the addition of cap-gc(3Me)TMP, 30 minutes after the addition of cap-gc(3Me)TMP, and 60 minutes after the addition of cap-gc(3Me)TMP, respectively. Also, Figures 10(e) to (h) are photographs of DG-expressing cells taken before the addition of pel-gc(3Me)TMP, 5 minutes after the addition of pel-gc(3Me)TMP, 30 minutes after the addition of pel-gc(3Me)TMP, and 60 minutes after the addition of pel-gc(3Me)TMP, respectively.

[0164] As a result, when cap-gc(3Me)TMP was added, localization of EGFP to the Golgi apparatus was observed. In contrast, when pel-gc(3Me)TMP was added, it became clear that the localization of EGFP to the Golgi apparatus was significantly reduced. From this result, in order to localize the compound represented by the above formula (25) to the Golgi apparatus, it was considered necessary that the number of carbon atoms in the group represented by R 2 be 9 or more.

[0165] [Experimental Example 7] (Examination of Localization by mgc(3Me)SLF * ) In HeLa cells, a fusion protein of a target protein and a tag protein was expressed. EGFP was used as the target protein, and FKBP(F36V) was used as the tag protein. FKBP(F36V) was placed on the N-terminal side of the fusion protein, and EGFP was placed on the C-terminal side. Hereinafter, this fusion protein may be referred to as "FKBP(F36V)-EGFP". The amino acid sequence of FKBP(F36V)-EGFP is shown in SEQ ID NO: 3.

[0166] 《Synthesis of mgc(3Me)SLF * 》 mgc(3Me)SLF was synthesized according to the following scheme (10). * Specifically, the same reaction as in Scheme (3) in Experimental Example 1 was carried out except that the compound represented by compound X2 (SLF * -COOH) was used instead of compound X1. Subsequently, the product was purified by reverse-phase HPLC to obtain mgc(3Me)SLF * as a white solid.

[0167]

Chemical formula

[0168] 11H NMR (400 MHz, CD3OD): δ 7.19 (1H, m), 6.88 - 6.84 (2H, m), 6.81 (1H, m), 6.73 (1H, m), 6.68 (1H, m), 6.59 (2H, s), 6.54 (1H, m), 5.58 (1H, m), 5.50 (1H, m), 5.39 (1H, m), 4.52 (2H, m), 4.42 (1H, m), 4.03 (2H, s), 3.99 (4H, s), 3.88 (m, 2H), 3.82 (6H, m), 3.78 (3H, s), 3.69 (6H, s), 3.66 (14H, m), 3.59 (6H, m), 3.45 (6H, m), 3.26 (2H, m), 3.10 - 2.89 (9H, m), 2.73 (2H, m), 2.70 (1H, m), 2.43 (2H, m), 2.25 (2H, m), 2.03 (2H, m), 1.73 (2H, m), 1.61 (4H, m), 1.59 (6H, m), 1.37 (2H, m), 1.28 (22H, m), 0.89 (6H, t).

[0169] Examination of Localization by mgc(3Me)SLF * mgc(3Me)SLF was added to the medium of HeLa cells expressing FKBP(F36V)-EGFP (hereinafter sometimes referred to as "FKBP(F36V)-EGFP-expressing cells") to a final concentration of 10 μM. Subsequently, the fluorescence of EGFP was observed with a confocal laser microscope. *

[0170] Figures 11(a) to (d) are confocal laser microscope photographs. The scale bar is 20 μm. Figure 11(a) is a photograph of FKBP(F36V)-EGFP-expressing cells before the addition of mgc(3Me)SLF. * Also, Figure 11(b) is a photograph of FKBP(F36V)-EGFP-expressing cells taken 5 minutes after the addition of mgc(3Me)SLF. * Also, Figure 11(c) is a photograph of FKBP(F36V)-EGFP-expressing cells taken 30 minutes after the addition of mgc(3Me)SLF. * Also, Figure 11(d) is a photograph of FKBP(F36V)-EGFP-expressing cells taken after the addition of mgc(3Me)SLF * ​This is a photograph of FKBP(F36V)-EGFP-expressing cells taken 60 minutes after the addition of

[0171] As a result, 5 minutes after the addition of mgc(3Me)SLF * it was revealed that EGFP was localized to the Golgi apparatus. Also, even 60 minutes after the addition of mgc(3Me)SLF * it was revealed that the localization of EGFP to the Golgi apparatus was maintained.

[0172] From the above results, it became clear that combinations of groups that function as tag proteins and ligands can localize tag proteins even when they are combinations other than the combination of eDHFR and TMP.

[0173] [Experimental Example 8] (Control of intracellular signal using mgc(3Me)TMP 1) Intracellular signals were controlled using mgc(3Me)TMP. First, an expression vector for a fusion protein of a target protein and a tag protein was prepared. As the target proteins, red fluorescent protein (RFP) and RasGEF were used. As the tag protein, eDHFR was used. RFP, eDHFR, and RasGEF were arranged in this order from the N-terminal side of the fusion protein. Hereinafter, this fusion protein may be referred to as "RD-RasGEF". The amino acid sequence of RD-RasGEF is shown in SEQ ID NO: 4.

[0174] Also, for comparison, an expression vector for a fusion protein in which RFP and eDHFR were arranged in this order from the N-terminal side was also prepared. Hereinafter, this fusion protein may be referred to as "RD". The amino acid sequence of RD is shown in SEQ ID NO: 5.

[0175] Also, an expression vector for a fusion protein of blue fluorescent protein (BFP) and Ras binding domain (RBD) (hereinafter sometimes referred to as "BFP-RBD") was prepared. The amino acid sequence of BFP-RBD is shown in SEQ ID NO: 6.

[0176] In addition, an expression vector for a fusion protein of EGFP and NRas (hereinafter sometimes referred to as "EGFP-NRas") was prepared. The amino acid sequence of EGFP-NRas is shown in SEQ ID NO: 7.

[0177] Subsequently, HeLa cells expressing RD-RasGEF, EGFP-NRas, and BFP-RBD were prepared. Also, for comparison, HeLa cells expressing RD, EGFP-NRas, and BFP-RBD were prepared.

[0178] Subsequently, mgc(3Me)TMP was added to the medium of each cell to a final concentration of 2.5 μM. Subsequently, the fluorescence of RFP, the fluorescence of BFP, and the fluorescence of EGFP were observed with a confocal laser microscope, respectively.

[0179] Figure 12 is a schematic diagram for explaining this experimental example. As shown in Figure 12, NRas is localized in the cell membrane and the Golgi apparatus. BFP-RBD is localized in the nucleus and the cytoplasm. When mgc(3Me)TMP is added to the medium to localize RasGEF in the Golgi apparatus, EGFP-NRas localized in the Golgi apparatus is activated. As a result, BFP-RBD binds to the activated NRas and translocates to the Golgi apparatus.

[0180] By observing the fluorescence of RFP, the localization of RD-RasGEF can be detected. Also, by observing the fluorescence of BFP, the localization of BFP-RBD can be detected. Also, by observing the fluorescence of EGFP, the localization of EGFP-NRas can be detected.

[0181] Figure 13(a) is a fluorescence micrograph of HeLa cells expressing RD-RasGEF, EGFP-NRas, and BFP-RBD. Figure 13(b) is a fluorescence micrograph of HeLa cells expressing RD, EGFP-NRas, and BFP-RBD. In Figures 13(a) and (b), "-" indicates a photograph before the addition of mgc(3Me)TMP, and "+mgcTMP_3Me" indicates a photograph 30 minutes after the addition of mgc(3Me)TMP. In Figures 13(a) and (b), the scale bar indicates 20 μm.

[0182] Figure 14(a) is a graph showing the results of measuring over time the ratio of the localization of RD-RasGEF to the Golgi apparatus and the ratio of the localization of BFP-RBD to the Golgi apparatus or nucleus in HeLa cells expressing RD-RasGEF, EGFP-NRas, and BFP-RBD (n = 5). In Figure 14(a), the values on the graph indicate the mean ± standard deviation. The left vertical axis indicates the ratio of localization to the Golgi apparatus or nucleus, and the higher the value, the higher the ratio of localization to the Golgi apparatus. The right vertical axis indicates the ratio of localization to the Golgi apparatus, and the higher the value, the more specifically localized to the Golgi apparatus. The horizontal axis indicates time (minutes).

[0183] Figure 14(b) is a graph showing the results of measuring over time the ratio of the localization of RD to the Golgi apparatus and the ratio of the localization of BFP-RBD to the Golgi apparatus or nucleus in HeLa cells expressing RD, EGFP-NRas, and BFP-RBD (n = 5). In Figure 14(b), the values on the graph indicate the mean ± standard deviation. The left vertical axis indicates the ratio of localization to the Golgi apparatus or nucleus, and the higher the value, the higher the ratio of localization to the Golgi apparatus. The right vertical axis indicates the ratio of localization to the Golgi apparatus, and the higher the value, the more specifically localized to the Golgi apparatus. The horizontal axis indicates time (minutes).

[0184] As a result, in HeLa cells expressing RD-RasGEF, EGFP-NRas, and BFP-RBD, it became clear that RD-RasGEF was localized to the Golgi apparatus and BFP-RBD was localized to the Golgi apparatus after the addition of mgc(3Me)TMP.

[0185] On the other hand, in HeLa cells expressing RD, EGFP-NRas, and BFP-RBD, although RD localized to the Golgi apparatus after the addition of mgc(3Me)TMP, the localization of BFP-RBD to the Golgi apparatus was not observed.

[0186] This result indicates that intracellular signals can be controlled using mgc(3Me)TMP.

[0187] [Experimental Example 9] (Control of Intracellular Signals Using mgc(3Me)TMP 2) Intracellular signals were controlled using mgc(3Me)TMP. First, an expression vector for a fusion protein of a target protein and a tag protein was prepared. EGFP and Sac1 were used as the target proteins. eDHFR was used as the tag protein. EGFP, eDHFR, and Sac1 were arranged in order from the N-terminal side of the fusion protein. Hereinafter, this fusion protein may be referred to as "GD-Sac1". The amino acid sequence of GD-Sac1 is shown in SEQ ID NO: 8.

[0188] Also, for comparison, a fusion protein in which EGFP, eDHFR, and Sac1 lacking phosphatase activity (hereinafter sometimes referred to as "Sac1-dead") were arranged in order from the N-terminal side was prepared. Hereinafter, this fusion protein may be referred to as "GD-Sac1-dead". The amino acid sequence of GD-Sac1-dead is shown in SEQ ID NO: 9.

[0189] Also, an expression vector for a fusion protein of a red fluorescent protein (RFP) and SidM (hereinafter sometimes referred to as "RFP-P4M-SidM") was prepared. The amino acid sequence of RFP-P4M-SidM is shown in SEQ ID NO: 10.

[0190] Subsequently, GD-Sac1 and RFP-P4M-SidM were expressed in COS-7 cells, which are cells derived from African green monkey kidneys. Also, COS-7 cells expressing GD-Sac1-dead and RFP-P4M-SidM were prepared for comparison.

[0191] Subsequently, mgc(3Me)TMP was added to the medium of each cell to a final concentration of 10 μM. Subsequently, the fluorescence of EGFP and the fluorescence of RFP were observed with a confocal laser microscope, respectively.

[0192] Figure 15 is a schematic diagram for explaining this experimental example. As shown in Figure 15, SidM binds to phosphatidylinositol-4-monophosphate (PI4P) and is localized in the Golgi apparatus. When mgc(3Me)TMP is added to the medium to localize Sac1 in the Golgi apparatus, Sac1 dephosphorylates PI4P, and as a result, SidM translocates from the Golgi apparatus to the cytoplasm.

[0193] By observing the fluorescence of EGFP, the localization of GD-Sac1 can be detected. Also, by observing the fluorescence of RFP, the localization of RFP-P4M-SidM can be detected.

[0194] Figure 16(a) is a fluorescence micrograph of COS-7 cells expressing GD-Sac1 and RFP-P4M-SidM. Also, Figure 16(b) is a fluorescence micrograph of COS-7 cells expressing GD-Sac1-dead and RFP-P4M-SidM. In Figures 16(a) and (b), "-" indicates a photograph before adding mgc(3Me)TMP, and "+mgcTMP_3Me" indicates a photograph 30 minutes after adding mgc(3Me)TMP.

[0195] Figure 17(a) is a graph showing the results of measuring the ratio of the localization of GD-Sac1 to the Golgi apparatus or cytoplasm over time in COS-7 cells expressing GD-Sac1 and RFP-P4M-SidM (n = 3). Figure 17(b) is a graph showing the results of measuring the ratio of the localization of RFP-P4M-SidM to the Golgi apparatus or cytoplasm over time in COS-7 cells expressing GD-Sac1 and RFP-P4M-SidM (n = 3). In Figures 17(a) and (b), the values in the graph indicate the mean ± standard deviation. Also, the vertical axis indicates the degree of the ratio of localization to the Golgi apparatus, and the higher the value, the more specifically localized to the Golgi apparatus. The horizontal axis indicates time (minutes).

[0196] Figure 17(c) is a graph showing the results of measuring the ratio of the localization of GD-Sac1 to the Golgi apparatus or cytoplasm over time in COS-7 cells expressing GD-Sac1-dead and RFP-P4M-SidM (n = 3). Figure 17(d) is a graph showing the results of measuring the ratio of the localization of RFP-P4M-SidM to the Golgi apparatus or cytoplasm over time in COS-7 cells expressing GD-Sac1-dead and RFP-P4M-SidM (n = 3). In Figures 17(c) and (d), the values in the graph indicate the mean ± standard deviation. Also, the vertical axis indicates the degree of the ratio of localization to the Golgi apparatus, and the higher the value, the more specifically localized to the Golgi apparatus. The horizontal axis indicates time (minutes).

[0197] As a result, in COS-7 cells expressing GD-Sac1 and RFP-P4M-SidM, it became clear that after the addition of mgc(3Me)TMP, GD-Sac1 was localized to the Golgi apparatus and RFP-P4M-SidM migrated to the cytoplasm.

[0198] On the other hand, in COS-7 cells expressing GD-Sac1-dead and RFP-P4M-SidM, although GD-Sac1-dead was localized to the Golgi apparatus after the addition of mgc(3Me)TMP, the migration of RFP-P4M-SidM to the cytoplasm was not observed.

[0199] This result further supports the fact that intracellular signals can be controlled using mgc(3Me)TMP.

[0200] [Experimental Example 10] (m D c NVOC TMP synthesis) A caged localization agent was synthesized. Specifically, m D cTMP, a compound in which a photodegradable protecting group is further bonded to the trimethoprim group of m D c NVOC TMP was synthesized. As the photodegradable protecting group, the NVOC group represented by the above formula (12) was used. The NVOC group can be deprotected, for example, by irradiating light with a wavelength of 405 nm. m D c NVOC The synthesis of c

[0201] [Chemical Formula]

[0202] 1 H NMR (400 MHz, d-DMSO): δ0.89 (t, 3H), 1.43 - 1.25 (m, 22H), 1.86 - 1.49 (m, 10H), 2.28 - 2.23 (m, 4H), 2.78 - 2.72 (m, 1H), 2.88 - 2.83 (m, 1H), 3.18 (t, 2H), 3.70 - 3.34 (m, 24H), 3.76 (s, 6H), 4.04 - 3.84 (m, 12H), 4.47 - 4.40 (m, 2H), 5.60 (s, 2H), 6.54 (s, 2H), 7.20 (s, 1H), 7.70 (s, 1H), 7.77 (s, 1H).

[0203] [Experimental Example 11] (Induction of Localization by Light Irradiation 1) In HeLa cells, a fusion protein of a target protein and a tag protein was expressed. As the target protein, mScarlet I, a fluorescent protein, was used, and as the tag protein, eDHFR was used. In addition, a K6 tag (a peptide tag with six linked lysine residues) was placed at the N-terminus of eDHFR.

[0204] Figure 18(a) is a schematic diagram showing the structure of the expressed fusion protein. Hereinafter, this fusion protein may be referred to as "K6-DS". The amino acid sequence of K6-DS is shown in SEQ ID NO: 11.

[0205] Subsequently, to the medium of HeLa cells (hereinafter sometimes referred to as "K6-DS-expressing cells") in which K6-DS was expressed, m D c NVOC TMP was added to a final concentration of 10 μM. Subsequently, m D c NVOC The cells were washed 5 minutes after the addition of TMP. Also, for comparison, K6-DS-expressing cells to which m D c NVOC TMP was not added were also prepared. Subsequently, light with a wavelength of 405 nm was irradiated for 2.6 seconds, and the fluorescence of mScarlet I was observed with a confocal laser microscope.

[0206] Figures 18(b) to (d) are confocal laser microscope photographs of K6-DS-expressing cells to which m D c NVOC TMP was added. Figure 18(b) is a photograph of K6-DS-expressing cells before light irradiation. Also, Figure 18(c) is a photograph of K6-DS-expressing cells 10 seconds after the start of the experiment. Also, Figure 18(d) is a photograph of K6-DS-expressing cells 60 seconds after the start of the experiment. Also, Figure 18(e) is a graph showing the experimental results in numerical values. In Figure 18(e), the horizontal axis indicates time (seconds), the vertical axis indicates the fluorescence intensity ratio of the cytoplasm, "+m D c NVOC TMP" indicates the result of K6-DS-expressing cells to which m D c NVOC TMP was added, and "-m D c NVOC TMP" indicates m D cNVOC It shows the results of K6-DS expressing cells without the addition of TMP.

[0207] As a result, m D c NVOC In K6-DS expressing cells with the addition of TMP, it was revealed that mScarlet I was localized to the cell membrane immediately after light irradiation.

[0208] From the above results, it became clear that by using a compound having a photocleavable protecting group, it is possible to control the localization induction of a tagged protein by light irradiation.

[0209] [Experimental Example 12] (Localization induction by light irradiation 2) Figure 19 is a graph showing the results of measuring the absorption spectrum of m D c NVOC TMP. As shown in Figure 19, it became clear that m D c NVOC TMP absorbs light with a wavelength of 405 nm but hardly absorbs light with a wavelength of 445 nm.

[0210] Subsequently, K6-DS expressing cells were treated with m D c NVOC TMP, light irradiation with a wavelength of 405 nm, light irradiation with a wavelength of 445 nm, and free trimethoprim (hereinafter sometimes referred to as "TMP") in various combinations, and the fluorescence of mScarlet I was observed with a confocal laser microscope. Subsequently, based on the confocal laser microscope photographs, the localization transfer efficiency of the tagged protein was measured. m D c NVOC TMP was added at a final concentration of 10 μM, light with a wavelength of 405 nm was irradiated for 2.6 seconds, light with a wavelength of 445 nm was irradiated for 2.6 seconds, and TMP was added at a final concentration of 100 μM.

[0211] Figure 20 is a graph showing the results of measuring the localization transfer efficiency of the tagged protein. In Figure 20, "-" represents m D c NVOCIndicates that TMP was not added, or that light irradiation at a wavelength of 405 nm or 445 nm was not performed. A "+" indicates m D c NVOC Indicates that TMP was added, or that light irradiation at a wavelength of 405 nm or 445 nm was performed.

[0212] Also, the vertical axis of the graph shows the localization transfer efficiency calculated by the following formula (F1). Localization transfer efficiency = 1 - (fluorescence intensity of cytoplasm 5 minutes after the start of the experiment / fluorescence intensity of cytoplasm at the start of the experiment (0 minutes)) …(F1)

[0213] As a result, m D c NVOC It was revealed that mScarlet I was localized to the cell membrane when TMP was added and light irradiation at a wavelength of 405 nm was performed. Also, it was revealed that the localization of mScarlet I to the cell membrane was suppressed in the presence of TMP.

[0214] [Experimental Example 13] (Induction of Localization by Light Irradiation 3) The induction of localization by light irradiation at the single-cell level was examined. First, m D c NVOC TMP was added to the medium of K6-DS-expressing cells at a final concentration of 10 μM, and observation was performed with a confocal laser microscope. Fig. 21(a) is a confocal laser microscope photograph at the start of the experiment. Subsequently, using the function of the confocal laser microscope, only the cell indicated as "Cell 1 " in Fig. 21(a) was irradiated with light at a wavelength of 405 nm for 2.6 seconds. Fig. 21(b) is a confocal laser microscope photograph 60 seconds after the start of the experiment. As a result, it was revealed that mScarlet I was localized to the cell membrane in the cell indicated as "Cell 1 ".

[0215] Fig. 21(c) is a confocal laser microscope photograph 600 seconds after the start of the experiment. Subsequently, using the function of the confocal laser microscope, only the cell indicated as "Cell 2Only the cells labeled "」 were irradiated with light of wavelength 405 nm for 2.6 seconds. Figure 21(d) is a confocal laser microscopy photograph taken 660 seconds after the start of the experiment. As a result, it was revealed that mScarlet I was localized to the cell membrane in the cells labeled "Cell 2 ".

[0216] Figure 21(e) is a confocal laser microscopy photograph taken 1200 seconds after the start of the experiment. Subsequently, using the function of the confocal laser microscope, only the cells labeled "Cell 3 " in Figure 21(e) were irradiated with light of wavelength 405 nm for 2.6 seconds. Figure 21(f) is a confocal laser microscopy photograph taken 1260 seconds after the start of the experiment. As a result, it was revealed that mScarlet I was localized to the cell membrane in the cells labeled "Cell 3 ". Also, the positions of the cells labeled "Cell 4 " are shown in Figure 21(f).

[0217] Figure 21(g) is a confocal laser microscopy photograph taken 1800 seconds after the start of the experiment. Subsequently, free TMP was added to the cell culture medium at a final concentration of 100 μM. Figure 21(h) is a confocal laser microscopy photograph taken 2400 seconds after the start of the experiment. As a result, it was revealed that the localization of mScarlet I to the cell membrane was abolished in the cells labeled "Cell 1 ", "Cell 2 ", and "Cell 3 ".

[0218] Figure 21(i) is a graph quantifying the change in the fluorescence intensity ratio of the cytoplasm in the cells labeled "Cell 1 ", "Cell 2 ", "Cell 3 ", "Cell 4 " in Figures 21(a) to (h). The horizontal axis indicates the time (seconds) from the start of the experiment.

[0219] [Experimental Example 14] (Induction of Localization by Light Irradiation 4) The control of cell movement signals by light irradiation was investigated. First, a fusion protein of a target protein and a tag protein was prepared. mScarlet I and Tiam1 were used as the target proteins. eDHFR was used as the tag protein. eDHFR, mScarlet I, and Tiam1 were arranged in order from the N-terminal side of the fusion protein. In addition, a K6 tag was further arranged on the N-terminal side of eDHFR. Hereinafter, this fusion protein may be referred to as "K6-DS-Tiam1". The amino acid sequence of K6-DS-Tiam1 is shown in SEQ ID NO: 12.

[0220] In addition, a fusion protein of Lifeact and mNeonGreen (hereinafter may be referred to as "Lifeact-mNeonGreen") was prepared. The amino acid sequence of Lifeact-mNeonGreen is shown in SEQ ID NO: 13. Lifeact is a peptide that specifically binds to F-actin.

[0221] Subsequently, K6-DS-Tiam1 and Lifeact-mNeonGreen were expressed in NIH3T3 cells, which are mouse embryo-derived fibroblasts. Subsequently, in the medium of NIH3T3 cells expressing K6-DS-Tiam1 and Lifeact-mNeonGreen (hereinafter may be referred to as "K6-DS-Tiam1 and Lifeact-mNeonGreen-expressing cells"), m D c NVOC TMP was added to a final concentration of 10 μM. Subsequently, m D c NVOC The cells were washed 5 minutes after the addition of TMP.

[0222] Subsequently, the fluorescence of mScarlet I and the fluorescence of mNeonGreen were observed with a confocal laser microscope, respectively. Figures 22(a) and (c) are confocal laser microscope photographs at the start of the experiment. Subsequently, using the function of the confocal laser microscope, the region surrounded by the square in Figure 22(a), that is, the entire cell, was irradiated with light of wavelength 405 nm for 2.6 seconds.

[0223] Figures 22(b) and (d) are confocal laser microscope photographs taken 300 seconds after the start of the experiment. Further, Figure 22(e) is an image showing regions where the areas of K6-DS-Tiam1- and Lifeact-mNeonGreen-expressing cells increased or decreased by comparing the cells at the start of the experiment with those 300 seconds after the start of the experiment. In Figure 22(e), "increase" indicates a region where the area increased compared to the start of the experiment, and "decrease" indicates a region where the area decreased compared to the start of the experiment.

[0224] As a result, it became clear that the areas of K6-DS-Tiam1- and Lifeact-mNeonGreen-expressing cells changed upon irradiation with light having a wavelength of 405 nm. From this result, it became clear that the cell motility signal can be controlled by light irradiation by using a compound having a photodegradable protecting group.

[0225] [Experimental Example 15] (Induction of Localization by Light Irradiation 5) m D c NVOC TMP was added to the medium of K6-DS-Tiam1- and Lifeact-mNeonGreen-expressing cells to a final concentration of 10 μM.

[0226] Subsequently, the fluorescence of mScarlet I and the fluorescence of mNeonGreen were observed with a confocal laser microscope, respectively. Figures 23(a) and (c) are confocal laser microscope photographs taken at the start of the experiment. Subsequently, using the function of the confocal laser microscope, in Figure 23(a), the region surrounded by a circle, that is, a part of the cell, was locally irradiated with light having a wavelength of 405 nm for 2.6 seconds.

[0227] Figures 23(b) and (d) are confocal laser microscope photographs taken 300 seconds after the start of the experiment. Further, Figure 23(e) is an image showing regions where the areas of K6-DS-Tiam1- and Lifeact-mNeonGreen-expressing cells increased or decreased by comparing the cells at the start of the experiment and 300 seconds after the start of the experiment. In Figure 23(e), "increase" indicates a region where the area increased compared to the start of the experiment, and "decrease" indicates a region where the area decreased compared to the start of the experiment.

[0228] As a result, it became clear that the areas of K6-DS-Tiam1- and Lifeact-mNeonGreen-expressing cells changed upon irradiation with light having a wavelength of 405 nm. From this result, it was further supported that by using a compound having a photocleavable protecting group, it is possible to control cell motility signals by light irradiation.

[0229] [Experimental Example 16] (Induction of Localization by Light Irradiation 6) m D c NVOC TMP was added to the medium of NIH3T3 cells expressing K6-DS-Tiam1 (hereinafter sometimes referred to as "K6-DS-Tiam1-expressing cells") so as to have a final concentration of 10 μM. Further, for comparison, m D c NVOC TMP was added to the medium of NIH3T3 cells expressing K6-DS (hereinafter sometimes referred to as "K6-DS-expressing cells") so as to have a final concentration of 10 μM.

[0230] Subsequently, the fluorescence of mScarlet I was observed with a confocal laser microscope. Subsequently, using the function of the confocal laser microscope, light having a wavelength of 405 nm was locally irradiated to the whole cell or a part of the cell for 2.6 seconds. Subsequently, changes in the cell morphology were observed with a confocal laser microscope.

[0231] Figures 24(a) to (d) are images showing regions where the area has increased or decreased, comparing K6-DS-Tiam1-expressing cells or K6-DS-expressing cells at the start of the experiment and 300 seconds after the start of the experiment. In Figures 24(a) to (d), "increase" indicates a region where the area has increased compared to the start of the experiment, and "decrease" indicates a region where the area has decreased compared to the start of the experiment.

[0232] Figure 24(a) shows typical results of irradiating the entire cell with light of wavelength 405 nm, and Figures 24(b) to (d) show typical results of locally irradiating a part of the cell with light of wavelength 405 nm. In Figures 24(a) to (d), "+m D c NVOC TMP" indicates that m D c NVOC TMP was added, "-m D c NVOC TMP" indicates that m D c NVOC TMP was not added, "+K6-DS-Tiam1" indicates the result of K6-DS-Tiam1-expressing cells, and "+K6-DS" indicates the result of K6-DS-expressing cells. Figures 24(c) and (d) are the results of negative controls. Also, in Figures 24(b) to (d), the regions surrounded by circles indicate the regions irradiated with light of wavelength 405 nm.

[0233] Also, Figure 25(a) is a graph showing the amount of area increase in the protruding region (region where the area has increased) of the cell, and Figure 25(b) is a graph showing the amount of area decrease in the degenerating region (region where the area has decreased) of the cell. Also, in Figures 25(a) and (b), "whole" indicates the result of irradiating the entire cell with light of wavelength 405 nm, "local" indicates the result of locally irradiating a part of the cell with light of wavelength 405 nm, "+" indicates that m D c NVOC TMP was added, the result of K6-DS-Tiam1-expressing cells, or the result of K6-DS-expressing cells, and "-" indicates that m D c NVOC TMP was not added, K6-DS-Tiam1-expressing cells were not used, or K6-DS-expressing cells were not used.

[0234] As a result, it was revealed that the area of K6-DS-Tiam1-expressing cells changed upon irradiation with light of a wavelength of 405 nm. From this result, it was further supported that by using a compound having a photodegradable protecting group, it is possible to control cell motility signals by light irradiation.

[0235] [Experimental Example 17] (m D c DEACM Synthesis of TMP) A caged localizer having a wavelength for deprotecting a photodegradable protecting group different from that of m D c NVOC TMP was synthesized. Specifically, according to the following scheme (12), m D cTMP, which is a compound in which a photodegradable protecting group is further bonded to the trimethoprim group of m D c DEACM TMP was synthesized. As the photodegradable protecting group, the DEACM group represented by the above formula (15) was used. The DEACM group can be deprotected, for example, by irradiating with light of a wavelength of 445 nm.

[0236] [Chemical Formula]

[0237] 11H NMR (400 MHz, d-DMSO): δ 0.85 (t, 3H), 1.12 (t, 6H), 1.18 - 1.41 (m, 22H), 1.45 - 1.75 (m, 10H), 2.03 - 2.22 (m, 5H), 2.58 - 2.80 (m, 2H), 2.99 (q, 2H), 3.50 - 3.62 (m, 15H), 3.69 (s, 6H), 3.75 (t, 2H), 3.80 (s, 2H), 3.85 - 3.95 (m, 6H), 4.22 - 4.36 (m, 2H), 5.37 (s, 2H), 6.15 (s, 1H), 6.51 (s, 2H), 6.56 (d, 1H), 6.69 (dd, 1H), 7.09 (s, 1H), 7.46 (d, 1H), 7.49 (s, 1H), 7.55 (d, 1H), 7.69 (q, 2H), 7.74 (t, 1H), 7.99 (t, 2H), 8.03 (t, 1H), 10.15 (brs, 1H).

[0238] Figure 26 shows m D c NVOC The absorption spectra of TMP and m D c DEACM This is a graph showing the results of measuring the absorption spectra of TMP. As shown in Figure 26, m D c NVOC TMP does not absorb light at a wavelength of 445 nm, but m D c DEACM it was revealed that TMP can absorb light at a wavelength of 445 nm.

[0239] [Experimental Example 18] (Localization induction by light irradiation 7) mTMP was added to the medium of HeLa cells expressing K6-DS (hereinafter sometimes referred to as "K6-DS expressing cells") to a final concentration of 10 μM. D c DEACM Subsequently, m D c DEACM the cells were washed 5 minutes after the addition of mTMP. Subsequently, the cells were irradiated with light at a wavelength of 445 nm for 2.6 seconds, and the fluorescence of mScarlet I was observed with a confocal laser microscope.

[0240] Figures 27(a) and (b) show m D c DEACMConfocal laser microscopy images of K6-DS-expressing cells to which TMP was added. Fig. 27(a) is a photograph of K6-DS-expressing cells before light irradiation. Fig. 27(b) is a photograph of K6-DS-expressing cells after light irradiation.

[0241] As a result, m D c DEACM It was revealed that in K6-DS-expressing cells to which TMP was added, localization of the tagged protein could be induced by irradiating light with a wavelength of 445 nm.

[0242] Fig. 27(c) is a graph showing the results of measuring the localization transfer efficiency of the tagged protein after irradiating K6-DS-expressing cells to which TMP was added with light having a wavelength of 405 nm or 445 nm. D c NVOC Fig. 27(d) is a graph showing the results of measuring the localization transfer efficiency of the tagged protein after irradiating K6-DS-expressing cells to which TMP was added with light having a wavelength of 405 nm or 445 nm.

[0243] Fig. 27(d) is a graph showing the results of measuring the localization transfer efficiency of the tagged protein after irradiating K6-DS-expressing cells to which TMP was added with light having a wavelength of 405 nm or 445 nm. D c DEACM Fig. 27(d) is a graph showing the results of measuring the localization transfer efficiency of the tagged protein after irradiating K6-DS-expressing cells to which TMP was added with light having a wavelength of 405 nm or 445 nm.

[0244] In Figs. 27(c) and (d), the horizontal axis of the graph indicates the intensity (%) of the laser output, and the vertical axis of the graph indicates the localization transfer efficiency calculated by the following formula (F1). Localization transfer efficiency = 1 - (fluorescence intensity of cytoplasm 5 minutes after the start of the experiment / fluorescence intensity of cytoplasm at the start of the experiment (0 minutes))... (F1)

[0245] As a result, m D c NVOC In K6-DS-expressing cells to which TMP was added, it was revealed that the tagged protein localizes to the cell membrane upon irradiation with 405 nm light, but there is little change in the localization of the tagged protein upon irradiation with 445 nm light. On the other hand, m D c DEACM In K6-DS-expressing cells to which TMP was added, it was revealed that the tagged protein localizes to the cell membrane regardless of whether it is irradiated with 405 nm light or 445 nm light.

[0246] From the above results, it became clear that it is possible to control the induction of localization of the tagged protein by using compounds having photodegradable protecting groups with different absorption wavelengths and controlling the wavelength of the irradiated light.

Industrial Applicability

[0247] According to the present invention, it is possible to provide a compound that specifically localizes in the Golgi apparatus.

Claims

1. A compound represented by the following formula (1). 【Chemical 1】 [In formula (1), Z represents a group represented by the following formula (2) or (3), and L is a divalent hydrocarbon group having 1 to 50 carbon atoms which may be interrupted by -CH ２ - being replaced by -O-, -CO-, -NH- or a group represented by the following formula (4), and R １ represents a group derived from a labeling substance or a group derived from a drug, a group that functions as a ligand for a tag protein, or a group in which a photodegradable protecting group is further bonded to a group that functions as a ligand for the tag protein. The labeling substance is a fluorescent substance, a Raman tag, a peptide tag or biotin. The drug is an anticancer agent or a vesicular transport inhibitor. In the following formulas (2) and (3), R ２ represents a linear hydrocarbon group having 9 or more carbon atoms that is saturated or unsaturated, and R ３ each independently represents a hydrogen atom or a methyl group, and at least one of the plurality of R ３ is a methyl group, and * indicates an asymmetric carbon atom. [Chemical Formula 2] ］

2. The combination of the tag protein and the group that functions as a ligand for the tag protein is a combination of the Escherichia coli dihydrofolate reductase (eDHFR) protein and the group represented by the following formula (5), a combination of the FK506-binding protein and the group represented by the following formula (6), a combination of the carbonic anhydrase I protein or the carbonic anhydrase II protein and the group represented by the following formula (7), a combination of the SNAP-tag (registered trademark) protein and the group represented by the following formula (8) or the following formula (9), a combination of the HaloTag (registered trademark) protein and the group represented by the following formula (10), or a combination of the photoactive yellow protein and the group represented by the following formula (11). The compound according to claim 1. [Chemical 3]

3. The compound according to claim 1 or 2, wherein the photocleavable protecting group is a group represented by any of the following formulas (12) to (20). 【Chemical 4】

4. Said R １ is a group derived from a labeling substance, and the Golgi body labeling agent comprising the compound according to claim 1.

5. Said R １ A Golgi apparatus-targeted drug delivery system comprising the compound according to claim 1, wherein R is a group derived from a drug.

6. Said R １ is a group that functions as a ligand for the tag protein, and is a localization agent for the Golgi apparatus of the tag protein, comprising the compound according to any one of claims 1 to 3.

7. A method for controlling the localization of a target protein in a cell in vitro, comprising: Into the interior of the cell, a localization agent according to claim 6 is introduced, wherein the R １ is a group that functions as a ligand for the tag protein, such that as a result, the group that functions as the ligand of the localization agent binds to the tag protein, and the tag protein localizes to the Golgi apparatus together with the target protein, including step (a). The cell is a cell that expresses a fusion protein of the target protein and the tag protein. The method.

8. After the step (a), the method further includes a step (b) of introducing a ligand of the tag protein into the cell, In the step (b), the group that functions as the ligand competes with the ligand, and the group that functions as the ligand and the ligand substitute in at least a part of the tag protein localized in the Golgi apparatus, and as a result, the target protein moves from the Golgi apparatus. The method according to claim 7.

9. A method for controlling the localization of a target protein in a cell in vitro, comprising: Into the interior of the cell, there is introduced a localization agent according to claim 6, wherein a photodegradable protecting group is further bonded to a group in which R １ functions as a ligand for the tag protein, in step (a1); After the step (a1), the cell is irradiated with light, and as a result, the photocleavable protecting group detaches from the localizing agent, and the group that functions as the ligand of the localizing agent binds to the tag protein, and the tag protein localizes in the Golgi apparatus together with the target protein. Step (a2). The cell is a cell that expresses a fusion protein of the target protein and the tag protein. The method.

10. After the step (a2), the method further includes a step (b) of introducing a ligand of the tag protein into the cell, ​ The method according to claim 9, wherein in the step (b), a group that functions as the ligand competes with the ligand, and the group that functions as the ligand and the ligand are substituted in at least a part of the tagged protein localized in the Golgi apparatus, and as a result, the target protein moves from the Golgi apparatus.

11. The method according to any one of claims 7 to 10, wherein the target protein is a signal transduction protein.

12. A kit for controlling the localization of a tagged protein in a cell, comprising the localization agent according to claim 6.

13. A cell expressing a fusion protein of a target protein and the tagged protein, an expression vector of a fusion protein of the target protein and the tagged protein, or, The kit according to claim 12, further comprising a vector for producing a fusion protein of the target protein and the tagged protein.

14. The kit according to claim 12 or 13, further comprising a ligand of the tagged protein.

15. A compound represented by the following formula (1). 【Chemical Formula 5】 [In formula (1), Z represents a group represented by the following formula (2) or (3), and L is a divalent hydrocarbon group having 1 to 50 carbon atoms which may be interrupted by -CH ２ - being replaced by -O-, -CO-, -NH- or a group represented by the following formula (4), and R １ represents a group that functions as a ligand for a protein or a group in which a photodegradable protecting group is further bonded to a group that functions as a ligand for the protein. In the following formulas (2) and (3), R ２ represents a linear hydrocarbon group having 9 or more carbon atoms that is saturated or unsaturated, R ３ each independently represents a hydrogen atom or a methyl group, and at least one of the plurality of R ３ is a methyl group, and * indicates an asymmetric carbon atom. [Chemical Formula 6] ］

16. The combination of the protein and the group that functions as the ligand of the protein is a combination of Escherichia coli dihydrofolate reductase (eDHFR) protein and the group represented by the following formula (5), FK506 binding protein and the group represented by the following formula (6), carbonic anhydrase I protein or carbonic anhydrase II protein and the group represented by the following formula (7), SNAP-tag (registered trademark) protein and the group represented by the following formula (8) or the following formula (9), HaloTag (registered trademark) protein and the group represented by the following formula (10), or a combination of photoactive yellow protein and the group represented by the following formula (11). The compound according to claim 15. 【Chemical Formula 7】

17. The compound according to claim 15 or 16, wherein the photodegradable protecting group is a group represented by any one of the following formulas (12) to (20). 【Chemical Formula 8】

18. A localization agent for the Golgi apparatus of the protein, comprising the compound according to any one of claims 15 to 17.

19. A method for controlling the localization of a protein in a cell in vitro, comprising: Into the interior of the cell, a localization agent according to claim 18 is introduced, wherein R １ is a group that functions as a ligand for the protein, and as a result, the group that functions as the ligand of the localization agent binds to the protein, and the protein is localized in the Golgi apparatus, including step (a).

20. after the step (a), further comprising a step (b) of introducing the ligand of the protein into the cell. The method according to claim 19, wherein in the step (b), a group functioning as the ligand competes with the ligand, and the group functioning as the ligand and the ligand are substituted in at least a part of the protein localized in the Golgi apparatus, and as a result, the protein moves from the Golgi apparatus.

21. A method for controlling the localization of a protein in a cell in vitro, comprising: Into the interior of the cell, a localization agent according to claim 18, wherein the R １ is a step (a1) of introducing a localization agent which is a group in which a photodegradable protecting group is further bonded to a group that functions as a ligand for the protein, a step (a2) of irradiating the cell with light after the step (a1), as a result, the photodegradable protecting group is detached from the localizing agent, a group functioning as the ligand of the localizing agent binds to the protein, and the protein is localized in the Golgi apparatus.

22. further comprising a step (b) of introducing a ligand of the protein into the cell after the step (a2), The method according to claim 21, wherein in the step (b), a group functioning as the ligand competes with the ligand, and the group functioning as the ligand and the ligand are substituted in at least a part of the protein localized in the Golgi apparatus, and as a result, the protein moves from the Golgi apparatus.

23. The method according to any one of claims 19 to 22, wherein the protein is a signal transduction protein.

24. A kit for controlling the localization of a protein in a cell, comprising the localizing agent according to claim 18.

25. The kit according to claim 24, further comprising a ligand of the protein.

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